Tree Physiology 24, 505515 2004 Heron PublishingVictoria, Canada

Induced responses to pathogen infection in Norway spruce phloem: changes in polyphenolic parenchyma cells, chalcone synthase transcript levels and peroxidase activity
NINA ELISABETH NAGY,1,2 CARL GUNNAR FOSSDAL,1 PAAL KROKENE,1 TRYGVE KREKLING,3 ANDERS LNNEBORG1,4 and HALVOR SOLHEIM1
1 2 3 4

Norwegian Forest Research Institute, Hgskoleveien 12, N-1432 s, Norway Corresponding author (nina.nagy@skogforsk.no) Institute of Chemistry and Biotechnology, Agricultural University of Norway, s, Norway Present address: DiaGenic A.S., Oslo, Norway

Received October 2, 2002; accepted September 21, 2003; published online March 1, 2004
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Summary Polyphenolic parenchyma cells (PP cells) in Norway spruce (Picea abies (L.) Karst.) stem phloem play important roles in constitutive and inducible defenses. To determine whether anatomical and molecular changes in PP cells are correlated with tree resistance, we infected two Norway spruce clones with the pathogenic fungus Ceratocystis polonica (Siem.) C. Moreau. The fungus induced significantly different lesion lengths in the two clones, indicating that one clone was more resistant to the fungus (short lesions) than the other (long lesions). After infection, the cross-sectional area of PP cells and their vacuolar polyphenol bodies increased in the three most recent annual rings of PP cells in both clones. The more resistant clone had larger PP cells with denser polyphenol bodies than the less resistant clone, whereas the less resistant clone accumulated relatively more polyphenols after infection. Compared with the less resistant clone, the more resistant clone contained higher starch concentrations before infection that were reduced more quickly after infection before returning to original values. Low transcript levels of chalcone synthase were detected in uninfected tissues of both clones, but the levels increased dramatically after infection. Transcript levels were higher and peaked 6 days earlier in the more resistant clone than in the less resistant clone. The activity of at least one highly basic peroxidase isoform was greatly enhanced after infection, and this increase occurred earlier in the more resistant clone. Keywords: Ceratocystis polonica, defense responses, phenol synthesizing enzymes, phenolic content, phloem parenchyma cells, Picea abies, starch concentration.

Introduction Plants produce various structural hindrances and toxic or re-

pellent metabolites to defend against challenges by insects and microorganisms (Karban and Baldwin 1997). Secondary plant metabolites in particular are important components of both the constitutive and inducible defense system of most plants (Harborne 1993, Grayer and Kokubun 2001). An increase in secondary metabolites such as phenolic compounds (flavonoids, tannins and the structural polymer lignin) seems to be an important inducible plant response to insect and pathogen attack (Halbrock and Scheel 1989, Dixon and Paiva 1995, Karban and Baldwin 1997). Phenolics are involved in both constitutive defenses and in inducible stress responses by deterring herbivores and pathogens, protecting against ultraviolet radiation, strengthening cell walls (lignin), tanning proteins, scavenging oxidative species and serving as signaling molecules (Dixon and Paiva 1995, Grace and Logan 2000). Many conifers, particularly in the family Pinaceae, are attacked and killed by scolytid bark beetles and their associated phytopathogenic fungi (Paine et al. 1997). One relatively wellstudied conifer defense system is that of Norway spruce (Picea abies (L.) Karst.). This species is attacked by the bark beetle Ips typographus L. and its fungal associate Ceratocystis polonica (Siem.) C. Moreau, which is capable of killing healthy trees (Horntvedt et al. 1983, Christiansen 1985). Generally, the bark of Norway spruce and other conifers contains layers of suberized, lignified cells and phenol-filled parenchyma cells (PP cells) that create a thick, constitutive defense barrier to potential invaders (Franceschi et al. 1998). The PP cells are also a major site for energy storage, in the form of starch and sugars. A new layer of PP cells is produced every year (Krekling et al. 2000). Attacks by insects or microorganisms induce a wound response in conifers that is characterized by morphological changes and accumulation of anti-fungal compounds that in-

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hibit infection and promote wound-healing (Berryman 1972, Woodward 1992). Recent evidence indicates that the two primary sites of inducible defenses in Norway spruce stems are the PP cells and traumatic resin ducts that form in the xylem after an attack. The size of the PP cells increases and their phenolic contents change in response to wounding, fungal inoculation and bark beetle attack (Franceschi et al. 2000, Krokene et al. 2003). Similar phenol-containing parenchyma cells develop in the xylem around the traumatic resin ducts (Nagy et al. 2000). In addition, local acquired resistance seems to be associated with PP cell activation and traumatic resin duct formation (Christiansen et al. 1999, Krokene et al. 1999, 2003, Franceschi et al. 2002). These studies suggest that phenolics play an important role in the defense strategy of Norway spruce. Stilbenes and flavonoids, together with the enzymes involved in their synthesis (e.g., PAL = phenylalanine ammonia lyase, CHS = chalcone synthase and STS = stilbene synthase; Lindberg et al. 1992, Brignolas et al. 1995a, 1995b, Franceschi et al. 1998, Chiron et al. 2000, Nagy et al. 2000, Viiri et al. 2001a), are among the most intensively studied anti-fungal phenolic compounds in Norway spruce bark. Stilbenes and flavonoids, which are constitutively present as glycosides, represent a primary chemical barrier to invasion and are directly involved in defense against injury and fungal infection (Nicholson and Hammerschmidt 1992, Brignolas et al. 1995a, 1995b, Evensen et al. 2000). Chemical analyses have shown that there are differences in phenolic composition and enzyme activities between resistant and susceptible clones of Norway spruce, both before and after fungal inoculation (Brignolas et al. 1995a, 1995b, Evensen et al. 2000). Phenylalanine ammonia lyase, a key enzyme in phenolic synthesis, is found in PP cells and ray cells of Norway spruce bark (Franceschi et al. 1998), in xylem and in epithelial cells of traumatic resin ducts (Nagy et al. 2000). The peroxidases constitute another important group of enzymes associated with phenolic chemistry that are involved in defense-related processes such as lignification, cross-linking of cell wall proteins, auxin catabolism, production of oxygen radicals, as well as direct defense against pathogens (Campa 1991, Mohan et al. 1993, Otter and Polle 1997). Increased accumulation of peroxidases has been reported in Norway spruce seedlings infected with a root pathogenic fungus (Asiegbu et al. 1999, Fossdal et al. 2001). The recently discovered role of PP cells in the defense of Norway spruce (Franceschi et al. 1998, 2000) prompted us to investigate the anatomical features of these cells in relation to phenolic synthesizing agents. By studying Norway spruce clones differing in resistance to fungal infection, we hoped to better understand the underlying defense mechanisms. We show that PP cell activation, which involves enlargement and changes in polyphenolic content, is preceded by up-regulation of chalcone synthase and highly basic peroxidases. These reactions occurred more rapidly in a relatively resistant clone than in a less resistant clone.

Materials and methods Plant material and fungal inoculation Field experiments were started on June 1, 1999 with 30-year-old Norway spruce trees from a clonal stand at the Hogsmark plantation of the Norwegian Forest Research Institute, s, Norway (see Franceschi et al. 1998 for a description of the stand). The studied trees were of Clone 579, with high resistance to C. polonica, and Clone 267, with limited resistance to C. polonica. Four trees of each clone were inoculated with pathogenic fungus growing on malt agar, which was applied with a 5-mm cork borer as described by Krokene and Solheim (1998). Each tree received 15 inoculations evenly spaced around the trunk in four rings, situated 1 m apart between 3 and 6 m above ground (six inoculations in the uppermost ring and three in each of the lower rings). Of the 15 inoculation sites per tree, five were inoculated with C. polonica (Isolate No. 93-208/115 from the Culture Collection of the Norwegian Forest Research Institute), five with Heterobasidion annosum (Fr.) Bref. (heterocaryotic strain 87-257/1, S intersterility group) and five with sterile agar as wounded controls. Only samples from C. polonica and sterile agar inoculated sites were included in the present study. Tissue samples were collected from each tree 3, 6, 10, 16 and 37 days after inoculation (starting with the uppermost ring) by removing rectangular strips of bark and sapwood (1.6 cm wide 10 cm long; Franceschi et al. 1998) immediately below and above the inoculation site. Upper samples were used for microscopy, and lower samples were used for molecular analysis. In addition, control samples of uninoculated tissues from the same trees were collected on the day of inoculation and each subsequent sampling day. Control samples were always taken at least 10 cm laterally from wounded sites. For microscopy, bark strips were stored in fixative until processed. For molecular analysis, whole tissue strips were frozen in liquid nitrogen immediately after sampling and stored at 80 C. Relative resistance of the two clones was determined by measuring the lengths of necrotic lesions in the inner bark of the samples for microscopy. Necrosis lengths were measured upward from the inoculation sites. The two clones were selected from a group of 15 clones that were tested for resistance in 1997 by measuring necrosis lengths 60 days after inoculation with C. polonica (eight inoculations per tree at 2 m height in 23 trees per clone; H. Solheim, unpublished data). Light microscopy and image analysis Subsamples, containing phloem, cambium, and at least one annual ring of xylem, were dissected 50 mm above the inoculation site on the upper samples. These samples were fixed in paraformaldehyde (2%) and glutaraldehyde (1.25%) in L-piperazine-N,N-bis (2-ethane sulfonic) acid buffer (50 mM, pH 7.2) for 12 h at room temperature, washed in the same buffer (3 15 min), dehydrated in an ethanol series, infiltrated in a resin:ethanol series and embedded in LR White acrylic resin (polymerization at 60 C for 24 h; TAAB Laboratories,

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Aldermarston, U.K.). Semi-thin cross sections (1.5 m) of the phloemcambium region were cut with a diamond knife, dried onto gelatin-coated glass slides, stained with periodic acid Schiffs (PAS) carbohydrate stain (Hotchkiss 1948, Nagy et al. 2000), and mounted with immersion oil. All reagents were from Sigma-Aldrich (St. Louis, MO). Sections were imaged at 16 with a Leica DC200 CCD camera on a Leitz Aristoplan photomicroscope. The images represented 540 435 m of the phloem, which included the 34 youngest rows of fully differentiated PP cells in the tangential direction. The current annual PP cell layer (PP99) was omitted from the analysis because the cells were undifferentiated at the time of the experiment. In the next three annual layers of PP cells (PP96, PP97, PP98), the contour of individual PP cells, and polyphenol bodies and starch grains within these cells, were outlined on transparencies. Cross-sectional areas were determined from scanned images of the transparencies using image analysis software (ImagePro Plus, Version 3.0, Media Cybernetics, Leiden, The Netherlands). For each sampling day, total cross-sectional areas of PP cells, polyphenol bodies and starch grains within the three annual layers of PP cells were expressed as proportions of the total image area. Changes over the course of the experiment are presented relative to Day 0 for each clone. RNA extraction and Northern analysis We extracted RNA from inoculated and control samples from two trees per clone and time, following the procedure of Lnneborg and Jensen (2000). Briefly, the outer bark was removed from the frozen samples, and 12 g of the inner bark was separated from the wood, ground with liquid N2 for 10 min, and extracted for 30 min in homogenizing buffer (0.5 M TRIS-buffered saline (pH 8.0), 300 mM NaCl, 5mM EDTA, 2% SDS, 0.5% polyvinylpyrrolidone (PVP), 0.5 mM aurintricarboxylic acid (ATA) and 14.3 mM 2-mercaptoethanol) (15 ml of homogenizing buffer per g fresh mass of inner bark). After multiple (67 times) chloroform:isoamyl alcohol extractions, the extracts were treated with 100 mg g 1 polyvinylpolypyrrolidone (PVPP) for 20 min at 75 C, and the RNA pellets were washed once with 2 M LiCl and once with 70% ethanol. The resuspended pellet was reprecipitated once with 2 M LiCl. For RNA gel blot hybridization, a total of 15 g total RNA was used and standard protocols (Ausubel et al. 1987) were followed. A 32P-labeled cDNA probe of Norway spruce chalcone synthase (CHS) was used for the hybridization. The CHS probe used (SPI3; GenBank accession number AF 417107) corresponds to the last 217 bp of the 546 partial cDNA. This probe, which contains the 3-untranslated region with < 30% similarity to other sequences, was used to avoid cross hybridization. The only known CHS-like sequence isolated from Norway spruce is SP13, and the 5 partial 346 bp coding region of this Norway spruce CHS shows 90% similarity to CHS sequences from other spruces and Pinus sylvestris L. (data not shown).

Filters were washed under stringent conditions with 0.1 SSC with 0.1% SDS at 65 C in the last two washes. Relative amounts of transcripts (measured as counts per minute, CPM) were determined from the 32P-signals with an Instant Imager (Packard Instrument, Meriden, CT) at an exposure time of 15 min in each case. The CPM data were obtained from three membranes hybridized in parallel and adjusted for the signal intensity from a 32P-labeled 18S rRNA probe as described by Fossdal et al. (2001). Film exposure was performed for 1624 h at 80 C after CPM data were obtained from the filters. All reagents were from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburg, PA) unless otherwise stated. Real time PCR Oligonucleotide primers and probes for real-time PCR were designed with Primer Express software Version 1.5a provided with Applied Biosystems (Foster City, CA) Real Time Quantitative PCR systems. Primers were designed for the Norway spruce CHS cDNA sequence, Gene Bank accession number AF417107. The designed CHS forward and reverse primers (5-CCGCCTCTCAAATAAATCGTATTAGT-3 and 5-ATT ATCAATTATTTGGGTTTCAGTTCTG-3, respectively) amplify only the 3 untranslated end of this partial cDNA (from base 354 to base 446). After amplification, only one peak was seen in the melting point analysis, verifying that only one PCR product originated from the CHS primer pairs. For each sample, a second internal control reaction was performed using the Norway spruce alpha-tubulin (aT ) transcript (Gene Bank accession number X57980) as a target under identical reaction conditions as for CHS. The aT 5-GGCATACCGGCAGCTCT TC-3 and 5-AAGTTGTTGGCGGCGTCTT-3 forward and reverse primers, respectively, amplify the cDNA region from base 187 to 252 of the aT cDNA. Only one peak appeared in the melting point analysis, verifying that one PCR product originated from the aT primer pairs. The primers were ordered from ABI PRISM Primers & TaqMan Probes Synthesis Service (Applied Biosystems). For cDNA synthesis, 2.5 g of total extracted RNA per sample was used. The cDNA synthesis was performed with the TaqMan reverse transcription reagents kit using a poly dT primer for cDNA synthesis. Equal volumes (5 l) of cDNA were used as the template and a 25-l PCR reaction was performed with a SYBR Green PCR Master Mix on 96-well PCR plates (Applied Biosystems). The master mix contained AmpliTaq Gold DNA polymerase, AmpErase uracil-N-glycosylase (UNG), dNTPs with dUTP, passive reference 1 and buffer components. The AmpErase UNG prevents the reamplification of any carryover PCR products containing uracil. The concentration of each primer was 50 mM. The samples were run as duplicates in each PCR run, and the CHS reaction and the aT control for the same sample were always run on the same plate. The PCR cycling parameters were 50 C for 3 min for UNG enzyme activity, 95 C for 10 min to denature the UNG enzyme and to activate the polymerase, and 40 cycles at 95 C for 15 s and 60 C for 1 min. The PCR reactions were run on agarose gels, and a single cDNA band of the ex-

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pected size was obtained after each run. No band was detected from the no template controls or the samples containing only the fungus (data not shown). Real-time PCR detection was performed on the ABI PRISM 7700 (Applied Biosystems). Data acquisition and analysis were performed with Sequence Detection System 1.7a Software Package (Applied Biosystems). For each sample, the critical threshold cycle value for CHS (dCt) was subtracted from the corresponding dCt value for aT, giving the relative ddCt value. This value gives the relative level of CHS compared with aT in the sample, and the higher the ddCt value, the more CHS transcript was present in the sample. For each time point, the ddCT values from the unwounded control samples were subtracted from the ddCT values from C. polonica and sterile agar inoculated samples, and the baseline of the graph was set at zero. Protein extraction and isoelectric focusing electrophoresis (IEF) of peroxidase isoforms Native, crude protein was extracted from 0.30.4 g of phloem tissue (prepared and ground with liquid N2 as described above) from inoculated and wounded control samples from two trees per clone and time, in low pH buffer (0.001 M sodium citrate, pH 5.0) with 3% CHAPS (3-[(3-cholamidopropyl) dimethyl amino]-1-propane-sulfonate). Insoluble material was removed by centrifuging twice at 10,000 g for 20 min. Total protein concentration was determined spectrophotometrically with Coomassie Plus Protein assay reagent (Pierce, Rockford, IL), and bovine serum albumin as the standard. To detect peroxidase isoforms, equal amounts of total protein (2.6 g) were applied to, and separated on, non-denaturing IEF gels (10% polyacrylamide gels, 1 mm thick, pH 3.59.5; Amersham Biosciences) prepared according to the manufacturers instructions. Isoelectric focusing was run at 4 C and 20 W for 2.5 h with an LKB 2117 Multiphor system (LKB, Bromma, Sweden). Peroxidase isoform activity was assayed as described by Kerby and Sommerville (1989), with 3-amino-9-ethylcarbazole (carbazol) and hydrogen peroxidase as substrates. All reagents were from Sigma-Aldrich unless otherwise stated. Statistical analysis Lesion length data and image quantification data were evaluated by analysis of variance by the mixed procedure of the SAS software package (SAS Institute, Cary, NC). Data from Days 10 and 37 after inoculation were analyzed on a single-tree basis, by considering contrasts between treatments within trees (pathogen treatment versus sterile agar or untreated bark controls) as the response variable. The statistical model was yij = + ci + eij ,where yij is the contrast of Clone i (i = Clone 267 or Clone 579) and ramet j ( j = 1, 2, 3, or 4) within Clone i, is the total mean, ci is the fixed effect of Clone i, eij is the random experimental error (residual) for all traits analyzed. The values of clones were tested for difference from 0 with t-tests (P < 0.05), and differences between clones were tested with an F-test (P < 0.05). In both cases, the residuals

were used as the experimental error term and values significantly different from 0 imply a treatment effect. Where differences between the analyses at Days 10 and 37 are significant, an effect of time is implied. Results Necrotic zone formation in response to inoculation Necrotic zones, observed as areas of yellow-brownish discoloration in the phloem, were produced in response to inoculation with C. polonica or with sterile agar, but were not observed in uninoculated control samples. In both inoculation treatments and clones, a narrow yellowish zone appeared around the inoculation point 3 days after inoculation. Later on, this zone became darker brown, and gradually increased in size, primarily in the longitudinal direction. Both clones and treatments had similar-sized necrotic zones during the first 16 days after inoculation (Figure 1), but the less resistant clone produced darker and significantly longer zones in response to the fungal inoculations compared with the sterile inoculations at Day 16 (P = 0.003). At Day 37, fungal inoculations had induced significantly longer reaction zones than sterile inoculations in the less resistant clone (P < 0.0001), but only marginally longer zones in the more resistant clone (P = 0.057). The difference between treatments was significantly greater in the less resistant Clone 267 than in the more resistant Clone 579 (Figure 1; P = 0.0009). Anatomical characterization of PP cells Light microscopy studies of phloem cross sections dissected 50 mm above the inoculation site revealed induced swelling of PP cells in both clones at Days 16 and 37 after inoculation with C. polonica (Figure 2). Very little or no swelling was observed in the corresponding samples inoculated with sterile agar or in uninoculated control samples. At the start of the experiment

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Figure 1. Phloem necrosis lengths in two Norway spruce clones ( = 579, = 267) inoculated with Ceratocystis polonica (filled symbols) or sterile agar (open symbols). Necrosis lengths were measured upward from the inoculation point on one sample per treatment per day after treatment, and are presented as means SE (n = 4 trees per clone).

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Figure 2. Light microscopy images of polyphenol-containing parenchyma (PP) cells in the 1996, 1997 and 1998 annual cell layers of the phloem before and after inoculation with the pathogenic fungus Ceratocystis polonica. All figures represent cross sections of tissue stained with periodic acid Schiffs stain. (A) At Day 0, the phloem consists of sieve cells and layers of small rounded PP cells with polyphenolic inclusions and starch grains. One cell thick parenchyma ray cells run radially through the rows of PP cells and sieve cells (400, bar = 25m). (B) At Day 37, the PP cells are swollen and the sieve cell layers are compressed because of the enlargement of the PP cells (400, bar = 25 m). (C) High magnification of the PP cell layer deposited in 1997 at Day 0 of the experiment showing PP cells filled with densely staining, evenly dispersed phenolic material in the vacuole and clusters of starch grains in the cytoplasm (800, bar = 12.5 m). (D) At Day 37, the PP cells are enlarged, phenols are of a porous-like type or dispersed around the surface of the vacuole, and starch grains are scattered (800, bar = 12.5 m).

(Figure 2A), the phloem contained regular annual layers of PP cells, with rows of sieve cells and radial oriented ray cells in between. At Day 37 after inoculation with C. polonica (Figure 2B), the PP cells in all observed rows had swollen and compressed the surrounding sieve cells into dense layers of cell walls. The rays appeared wavy because of this compression and they also contained starch grains. In all observed PP cell layers, the cells were alive at all sampling times, as indicated by the presence of intact cytoplasm containing nuclei, lipid bodies and starch grains. In both clones, the starch pool and polyphenol concentration of the PP cells were affected by infection with C. polonica (Figure 2). At Day 0, polyphenols were observed as large homogeneous bodies within the vacuole of the more resistant clone (Figure 2C), but appeared as scattered droplets in the less resistant clone (data not shown). At Day 37 after infection

(Figure 2D), the polyphenol bodies in the swelling PP cells in both clones appeared as porous aggregates that filled the whole vacuole, as distinct dense rings along the cell periphery, or as droplets. Starch grains appeared clustered before inoculation in both clones (Figures 2A and 2C), but were more scattered and less prominent after infection (Figure 2D). In the more resistant clone, starch grains were less pronounced 16 days after infection (Figure 2B and 2D), but seemed to be more prominent again 37 days after infection, whereas they continued to be less obvious in the less resistant clone. In control tissue at Day 37, only small and scattered starch grains were observed in both clones (data not shown). Quantification of morphological changes in PP cells To quantify morphological changes in PP cells, we measured the cross-sectional areas of PP cells, polyphenol bodies and

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starch grains within the 19961998 annual layers (PP1996 PP1998). Because there was no increase in PP cell size (P = 0.490.77) or polyphenol body size (P = 0.150.61) in uninoculated control samples at 10 or 37 days after the start of the experiment (Figures 3A and 3B), we assume there were no whole-tree systemic effects in either clone after treatment. Up to Day 10, the mean cross-sectional area of PP cells in all three annual layers was comparable between clones and treat-

ments (~600 m2 per cell, or ~10% of the imaged areas) (Figure 3A). At Day 37 after fungal inoculation, all PP cells in both clones had enlarged significantly compared with values at Day 0 (P = 0.0001 for both clones) (Figure 3A), and PP cells had a mean cross-sectional area of about 1500 m2 in the more resistant clone and 1200 m2 in the less resistant clone. Inoculations with C. polonica induced much more swelling than sterile inoculations at Day 37 (P = 0.01 and 0.057 for the more and the less resistant clone, respectively), and the difference between the treatments did not differ significantly between clones (P = 0.39). Sterile inoculation induced little increase in PP cell size 50 mm from the inoculation site and did not differ significantly (P = 0.430.79 for the two clones) from the uninoculated controls (Figure 3A). The area of the polyphenolic bodies within PP cells increased following inoculation with C. polonica (Figure 3B). At Day 0, the mean cross-sectional area of phenolic bodies per PP cell was ~240 m2 in all three annual layers. Thirty-seven days after inoculation with C. polonica, this area had increased significantly to about 1000 m2 in both clones (P < 0.0005). At this time, the phenolic bodies were significantly larger in response to fungal inoculation than in response to sterile inoculation (P < 0.009 for both clones) (Figure 3B). The difference in phenolic body size in fungal and sterile inoculated samples did not differ significantly between clones (P = 0.90). Phenolic body size in sterile inoculated samples did not increase after inoculation, and was not significantly larger than in uninoculated controls 37 days after inoculation (P > 0.99 for both clones). The area within PP cells that was covered by starch grains showed an initial increase during the first 10 days after inoculation, followed by a large decrease in most cases (Figure 3C). Starch concentrations decreased more and sooner after infection in the more resistant clone (Figure 3C), but then showed a significant increase in fungal-inoculated tissues between Days 16 and 37 (P < 0.04). In the less resistant clone, there was a significant reduction in starch concentrations over the same interval (P < 0.03). In both clones, however, fungal inoculation induced significantly less starch depletion than sterile inoculations 37 days after inoculation (P < 0.02). Starch concentration decreased significantly in uninoculated control tissues of both clones from Day 0 to Day 37 (P < 0.03). Temporal levels of chalcone synthase transcripts after infection Northern blotting with the CHS probe detected low transcript levels in phloem extracts of uninoculated control tissues. After infection, CHS transcripts increased dramatically in both clones (Figures 4AC). Transcript levels started to increase from Day 3, peaked at Days 1016, and then declined toward Day 37 (Figure 4B). Transcripts increased to a higher level and peaked earlier in the more resistant clone than in the less resistant clone (Figure 4B). In the more resistant clone, maximum transcript intensity was detected at Day 10, whereas in the less resistant clone, maximum intensity was detected at Day 16.

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Figure 3. Changes in cross-sectional areas of polyphenolic parenchyma (PP) cells and cell components in three annual cell layers (PP1996PP1998) in the phloem of Norway spruce. Data are presented as percent change in total image area that is covered by (A) PP cells, (B) polyphenol bodies and (C) starch grains, relative to the areas measured at the start of the experiment. Cross sections were taken from two clones ( = 579, = 267) inoculated with Ceratocystis polonica (filled symbols) or sterile agar (open symbols). Ceratocystis polonica inoculated tissue sites were analyzed 3, 6, 10, 16 and 37 days after treatment. Sterile agar and uninfected control tissue were analyzed at Days 10 and 37 (open symbols without continuous line). On each cross section, PP cells extending 0.5 mm in the tangential direction of the tissue were included in the analysis. The data are presented as means + or SE, n = 4 individual trees per clone.

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Figure 4. (A) Northern-blot analysis showing chalcone synthase (CHS) transcripts in uninoculated bark (Control) and in bark inoculated with Ceratocystis polonica (Infected) from two Norway spruce clones (579 and 267) at 0, 3, 6, 10, 16 and 37 days after inoculation. The RNA transcripts were detected with 32P-labeled cDNA probes for CHS. Hybridization with 32P-labeled 18S rRNA was carried out as a control for loading. (B) Relative 32P-activity (counts per minute, CPM) of the transcripts was detected by Instant Image quantification (relative to 18S rRNA). Values represent means of n = 3 blots (three replicates from the same extraction) per clone ( = 579, = 267). Open and filled symbols indicate uninoculated control samples and samples inoculated with C. polonica, respectively. (C) Real-time PCR analysis of CHS transcripts in clone 579 ( ) and 267 ( ). Open and filled symbols indicate samples inoculated with sterile agar and C. polonica, respectively. The CHS transcript was detected by real-time PCR and subtracted from alpha-tubulin (aT ), to express relative levels of CHS (ddCT) at 3, 6, 10, 16 and 37 days after treatment. The ddCT values obtained from the unwounded control samples were subtracted from the ddCT values from C. polonica and sterile agar inoculated samples at each time, and the baseline of the graph was set at 0. Data are presented as means SE, n = 2 trees per clone.

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To increase the temporal resolution and to compare wounded and inoculated samples we did a real-time PCR analysis of CHS transcripts relative to a tubulin transcript (ddCt). The relative ddCT-levels showed the same trend as the Northern analyses (Figure 4); CHS transcript levels increased faster and to a greater extent after infection in the more resistant clone than in the less resistant clone. There was a clear effect of wounding on CHS transcript levels, but the response was weaker than with fungal infection. Temporal change in peroxidase isoforms after infection Non-denaturing IEF gels of peroxidases revealed the existence of multiple isoforms in the bark of both clones (Figure 5). Ten to 11 peroxidase activity bands were observed in control tissues of both clones. An extra basic band (pI ~8.6) was observed only in the less resistant clone, and an acidic band (pI ~3.4) was seen only in the more resistant clone. After infection, the intensity of two highly basic isoforms (pI ~9.5) increased relative to the control, whereas none of the acidic isoforms were affected by infection (Figure 5). The increase

was most pronounced 10 and 16 days after infection in the more resistant clone, whereas in the less resistant clone the same bands were only weakly detected at Day 16 (Figure 5). One weak band (pI ~ 9.0) became less intense after infection in both clones. Discussion Cellular and tissue reactions Different types of stress (e.g., wounding, pathogen infection, drought) may induce similar responses in plants. This raises the question of whether it was wounding, fungal infection or both, that induced the responses we observed. Responses to mechanical wounding are well characterized in conifers (Wainhouse et al. 1998) and are generally weaker and more restricted than those observed when pathogenic fungi are introduced into the wound (Solheim 1988). Similarly, we observed that control samples from sterile agar inoculations showed limited responses compared with responses of samples infected with C. polonica, indicating that the changes we ob-

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Figure 5. Isoelectric focusing electrophoresis (IEF) analysis of peroxidase isoforms in uninfected bark (Control) and bark infected with Ceratocystis polonica (Infected) from two Norway spruce clones (579 and 267) at 0, 3, 6, 10, 16 and 37 days after inoculation. Native proteins were extracted in low pH buffer containing CHAPS. Crude protein extracts from 0.1 g of bark tissue were separated on a 10% IEF gel (pH 3.510). Equal amounts of total protein (5 g) were loaded in each lane. The IEF gel is a representative of the analysis of samples from n = 2 trees per clone. Estimated pI values and two highly basic peroxidase isoforms, one with increased intensity ( +) and one with decreased intensity ( ) after infection, are indicated.

served were primarily an effect of pathogen infection. When individual trees are sampled repeatedly over time, as in our study, there is a possibility of eliciting whole-tree systemic responses (Bonello et al. 2001). However, control samples from unwounded fresh bark appeared normal, indicating that the responses we observed were primarily local. Similar conclusions have been reached in other studies based on a comparable experimental design (Franceschi et al. 2000, Nagy et al. 2000, Krekling et al. 2003, Krokene et al. 2003). Defense responses of coniferous trees to pathogen infection include rapid cell death and accumulation of secondary metabolites at the infection site (e.g., phenolic and resinous compounds), resulting in discrete necrotic lesions (Berryman 1972, Nicholson and Hammerschmidt 1992, Krokene and Solheim 1997). Tissue discoloration and necrosis are associated with oxidation and polymerization of accumulating phenols. These processes make the attacked tissues resistant to microbial decay, constrain fungal invasion, and may lead to acquired resistance against future attacks (Klement and Goodman 1967, Nicholson and Hammerschmidt 1992, Krokene et al. 1999). The PP cells form the major living tissue in the phloem, and are a target for invasive organisms as well as an important site for resistance to infection (Franceschi et al. 1998, 2000, Krekling et al. 2000, Krokene et al. 2003). When an intruder damages PP cells, it may cause the release of stored phenols and activate inducible defense responses (Franceschi et al. 1998, 2000). We observed that pathogen infection resulted in pronounced expansion of PP cells, as has been reported for other Norway spruce clones (Franceschi et al. 1998, Krokene et al. 2003). Expansions of PP cells might be a secondary response induced by growth substances liberated from dying cells (Biggs 1992, Woodward 1992), or result from changes in turgor pressure caused by an accumulation of osmotically active substances like soluble phenols in the vacuole (Franceschi et al. 1998). In our study, PP cell expansion was accompanied by an increased area of polyphenols within cells and changes in the appearance of the phenols from large homogeneous bodies and scattered droplets to porous aggregates and ring-

like structures along the cell periphery. The quantitative and qualitative changes observed in the phenolic bodies may be related to alterations in their chemical state, such as production and accumulation of insoluble phenol polymers or phenol monomers with higher solubility. In support of this, other studies have demonstrated both quantitative and qualitative changes in phenolic chemistry in the reaction zones of Norway spruce phloem. Trees inoculated with C. polonica show an early increase in concentrations of flavonoids (e.g., catechin and stilbene phenolic monomers), and it has been suggested that flavonoids are gradually converted to tannins and other insoluble compounds (Brignolas et al. 1995a, Evensen et al. 2000). The increase in flavonoids and tannins is interpreted as the formation of a chemical barrier, because these compounds can inactivate fungal enzymes and have fungistatic effects (Brignolas et al. 1995c, Evensen et al. 2000). The ensuing polymerization may lead to stiffening of the cell matrix, which may inhibit the progress of invading pathogens (Brignolas et al. 1995b). Different clones of Norway spruce show highly variable resistance to colonization, and clonal differences in phenolic content have been reported. Brignolas et al. (1995a) found that resistant clones had a higher mean concentration of flavonoids, whereas Evensen et al. (2000) concluded that the concentration of stilbene was more closely related to resistance. We found a larger relative increase in polyphenolic concentration in the less resistant clone, which initially had a lower concentration of polyphenols than the more resistant clone. This finding could be explained by the need to produce additional amounts of phenols to reach a high enough concentration to provide efficient defense in the less resistant clone (cf. Krokene et al. 2003). We note that, because of the low number of inoculations per tree in our study, all trees were able to defend themselves and contain the pathogen within discrete lesions. Concomitantly with phenol accumulation in PP cells, starch concentrations decreased in both clones after infection. This observation is in accordance with the findings of Viiri et al. (2001b) that the concentration of soluble carbohydrates in

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Norway spruce phloem decreased considerably after C. polonica inoculation. Starch reserves in the phloem constitute a carbon source that may be mobilized rapidly during defense reactions for use in the synthesis of secondary metabolites such as resin (Bordasch and Berryman 1977) and polyphenols. Starch is hydrolyzed in cells bordering lesions induced by mechanical wounding, and the resulting sugars are used for increased respiration during repair reactions, for production of protecting substances (e.g., phenols, tannin) and for additional cell wall material (Wardell and Hart 1970, Johansson and Stenlid 1985). After infection, the reduction in starch concentrations was faster in the more resistant clone than in the less resistant clone. However, after Day 16, starch concentrations increased again in the more resistant Clone 579, whereas they continued to decrease in the less resistant Clone 267. An explanation for the lower starch concentrations in the less resistant clone may be that more carbon was used for defense. Alternatively, a re-accumulation of starch reserves may have occurred in the more resistant clone after the most active phase of the defense response was completed. These renewed starch reserves could potentially contribute to counteract later attacks and thus form part of the acquired resistance response observed in Norway spruce (e.g., Krokene et al. 1999). In the controls of both clones there was a continuous decrease in starch concentrations, which is expected in healthy tissue as a result of normal consumption during the growing season (Christiansen and Ericsson 1986). Changes in CHS transcript levels and peroxidase activity Although transcript levels were analyzed from only two trees per clone per time after inoculation, our results highlight important temporal aspects of defense responses in trees with different degrees of resistance to pathogen infection. Transcripts of CHS, which were found at low constitutive levels in unwounded control tissues, were strongly up-regulated 3 16 days after fungal inoculation in both clones. This is consistent with other studies showing that infection results in elevated transcript levels, enzyme activity, and amounts of flavonoids and stilbenes (Lindberg et al. 1992, Schwekendiek et al. 1992, Brignolas et al. 1995a, 1995b, Dixon and Paiva 1995, Viiri et al. 2001a). The changes in CHS transcript levels occurred about a week before observable morphological changes appeared in the phenolic bodies of the PP cells. This delay may correspond to the time required for increased enzyme activity to produce observable morphological changes in phenol bodies. Transcript levels peaked sooner after infection in the more resistant clone (Day 10) than in the less resistant clone (Day 16). We also detected an earlier increase after infection in a basic peroxidase isoform (pI 9.5) in the more resistant clone. Brignolas et al. (1995a) found higher activities of chalcone synthase and stilbene synthase in response to C. polonica infection in a more resistant clone than in a less resistant clone of Norway spruce. Based on this observation, they proposed that resistant clones are better able to activate the phenolic pathway than less resistant clones. Our results on CHS transcript levels and peroxidase activities indicate that differences in resistance

are related to the speed of expression of these enzymes. In general, the more rapidly a defense response develops, the lower the possibility for pathogen invasion (Biggs 1992, Woodward 1992, Woodward and Pocock 1996). A decline in transcript levels and peroxidase activity was observed by Day 37 after infection. This decline is in accordance with observations in angiosperm systems, suggesting that defense responses to infection are transient (Dixon and Harrison 1990) and cease when the further accumulation of metabolites becomes unnecessary. The basic peroxidase that increased in the more resistant clone after infection may be similar to the defense-related spruce peroxidase (SPI2, pI 9.5) that accumulates in Norway spruce seedlings after infection with the pathogen Pythium dimorphum Hendrix and Campbell (Fossdal et al. 2001). Spruce peroxidase SPI2 accumulated in the shoot as a systemic response to infection in the roots, indicating that up-regulation of these peroxidases forms part of the host defense system (Fossdal et al. 2001). This is consistent with the active role peroxidases play in defense reactions and fundamental resistance mechanisms in plants. Previous studies suggest that basic peroxidases participate in lignin formation during needle development in Norway spruce (Polle et al. 1994, Otter and Polle 1997), and our study demonstrates that they also accumulate in stems of mature Norway spruce after fungal infection. In conclusion, PP cells, vacuolar polyphenolic bodies, starch conversion, and activity of phenol-synthesizing metabolites converge to enhance resistance to pathogen infection in Norway spruce. Taken together, these responses probably form an important part of the resistance capacity of individual clones. Rapid mobilization of defense responses appears to be crucial for resistance, and an early response was typical of the more resistant clone where CHS transcript levels changed more rapidly, peroxidase activity appeared earlier, and polyphenol and starch pools of PP cells were more dynamic than in the less resistant clone.
Acknowledgments The authors gratefully acknowledge Drs. Erik Christiansen and Vince R. Franceschi for critical review of the manuscript and helpful discussions, and Marianne Jensen and Inger Heldal for technical assistance. We also thank Dr. ystein Johnsen for valuable suggestions and help with the statistical analysis. This research was funded by The Research Council of Norway under Project No. 104023/110, 113754/ 111, 117925/140 and 133338/110.

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