Anatomical-based defense responses of Scots pine (Pinus sylvestris) stems to two fungal pathogens

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1 Tree Physiology 26, Heron Publishing Victoria, Canada Anatomical-based defense responses of Scots pine (Pinus sylvestris) stems to two fungal pathogens NINA ELISABETH NAGY, 1 PAAL KROKENE 1,2 and HALVOR SOLHEIM 1 1 Norwegian Forest Research Institute, Høgskoleveien 8, N-1432 Ås, Norway 2 Corresponding author (paal.krokene@skogforsk.no) Received January 28, 2005; accepted April 9, 2005; published online November 8, 2005 Summary We investigated the cellular responses of stem tissues of mature Scots pine (Pinus sylvestris L.) trees to inoculations with two fungal pathogens. The bark beetle vectored fungus, Leptographium wingfieldii Morelet, induced longer lesions in the bark, stronger swelling of polyphenolic parenchyma cells, more polyphenol accumulation and increased ray parenchyma activity compared with the root rot fungus, Heterobasidion annosum (Fr.) Bref., or mechanical wounding. Axial resin ducts in the xylem are a general feature of the preformed defenses of Scots pine, but there was no clear induction of additional traumatic axial resin ducts in response to wounding or fungal infection. The anatomical responses of Scots pine to pathogen infection were localized to the infection site and were attenuated away from bark lesions. The responses observed in Scots pine were compared with published studies on Norway spruce (Picea abies (L.) Karst.) for which anatomically based defense responses have been well characterized. Keywords: calcium oxalate crystals, Heterobasidion annosum, Leptographium wingfieldii, polyphenolic parenchyma cells, ray parenchyma cells, resin ducts, starch. Introduction Pines and other conifers are attacked by a variety of insects and pathogens, which may cause tree death or reduce wood quality. For example, pine trees attacked by bark beetles and their phytopathogenic fungal associates develop a bluish discoloration in the sapwood that reduces the value of the timber. The blue stain fungus, Leptographium wingfieldii Morelet, is the most virulent fungus associated with the pine shoot beetle, Tomicus piniperda L., and can kill healthy Scots pine, Pinus sylvestris L., trees in experimental mass inoculations (Långström et al. 1993, Solheim et al. 1993, 2001). Root rot fungi are another serious problem in Scots pine, causing wood decay and large economic losses. The root rot fungus, Heterobasidion annosum (Fr.) Bref., is the most important disease of pines and other conifers in northern temperate regions (Woodward et al. 1998). It normally enters trees through stumps and root grafts, but can also colonize stem wounds in various conifers (Redfern and Stenlid 1998). In general, conifers respond to attack by bark-boring insects and pathogenic fungi by forming biochemical and anatomical barriers that compartmentalize the attacked area (Reid et al. 1967). An important defense response that serves multiple functions is resin production and secretion. Pines have a welldeveloped preformed resin duct system (Christiansen et al. 1987). Resin can flush out or trap invading organisms, has toxic or inhibitory effects on insects and pathogens and is involved in wound sealing (Johnson and Croteau 1987). In response to an attack, pines produce resin in the preformed resin duct system (Christiansen et al. 1987), as well as in newly formed axial traumatic resin ducts (TD) in the xylem (Lombardero et al. 2000). Phenolics are another important group of defensive compounds. Polyphenols are stored constitutively in the vacuoles of polyphenolic parenchyma cells (PP cells) in the secondary phloem of most Pinaceae species (Franceschi et al. 1998, Krekling et al. 2000, Hudgins et al. 2003). The PP cells increase in size in response to wounding and pathogen infection and probably mobilize their phenolic content into surrounding tissues (Franceschi et al. 1998, 2000, Krokene et al. 2003, Nagy et al. 2004). Chemical changes in Scots pine after wounding or infection include quantitative rather than qualitative changes in terpenes (Delorme and Lieutier 1990, Långström et al. 1992) and qualitative changes in phenols (Lieutier et al. 1996, Bois and Lieutier 1997). Certain phenols that occur constitutively in low concentrations considerably increase in concentration after infection, whereas the concentrations of other phenols decrease, and these changes seem to be correlated with the degree of host resistance (Bois and Lieutier 1997). Anatomical and molecular responses to insect and pathogen attack and wounding have been extensively investigated in Norway spruce (Picea abies (L.) Karst) stems (Brignolaset al. 1995a, 1995b, Franceschi et al. 1998, 2000, Krekling et al. 2000, 2004, Nagy et al. 2000, 2004, Krokene et al. 2003, Hudgins and Franceschi 2004). These studies indicate that protection against entry and spread of the bark beetle, Ips typographus (L.), and its pathogenic fungal associate, Ceratocystis polonica (Siem.) C. Moreau, are associated with features of the resin duct system and PP cells. Here we report a study of anatomical defenses in Scots pine, a species that differs from

2 160 NAGY, KROKENE AND SOLHEIM Norway spruce in both anatomy and in its beetle pests and fungal pathogens. Materials and methods Plant material and fungal inoculation The experiment was carried out on Scots pine trees about 60 years old growing at Åsmyra in Akershus, SE Norway. On June 24, 1999, three randomly selected, healthy trees (with diameters 1.3 m above ground of 23.4, 26.1 and 28.0 cm) were inoculated four times each with L. wingfieldii (Isolate No /11), H. annosum (heterocaryotic strain /8,Pintersterility group) and sterile agar. The fungi were from the culture collection of the Norwegian Forest Research Institute. Inoculations were made by removing a bark plug with a 5-mm cork borer, inserting an inoculum in the wound and replacing the plug. Inocula consisted of actively growing mycelia on malt agar (2.0% malt, 1.5% agar) or sterile malt agar. The 12 inoculation sites per tree were in four rings encircling the trunk at about 1, 1.75, 2.75 and 3.75 m above ground. In each ring, there was a site for sampling uninoculated control tissue, except in the lowermost ring, where there were two uninoculated control sites. Each type of inoculum occurred once in each ring, the inoculation and control sites were evenly distributed within rings and the sequence of treatments was the same in all rings. One ring was sampled 6, 15, 36 and 102 days after inoculation. An uninoculated control sample was taken from the lowermost ring of each tree at the time of inoculation (Day 0). Sampling started at the lowermost ring and proceeded up the stem. Samples, consisting of rectangular pieces of bark and sapwood (1 cm wide, 5 cm high and about 1 cm deep) were placed directly in fresh fixative (see below for description of content). Samples taken at the inoculation sites included the cork borer wound at the lower end. No attempts were made to re-isolate fungus from the inoculated tissues. On the last sampling day, trees had developed extensive symptoms in response to inoculations with L. wingfieldii. In order to include the full host response and to study responses further away from the inoculation sites, additional bark samples were taken directly above the standard sample and 30 cm above the inoculation sites for all treatments. Lesion length at the different inoculation sites was determined by measuring the length of necrosis in the inner bark of the samples. Necrosis lengths were measured upward from the inoculation sites. Light microscopy In the laboratory, 4-mm-wide subsamples of bark and wood were removed from the samples collected at the beginning (Day 0) and end (Day 102) of the experiment. The samples collected on Days 6 36 were used only for lesion length measurements and were not processed for anatomical studies. All subsamples were placed in a drop of fresh fixative of 2% paraformaldehyde and 1.25% glutaraldehyde in 50 mm L-piperazine-N-N -bis (2-ethane sulfonic) acid buffer (ph 7.2). For samples taken above the inoculation sites, 1-mm-wide subsamples were cut from about 2 mm above the upper edge of the inoculation site and about 2 (2.3 ± 0.6, mean ± SD) and 27 cm (26.9 ± 3.0) above the upper margin of the necrotic lesions. For uninoculated control samples, a similar subsample was cut from the middle of each sample. The slices were placed in fixative overnight at room temperature and rinsed with the same buffer (3 15 min), dehydrated in an ethanol series (70, 80, 90, 96 and 4 100%; 15 min in each), infiltrated (resin:ethanol series 1:3, 1:2, 1:1, 100%; 12 h in each) and embedded with LR White acrylic resin (polymerization for 24 h at 60 C; TAAB Laboratories, Berkshire, U.K.). Cross sections (1.5 µm thick) were cut with a diamond knife using an ultramicrotome (Ultracut E. Reichert Jung, C. Reichert A.G., Vienna, Austria), and dried on gelatin-coated slides. All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Sections were stained with Stevenel s blue (del Cerro et al. 1980) for visualization of general anatomy, PP cells, polyphenolic bodies within PP cells and calcium oxalate crystals, and with Schiff s periodic acid stain for visualization of carbohydrates and starch grains (Hotchkiss 1948). The presence of starch was confirmed by staining with iodine potassium iodide. Effects of pathogen infection on the size and number of radial rays in the wood were analyzed on tangential sapwood sections (15 µm thick) stained with Stevenel s blue. Tangential sections were cut close to the cambium (latewood) and deeper into the current sapwood growth (earlywood) with a cryotome (Cryo-Star HM 560, Microm International GmbH, Walldorf, Germany). All stained sections were mounted with immersion oil and examined and photographed with a Leitz Aristoplan light microscope (Ernst Leitz GmbH, Wetzlar, Germany) fitted with a Leica DC300 CCD camera, using bright field optics. Light microscopy with polarized light was used for observations of calcium oxalate crystals. The phenolic nature of the bodies inside the PP cells was confirmed by fluorescence microscopy (Franceschi et al. 1998). Image analysis For quantification of PP cells, polyphenolic bodies, starch grains and calcium oxalate crystals, cross sections were imaged at 16 magnification. The images represented µm of the phloem and included the three most recent annual layers of fully differentiated PP cells (the current annual PP cell layer was omitted from the analysis because the cells were undifferentiated at the time of the experiment). Within these PP cell layers, we outlined the contour of individual PP cells as well as polyphenolic bodies, starch grains and calcium oxalate crystals on transparencies. Cross-sectional areas were determined from scanned images of the transparencies with image analysis software (ImagePro Plus, Version 3.0, Media Cybernetics, Leiden, The Netherlands). For each sampling day, total cross-sectional areas of PP cells, polyphenolic bodies, starch grains and crystals were expressed as proportions of the total image area. For all treatments (including uninoculated controls), changes over the course of the experiment were presented relative to uninoculated samples on Day 0 for each tree. To quantify the size and number of uni- and multi- TREE PHYSIOLOGY VOLUME 26, 2006

3 serate ray parenchyma in the current-year sapwood at the end of the experiment, we used the procedure described for the phloem, except that sections were imaged at 4 magnification, representing µm of the xylem. Xylem resin ducts at the end of the experiment were quantified by measuring the percent coverage of ducts (including the epithelial cells lining the ducts) across 1380 µm in the tangential direction on sections imaged at 5 magnification. INDUCED DEFENSE RESPONSES IN SCOTS PINE 161 Statistical analysis Lesion lengths and image quantification data were analyzed on a single tree basis by using the calculated treatment difference within trees (pathogen treatment versus sterile agar inoculated or untreated bark controls) as the response variable. To test if treatments differed significantly from the control, data were subjected to one-sample t tests with SYSTAT (SPSS, Chicago, IL). In addition, lesion lengths were analyzed by repeated measures analysis of variance (ANOVA), with treatment nested within sampling time, to detect differences over time. The ANOVA was performed with the general linear models procedure in SAS (SAS Institute, Cary, NC). Actual lesion length measurements are presented in Figure 1. Results Despite the small number of replicates (n = 3), we detected significant treatment effects for most variables. When no significant differences could be detected, this was generally because the actual differences were small, except for starch grains and calcium oxalate crystals where great variation between trees masked any treatment effects. Length of necrotic lesions Inoculation with L. wingfieldii induced significantly longer necrotic lesions in the phloem than inoculation with H. annosum or sterile agar (Figure 1) (sterile control: P = 0.004; H. annosum: P = 0.020; 102 days after inoculation). After inoculation, the response to H. annosum did not differ significantly from the response to sterile agar (P = ). Anatomical characterization of PP cells At the start of the experiment, the secondary phloem of uninoculated tissue consisted of regular annual layers of PP cells, with rows of sieve cells and radially oriented ray cells in between (Figure 2A). At the end of the experiment, there were clear differences between treatments in the appearance of PP cells and their polyphenolic bodies. In sterile inoculated tissue, some compression of the sieve cell rows was observed 2 cm above the lesions (Figure 2B). Above H. annosum and L. wingfieldii lesions, the secondary phloem appeared more disorganized, with swollen and irregularly shaped PP cells and surrounding sieve cells that were compressed into dense layers of cell walls (Figures 2C and 2D). The polyphenolic bodies within the PP cells were denser and stained more intensely in uninoculated and sterile agar controls than in tissues inoculated with H. annosum or L. wingfieldii, where polyphenols were of a pumice-like, porous type with light staining. In Figure 1. Necrotic lesion length in the phloem of Scots pine following inoculation with L. wingfieldii ( ), H. annosum ( ) or sterile agar ( ). Lesions were measured upward from the inoculation point. Values are means ± SE, n = 3 trees per treatment. young PP cell layers from pathogen-inoculated tissue, the polyphenolic bodies were of a ring-like type, following the vacuolar periphery (Figures 2C and 2D). At the end of the 102-day experiment, the degree of swelling of PP cells and the size of their phenolic bodies differed between treatments and with distance from the lesions (Figure 3). In uninoculated control samples, there were no significant changes in the size of PP cells or their phenolic bodies relative to samples at the start of the experiment (P = ). Leptographium wingfieldii was the only treatment that induced significant swelling of PP cells and phenolic bodies close to the lesions (96 100% increase relative to uninoculated control, P = 0.04 for both variables; other treatments: 2 42% increase, P = ) (Figures 3A and 3B). Further away from the lesions (27 cm), L. wingfieldii induced a small but significant increase in PP cell size (23% increase, P = 0.04; Figure 3A), whereas none of the other treatments significantly increased phenolic body size at 27 cm from the lesions (1 20% increase, P = ; Figure 3B). Starch granules were present in the cytoplasm of PP cells sampled 2 cm above the lesions. At the start of the experiment in late June, the starch grains appeared as dots clustered along the inner margin of the cell wall. At the last sampling date in early November, little starch was left in any sample and only tiny grains remained visible. The area covered by starch grains varied greatly among trees and the observed differences between inoculated and uninoculated tissues at Day 102 were not significant for any comparisons (56 184% difference between the various treatments, P = ). However, relative to the area covered by starch grains in uninoculated tissue at the start of the experiment (1.51% of cross-sectional area), there was a clear and significant reduction (3- to 8-fold reduction, P = ) in all treatments at the end of the experiment, including the uninoculated control. Calcium oxalate crystals were found scattered evenly throughout the secondary phloem (Figure 2). These crystals occurred intracellularly in crystalliferous parenchyma cells lo- TREE PHYSIOLOGY ONLINE at

4 162 NAGY, KROKENE AND SOLHEIM Figure 2. Light photomicrographs of cross sections of Scots pine phloem showing the most recent annual layers of polyphenolic parenchyma (PP) cells. (A) Uninoculated tissue at the beginning of the experiment. Occasional calcium oxalate crystals are found scattered throughout the phloem (arrowheads). (B D) Inoculated tissue sampled 2 cm above the edge of necrotic lesions 102 days after treatment. (B) Tissue inoculated with sterile agar is similar to uninoculated tissue, and only slight swelling is observed. (C) In tissue inoculated with H. annosum, the PP cells are swollen and the sieve cells have been compressed by the expanding PP cells. Calcium oxalate crystals are more abundant than in (A) and (B). (D) Tissue inoculated with L. wingfieldii is similar to (C), except that the phenolic content of the PP cells is of a porous-like type, or dispersed around the surface of the vacuole. The higher magnification image (inset) shows calcium oxalate crystals in crystalliferous parenchyma cells located between the PP cells. The crystalliferous parenchyma cells are lined with polyphenolic substances. Abbreviations: C = cambium; R = ray cells; and S = sieve cells. TREE PHYSIOLOGY VOLUME 26, 2006

5 INDUCED DEFENSE RESPONSES IN SCOTS PINE 163 Figure 3. Size of polyphenolic parenchyma cells in the current annual phloem increment (A) and the polyphenolic bodies within these cells (B) in Scots pine at 102 days after inoculation with pathogenic fungi or sterile agar. Cell size was measured on 0.23-mm 2 sections sampled ~2 (black bars) and 27 cm (white bars) above necrotic lesions, and is expressed as percent increase in cross-sectional areas of polyphenolic parenchyma (PP) cells and PP cell content relative to uninoculated control samples at the start of the experiment. Values are means + SE, n = 3 trees per treatment. cated between the PP cells (Figure 2D, inset) and were present in both younger and older PP cell layers (Figure 2). Calcium oxalate crystals seemed more abundant in H. annosum and L. wingfieldii inoculated tissues (0.96 and 0.70% of cross-sectional areas, respectively) than in uninoculated controls (0.41% of cross-sectional area), but these differences were not statistically significant because of high variation between trees (L. wingfieldii: P = 0.17; H. annosum: P = 0.09). Anatomical characterization of radial rays in the xylem Numerous radial rays were observed in tangential sections of early- and latewood of the current sapwood growth (Figure 4). Radial rays appeared in uniseriate and multiseriate forms evenly distributed throughout the xylem. The numerous uniseriate rays typically consisted of 5 8 cells in a row. The occasional multiseriate rays had several cell layers in the middle, including a resin canal and single cells at the ends. Rays were narrow in earlywood from both uninoculated and inoculated material (Figure 4A), but had a swollen appearance in latewood. This was particularly prominent close to L. wingfieldii inoculations (Figure 4B), where the area covered by rays was 68% greater than in uninoculated control samples (P = 0.06). There were no significant differences for other combinations of treatments and sapwood depths, either close to the lesions ( 18 to 25% increase, P > 0.12) or further away ( 24 to 1% increase, P > 0.46). Inoculation did not appear to induce production of new rays, because the number of uni- or multiseriate rays was not significantly higher in inoculated than in uninoculated samples (99 versus 112 for uniseriate across all treatments, sapwood depths and sampling positions, 2.2 versus 2.7 for multiseriate; P > 0.15). Anatomical characterization of axial resin ducts in the xylem Axial resin ducts were observed in cross sections of the current sapwood growth. They appeared both as single, scattered ducts and as tangentially oriented rows of ducts. Both arrangements were found together in uninoculated material, as well as in sterile agar and pathogen-inoculated material. There were no significant differences between inoculated and untreated control samples at either 2 or 27 cm above the lesions (L. wingfieldii: P > 0.11; H. annosum: P > 0.33; sterile agar: P > 0.26). However, in the immediate vicinity of the pathogen-inoculated sites and particularly, close to the sites of the L. wingfieldii inoculations, extensive rows of traumatic axial resin ducts were occasionally observed inside callus-like structures in the sapwood (Figure 5A). These structures also contained abnormal swollen tissue with large aggregates of parenchyma cells with large vacuolar polyphenolic bodies and abundant starch grains (Figures 5A and 5B). Normal axial resin ducts that occurred further away from the inoculation sites (Figure 5C) had smaller epithelial cells, the surrounding cells were smaller with fewer polyphenolic bodies and starch grains, and their general appearance was similar to that of resin ducts from untreated samples (Figure 5D). Discussion Anatomical responses to pathogen infection in the secondary phloem of Scots pine included swelling of PP cells and changes in phenolic bodies and starch grains. Such cellular responses are characteristic of conifer defense responses against pathogen attack and subsequent wound sealing. However, the anatomical responses in Scots pine were generally weaker and more localized to the vicinity of the inoculation site than in, for example, Norway spruce (Franceschi et al. 1998, 2000, Krekling et al. 2000, 2004, Nagy et al. 2000, Krokene et al. 2003). Formation of TDs in response to wounding or pathogen attack is a characteristic defense response in the xylem of many conifers (Christiansen et al. 1999, Nagy et al. 2000, Hudgins et al. 2004). Scots pine, however, showed a weak and localized TD response, because no induction of TDs was observed outside the areas of necrotic lesions more than 3 months after pathogen infection. In comparison, the TD response in Norway spruce gradually spreads several meters from a site of infection (Christiansen et al. 1999, Krekling et al. 2004). The weaker and more local response in Scots pine compared with that in Norway spruce might reflect a greater reliance on constitutive anatomical defenses, such as preformed axial resin ducts, PP cells and calcium oxalate crystals. In contrast, many other Pinaceae species, like Norway spruce, have a highly in- TREE PHYSIOLOGY ONLINE at

6 164 NAGY, KROKENE AND SOLHEIM Figure 4. Radial ray parenchyma cells in earlywood (A) and latewood (B) of Scots pine after inoculation with L. wingfieldii. Most of the rays are uniseriate (small arrowhead), with a few multiseriate rays (large arrowhead) with a resin canal in the center. The latewood rays are larger and their cytoplasm appears more active. A large axial resin duct (AD) passes through section B. ducible anatomical defense strategy and less abundant calcium oxalate crystals (Hudgins et al. 2003), but an extensive TD response (Nagy et al. 2000). Inoculation with L. wingfieldii induced much longer lesions in Scots pine than inoculation with H. annosum or mechanical wounding. This agrees well with previous inoculation studies showing that L. wingfieldii is more virulent to Scots pine than other pine-infesting fungi (Lieutier et al. 1989, Långström et al. 1993, 2001, Solheim et al. 1993, 2001). Because L. wingfieldii is a specialized canker fungus, it is expected to be more virulent in stem inoculations than H. annosum, which is normally a root pathogen in pines. Our results agree with previous studies showing H. annosum is a weak wound colonizer in Scots pine (Rishbeth 1951) compared with Norway spruce, where it may be important in wounds made during summer (Redfern and Stendlid 1998). There was also a differential anatomical response to the two pathogens in Scots pine. The more virulent pathogen, L. wingfieldii, induced a stronger activation of PP cells than H. annosum or mechanical wounding. Thus, the Scots pine trees seem to be able to grade the strength of their resistance responses according to the severity of the challenge presented. Many plants produce calcium oxalate crystals as a defense against herbivores or to sequester excess calcium (Arnott and Pautard 1970, Franceschi and Horner 1980, Franceschi 2001). In a recent study of the distribution of calcium oxalate crystals in the secondary phloem of conifers, all Pinaceae species were found to accumulate crystals intracellularly in crystalliferous parenchyma of the bark secondary phloem (Hudgins et al. 2003). Accumulation of calcium oxalate crystals has recently been proposed as a constitutive defense mechanism in the Pinaceae against bark-boring insects (Hudgins et al. 2003). Starch reserves in the phloem declined from June to November, in both inoculated tissues and in untreated control tissues. This is a normal seasonal development, because starch reserves are used for growth (Christiansen and Ericsson 1986) and production of soluble carbohydrates during the frost hardening process in the fall. Phloem starch reserves may also be consumed by defense reactions, such as the formation of reaction zones in the phloem (Reid et al. 1967, Christiansen and Ericsson 1986). After infection, starch reserves are sometimes rebuilt in the vicinity of successful defense reactions (Nagy et al. 2004). In Scots pine, we observed large accumulations of starch grains in the callus-like regions close to the fungal inoculation sites. Rebuilding of starch reserves may be important for local continuation of defense responses and to ensure raw materials for further defense reactions. Such starch reserves could contribute to counteracting later attacks and thus form part of the acquired resistance response observed in Norway spruce and Scots pine (Krokene et al. 2000, Nagy et al. 2004). Pathogen infection induced changes in radial rays. The total ray area increased after L. wingfieldii infection as a result of enlargement of existing rays, not as a result of an increase in the number of rays. The activation of ray parenchyma cells was particularly pronounced in latewood areas of the xylem with abundant axial resin ducts. Enlargement of ray cells may be related to increased cellular activity and resin production. Pine and spruce trees previously exposed to pathogen infection have been shown to develop enhanced resistance to subsequent infections (Krokene et al. 2000, 2001, Bonello et al. 2001). In Norway spruce, this induced disease resistance seems to depend on local activation of PP cells and the formation of traumatic resin ducts (Krokene et al. 2003). Compared with Norway spruce, the strength of the induced disease resistance is weaker in Scots pine and seems unrelated to the TREE PHYSIOLOGY VOLUME 26, 2006

7 INDUCED DEFENSE RESPONSES IN SCOTS PINE 165 Figure 5. Axial resin ducts in the current annual sapwood growth of Scots pine 102 days after inoculation with L. wingfieldii. (A) Appearance of traumatically formed resin ducts in abnormal swelling tissue 2 mm above inoculations with L. wingfieldii. (B) A higher magnification of (A) showing epithelial cells lining the traumatic resin canals and surrounding parenchyma cells filled with starch grains and with polyphenols in the vacuoles. (C) Resin ducts and nearby tissue 27 cm above necrotic lesions. (D) Resin ducts from an uninoculated control sample. Abbreviations: C = cambium; P = phloem; PP = polyphenolic parenchyma cells; R = radial ray; TD = traumatic axial resin duct; and X = xylem. amount of host tissue destroyed by the pretreatment (Krokene et al. 2000, 2001). The weaker induced response in Scots pine parallels the weaker anatomically based defense reactions of this species and it may reflect the difference between Scots pine and Norway spruce in the species of bark beetles that attack them. Unlike Norway spruce and many other conifers, Scots pine is not subject to large-scale outbreaks of aggressive tree-killing bark beetles. The major bark beetle pest of Scots pine under Nordic conditions is the pine shoot beetle Tomicus piniperda that colonizes trees only when they are severely weakened by defoliation or other stress factors (Annila et al. 1999, Långström et al. 2001). The presence of systemic defense responses in pines (Bonello et al. 2001, Bonello and Blodgett 2003) suggests that there might be interference between inoculation sites when trees are given multiple fungal inoculations as in the present study. However, interactions between inoculation sites did not appear to be important in our study, because no significant anatomical changes were observed in unwounded tissues during the experiment. Although all members of the Pinaceae that have been closely examined show a distinct response to fungal infection or artificial induction, our findings indicate that the response may vary significantly among species and that it is regulated based on the virulence of the attacking organism. These inter- and intraspecific differences in defense responses need to be further characterized because they could provide important information on relative pest and disease resistance. Acknowledgments The Research Council of Norway and the Norwegian Forest Research Institute financed this study. Olaug Olsen and Gry Alfredsen provided excellent technical assistance. Sample processing and microscopy was performed at the Electron Microscopy Laboratory at The Agricultural University of Norway under the assistance of Trygve Krek- TREE PHYSIOLOGY ONLINE at

8 166 NAGY, KROKENE AND SOLHEIM ling. We thank the above-mentioned persons and institutions. We are also grateful to Dr. Vincent R. Franceschi, Washington State University, for helpful comments on the manuscript. References Annila, E., B. Långström, M. Varama, R. Hiukka and P. Niemelä Susceptibility of defoliated Scots pine to spontaneous and induced attack by Tomicus piniperda and Tomicus minor. Silva Fenn. 33: Arnott, H.J. and F.G.E. Pautard Calcification in plants. In Biological Calcification. Ed. H. Schraer. Appleton-Century-Crofts, New York, pp Bois, E. and F. Lieutier Phenolic response of Scots pine clones to inoculation with Leptographium wingfieldii, a fungus associated with Tomicus piniperda. Plant Physiol. Biochem. 35: Bonello, P. and J.T. Blodgett Pinus nigra Sphaeropsis sapinea as a model pathosystem to investigate local and systemic effects of fungal infection of pines. Physiol. Mol. Plant Pathol. 63: Bonello, P., T.R. Gordon and A.J. Storer Systemic induced resistance in Monterey pine. For. Pathol. 31: Brignolas, F., B. Lacroix, F. Lieutier, D. Sauvard, A. Drouet, A.C. Claudot, A. Yart, A.A. Berryman and E. Christiansen. 1995a. Induced responses in phenolic metabolism in two Norway spruce clones after wounding and inoculation with Ophiostoma polonicum, a bark beetle-associated fungus. Plant Physiol. 109: Brignolas, F., F. Lieutier, D. Sauvard, A. Yart, A. Drouet and A.C. Claudot. 1995b. Changes in soluble-phenol content of Norway spruce (Picea abies) phloem in response to wounding and inoculation with Ophiostoma polonicum. Eur. J. For. Pathol. 25: Christiansen, E. and A. Ericsson Starch reserves in Picea abies in relation to defence reaction against a bark beetle transmitted blue-stain fungus, Ceratocystis polonica. Can. J. For. Res. 16: Christiansen, E., R.H. Waring and A.A. Berryman Resistance of conifers to bark beetle attack: searching for general relationships. For. Ecol. Manage. 22: Christiansen, E., P. Krokene, A.A. Berryman, V.R. Franceschi, T. Krekling, F. Lieutier, A. Lönneborg and H. Solheim Mechanical injury and fungal infection induce acquired resistance in Norway spruce. Tree Physiol. 19: del Cerro, M., J. Cogen and C. del Cerro Stevenel s blue, an excellent stain for optical microscopical study of plastic embedded tissue. Microsc. Acta 83: Delorme, L. and F. Lieutier Monoterpene composition of the preformed and induced resins of Scots pine and their effect on bark beetles and associated fungi. Eur. J. For. Pathol. 20: Franceschi, V.R Calcium oxalate in plants. Trends Plant Sci. 6:331. Franceschi, V.R. and H.T. Horner Calcium oxalate crystals in plants. Bot. Rev. 46: Franceschi, V.R., T. Krekling, A.A. Berryman and E. Christiansen Specialized phloem parenchyma cells in Norway spruce (Pinaceae) bark are an important site of defense reactions. Am. J. Bot. 85: Franceschi, V.R., P. Krokene, T. Krekling and E. Christiansen Phloem parenchyma cells are involved in local and distant defense responses to fungal inoculation or bark beetle attack in Norway spruce (Pinaceae). Am. J. Bot. 87: Hotchkiss, R A microchemical reaction resulting in the staining of polysaccharide structures in fixed tissue preparation. Arch. Biochem. 16: Hudgins, J.W. and V.R. Franceschi Methyl jasmonate-induced ethylene production is responsible for conifer phloem defense responses and reprogramming of stem cambial zone for traumatic resin duct formation. Plant Physiol. 135: Hudgins, J.W., T. Krekling and V.R. Franceschi Distribution of calcium oxalate crystals in the secondary phloem of conifers: a constitutive defense mechanism? New Phytol. 159: Hudgins, J.W., E. Christiansen and V.R. Franceschi Induction of anatomically based defense responses in stems of diverse conifers by methyl jasmonate: a phylogenetic perspective. Tree Physiol. 24: Johnson, M.A. and R. Croteau Biochemistry of conifer resistance to bark beetles and their fungal symbionts. ACS Symp. Ser. 325: Krekling, T., V.R. Franceschi, A.A. Berryman and E. Christiansen The structure and development of polyphenolic parenchyma cells in Norway spruce (Picea abies) bark. Flora 195: Krekling, T., V.R. Franceschi, P. Krokene and H. Solheim Differential anatomical responses of Norway spruce stem tissues to sterile and fungus infected inoculations. Trees 18:1 9. Krokene, P., H. Solheim and B. Långström Fungal infection and mechanical wounding induce disease resistance in Scots pine. Eur. J. Plant Pathol. 106: Krokene, P., H. Solheim and E. Christiansen Induction of disease resistance in Norway spruce (Picea abies) by necrotizing fungi. Plant Pathol. 50: Krokene, P., H. Solheim, T. Krekling and E. Christiansen Inducible anatomical defense responses in Norway spruce stems and their possible role in induced disease resistance. Tree Physiol. 23: Långström, B., C. Hellqvist, A. Ericsson and R. Gref Induced defence reactions in Scots pine following stem attacks by Tomicus piniperda. Ecography 15: Långström, B. H. Solheim, C. Hellqvist and R. Gref Effects of pruning young Scots pine on host vigor and susceptibility to Leptographium wingfieldii and Ophiostoma minus, two blue-stain fungi associated with Tomicus piniperda. Eur. J. For. Pathol. 23: Långström, B., E. Annila, C. Hellqvist, M. Varama and P. Niemelä Tree mortality, needle biomass recovery and growth losses in Scots pine following defoliation by Diprion pini (L.) and subsequent attack by Tomicus piniperda (L.). Scan. J. For. Res. 16: Lieutier, F., A. Yart, J. Garcia, M.C. Ham, M. Morelet and J. Levieux Phytopathogenic fungi associated with two bark beetles of Scots pine (Pinus sylvestris L.), and preliminary study of their aggressiveness for the host. Ann. Sci. For. 46: Lieutier, F., D. Sauvard, F. Brignolas, V. Picron, A. Yart, C. Bastien and C. Jay-Allemand Changes in phenolic metabolites of Scots pine phloem induced by Ophiostoma brunneo-ciliatum, a bark-beetle-associated fungus. Eur. J. For. Pathol. 26: Lombardero, M.J., M.P. Ayres, P.L. Lorio and J.J. Ruel Environmental effects on constitutive and inducible resin defences of Pinus taeda. Ecol. Lett. 3: Nagy, N.E., V.R. Franceschi, H. Solheim, T. Krekling and E. Christiansen Wound-induced traumatic resin duct development in stems of Norway spruce (Pinaceae): anatomy and cytochemical traits. Am. J. Bot. 87: Nagy, N.E., C.G. Fossdal, P. Krokene, T. Krekling, A. Lönneborg and H. Solheim Induced responses to pathogen infection in Norway spruce phloem: changes in polyphenolic parenchyma cells, chalcone synthase transcript levels and peroxidase activity. Tree Physiol. 24: TREE PHYSIOLOGY VOLUME 26, 2006

9 INDUCED DEFENSE RESPONSES IN SCOTS PINE 167 Redfern, D.B. and J. Stenlid Spore dispersal and infection. In Heterobasidion annosum: Biology, Ecology, Impact and Control. Eds. S. Woodward, J. Stenlid, R. Karjalainen and A. Hüttermann. CAB International, Wallingford, U.K., pp Reid, R.W., H.S. Whitney and J.A. Watson Reactions of lodgepole pine to attack by Dendroctonus ponderosae Hopkins and blue stain fungus. Can. J. Bot. 45: Rishbeth, J Observations on the biology of Fomes annosus, with particular reference to East Anglian pine plantations. II. Natural and experimental infection of pines and some factors affecting severity of the disease. Ann. Bot. 15: Solheim, H., B. Långström and C. Hellqvist Pathogenicity of the blue-stain fungi Leptographium wingfieldii and Ophiostoma minus to Scots pine: effect of tree pruning and inoculum density. Can. J. For. Res. 23: Solheim, H., P. Krokene and B. Långström Effects of growth and virulence of associated blue-stain fungi on host colonization behavior of the pine shoot beetles Tomicus minor and T. piniperda. Plant Pathol. 50: Woodward, S., J. Stenlid, R. Karjalainen and A. Hüttermann Heterobasidion annosum: biology, ecology, impact and control. CAB International, Wallingford, U.K., 589 p. TREE PHYSIOLOGY ONLINE at