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Host responses in the xylem of trees after inoculation with six wood-decay fungi differing in invasiveness

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Host responses, i.e. formation of reaction and barrier zones, were studied in the xylem of Douglas fir, beech, oak, and sycamore trees after wounding and artificial inoculation with brown, soft, and white rot fungi. The objective of this study was to
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  Host responses in the xylem of trees afterinoculation with six wood-decay fungi differing ininvasiveness 1 Giuliana Deflorio, Erwin Franz, Siegfried Fink, andFrancis Willis Mathew Robert Schwarze Abstract: Host responses, i.e., formation of reaction and barrier zones, were studied in the xylem of Douglas-fir, beech,oak, and sycamore trees, after wounding and artificial inoculation with brown-, soft-, and white-rot fungi. The objective of this study was to determine whether strongly invasive wood-decay fungi trigger a higher magnitude of host response thanweakly invasive fungi. Differences in active host response, observed microscopically, depended on wood anatomy. Restric-tion of discoloration and decay by reaction zones was primarily influenced by the content and distribution of parenchymacells within the sapwood of each host. By contrast, barrier-zone anatomy showed similarities to the basic xylem structureof each host, except for some cell types that were either reduced in number or absent. Regardless of the decay fungus ino-culated, individual trees of each host responded differently. With the exception of beech trees inoculated with the soft-rotfungus Kretzschmaria deusta (Hoffm.: Fr.) P. Martin and the white-rot fungus Trametes versicolor  (L.: Fries) Pila´t, hostresponse appeared to be nonspecific, as the degree of fungal invasiveness did not influence the magnitude of host responsewithin the xylem of investigated trees. Key words: xylem, wood decay fungi, reaction zone, barrier zone, hardwood, softwood. Re´sume´: Les auteurs ont e´tudie´les re´actions de l’hoˆte, i.e. la formation de zones de barrage et de re´action, dans le xy-le`me de tiges le Douglas, de heˆtre, de cheˆne et de sycomore apre`s blessures et inoculations artificielles avec des champi-gnons de carie, brune, molle et blanche. L’e´tude visait a`de´terminer si les champignons de carie tre`s envahissantsde´clenchent une plus forte re´action de la part de l’hoˆte que les champignons faiblement envahissants. L’observation mi-croscopique montre que les re´actions actives de l’hoˆte de´pendent de l’anatomie du bois. Les restrictions de la de´colorationet de la pourriture par les zones de re´action sont surtout influence´es par le contenu et la distribution des cellules de paren-chyme dans l’aubier de chaque hoˆte. Au contraire, l’anatomie des zones de barrage montre des similitudes dans la struc-ture de base du xyle`me de chaque hoˆte, sauf pour certains types de cellules qui sont soient re´duits ou soient absents.Inde´pendamment du champignon de carie inocule´, les individus de chaque hoˆte re´agissent diffe´remment. Avec l’exceptiondes heˆtres inocule´s avec le champignon de la pourriture molle, Kretzschmaria deusta (Hoffm.: Fr.) P. Martin et le champi-gnon de la carie blanche Trametes versicolor  (L.: Fries) Pila´t, les re´actions de l’hoˆte ne semblent pas spe´cifiques puisquele degre´de capacite´d’envahissement fongique n’a pas influence´l’amplitude de la re´action de l’hoˆte dans le xyle`me desarbres observe´s.  Mots-cle´ s : xyle`me, champignons de carie blanche, zone de re´action, zone de barrage, bois franc, bois mou.[Traduit par la Re´daction] Introduction Host responses in the xylem have been studied in severalhost species (Sharon 1973; Moore 1978; Mulhern et al.1979; Bauch et al. 1980; Smith 1980; Tippett and Shigo1980; Blanchette 1982; Rademacher et al. 1984; Lowerts etal. 1986; Torelli et al. 1994). These studies have describedchemical and physical properties of reaction and barrierzones in gymnosperm and angiosperm trees, either afterwounding or after wounding and fungal inoculation. Differ- Received 15 July 2008. Published on the NRC Research Press Web site at botany.nrc.ca on 23 December 2008. G. Deflorio, 2,3 E. Franz, and S. Fink. Institute of Forest Botany, University of Freiburg, Bertoldstrasse 17, D-79085 Freiburg imBreisgau, Germany. F.W.M.R. Schwarze. Institute of Forest Botany, University of Freiburg, Bertoldstrasse 17, D-79085 Freiburg im Breisgau, Germany;Swiss Federal Laboratories for Materials Testing and Research (Empa), Wood Laboratory, Lerchenfeldstrasse 5, CH-9014 St. Gallen,Switzerland. 1 This article is one of a collection of papers based on a presentation from the Stem and Shoot Fungal Pathogens and Parasitic Plants:the Values of Biological Diversity session of the XXII International Union of Forestry Research Organization World Congress meetingheld in Brisbane, Queensland, Australia, in 2005. 2 Corresponding author (e-mail: giulianade@gmail.com). 3 Present address: Department of Plant and Soil Science, School of Biological Sciences, University of Aberdeen, Cruikshank Building,23 St. Machar Drive, Aberdeen, AB24 3UU, UK. 26 Botany 87 : 26–35 (2009) doi:10.1139/B08-113 Published by NRC Research Press  ent host responses have sometimes been reported to be asso-ciated with fungal infection (Tippett et al. 1982; Bonsen etal. 1985; Biggs 1986; Rioux and Ouellette 1991; Barry etal. 2002; Krekling et al. 2004). Other investigations havefound similar host responses after wounding and fungal in-oculation (Biggs 1984). None of these studies have com-pared interactions at the host–fungus interface amongdifferent trees inoculated with the same wood decay fungusor, vice versa, different wood decay fungi inoculated in thesame host species.The importance of host defence mechanisms such as lig-nification and suberization has been emphasized in previousstudies on host–fungus interactions (Pearce and Rutherford1981; Biggs 1984; Pearce and Holloway 1984; Pearce andWoodward 1986; Schmitt and Liese 1991, 1993; Pearce1996; Woodward and Pocock 1996). In contrast, fungal in-vasiveness, i.e., ability of fungal hyphae to penetrate hostboundaries and thus bring about lesion expansion, has re-ceived little attention. The mechanisms by which decayfungi overcome these boundaries have been the subject of only a few microscopical studies (Schwarze and Fink 1997;Schwarze et al. 2000; Baum and Schwarze 2002; Schwarzeet al. 2004; Schwarze 2007). As a consequence, littleknowledge exists on the effect of wood-decay fungi withdifferent degrees of invasiveness on the magnitude of hostresponse.The objective of the present study was to investigatewhether host responses in the xylem (including accumula-tion of polyphenols and degree of blocked cell lumina inthe reaction zone and barrier zone) are influenced by thepresence of weakly or strongly invasive wood-decay fungi.For this purpose, Douglas-fir ( Pseudotsuga menziesii (Mirb.) Franco), beech ( Fagus sylvatica L.), oak ( Quercusrobur  L.), and sycamore (  Acer pseudoplatanus L.) treeswere inoculated with five basidiomycetes ( Fomitopsis pini-cola (Sw.: Fr) P. Karst., Ganoderma resinaceum Boud. inPat., Ganoderma adspersum (Schulz.) Donk, Ganoderma applanatum (Pers.) Pat., Trametes versicolor  (L.: Fries) Pila´t, and one ascomycete ( Kretzschmaria deusta (Hoffm.: Fr.) P. Martin). Comparative observations with acontrol treatment were used to assess the magnitude of ac-tive host response. With the exception of  F. pinicola , all of these decay fungi are capable of degrading both lignin andcellulose, and are able to invade living sapwood (Wilkins1934, 1936, 1939, 1943; Pearce and Woodward 1986;Pearce 1991; Schwarze and Baum 2000). Materials and methods Identification and cultivation of fungal material Pure cultures of six fungal species (Table 1) were isolatedfrom sporocarps. Pure cultures of the basidiomycetes weregrown on a selective medium consisting of 2% malt-extractagar (MEA) and 230 mg/L of thiabendazole; K. deusta wasalso grown on 2% MEA. To avoid bacterial contamination,1 mL/L of a 0.1% solution of the antibiotics penicillin,streptomycin, and tetracycline was added before pouring themedium. Fresh cultures of each isolate were stored in thedark in 90 mm diameter Petri dishes at 25 8 C and 65% RH. Degree of fungal invasiveness Degree of fungal invasiveness (i.e., weak, moderate,strong) was related to the extent of dysfunctional sapwood(expressed in centimetres) that developed from inocula inthe longitudinal, radial, and tangential wood direction of trees felled and dissected after 28 months (Deflorio et al.2008). Since radial and tangential extent of dysfunctionalsapwood appeared to be more accurate than longitudinal ex-tent, Table 2 summarizes fungal invasiveness with referenceto the former response variables. Inoculations of standing trees Pine and beech sapwood dowels, 70 mm long and 20 mmin diameter, were axially split into two and re-wetted untilthey reached 50%–70% RH. They were subsequently auto-claved at 121 8 C for 30 min. Wood dowels were incubatedon 14 d old fungal cultures for 5 weeks at 25 8 C and 50%–70% RH. Dominant and co-dominant Douglas-fir, beech,oak, and sycamore stems (Table 1) at a site in the Mooswaldforest (Freiburg im Breisgau, Germany) were inoculatedwith dowels in May 2002, following the procedure describedin Deflorio et al. (2008). Table 1. Tree and fungal material used for stem inoculations in Mooswald (Freiburg im Breisgau, Germany).TreeDiameter(mean±SE)Height(mean±SE) Age a (years) Pseudotsuga menziesii 42.0±3.4 23.3±1.3 40 Fagus sylvatica 49.8±1.6 29.1±0.5 65 Quercus robur  46.9±3.1 26.7±1.3 70  Acer pseudoplatanus 41.4±1.6 24.3±0.4 55Fungus Decay type b Isolate No. Host Location Fomitopsis pinicola BR 150801.1 ex F. sylvatica Wutach Kretzschmaria deusta SR 271098.3 ex A. pseudoplatanus Freiburg Ganoderma resinaceum WR 250501.1 ex Quercus rubra L. Freiburg Ganoderma adspersum WR 99.2 ex F. sylvatica Freiburg Ganoderma applanatum WR 260202.1 ex Q. rubra Freiburg Trametes versicolor  WR 260202.1 ex F. sylvatica Freiburg Note: Diameter was measured in centimetres; height was measured in metres. a The average age was determined by counting tree rings after felling. b Decay type: BR, brown rot; SR, soft rot; WR, white rot. Deflorio et al. 27 Published by NRC Research Press  Reisolation of the causal agent Three small wood fragments (5 mm  5 mm) were ran-domly taken from the inoculum, decayed wood, and discol-oured wood (nine isolations from each wound). These wereplated onto MEA and incubated in the dark at 25 8 C and65% RH. Isolates were subcultured after 2 weeks and identi-fied. For the basidiomycetes, identity was confirmed usingmycelial characters observed on MEA, as well as enzymetests (Stalpers 1978). The successful reisolation of each fun-gal species was recorded along with the occurrence of otherfungal isolates. Light microscopy Four wood samples that contained the host–fungus inter-face (Fig. 1) of each host–fungus combination were ran-domly selected from the xylem of trees felled after28 months, for light microscopy. Samples, approximately10 mm  5 mm  5 mm, were fixed in 2% glutaraldehydein 0.2 mol/L phosphate buffer (pH 7.2), dehydrated in 2-propanol and embedded in a methacrylate medium accord-ing to the methods described in Schwarze and Fink (1997).Transverse and radial sections of approximately 3 m m wereprepared using a rotary microtome (Leica 1 2040 Supercut)fitted with a diamond knife. Histological staining The wood anatomy features of each host–fungus combina-tion were observed with light microscopy (Zeiss 1 Axiophot)after treatment with several stains. Sections were firststained in 1% acriflavin and 2% safranin for 12 h, and thencounter-stained in 1% auramin for 30 min, before beingplaced in 1% methylene blue and 1% astra-blue for 5 min.For the detection of lignin, additional wood sections werestained in 0.05% toluidine blue O solution for 5 min, beforebeing placed in a CaCl 2 solution for 20 min (Herr 1992).Acridine Orange was used to detect lignin under secondaryfluorescence. For this purpose, 1 m m thin wood sectionswere stained for 60 min in an Acridine-Orange solution      T    a     b     l    e      2  .     D   e   g   r   e   e   o    f    f   u   n   g   a    l    i   n   v   a   s    i   v   e   n   e   s   s    (   w   e   a    k ,   m   o    d   e   r   a    t   e ,   s    t   r   o   n   g    )   a   s   r   e    l   a    t   e    d    t   o    t    h   e   r   a    d    i   a    l   a   n    d    t   a   n   g   e   n    t    i   a    l   e   x    t   e   n    t   o    f    d   y   s    f   u   n   c    t    i   o   n   a    l   s   a   p   w   o   o    d   o    f    t   r   e   e   s   s   a   m   p    l   e    d   a    f    t   e   r    2    8   m   o   n    t    h   s    (    n    =    1    2    ) .    D   o   u   g    l   a   s  -    f    i   r    B   e   e   c    h    O   a    k    S   y   c   a   m   o   r   e    D   e   g   r   e   e   o    f    f   u   n   g   a    l    i   n   v   a   s    i   v   e   n   e   s   s    R   a    d    i   a    l    T   a   n   g   e   n    t    i   a    l    R   a    d    i   a    l    T   a   n   g   e   n    t    i   a    l    R   a    d    i   a    l    T   a   n   g   e   n    t    i   a    l    R   a    d    i   a    l    T   a   n   g   e   n    t    i   a    l    W   e   a    k    A    l    l   o    t    h   e   r    f   u   n   g   a    l    i   s   o    l   a    t   e   s    A    l    l   o    t    h   e   r    f   u   n   g   a    l    i   s   o    l   a    t   e   s    A    l    l   o    t    h   e   r    f   u   n   g   a    l    i   s   o    l   a    t   e   s    A    l    l   o    t    h   e   r    f   u   n   g   a    l    i   s   o    l   a    t   e   s    A    l    l   o    t    h   e   r    f   u   n   g   a    l    i   s   o    l   a    t   e   s    A    l    l   o    t    h   e   r    f   u   n   g   a    l    i   s   o    l   a    t   e   s    M   o    d   e   r   a    t   e    N   o   s    i   g   n    i    f    i   c   a   n    t    d    i    f    f   e   r   e   n   c   e      T .   v   e   r   s     i   c   o     l   o   r     N   o   s    i   g   n    i    f    i   c   a   n    t    d    i    f    f   e   r   e   n   c   e    S    t   r   o   n   g      F .   p     i   n     i   c   o     l   a     K .     d   e   u   s    t   a     K .     d   e   u   s    t   a     K .     d   e   u   s    t   a     K .     d   e   u   s    t   a  ,      T .   v   e   r   s     i   c   o     l   o   r     K .     d   e   u   s    t   a  ,      T .   v   e   r   s     i   c   o     l   o   r      N    o     t    e    :     O   r    i   g    i   n   a    l    d   a    t   a   a   r   e   s    h   o   w   n    i   n    D   e    f    l   o   r    i   o   e    t   a    l .    (    2    0    0    8    ) . Fig. 1. Longitudinal section of an inoculated beech stem showing( a ) schematic drawing of the dysfuctional sapwood (shaded) formedafter artificial inoculation with the soft rot fungus Kretzschmariadeusta ; ( b ) regions (within broken line) from which reaction zone(RZ) and barrier zone (BZ) were excised for histological investiga-tion. Scale bar = 2 cm. I, inoculum dowel; S, sound sapwood; D,decayed sapwood. 28 Botany Vol. 87, 2009 Published by NRC Research Press  (0.05%). Observations were made by fluorescence micro-scopy using the BP 450/490 – FT 510 – LP 520 filter com-bination (Zeiss 1 Axiophot). For the detection of suberin,sections were first treated with trichlorethylene for 30 min,and subsequently stained in a 0.5% solution of Sudan IVfor 10 min (Schmitt and Liese 1993). Determination of barrier-zone width Maximum barrier-zone width was measured with a Zeiss 1 Axiophot microscope fitted with a Zeiss 1 AxioCam MRc5      T    a     b     l    e      3  .     S   u   c   c   e   s   s    f   u    l    f   r   e   q   u   e   n   c   y   o    f    f   u   n   g   a    l   r   e    i   s   o    l   a    t    i   o   n   s    (    %    )    f   r   o   m    i   n   o   c   u    l   u   m ,   a   n    d    d    i   s   c   o    l   o   r   e    d    /    d   e   c   a   y   e    d   w   o   o    d    i   n    t   r   e   e   s   s   a   m   p    l   e    d   a    f    t   e   r    2    8   m   o   n    t    h   s .    D   o   u   g    l   a   s  -    f    i   r    B   e   e   c    h    O   a    k    S   y   c   a   m   o   r   e    T   r   e   a    t   m   e   n    t    I    D    i   s    /    D   e   c    O    t    h   e   r    a     I    D    i   s    /    D   e   c    O    t    h   e   r    I    D    i   s    /    D   e   c    O    t    h   e   r    I    D    i   s    /    D   e   c    O    t    h   e   r      G .   r   e   s     i   n   a   c   e   u   m     0    0    6    7    3    3    5    8    3    3    2    5    1    3    4    2    7    5    3    8    5    0      G .   a     d   s   p   e   r   s   u   m     2    5    0    6    7    1    7    2    5    5    6    0    1    3    5    0    2    5    6    3    5    0      G .   a   p   p     l   a   n   a    t   u   m     0    3    8    5    0    3    3    4    2    5    6    5    0    5    0    3    3    5    0    5    0    5    0      T .   v   e   r   s     i   c   o     l   o   r     0    2    5    4    2    1    7    3    3    6    7    2    5    2    5    5    0    7    5    8    8    1    7      K .     d   e   u   s    t   a     2    5    0    5    0    5    0    5    0    1    7    2    5    5    0    4    2    5    0    6    3    3    3      F .   p     i   n     i   c   o     l   a     2    5    3    8    1    7    3    3    2    5    5    0    0    0    7    5    0    2    5    5    0    C   o   n    t   r   o    l   n   a      b    n   a    5    0   n   a   n   a    6    7   n   a   n   a    5    0   n   a   n   a    5    0      N    o     t    e    :     D    i   s ,    d    i   s   c   o    l   o   u   r   e    d   w   o   o    d   ;    D   e   c ,    d   e   c   a   y   e    d   w   o   o    d .    a     O    t    h   e   r ,   o    t    h   e   r    i   s   o    l   a    t   e    d    f   u   n   g    i .      b    n   a ,   n   o    t   a   v   a    i    l   a    b    l   e . Fig. 2. Comparison of anatomy and host response of Douglas-fir insound (no treatment), wounded, and artificially inoculated sapwood.( a ) Transverse section (TS) of sapwood consisting of tracheids anduniseriate xylem rays. Scale bar = 20 m m. ( b ) Weakly developedbarrier zone (TS) with one row of traumatic resin canals (TRC).Scale bar = 20 m m. ( c ) TS of strongly developed barrier zone withclosely aligned, tangential bands of traumatic resin canals (TRC).Note: lumina of tracheids in close proximity to resin canals arestrongly occluded with polyphenols (arrowheads). Scale bar =50 m m. ( d  ) Radial longitudinal section (RLS) of thick-walled xylemray parenchyma cells (arrows) and tracheids with reduced cellwidth and length (arrowheads). Scale bar = 10 m m. ( e ) TS of axialparenchyma cells (multiple rows; arrowheads) strongly occludedwith polyphenols in close proximity to TRC. Scale bar = 10 m m.(  f  ) TS of resin canals (RC) in sound sapwood with thin-walled,rectangular epithelial cells (EP). Scale bar = 10 m m. ( g ) TS of trau-matic resin canals (TRC) in the barrier zone, with thick-walled,round epithelial cells (EP). Scale bar = 5 m m. ( a – e ) Standard stain;(  f  and g ) Acridine-Orange stain. Deflorio et al. 29 Published by NRC Research Press  digital camera system. Zeiss 1 AxioVision AC digital imageanalysis software was operated by using the software scale bar. Statistical analysis Eight trees, two per tree species, were used to investigatebarrier-zone width. Four replicates of each treatment (i.e.,there were two replicate areas on each tree, at 70 and140 cm above ground) were set up in May 2001 as a com-plex split-plot design with two main effects, namely ‘‘host’’(4 levels) and ‘‘fungus’’ (7 types including the control), with‘‘tree’’ nested within ‘‘host’’ as block effect. Host and funguswere all fixed effects, while ‘‘tree-within-host’’ was random.An analysis of variance (ANOVA) was undertaken on theresponse variable barrier-zone width. Assumptions of nor-mality and homoscedasticity were tested, and transforma-tions to stabilize variances were determined from therelationship between group standard deviations and groupmeans (Draper and Smith 1998). The ‘‘Type III SS’’ wasconsidered for the analysis of unbalanced data sets.Where the overall ANOVA indicated significant effects,the nature of significant differences was explored using thea posteriori Ryan–Einot–Gabriel–Welsch (REGWQ) multi-ple range test. All analyses were undertaken with the SASstatistical package (SAS Institute Inc. 1999), using a signifi-cance level of  a = 0.05. Results Reisolation of the causal agent Not all fungal species were consistently reisolated fromtheir inoculated hosts (Table 3). In all hosts, reisolation failurewas frequently associated with the occurrence of other fungalcompetitors, mainly Penicillium spp. and Trichoderma spp.Reisolation was more successful in beech and sycamorethan in Douglas-fir and oak. In Douglas-fir, a low reisolationfrequency was recorded for all of the white rot fungi (withthe exception of  G. applanatum ), whereas the brown-rot fun-gus F. pinicola was frequently reisolated (Table 3). In oak, F. pinicola was never reisolated, and the white-rot fungi G. resinaceum and G. adspersum were infrequently found.In comparison, beech and sycamore had greater reisola-tion frequencies. For some fungal isolates, such as G. resinaceum and K. deusta in beech, as well as G. adspersum , T. versicolor  , and K. deusta in sycamore,high frequencies were recorded (Table 3). Barrier-zone width In each host, barrier-zone width was highly affected bythe individual tree being inoculated ( P = 0.002). This find-ing suggests that within a host species, trees responded dif-ferently to wounding and inoculation. A significant host  fungus interaction ( P = 0.04) was also recorded: some hostsresponded differently to inoculation with the different fungalspecies, while others did not. Subsequent REGWQ test foreach host revealed that the barrier zone extended greatest inbeech trees inoculated with the soft-rot fungus K. deusta andthe white-rot fungus T. versicolor  , for which barrier-zonewidth was, respectively, 1277 ± 556 m m and 852 ± 434 m m(Table 4). Thus a relationship between barrier-zone widthand degree of fungal invasiveness was confirmed only inbeech.Since a high tree-within-host variation was detected, ex-ample sections of reaction and barrier zones are illustrated(Figs. 2–5) and summarized (Table 5) for each host, withemphasis on weak and strong compartmentalization. Theseexamples are compared with features of sound xylem. Sound sapwood Douglas-fir wood is homogeneous in structure (Fig. 2 a ).The xylem consists predominantly of tracheids, which arethin-walled in the earlywood, thick-walled in the latewood,and have numerous bordered pits. A typical feature of thetracheid’s cell wall of this host species is the helical thicken-ings (Fig. 2 d  ). Xylem rays are uniseriate (Fig. 2 a ). Resinducts (30–35 m m in diameter) are often present in the xylemand appear more frequently in the latewood (Fig. 2  f  ). Livingepithelial cells surround the resin canals and synthesize resin(Fig. 2  f  ). Epithelial cells are thin-walled and rectangular inform (approx. 2 m m  10 m m). Epithelial cells are enclosedby 1–2 rows of parenchyma cells which are living, thick-walled, and strongly lignified (Fig. 2  f  ).Beech is a diffuse-porous wood species in which vesselsare embedded in a matrix consisting of fiber-tracheids(Fig. 3 a ). Generally, fiber-tracheids are thick-walled.Usually, the last few cell rows in the latewood arethicker-walled and more highly lignified than the remain-ing fibre-tracheids. Axial parenchyma cells are eitherdiffuse-aggregate or apotracheal-reticulate and arranged inshort tangential cell rows between uniseriate and multiseri-ate xylem rays (Fig. 3 a ).Ring-porous wood of oak is a more evolved wood charac-terized by cell types with specific functions (Fig. 4 a ).Water-conducting vessels are radially distributed and havelarger cell lumina in the earlywood than in the latewood(Fig. 4 a ). Fiber-tracheids have a dual function: they conductwater and provide strength and support, whereby libriform Table 4. Average barrier-zone width in micrometres (mean ± SE) in Douglas-fir, beech, oak, and sycamoretrees, sampled 28 months after artificial inoculation ( n = 4).Treatment Douglas-fir Beech Oak Sycamore G. resinaceum 413±38a 280±68b 285±38a 337±68a G. adspersum 425±42a 356±39b 345±118a 350±54a G. applanatum 547±51a 233±24b 345±91a 400±87a T. versicolor  325±50a 852±434ab 322±64a 320±62a K. deusta 420±111a 1277±556a 237±38a 332±69a F. pinicola 270±95a 226±38b 250±44a 422±43aControl 395±40a 328±77b 593±58a 397±71a Note: Means within columns followed by the same letter are not significantly different ( P = 0.05); REGWQ multiplerange test. 30 Botany Vol. 87, 2009 Published by NRC Research Press
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