Plant Pathology (2009) 58, 982–989 Doi: 10.1111/j.1365-3059.2009.02100.x

Basalstemrotofoilpalm(Elaeisguineensis);modeof rootinfectionandlowersteminvasionbyGanodermaboninense

R. W. Reesa, J. Floodb, Y. Hasanc, U. Potterd and R. M. Coopera*aDepartment of Biology and Biochemistry, University of Bath, Claverton Down, Bath, BA2 7AY; bCABI Europe-UK, Bakeham Lane,

Egham Surrey, TW20 9AY UK; cBah Lias Research Station, PTPP London Sumatra Indonesia Tbk, PO Box 1154, 20011 Medan, Indonesia;

and dCentre for Electron Optical Studies, University of Bath, Bath BA2 7AY, UK

Reproducible infection of intact roots of oil palm (Elaeis guineensis) with Ganoderma boninense, the cause of basal stem

rot, showed penetration followed by rapid longitudinal progression of hyphae and colonization of the lower stem (bole).

Light and transmission electron microscopy showed invasion of the root cortex, with no evidence of selective progression

through the vascular system or lacunae. In newly colonized tissue the fungus behaved as a hemibiotroph, with numerous,

wide, intracellular hyphae occupying entire host cells that possessed intact cell walls and contained discernible cytoplasm

and organelles. In the bole this phase coincided with a complete depletion of previously abundant starch grains in advance

of invasion. Subsequently, in the roots and colonized stem base, widespread necrotrophic, enzymatic attack of all layers of

the host cell walls occurred. Hyphae were intra- and intercellular and intramural and associated host cell wall degradation

was often at a distance from hyphae, resulting in cavities within cell walls. A third developmental stage was the formation of

an extensive, melanized, tough mycelium or pseudo-sclerotium which surrounded roots and comprised many very thick-

walled cells encasing more typical thin-walled hyphae. Macroscopic observation of and isolation from the bole of randomly

felled, commercial palms provided confirmatory evidence that multiple infections originated in the roots before spreading

into the base of long-established palms.

Keywords: cell wall degradation, hemibiotrophy, root infection, starch degradation, white rot

Introduction

The greatest threat to oil palm (Elaeis guineensis)production in South East Asia is from stem rot, caused bythe white-rot fungus Ganoderma boninense. The mostcommon manifestation of the disease is basal stem rot(BSR), where progressive decay of the root system andultimately of the lower stem induces disease symptoms.Symptoms comprise water stress, one-sided mottling ofthe canopy, flattening of the crown, multiple unopenedspears and production of basidiocarps on the lower stem(Turner, 1981). Upper stem rot (USR) occurs much lessfrequently, with stem decay and basidiocarp productionat higher elevations (Flood et al., 2003), sometimesresulting in fracture and toppling of the stem above thepoint of infection.

The primary route of infection appears to be throughroot contact with inoculum sources in the soil. Numerousinfection trials using oil palm seedlings and often largeGanoderma-colonized rubber-wood blocks have provided

*E-mail: bssrmc@bath.ac.uk

Published online 8 June 2009

982

data supporting this view (Navaratnam & Chee, 1965;Lim et al., 1992; Sariah et al., 1994; Hasan & Turner,1998; Lim & Fong, 2005; Breton et al., 2006). Rees et al.(2007) showed that by attaching infested wood blocksto roots much smaller inoculum can be used, allowinginfection to occur through unwounded roots and progres-sion and rate of invasion to be followed. Root infectionalso occurs by several other root-infecting basidiomycetediseases of trees and perennial crops, which are spread byvegetative growth. Heterobasidion annosum infects liv-ing roots if they contact colonized dead roots of pinestumps initially infected by basidiospores (Woodwardet al., 1998). Root infection by Armillaria species occursprimarily by means of rhizomorphs or by direct mycelialgrowth from diseased to healthy roots (Onsando, 1997).

Argument against root infection as the primary meansof BSR comes from genetic studies that show a high degreeof heterogeneity of G. boninense in oil palm plantations(Miller et al., 1999; Pilotti et al., 2004; Pilotti, 2005). Thisis atypical of tree-pathogenic basidiomycetes known to bespread by root contact (Woodward et al., 1998). However,heterogeneity may originate from the saprotrophiccomponent of the G. boninense life cycle on oil palm debrisin the field that might initiate infection (Flood et al.,

ª 2009 The Authors

Journal compilation ª 2009 BSPP

Ganoderma boninense infection of oil palm 983

2003). Nevertheless, how this colonization of oil palmdebris occurs remains uncertain, because G. boninense isa poor competitor in non-sterile soil or in the organicdebris that accumulates at the frond-stem junctions (Reeset al., 2007). Also, infection from a basidiospore inoculumhas never been reported from the field, althoughmonokaryotic mycelium derived from a single basidios-pore can colonize sterilised oil palm wood blocks (Hasan& Flood, 2003).

The initial stagesof infectionand the routeof colonizationare poorly understood and are remarkably controversial,as is the status of Ganoderma as a true pathogen (Pater-son, 2007). Inoculation procedures described by Reeset al. (2007) were used to investigate the mode of infec-tion in roots under controlled conditions. Roots and basalstems of naturally infected, excavated palms were exam-ined under field conditions in commercial plantings inIndonesia. This paper describes different stages bothmacroscopically and microscopically, of the developmentof Ganoderma on and in oil palm roots and its progressvia the root cortex to the stem base.

Materials and methods

Fungal strain and isolation from palms

Ganoderma boninense isolate (GMR3) (Rees et al., 2007)was used for infection of palm seedlings in the UK and fornursery tests at Bah Lias Research Station (BLRS), NorthSumatra, Indonesia. It was isolated from an infected oilpalm in Gunung Malayu Estate in North Sumatra usingGanoderma selective medium (GSM) (Ariffin & Seman,1991) and was maintained on potato dextrose agar asdescribed by Rees et al. (2007). All isolations from rootsand boles of plantation palms were onto GSM. Carefulexcision of palm tissue combined with the medium’sselectivity excludes most contamination. Colonymorphology and characteristic brown staining of thesurrounding agar aided identification of G. boninense.Sub-culturing onto PDA was performed if further confir-mation was required.

Oil palm seedlings

Twelve-month-old oil palm seedlings used for infectionstudies were commercial Deli dura · AVROS pisiferacrosses supplied by BLRS. Progeny from two crosses wereused: A1121/03-13 · BL148/05-08 and A1123/33-05 ·BL148/05-08. Palms were maintained under glasshouseconditions as described by Rees et al. (2007).

Preparation of wood blocks and inoculation of oilpalm seedlings

Oil palm wood blockswere obtained from BLRSand treatedby a method adapted from Adaskaveg et al. (1990) asdescribed by Rees et al. (2007). Briefly, wood blocks werecut to 3 cm3, sterilized before inoculation with G. boninenseand incubated for 4 weeks. Colonized blocks were then

Plant Pathology (2009) 58, 982–989

physically attached to oil palm primary roots withParafilm. Progress of colonization during 5 months wasmonitored as described (Rees et al., 2007) and images ofmacromorphological symptoms were taken and micro-scopy was performed on fixed and embedded sectionsfrom diseased tissue.

Random felling of mature palms

All observations of mature palm infections were fromnatural infections in the field. Palms from GunungMalayu Estate were planted in 1989 following previousoil palm plantings in 1952 (Fig. 2). Trees of 5, 7, 9, 13 and15 years, without symptoms, were identified at randomlocations. Six replicates at each sampling age weremarked and the boles of the trees were excavated, and theroot mass was observed for characteristic Ganoderma-induced lesions. The root mass was then removed by axeand the resulting bole surface examined for basal stemrot. A transverse section was also taken across each bole.The boles and adjacent trunk were cut into longitudinalsections to examine the extent of lesions. Routineisolations from root, bole and stem lesions during thisstudy confirmed their origin as Ganoderma. The isolateused here, GMR3, was isolated in this way. It should benoted that in the 15 year age class, there were someinfected trees with Ganoderma basidiocarps and foliarsymptoms; some of these were also destructively har-vested in addition to the experimental replicatesdescribed above (see Results).

Light microscopy of infected oil palm seedling roots

Samples for light, UV and transmission electron microscopy(TEM) were taken from root and bole tissues showingdifferent stages of invasion, i.e. primarily from the non-necrotic zone in advance of visible infection, the necroticborder and from tissue with advanced necrosis. Semi-thinsections (0Æ5 lm) were prepared from fixed, embeddedbut unstained tissue prepared exactly as for electronmicroscopy (see below). Unless stated separately, allstains were made up as 10 g L)1 aqueous solutions indistilled water. Sudan IV (Fisher Scientific Ltd) was usedto assess the degree of suberinization. Epoxy Tissue Stain(Electron Microscopy Sciences) was used as a generaltissue stain: it contains toluidine blue and basic fuchsin toshow areas of lignin. The fluorochrome, calcofluor white(Sigma-Aldrich), stains 1,4-linked glucan polymers(cellulose, chitin) and causes hyphal and plant cell wallsto fluoresce under UV-light.

Transmission electron microscopy

Samples were collected from representative tissues asdescribed and processed with some adaptations from themethod described by Bishop & Cooper (1983): 1 mm3

samples were fixed in glutaraldehyde in 0Æ05 M pipera-zine-N, N ‘-bis (2-ethanesulfonic acid) buffer at pH 8Æ0for 16–20 h with vacuum treatment. Post-fixation was in

Figure 1 Penetration of intact roots of oil palm

(Elaeis guineensis) by Ganoderma boninense

prior to bole infection. (a) Ganoderma-

colonized rubber-wood block (rwb) inoculum

firmly attached to an oil palm root with mycelial

growth around the root (arrow). (b) G.

boninense white mycelium protruding through

the epidermis of an infected root (arrows). (c)

Infection at the root-stem interface. Two roots

are infected (discoloured brown, arrows) but

with no visible necrosis in the stem base. (d)

Advance of the fungus into the stem base (bole)

tissue from infected roots (arrows). Infected

tissue appears brown; the perimeter of this

region is typically dark brown and immediately

in advance of the infected area is a narrow area

of yellow tissue; these have been termed the

‘reaction zone’ (rz) and ‘transition zone’ (tz),

respectively, by Darmono (1998) (see

Discussion). Uninfected tissue is cream-

coloured. (e) Transverse section showing

spread of necrosis into bole parenchyma from

one infected root (arrow). (f) Infected root with

fungal, melanized pseudo-sclerotium

covering the surface of the root connecting with

a basidiocarp (bas) which has emerged from

the stem base. (g) Transmission electron

micrograph of melanized pseudo-sclerotium

cells. Note some hyphae have extremely thick,

melanized, lamellate, cell walls (*) with little or no

cytoplasm whilst others retain thin walls and

cytoplasm (arrows).

984 R. W. Rees et al.

osmium tetroxide followed by dehydration through anacetone series. Samples were embedded in Spurr’s epoxyresin.One hundrednanometre sectionswere cutand stainedwith uranyl acetate and lead citrate prior to examinationwith a Jeol 1200EX transmission electron microscope.

Results

Root infection by G. boninense

During 5 months, infection by G. boninense of the rootand then basal stem tissue followed a typical pattern aspreviously described (Rees et al., 2007). Root infectionoccurred subsequent to firm attachment of hyphae tothe root surface (Fig. 1a,b). The attachment was eitherlocalized to the initial point of contact or sometimes thefungus completely enveloped the root. Following intimatecontact with the root surface, penetration of the epidermis

and exodermis occurred, as a prelude to internal invasion.On sectioning, heavily infected root tissue was evident bybrown discoloration, primarily in the cortex, and in someinstances G. boninense mycelium re-emerged throughruptures in the epidermis of infected roots (Fig. 1b).

The advancing edge of infection was determined bytransferring sections onto GSM agar, thereby allowingprogression of infection to be followed through the rootsand into the lower stem or bulb (bole in mature palms)together with sampling for microscopy of differentinfection stages. The pathogen could be isolated fromsymptomless tissue just in advance (< 3 mm) of necrosis.Fig. 1c shows an infected root, distinguishable fromadjacent healthy roots by its brown discoloration,connected to the stem base, where the infection is not yetapparent. A later stage where infection has progressedinto the bole from infected roots is shown in Fig. 1d,e. Thefungal mass often became encrusted and pigmented with

Plant Pathology (2009) 58, 982–989

Figure 2 Trunk bases from plantation oil palms

(Elaeis guineensis) from which roots have been

excised (a–c) showing multiple infections with

Ganoderma boninense. (a) Uninfected bole

tissue from a 13-year-old palm is uniformly

cream-coloured. (b) Numerous necrotic lesions

(arrows) indicate multiple root infections on a

15-year-old palm. (c) Root-bole interface

showing a severely infected, degraded root

(R), and progress of the pathogen into the

basal tissue. Infection is seen extending in

waves delimited by melanized mycelium

(arrows). (d) Transverse section through the

trunk base of a palm (selected with

basidiocarps, present but not shown) and with

minimal foliar symptoms; coalesced lesions

(arrows) show advanced infection on one side

of the basal tissue, indicating the probable entry

point of infection was via one or few adjacent

roots.

Ganoderma boninense infection of oil palm 985

melanin, forming extremely tough pseudo-sclerotia, whichwere frequently connected to basidiocarps that formedfrom infected stem bases (Fig. 1f). Examination of thistissue by TEM showed that some of the cells had devel-oped massive, lamellated walls and appeared to have losttheir cytoplasm, whilst others had normal cell walls andcontained typical cytoplasmic organization (Fig. 1g).

The recalcitrant nature of the endodermis and vascularcylinder resulted in roots with advanced infection oftenwith intact outer cell layers disconnected from a non-degraded stele. Nevertheless, ultimately the thickenedcell walls of the outer cortex and the stele were alsopartially destroyed as colonization progressed leading todisintegration of remaining root fragments.

Natural infection of mature palms within plantations

Progress of infection from the roots into the bole was alsoobserved by felling of symptomless commercial palms,and removal of roots. Random felling of palms aged 5 to13 years from different aged plantings indicated that allpalms remained uninfected (as judgedbyvisual inspectionof roots and boles) (Fig. 2a). However, one out of sixselected, 15-year-old palms not showing symptoms wasinfected with G. boninense. Fig. 2b shows multiple,brown, discrete lesions on the bole tissue revealing thatnumerous roots have been infected. Dissections revealedthat lesionsclearlyprogressed frominfectedroots (Fig.2c)to establish in the bole tissue and isolation confirmed thepresence of G. boninense. No infection from excisedfrond bases originating near the stem base was observed.Examination of other trees with basidiocarps showed thatinternal symptoms as described above were typicallyassociated with the onset of foliar symptoms and basidio-

Plant Pathology (2009) 58, 982–989

carp formation; in one case infection was localized toone side of the palm, presumably indicative of infection ofone or few localized roots (Fig. 2d). Separate lesionssometimescoalescedtocovermuchof thebole tissue.

Microscopy of root infection

Palm root anatomy has been described by Seubert (1997)and Jourdan & Rey (1997) and is shown for oil palm inFig. 3a,b.

Initial penetration of the outer layers of the root byG. boninense was not easily viewed microscopicallybecause of the chronic and non-synchronous nature ofinfection. Subsequent infection comprised ingress of thehyphae into the more easily degraded inner cortex andlongitudinal progression along roots. Light microscopyshowed that during early colonization of host tissue,G. boninense produces abundant, enlarged, intracellularhyphae, mainly in the inner cortical cells (Fig. 3c). Thiswas followed by considerable breakdown of cortical cellwalls (Fig. 3d) as was also revealed by TEM (Fig. 3e,f).The cell wall is degraded in multiple, localized areasassociated with hyphae but also often at some distancefrom invading hyphae. All wall layers were attacked,ultimately resulting in complete breakdown of the cellwall including the middle lamella, thus facilitating inter-and intracellular and intramural colonization of the oilpalm root. The stele and lacunae were not invaded to anyextent during the early stages of root infection.

Microscopy of infection of the basal stem

Cells of the basal stem of uninoculated seedlings wereshown by light microscopy and TEM to contain abundant

Figure 3 Microscopy of uninfected (a,b) and infected (c–f) oil palm (Elaeis guineensis) roots showing anatomy and fungal colonization by

Ganoderma boninense. The epidermis (ep) or rhizodermis comprises one cell layer with a thickened outer wall and with no evidence of root hairs.

Below this is the exodermis (ex) with secondary cell walls. There follows an outer cortex (oc) comprising a thin-walled layer about three cells thick

before several layers of cells with suberinized secondary walls. The inner cortex (ic) has thin primary walls and contains multiple, large air spaces

or lacunae (la) which run longitudinally. A suberinized endodermis (en) surrounds the stele (s) which comprises typical monocotyledonous

vascular architecture. (a) Stele and inner cortex. Transverse section (TS) stained with calcofluor viewed under UV-light; the vascular cylinder

appears bright, indicative of thick, cellulose-rich, cell walls. (b) TS. Epidermis (rhizodermis), exodermis and outer cortex. Darkly staining regions

with Sudan IV indicate cell walls enriched with suberin. (c) Initial intensive colonization of inner cortical cells by wide hyphae (h) (toluidine blue

and basic fuchsin stain). Secondary thickening pattern suggests radial vessels in this section. (d) Later infection results in disruption of outer

cortex, and to a lesser extent exodermis and epidermis, but primarily the inner cortex including lacunae lose integrity. Endodermis and the stele

appear still intact (calcofluor white stain). (e,f) Transmission electron microscopy of infected tissue corresponding to Fig. 3d, showing intracellular

and transmural hyphae (h) and enzymatic decay of cortical cell walls (arrows). Note the localized nature of the decay regions and that the

degradation is not confined to any particular cell wall layer.

986 R. W. Rees et al.

starch grains (Fig. 4a–c) and large central vacuoles (Fig. 4a).However, when infected with G. boninense, host cellsimmediately in advance of the invasion front were filledwith cytoplasm with apparent breakdown of starch grainsand extensive vesicular budding of the plasma membrane(Fig. 4c). Consistent with microscopic observations fromcolonized root tissue, infected cells in basal palm tissuewere first colonized by numerous, enlarged, fungalhyphae (Fig. 4d). Invaded cells often containeddiscerniblecytoplasm and organelles, but starch grains were invari-ably absent. Subsequently, widespread destruction of thehost cell walls occurred (Fig. 4e).

Discussion

This study provides further evidence for the initiation ofinfection of oil palm by G. boninense from a point ofprimary contact with the roots, ultimately leading tofungal penetration into the basal stem. Root infectionwas observed both from controlled inoculation underglasshouse conditions in the UK and from felling ofcommercial palms in North Sumatra.

Colonization by G. boninense can occur throughunwounded roots then progresses mainly through theinner, thin-walled cortex. A previous study on root

Plant Pathology (2009) 58, 982–989

Figure 4 Cells from stem base of oil palm

(Elaeis guineensis) uninfected (a,b) and

infected with Ganoderma boninense (c,d,e)

showing starch depletion and wall degradation.

(a) Light microscopy reveals abundant starch

grains (s) in uninfected palms. (b) Transmission

electron microscopy reveals cells with a large

vacuole and containing numerous starch grains

(s); cell wall (cw). (c) In the ‘transition zone’

(sensu Darmono, 1998) beyond G. boninense

infection, the cytoplasm expands to fill the cell

with early stages of loss of structure of the starch

grains (s) and extensive extra-vesicular

budding (arrows) at the plasma membrane. (d)

G. boninense initially colonizes the host cell with

broad hyphae (h) and starch grains are no

longer present. Note that cell walls appear intact

and cytoplasm containing organelles is still

discernible. (e) As the infection progresses,

there is widespread attack of the host cell walls

(arrows) from intracellular and transmural

hyphae (h).

Ganoderma boninense infection of oil palm 987

infection by G. boninense considered that infectionof vascular tissue predominated, although colonizationof all other tissues was observed too (Sariah et al., 1994).In contrast, in the present study no initial colonizationwas observed in the vascular tissue, or lacunae. Lacunaeare wide, long,axiallyorientatedcylindrical spaces surroundedby thin-walled cells and prima facie offer an ideal route forlongitudinal spread, but these channels were not exploited.

Melanized pseudo-sclerotia, which enveloped someroots (and formed on decayed inoculum wood blocks),represent an alternative source of infection, or conversely,they may reflect internal colonization and be an externalmanifestationoffungal invasion;however, itwasnotprac-ticable todistinguish the sequenceof events.The functionsof this melanized structure are likely to be manifold, suchasprotectionfromhostdefences,dehydrationandcompet-ing microorganisms. Survival functions may result by theempty cells with massive, melanized cell walls enclosingthin-walled hyphae with active cytoplasm; G. boninensecouldberoutinelyreisolatedfrompseudosclerotia.

Infection appears to involve developmental switches.There was an initial apparent biotrophic phase in infectionof both root cortex and stem base, involving largelyintracellular colonization by wide hyphae of host cells

Plant Pathology (2009) 58, 982–989

with fully intact cell walls and in some cases containingrecognizable cytoplasm. This mode of invasion is reminis-cent of the well-characterized hemibiotrophic phase ofsome Colletotrichum spp. (Perfect et al., 1999; Latunde-Dada & Lucas, 2007) and even of Magnaporthe grisea(Heath et al., 1992). Hemibiotrophy is a strategy deployedby certain ascomycetes, but to the authors’ knowledge is notwell described for basidiomycetes with woody hosts,other thanMoniliophthoraperniciosaonTheobromacacao(Gesteira et al., 2007). Further suggestion that G. boninensemight be capable of subtle invasion of hosts comes fromAbdullah (2000) who described the fungus as an endophytein coconuts and Panchal & Bridge (2005) who detectedGanoderma DNA in oil palms with no symptoms.

The biotrophic mode of invasion was followed by anaggressive necrotrophic phase associated with extensivehost cell wall degradation. The formation of melanizedmycelium might be considered as a third phase andoccurred both within host tissues (possibly in responseto host defences) and on a much larger scale external toroots in the form of very tough pseudo-sclerotia.

For successful penetration and degradation of intactroots, production of an array of cell wall-degradingenzymes (CWDEs) (Cooper, 1984) is likely to be required

988 R. W. Rees et al.

in order to penetrate the outermost tissues, comprising therecalcitrant polymers cellulose, lignin and suberin. Onemonth after inoculation, removal from roots of the adher-ing mycelium revealed bleaching underneath (data notshown), probably indicative of the oxidative breakdownof lignin and the white rot status ascribed to this genus(Adaskaveg et al., 1990; Paterson, 2007). Macroscopicevidence of enzymic degradation of the tough root outercell layers and all subsequent invaded tissues of root andstem was confirmed by light and transmission electronmicroscopy (TEM).

Invasion of root cortex and stem parenchyma resultedin development of holes through all cell wall layers. Similarpatterns of simultaneous cell wall attack have beenobserved from degradation of Laurelia philipiana woodby a Ganoderma sp. (Agosin et al., 1990) and of date palmwoodbybothPhanerochaetecolossumandP.chrysosporium(Blanchette, 1984; Adaskaveg et al., 1991). This contrastswith selective lignin degradation, evident ultrastructu-rally by the loss of electron density from the middlelamella, with less degradation of the polysaccharidecomponents of the S1 and S2 cell wall layers (Agosinet al., 1990). Areas of cell wall attack were sometimes notadjacent to fungal hyphae. Lignin degradation can occurat a distance as the necessarily oxidative nature of ligninbreakdown produces reactive oxygen species and theresulting phenoxy radicals may be damaging to the fungalcell wall (Kirk, 1987). Manganese peroxidase (MnP) andlaccase production have been reported for G. lucidum byd’Souza et al. (1996) and aggressiveness of H. annosumhas been related to laccase production (Johansson et al.,1999). Also laccases can contribute to melanin forma-tion, as occurs in pseudo-sclerotia (Johansson et al.,1999). Enzyme activities from G. boninense correspond-ing to the presence of lignin and all other major structuralcell wall polymers have been detected together with theireffect on host wall composition during G. boninenseinfection (Rees, 2006).

Along with cell wall polymers, starch representsanother major polysaccharide that could be utilized as asignificant source of carbon during basal stem rot of oilpalm by G. boninense. Adaskaveg et al. (1991) recordedhigh levels of starch in date palm wood and similarlyvery high starch content of oil palm seedlings was detectedthat was rapidly depleted during early infection (Rees,2006) and was confirmed here by TEM of bole tissues.Depletion of starch could result from host and/or patho-gen metabolism (G. boninense readily utilizes insolublestarch (Rees, 2006)), but it is apparent that a very signifi-cant energy resource is contributing to this host-pathogeninteraction.

Typical defence responses of woody tissues, such asreinforcement of cell walls by suberinization and/orlignification or periderm formation (Pearce, 1996), werenot evident ultrastructurally in roots or stems. Distinctmelanized margins comprising Ganoderma myceliumwere observed and have often been found in both basaland upper stem rot of oil palm (Darmono, 1998; Hasanet al., 2005) and also were described for Ganoderma

tsugae infection of Tsuga canadensis (Blanchette, 1984).These pathogen responses might reflect the formation ofputative barrier zones, described as reaction zones andtransition zones by Darmono (1998) for G. boninenseinfection of oil palm. Zonal decay patterns such as inFig. 2c suggest barriers are formed but frequently breached.Possibly defences in oil palm are more reliant on antimi-crobial compounds, as found in the sapwood of many treespecies (Pearce, 1996), than on physical, wall-associatedresponses. In the absence of oil palm genotypes with sig-nificant resistance to G. boninense (Rees et al., 2007),mechanisms of defence in oil palm must remain a matterfor speculation.

Overall this study provides confirmation of root invasionas probably the main mode of infection by G. boninense.This basic information is important in any considerationof disease control strategies. Also the inoculation methodhas facilitated microscopic investigation of distinct infec-tion stages, revealing the route and nature of infection. Itis particularly apparent that CWDEs play a key role inpathogenicity. However, analysis of putative pathogeni-city factors in this poorly understood pathosystem shouldbenefit from the methods described here and by Rees et al.(2007) and should enable a study equivalent to thatdescribed by Karlsson et al. (2003) for H. annosum. In thelatter study more than 900 expression sequence tags(ESTs) from total RNA from mycelium during early infec-tion of Scots pine roots were obtained. Such an analysiswith Ganoderma would be enhanced by the existing gen-ome sequence from the lignocellulose-degrading basidio-mycete P. chrysosporium (Martinez et al., 2004).

Acknowledgements

R.W.R. was supported by a BBSRC Industrial CASEStudentship with CABI, linked with P.T.P.P. LondonSumatra Indonesia Tbk (LONSUM). We thank HughFoster and Steven Nelson for their considerable supportand advice and for providing facilities and support staffat Bah Lias Research Station (BLRS), Sumatra. We wishto thank LONSUM for permission to publish this paper.

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