INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Acquired resistance to pathogen infection has been observed in a number of angiosperms, including tobacco, cucumber, and different monocots (Kessmann et al., 1994; Hammerschmidt and Smith Becker, 1997). When plants are pretreated with a necrotizing pathogen, long-lasting, broad-spectrum resistance may be induced to subsequent pathogen infections (Ryals et al., 1994). Such acquired resistance can be expressed locally at or very near the pretreatment site (Ross, 1961a), or systemically (e.g. in another leaf, Ross, 1961b; Ryals et al., 1996). Activation of acquired resistance seems to depend on the development of pathogen-induced cell death (Dangl et al., 1996; Ryals et al., 1996).

Although much is known about the mechanisms of acquired resistance in herbaceous plants (Ryals et al., 1995), information is scarce for conifers, probably due to the difficulties of working with large woody species. Conifers have well-developed constitutive and inducible defenses against insects and pathogens (Berryman, 1972). Important constitutive defenses include resin stored in ducts or blisters in the bark and sapwood (Bannan, 1936; Berryman, 1972) and phenol-rich parenchyma cells in the phloem (Franceschi et al., 1998). The inducible defense includes a hypersensitive response triggered by wounding or infection of the phloem, followed by accumulation of terpenes and phenolics in the cells surrounding the site of attack and the formation of a necrotic reaction zone (Reid et al., 1967; Raffa, 1991). Resistance mechanisms have been induced in suspension cultures of different pines (Lesney, 1989; Campbell and Ellis, 1992; Hotter, 1997), and foliar treatment with 5-chlorosalicylic acid increased the resistance of Pinus radiata seedlings to the pathogenic fungus Sphaeropsis sapinea (Reglinski et al., 1998).

Induction of acquired resistance following pretreatment with wounding or pathogen infection, to our knowledge, has never been clearly demonstrated in large conifer trees. However, in a previous study we observed that Norway spruce (Picea abies [L.] Karst.) trees pretreated with 12 mechanical bark wounds (100 × 16 mm) on a 0.8-m band on the lower bole exhibited strongly enhanced resistance to subsequent challenge inoculation with the phytopathogenic bluestain fungus Ceratocystis polonica (Siem.) C. Moreau, a virulent associate of the Eurasian spruce bark beetle Ips typographus L. (Christiansen et al., 1999). A subsequent study indicated that tree resistance could also be enhanced by pretreatment with a few fungal inoculations or small (approximately 20 mm²) mechanical wounds (Evensen, 1998). Because of limitations to experimental design with larger trees, there is an inherent problem in distinguishing wound effects from fungal effects in these previous experiments, in addition to problems with genetic variability of the trees. In the present study we take advantage of a stand of genetically identical trees to further substantiate that acquired resistance can be induced in Norway spruce. We wanted to: (a) test the hypothesis that previous exposure to a sublethal number of inoculations with a fungal pathogen is more efficient in inducing resistance than a similar number of sterile wounds, (b) determine whether the induced resistance was expressed locally or systemically, and (c) determine whether it was specific to the pathogen used in the challenge inoculation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Three experiments were carried out from 1997 to 1998 in a monoclonal stand of Norway spruce planted by the Norwegian Forest Research Institute at Overud near Kongsvinger, SE Norway. The trees originated from 3-year-old rooted cuttings planted in 1966 in a regular 2- × 2-m array and were about 16 m high at the start of the experiment.

On June 17, 1997, 42 trees (diameter at 1.3 m height: 14.95 ± 1.18 cm [mean ± SD]) were selected for a dose-response experiment. Groups of six trees each were randomly assigned to six different pretreatments of fungal or sterile inoculations: high (100 inoculations/m² or an average of 56 inoculations/tree), medium (50 inoculations/m², approximately 28 inoculations/tree), or low (10 inoculations/m², approximately six inoculations/tree) dosages. Six trees were left untreated as controls. Three weeks after pretreatment, all 42 trees were mass inoculated with Ceratocystis polonica (400 inoculations/m², approximately 225 inoculations/tree) to determine tree resistance. This dosage has previously been shown to kill susceptible trees in the same stand (Sandnes, 1997). All inoculations were done by removing a bark plug with a 5-mm cork borer, inserting inoculum in the wound, and replacing the plug. Inoculum consisted of actively growing mycelium of C. polonica (isolate no. NISK 93-208/115) on malt agar (2% [w/v] malt and 1.5% [w/v] agar) or sterile malt agar. Both pretreatment inoculations and challenge inoculations were evenly spaced, using a template, over a 1.2-m section of the stem from about 0.8 to 2 m in height. Challenge inoculations were always made at least 3 cm away from the nearest pretreatment inoculation.

On June 3, 1998, 24 trees (diameter: 18.03 ± 1.18 cm) were inoculated to test whether the enhanced resistance was expressed systemically or only locally. Eighteen randomly chosen trees were pretreated with the medium dosage of fungal inoculations (i.e. 50 inoculations/m²) between 0.8 and 2.0 m in height and six trees were left untreated as controls. Three weeks later, the control trees and six pretreated trees were challenge inoculated (i.e. 400 inoculations/m²) in the same section (0.8-2.0 m), and six trees were challenge inoculated between 2.0 to 3.2 m and 3.2 to 4.4 m, respectively.

On the same day, eight trees (diameter: 17.59 ± 0.77 cm) were inoculated to test if the enhanced resistance was specific to the pathogen used in the challenge inoculation. Four trees each were randomly assigned to pretreatment with 50 inoculations/m² of the root rot fungus Heterobasidion annosum (Fr.) Bref. (isolate no. NISK 87-257/1) or sterile malt agar between 0.8 and 2.0 m in height. Three weeks later, all trees were challenge inoculated (i.e. 400 inoculations/m²) with C. polonica in the same section.

Both years, all trees were harvested 15 weeks after challenge inoculation and two thin discs (approximately 5 mm) were cut 0.4 m from the lower and upper ends of the challenge-inoculated stem section. From the trees that were challenge inoculated above the pretreatment band in 1998, we also cut a disc at the lower and upper ends of the challenge-inoculated section. On each disc we measured the proportion of the sapwood that had been bluestained by the fungus and the proportion of dead cambium (Krokene and Solheim, 1998). Re-isolation of fungus was made from three challenge inoculation points per tree, except for the experiment with H. annosum, where no re-isolations were made. On harvesting, we measured the vertical extension of four of the uppermost and four of the lowermost phloem necroses extending from challenge inoculation sites on each tree. Because necroses tended to coalesce within challenge inoculated sections in susceptible trees, their lengths were measured upwards from upper inoculation points, and downwards from lower points.

Data were subjected to ANOVA or Wilcoxon rank sum tests (SAS Institute, 1987). If treatments were significantly different (P < 0.05) after ANOVA, means were separated using LSD at P = 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

1997 Experiment: Dose-Response Relationship

By all three measures of fungal colonization used, pretreatment with fungal or sterile inoculations enhanced the resistance of Norway spruce to subsequent challenge inoculation with C. polonica, but the effect was much stronger with fungal inoculations (Fig. 1). For each pretreatment dosage, fungal inoculations had a significantly stronger effect than sterile inoculations. For example, the low fungal dosage was as effective as the medium dosage of sterile inoculations. Since the low dosage was only one-fifth that of the medium dosage, this indicates that fungal inoculations were about five times more effective in inducing enhanced resistance than sterile inoculations. The very low threshold for inducing enhanced resistance following fungal inoculations was surprising; as few as five to seven inoculations gave a 20% to 40% reduction in host symptoms to challenge inoculation compared with the control. The medium fungal pretreatment dosage protected the trees very efficiently, and reduced host symptoms by 80% to 97% relative to the untreated control trees. The low dosage of sterile inoculations did not enhance tree resistance (Fig. 1).

View larger version (41K): [in this window] [in a new window]   Figure 1.   Symptoms of fungal infection in Norway spruce trees pretreated with a high (100 inoculations/m2, ), medium (50 inoculations/m2, ), or low (10 inoculations/m2, ) dosage of C. polonica or sterile malt agar inoculations 3 weeks before challenge inoculation with C. polonica (400 inoculations/m2). Control trees () did not receive any pretreatment before challenge inoculation. For each tree symptom, bars with different letters were significantly different by the LSD test (P = 0.05) following ANOVA.

For sterile pretreatment inoculations there was a straightforward dose-response relationship, with higher dosages having a stronger effect on tree resistance (Fig. 1). For fungal inoculations the high pretreatment dosage did not give better protection than the medium dosage (Fig. 1). On the contrary, the high dosage seemed to be somewhat less efficient in enhancing resistance than the medium dosage, but the difference was not significant at the 0.05 level. C. polonica was reisolated from 63% of all inoculation points, and from 83% of all trees.

1998 Experiment: Localized or Systemic Acquired Resistance?

Trees that were pretreated and challenge inoculated in the same area showed strongly enhanced resistance to infection (Fig. 2). The protection of these trees was similar to that found in trees pretreated with the same fungal dosage in 1997, although trees suffered more bluestaining in 1998 (14.8% versus 3.2%; Wilcoxon test: n = 12, Z = 1.84, P = 0.07). Much of the difference between years was caused by a single tree with extensive bluestain in 1998 (percentage bluestain with this tree excluded: 9.0% versus 3.2%; Wilcoxon test: n = 11, Z = 1.56, P = 0.12).

View larger version (50K): [in this window] [in a new window]   Figure 2.   Symptoms of fungal infection in Norway spruce trees pretreated with a medium dosage (50 inoculations/m2) of C. polonica 3 weeks before challenge inoculation (400 inoculations/m2) with the same fungus at different heights. Trees were pretreated on the lower bole (between 0.8 and 2.0 m above ground) and challenge inoculated either in the same area (), immediately above (2.0-3.2 m, ), or further up (3.2-4.4 m, ) the stem. Control trees () did not receive any pretreatment before challenge inoculation. For each tree symptom, bars with different letters were significantly different by the LSD test (P = 0.05) following ANOVA.

Trees that were challenge inoculated above the pretreatment band did not show enhanced resistance to infection and had symptoms similar to the control trees (Fig. 2). However, trees that were challenge inoculated immediately above the pretreatment band (between 2.0 and 3.2 m) had shorter phloem necroses and less necrotic cambium close to the band than further away from it (Table I), indicating a restricted spread of the enhanced resistance from the pretreatment band. This pattern was not seen in trees that had been challenge inoculated farther away from the pretreatment band (Table I). C. polonica was reisolated from 60% of all inoculation points, and from 88% of all trees.

1998 Experiment: Nonspecific Induction of Acquired Resistance?

Trees that were pretreated with H. annosum showed strongly enhanced resistance to infection compared with untreated control trees and trees pretreated with sterile agar (Fig. 3). The protection offered by pretreatment with H. annosum was at least as good as that offered by C. polonica (Fig. 3).

View larger version (39K): [in this window] [in a new window]   Figure 3.   Symptoms of fungal infection in Norway spruce trees pretreated with a medium dosage (50 inoculations/m2) of H. annosum (), C. polonica (), or sterile malt agar () inoculations 3 weeks before challenge inoculation with C. polonica (400 inoculations/m2). Control trees () did not receive any pretreatment before challenge inoculation. For each tree symptom, bars with different letters were significantly different by the LSD test (P = 0.05) following ANOVA.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The experimental results clearly show that resistance to the pathogenic bluestain fungus C. polonica can be induced in Norway spruce, that this induced disease resistance follows a dose-response dynamic, that it is not specific to the pretreatment fungus, and that it is nonsystemic and restricted to the pretreated area of the stem. In addition, the present experiment is the first demonstration, to our knowledge, of a significant differential effect between mechanical wounding alone (i.e. sterile inoculations) and fungus-infected wounding on subsequent resistance. This indicates that the presence of the fungus induces additional defensive responses that may be specifically targeted at fungal invasion. Alternatively, if enhanced resistance is elicited by destruction of host tissues, the differential effect between wounding and wounding plus pathogen could be due to the fact that fungal infection destroys more tissue than sterile wounding.

The protection induced by fungal inoculation did not follow strict dose-response dynamics, since the high dosage appeared to be less effective in enhancing resistance than the medium dosage. However, most of the infection in trees pretreated with the high dosage may have been caused by the pretreatment itself (which corresponded to 25% of the lethal challenge dosage). The observation of characteristic wedges of blue-stained sapwood inside pretreatment inoculation points (but not under challenge inoculation points) in some of these trees substantiates this conclusion. The short necroses induced by the challenge inoculations also suggest that the high pretreatment dosage was as equally effective as the medium dosage in protecting the trees.

Acquired resistance in angiosperms is typically nonspecific, i.e. pretreatment with a particular pathogen normally induces resistance against a broad spectrum of other pathogens, and a number of different pathogens are often able to induce resistance in a given host plant (Ryals et al., 1996; Hammerschmidt and Smith Becker, 1997). The induced disease resistance observed in Norway spruce also seemed to be nonspecific, as pretreatment with H. annosum induced a similar level of resistance as C. polonica. Furthermore, the observation that mechanical wounding (i.e. sterile inoculations) induced resistance suggests that some major component of induced disease resistance in Norway spruce is nonspecific. Mechanical wounding does not appear to induce acquired resistance to pathogens in most angiosperms (Hammerschmidt, 1993).

The mechanism responsible for induced resistance in Norway spruce may involve a spread of inducible defense reactions beyond the local lesion, such as enhanced resin production or activation of phenol-rich parenchyma cells (Franceschi et al., 1998, 2000). In support of this is our observation that traumatic resin ducts are initiated at distances of 5 to 10 cm from a local wound 1 to 2 weeks after wounding (Nagy et al., 2000), and that such ducts are formed several meters away from the inoculation band in surviving trees 3 months after a massive C. polonica inoculation (Christiansen et al., 1999). The fact that initiation and spread of traumatic resin ducts is a relatively slow process may explain why enhanced resistance in Norway spruce was observed only locally 3 weeks after pretreatment. The reduced host symptoms close to the pretreatment band in trees that were challenge inoculated immediately above the pretreatment band indicates a restricted spread of enhanced resistance after 3 weeks. Perhaps this effect would have spread further from the pretreated stem section if we had extended the interval between pretreatment and challenge inoculation.

The presence of induced disease resistance in Norway spruce is relevant to tree resistance against attacks by Ips typographus. This bark beetle is a major pest of Norway spruce and kills healthy trees through pheromone-mediated mass attacks (Christiansen and Bakke, 1988). Phytopathogenic fungal associates seem to play an important part in tree killing by enhancing the virulence of each beetle attack (Horntvedt et al., 1983; Krokene and Solheim, 1998). Sublethal beetle/fungus attacks could be expected to weaken trees and render them more susceptible to attacks later in the season or the following year. However, our observations of experimentally induced disease resistance suggest the opposite: Sublethal attacks may enhance tree resistance and render trees less susceptible to subsequent beetle attacks. This has important implications toward our understanding of the population dynamics of bark beetles, since such an inducible resistance system could hasten the collapse of outbreaks once the beetle population starts to decline and fewer beetles can be summoned to mass-attack trees. Research is currently underway to investigate the effect of pretreatment inoculations with C. polonica on tree resistance toward I. typographus attacks, as well as to characterize the actual mechanisms that produces induced disease resistance in Norway spruce.