Susceptibility evaluation of Picea abies and Cupressus lusitanica to the pine wood nematode (Bursaphelenchus xylophilus)

Authors


Abstract

Pine wilt disease (PWD), recently introduced into Europe, is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus and is a devastating illness that affects mainly pine trees. It is known that the PWN is capable of infecting other conifers; however, there is currently no information on which other plant species may be susceptible to PWD. In this study, the potential susceptibility of two common species of European forests, Picea abies and Cupressus lusitanica, to PWN was assessed through the monitoring of visual external symptoms, dimension and localization of the nematode population in stems, quantification of total chlorophyll, total soluble phenolics and lignin, at 7, 14, 21 and 28 days after inoculation. The degree of susceptibility was established through the comparison of symptoms with Pinus pinaster, a well-known PWN host. Furthermore, the stem ultrastructure of P. abies, C. lusitanica and Pn. pinaster was analysed by scanning electron microscopy. The results suggest that P. abies and C. lusitanica are resistant to PWN, and that lignin biosynthesis in these species is affected at an early stage of the infestation. Nevertheless, P. abies seems to be a compatible host that could act as a repository for PWN.

Introduction

Globalization, particularly increased human migration and the increase of commercial exchanges at a global level, has been responsible for the introduction of non-indigenous organisms into several ecological communities. Many times, when no natural predators are present and the environmental conditions are favourable, these exotic organisms may become harmful to the ecosystems they are introduced into. This seems to be true in the case of the pine wood nematode (PWN), Bursaphelenchus xylophilus, in Portuguese pine forests. This nematode is the causal agent of pine wilt disease (PWD), a devastating infirmity characterized by high tree mortality within short periods of time. The PWN is native to North America, where it is not associated with high prevalence of the disease but, after introduction to Japan, China, Taiwan and Korea, it became damaging to these Far East forests (Shin & Han, 2006). In 1999, PWN was identified for the first time in Europe, specifically in Portugal, where it was detected only in a small area in the Setubal Peninsula (Mota et al., 1999). However, despite the restraining measures taken at the time, PWN was later detected in other geographic areas (Fonseca et al., 2012), until the entire Portuguese territory was classified as an ‘affected area’ (Rodrigues, 2008). PWN was recently detected in Spain (EPPO, 2009; Robertson et al., 2011), demonstrating that the restriction measures taken were not sufficient to prevent the spreading of PWN.

The infection of host trees occurs during the summer, when cerambycid beetles of the genus MonochamusM. galloprovincialis in Portugal (Sousa et al., 2001) – transporting PWNs in their tracheas feed on healthy trees. The nematodes invade tree tissues through the beetles' feeding wounds and start to migrate through cortex and xylem resin ducts, where they develop and reproduce, eventually leading to host tree death. When female vector beetles lay eggs in these PWN-infested dead trees, PWN juveniles aggregate around the beetles' pupal chambers and invade the beetles' tracheal system when the young beetles emerge. As beetles leave dead trees and go to feed on healthy tree branches, they carry the PWNs, initiating a new infection cycle.

After PWN infection of pines, the disease progresses in two stages. In the early stage, nematodes feed on the host's resin duct parenchyma cells, leading to destruction of cambium, cortex, phloem and resin duct tissues, formation of wound periderm in cortex parenchyma cells around resin ducts and enhanced ethylene production. At this stage, the nematode population remains small and no external symptoms are visible. During the advanced stage, the nematode population increases and continues migrating and feeding on host tissues, leading to the accumulation of suberin-like substances, xylem occlusion, cavitation and embolism, possibly resulting from terpenoid (particularly oleoresins) synthesis in xylem cells (Myers, 1988). These modifications in the xylem translate into dysfunction of water conduction in the stem, resulting in decreased water potential, transpiration and photosynthesis, further leading to the manifestation of external symptoms, namely leaf chlorosis (Fukuda, 1997; Hara & Takeuchi, 2006). In fact, a decrease in photosynthetic rate has been associated with accentuated nematode reproduction in Pinus (Pn.) thunbergii seedlings, which eventually leads to a drastic advance of PWD from the early to the advanced stage (Kawaguchi, 2006). During the advanced stage there is also increased lignin production, which is thought to be associated with disease resistance. Constitutive lignin in particular has already been shown to be related to defence mechanisms against nematodes (Kawaguchi, 2006).

Soluble polyphenols appear to have an important role in PWD; however, contradictory information concerning these compounds has been presented. It has been suggested that browning of pine tissues during PWD is a result of the accumulation of polyphenols and other metabolites, caused by the action of B. xylophilus (Futai, 2003), and that, to some extent, PWD may result from the production of such metabolites. Nevertheless, more recently, Kuroda et al. (2011) reported greater production of phenolic metabolites in a PWD-resistant variety of Pinus densiflora, suggesting that it may be responsible for tree resistance against PWNs.

Pine wood nematodes are thought to be transported in timber, both separate from their vectors or within the beetles inside the wood. Consequently, in countries where the PWN is associated with epidemic disease, the social and economic effects are very serious because the timber industry generates huge revenues and provides jobs for thousands of people. In Portugal in particular, Pinus pinaster is one of the most important pine timbers; unfortunately this pine species is also the preferred PWN-host (Webster & Mota, 2008; Franco et al., 2011).

In spite of much research effort in the past century, no effective strategies to combat PWN have been identified so far. Moreover, other genera of the order Pinales, including Larix, Pseudotesuga, Picea, Chamaecyparis, Abies and Cedrus are also considered to be suitable PWN hosts (Kishi, 1995; Aikawa et al., 2009; EPPO, 2009). Nonetheless, the available information on the susceptibility of these and other species to PWN is rare; very few reports exist on PWN infection of non-Pinus species belonging to the family Pinaceae, such as Picea (Kishi, 1995; works cited in Takeuchi, 2008; Aikawa et al., 2009). Picea abies (Norway spruce or European spruce) is a species of spruce native to Europe and commonly planted in Norway, Poland, Greece, Russia and Portugal. Cupressus lusitanica (white cedar or Mexican cypress) was one of the first New World conifers to be brought to Europe, having been planted in Portugal since 1634. Given the recent introduction of PWN to the European continent, this study aimed to investigate the potential susceptibility of conifer plants other than pines to PWN, and to understand the mechanisms involved in resistance to PWD.

The susceptibility to PWN was evaluated for 4 weeks after inoculation by monitoring visual external symptoms (leaf chlorosis and tree death), dimension and localization of the nematode population and quantification of total chlorophyll content. The degree of susceptibility was established through the comparison of symptoms with Pn. pinaster plants treated and analysed as described for P. abies and C. lusitanica plants. Total soluble phenolic compounds and lignin were also analysed in all species to assess if their biosynthesis is involved in PWN resistance. Furthermore, as nematode progression and reproduction is dependent on their ability to migrate and feed on epithelial cells of host's resin ducts, the ultrastructure of P. abies and C. lusitanica stems was visualized and compared with the stem morphology of the known PWN host Pn. pinaster using scanning electron microscopy (SEM). This study thus integrates anatomical, biochemical and nematode progression data. Considering the current worldwide expansion of this pest, including Europe, and the distribution of these two plant species, the topic is relevant to the forest sector in general.

Materials and methods

Plant material

One-year old P. abies (25–30 cm tall), C. lusitanica (50–55 cm tall) and Pn. pinaster (35–40 cm tall) seedlings were obtained from Anadiplanta Nursery (Anadia, Aveiro, 40°26′27″ N, 8°25′47″ W) and kept in an environmental growth chamber (Fitoclima 10 000 EHF, Aralab) under a 16-h light/8-h darkness photoperiod at 25°C/18°C, respectively, and 80% relative humidity. Photon flux density during the day was 380 μmol m−2 s−1. Plants were watered every other day and fertilized every week throughout the experimental period.

PWN maintenance and inoculum preparation

The virulent B. xylophilus strain HF, isolated from Setubal region (Portugal), was used (Mota et al., 2006). Bursaphelenchus xylophilus cultures were grown on barley grains with Botrytis cinerea mycelium at 25°C in the dark for 7 days. Juveniles and adult nematodes were extracted overnight at room temperature using the Baermann funnel technique (Baermann, 1917).

A known volume of the nematode suspension obtained from the Baermann funnel was placed in a nematode counting dish and living nematodes were counted under a transmitted light microscope (Axiostar Plus, Carl Zeiss) and estimated for the initial suspension. The nematode suspension was prepared in autoclaved deionized water in order to obtain a final concentration of approximately 1000 nematodes mL−1 (Roriz et al., 2011). Nematode recovery and suspension preparation occurred on the day of inoculation.

Inoculation

Eighty P. abies and C. lusitanica seedlings and 40 Pn. pinaster seedlings were inoculated with PWN or with autoclaved deionized water (control) using the technique described by Asai & Futai (2001) with slight modifications. Briefly, after peeling off a 1-cm portion of the bark, 15 cm above the stem base, a tissue paper swab was placed on the wound and a freshly prepared aqueous suspension with 1000 nematodes or the deionized water (control) was pipetted onto the swab. The inoculation site was sealed with Parafilm to prevent desiccation.

Sample collection and processing

On the day of inoculation, leaves and stems of 10 additional plants of each species were collected and stored at −80°C until analysis of total chlorophyll, lignin and total soluble phenolics. Seven, 14, 21 and 28 days after inoculation (dai), 40 P. abies and C. lusitanica plants were sacrificed for subsequent analysis: 20 plants of each species (10 PWN-inoculated and 10 controls) were used for nematode quantification and the leaves and stems from another set of 20 plants of each species (10 PWN-inoculated and 10 controls) were collected and stored at −80°C for total chlorophyll, lignin and total soluble phenolics analysis. Seven and 21 dai, 40 Pn. pinaster plants were sampled as described above for the same purposes.

Visual external symptoms

Prior to sample collection, the extent of external symptoms (leaf chlorosis/necrosis and tree death) was assessed 7, 14, 21 and 28 dai. External symptoms were classified as: I, no leaf chlorosis; II, <10% brown leaves; III, 10–25% brown leaves; IV, 25–50% brown leaves, some lateral branches dead; and V >50% brown leaves, tree death.

Nematode extraction and quantification

For assessment of nematode survival and progression, the stems of PWN-inoculated and water-inoculated plants of each species, collected as previously described at 0, 7, 14 and 21 dai, were cut into five segments corresponding to the inoculation site and the segments up to 2 and 4 cm above and below the inoculation site. Each segment was cut into smaller pieces, weighed and placed in microcentrifuge tubes with 1 mL deionized water for 24 h at 25°C for nematode extraction. The remaining stem segments (above and below the 4 cm limits) were placed in individual 15 mL centrifuge tubes with 3 mL deionized water for the same purpose. After 24 h, extracted nematodes were counted as previously described. The amount of nematodes present in each stem segment was determined taking into account the weight of each sampled segment (nematodes g−1).

Chlorophyll extraction and quantification

Leaves collected as previously described were used for chlorophyll quantification. Total chlorophyll was extracted and quantified according to a modified protocol of Abadía et al. (1984). Briefly, 12·5 mL of 0·4% (w/v) calcium carbonate in methanol were added to 0·5 g leaves. After 48 h, the supernatant was collected and absorbance was measured at 663 nm and 645 nm using a NanoPhotometer (Implen GmbH). The amount of extracted chlorophyll (mmol g−1) was calculated as: total chlorophyll = ((8·02 × A663) + (20·21 × A645)) × (0·0125 × dilution factor)/fresh weight (g) (Abadía et al., 1984).

Lignin extraction and quantification

Stems were lyophilized for 72 h and ground to a fine powder. Lignin concentration of stems was determined using the acetyl bromide method, adapted from Fukushima & Hatfield (2001). Approximately 150 mg of powder were transferred to a 15 mL centrifuge tube with 5 mL methanol and extracted for 24 h in the dark at 4°C. The methanolic extract was recovered by centrifugation (5000 g for 5 min) and stored at 4°C for further phenolics analysis. The pellet was extracted once with water, then with acetone and finally with hexane, discarding the supernatant. After the last extraction, the pellet was dried overnight at 60°C. Ten milligrams of dried pellet were added to 500 μL acetic acid and 500 μL of 25% (v/v) acetyl bromide in acetic acid. The mixture was incubated at 50°C for 2 h with agitation. Samples were spun for 30 s and 100 μL of the supernatant were mixed with 200 μL acetic acid, 150 μL 3 m NaOH, 50 μL 0·5 m hydroxylamine hydrochloride and 500 μL acetic acid. Each sample was analysed in triplicate. The absorbance was measured at 280 nm using a NanoPhotometer (Implen GmbH) and lignin was quantified using a lignin (Aldrich) standard calibration curve.

Total soluble phenolics extraction and quantification

Concentration of total soluble phenolics was determined using the methanolic extract obtained as described above by the Folin method (Azevedo et al., 2010). Five millilitres of ultrapure water and 0·5 mL Folin–Denis' reagent (Fluka) were added to 0·1 mL of the methanolic extract. The mixture was shaken thoroughly and allowed to stand for 5 min, after which 1·5 mL of 35% (w/v) sodium carbonate was added. The reaction was performed in the dark for 2 h, at the end of which 2·9 mL ultrapure water were added. Three replicates per sample were analysed. The absorbance was measured at 760 nm using a NanoPhotometer (Implen GmbH) and total phenolics were quantified using a quercetin (Sigma) standard calibration curve.

Scanning electron microscopy (SEM)

Manual cuts were made as thinly as possible (c. 2 mm) using a razor blade 15 cm above the base of the stem and each sample was attached to a support with double-sided duct tape. Samples were visualized and photographed in a low-vacuum (11 Pa) SEM (5600LV, JEOL), operating at 15 kV. Resin ducts were counted and their diameter measured using image tool v. 3.0 (IT3) image analysis software (University of Texas Health Science Center).

Statistical analysis

Averages and standard deviations were calculated using Microsoft excel 2010 software. Significant differences were determined by one-way anova for the resin duct analysis and two-way anova for the remaining analysis, followed by Tukey's post hoc test when the anova indicated significant differences (graphpad prism, v. 6.01). Statistical significance was considered at < 0·05.

Results

External symptoms

Table 1 illustrates the evolution of visual symptoms in PWN-inoculated seedlings of P. abies, C. lusitanica and Pn. pinaster during the experimental period. Until 14 dai, no visible symptoms were observed in P. abies control and PWN-inoculated seedlings. After 21 dai, partial leaf chlorosis was observed in both control and PWN-inoculated seedlings. No chlorosis was observed in C. lusitanica control and PWN-inoculated seedlings during the experimental period. Pinus pinaster seedlings inoculated with PWN presented partial leaf chlorosis 14 dai and at the end of 28 dai evident leaf chlorosis was observed; Pn. pinaster control seedlings showed no signs of chlorosis throughout the experiment.

Table 1. External visual symptoms observed in Picea abies, Cupressus lusitanica and Pinus pinaster control and pine wood nematode (PWN)-inoculated seedlings
SpeciesTreatmentDays after inoculation
7142128
  1. I, no leaf chlorosis; II, <10% brown leaves; III, 10–25% brown leaves; IV, 25–50% brown leaves, some lateral branches dead; V, >50% brown leaves, tree death.

P. abies ControlIIIIII
PWN-inoculatedIIIIII
C. lusitanica ControlIIII
PWN-inoculatedIIII
Pn. pinaster ControlIIII
PWN-inoculatedIIIIIIIV

Nematode population

Average changes in nematode population in PWN-inoculated P. abies, C. lusitanica and Pn. pinaster seedlings are presented in Figure 1. In P. abies, 7 dai, the nematodes had spread over a considerable portion of the stem, covering an extension of about 10 cm. This pattern of dispersal was maintained for 14 dai; nevertheless, a regression in the nematode dispersal pattern was registered at 21 dai. Generally, the number of nematodes was higher in the segment corresponding to the inoculation site than in the other stem segments. Overall, the number of nematodes decreased with time, with the majority of nematodes detected at 21 dai being restricted to the inoculation site. In C. lusitanica, 7 dai, the nematode population was located in the inoculation site and the segments up to 2 cm above and below the inoculation site. By 14–21 dai, nematode distribution was restricted to the inoculation site. As in P. abies, the number of nematodes was greater at the inoculation site than in the other stem segments and decreased over time. Moreover, the number of nematodes recorded in C. lusitanica was always fewer than the number registered in P. abies. In Pn. pinaster seedlings, 7 and 21 dai, the nematodes had spread over the entire stem. At both time points, Pn. pinaster seedlings contained a considerably greater nematode density in all portions of the stem compared to P. abies and C. lusitanica. At 7 dai, the nematodes were evenly distributed along the whole stem, whereas by 21 dai nematodes preferentially concentrated near the inoculation site. Nevertheless, nematode numbers increased with time in all stem segments analysed, especially at the inoculation site, with the exception of the upper segment which presented a slight decrease. In general, in both P. abies and C. lusitanica, nematodes migrated preferentially upwards, whereas in Pn. pinaster nematodes migrated preferentially downwards.

Figure 1.

Nematode population per gram of stem fresh weight in Picea abies, Cupressus lusitanica and Pinus pinaster pine wood nematode (PWN)-inoculated seedlings, 7, 14 and 21 days after inoculation (dai), in relation to the stem inoculation site. Each symbol represents the mean of 10 seedlings.

Stem morphology

Visual inspection of stem morphology (Fig. 2) revealed that the P. abies stem most closely resembles the Pn. pinaster stem, with easily visible resin ducts in both the xylem and cortex, whereas in C. lusitanica stems no such resin ducts were observed. Moreover, resin ducts were significantly more numerous (c. 1·6-fold) and wider (c. 2·3-fold) in Pn. pinaster than in P. abies (Fig. 3).

Figure 2.

Scanning electron microscopy imaging of (a) Picea abies, (b) Cupressus lusitanica and (c) Pinus pinaster stems. E, epidermis; C, cortex; RD, resin duct; PH, phloem; X, xylem; P, pith.

Figure 3.

Number (a) and diameter (b) of resin ducts in Picea abies and Pinus pinaster seedlings. Each value is the mean of three seedlings. Error bars represent standard deviation. Bars showing the same letter are not significantly different ( 0·05, one-way anova).

Chlorophyll concentration

In P. abies, total chlorophyll content significantly decreased in both control and PWN-inoculated seedlings in general in comparison to initial levels (Fig. 4). At each experimental time point, there were no significant differences in total chlorophyll content between control and PWN-inoculated seedlings, except at 21 dai when PWN-inoculated seedlings contained significantly less chlorophyll than controls. In C. lusitanica, chlorophyll concentration of PWN-inoculated plants was not significantly different from the control and remained approximately constant throughout the experiment. Pinus pinaster seedlings presented no significant differences in chlorophyll concentration between initial, control and PWN-inoculated seedlings 7 dai; however, significant total chlorophyll decrease in PWN-inoculated seedlings occurred by 21 dai.

Figure 4.

Total chlorophyll concentration in Picea abies, Cupressus lusitanica and Pinus pinaster control and pine wood nematode (PWN)-inoculated seedlings, 7, 14, 21 and 28 days after inoculation (dai). Each value is the mean of 10 seedlings. Error bars represent standard deviation. Bars showing the same letter are not significantly different ( 0·05, two-way anova).

Lignin concentration

Control plants of each species did not show significant alterations in lignin concentration during the experiment (Fig. 5). Seven dai, PWN-inoculated P. abies and C. lusitanica had significantly greater lignin concentration than control plants. However, in P. abies lignin content significantly decreased by 14 dai. In C. lusitanica, no statistically significant differences in lignin concentration were found during the period of the experiment. Similarly, Pn. pinaster seedlings presented no significant differences between initial, control and PWN-inoculated seedlings during the experiment.

Figure 5.

Lignin concentration in Picea abies, Cupressus lusitanica and Pinus pinaster control and pine wood nematode (PWN)-inoculated seedlings, 7, 14, 21 and 28 days after inoculation (dai). Each value is the mean of 10 seedlings. Error bars represent standard deviation. Bars showing the same letter are not significantly different ( 0·05, two-way anova).

Total soluble phenolics content

In general, no significant changes in total phenolic compounds occurred between initial, control and PWN-inoculated plants of the three plant species (Fig. 6). Nevertheless, concentrations of total soluble phenolics were significantly greater in P. abies and C. lusitanica plants than in Pn. pinaster.

Figure 6.

Concentration of total soluble phenolic compounds in Picea abies, Cupressus lusitanica and Pinus pinaster control and pine wood nematode (PWN)-inoculated seedlings, 7, 14, 21 and 28 days after inoculation (dai). Each value is the mean of 10 seedlings. Error bars represent standard deviation. Bars showing the same letter are not significantly different ( 0·05, two-way anova).

Discussion

In this work the susceptibility of two common conifer species of the European flora, P. abies and C. lusitanica, to PWN was investigated, comparing their symptoms after PWN inoculation to those observed in Pn. pinaster, the most susceptible host (Webster & Mota, 2008; Franco et al., 2011). Pinus pinaster seedlings were, therefore, used mainly as positive controls at critical time points of the experiment. Franco et al. (2011) reported that at 10 and 20 dai important molecular alterations occur in PWN-inoculated Pn. pinaster seedlings, resulting in several biochemical changes and, eventually, tree death at 20 dai. Thus, in this experiment, Pn. pinaster seedlings were analysed 7 and 21 dai.

The activity of the nematode population inside the host's tissues induces several histological and biochemical modifications that eventually lead to leaf discoloration, the only visual symptom that allows the identification of a diseased tree, which is probably due to the impairment of water transport inside the plant (Fukuda, 1997; Hara & Takeuchi, 2006). The results showed that P. abies plants (both control and PWN-inoculated) presented the same pattern of external symptoms (leaf chlorosis), with mild chlorosis appearing 21 dai, whereas C. lusitanica plants presented no external symptoms throughout the experiment (Table 1). Given that both control and PWN-inoculated P. abies plants presented the same pattern of external symptoms, leaf chlorosis was probably a result of the inoculation procedure, rather than the activity of the nematode population. However, in PWN-inoculated Pn. pinaster plants leaf chlorosis was evident 14 dai and the extent of chlorotic leaves increased gradually until 28 dai. Moreover, the PWN population in both P. abies and C. lusitanica plants decreased over time, whereas in Pn. pinaster the nematode population substantially increased from 7 to 21 dai (Fig. 1), suggesting that in Pn. pinaster the disease developed from the early to the advanced stage, thus inducing leaf chlorosis, whereas in P. abies and C. lusitanica that did not occur (Fukuda, 1997). These results seem to indicate that P. abies and C. lusitanica may be resistant to PWD. Furthermore, the dispersion of inoculated PWNs in the stem of both species also reduced over time following PWN-inoculation, whereas in Pn. pinaster the nematodes spread to the whole stem 7 dai and the dispersion pattern was maintained until 21 dai. Inhibition of both nematode reproduction (preventing the increase of nematode population) and nematode migration through host tissues have been related with resistance to PWD (Mori et al., 2008). Nevertheless, the size of the nematode population and the extent of migration of PWNs in P. abies were greater than in C. lusitanica. This seems to indicate that P. abies susceptibility to PWN is greater than C. lusitanica, which is not surprising because P. abies belongs to the family Pinaceae and is therefore phylogenetically closer to the genus Pinus, the known PWN-host than C. lusitanica, in the Cupressaceae family. Accordingly, SEM results showed higher morphological similarity between P. abies and Pn. pinaster than between C. lusitanica and Pn. pinaster, namely concerning the easily visible resin ducts, which are suggested to be the main pathways of PWN migration through host tissues (Kawaguchi, 2006; Roriz et al., 2011). This is probably the reason why nematodes were more numerous and more widely distributed within the stem in P. abies than in C. lusitanica. Additionally, resin ducts were more abundant and wider in Pn. pinaster plants than in P. abies (Fig. 3). Moreover, resin ducts were more evenly distributed in Pn. pinaster, appearing in both cortex and xylem, than in P. abies, in which they appeared only in the xylem. These factors may be the reason why the nematode population was able to reproduce and migrate more successfully in Pn. pinaster plants than in Pabies (Kawaguchi, 2006). However, these studies were conducted in 1-year old trees; the possibility that PWN-susceptibility could be different in older, more mature trees should not be overlooked. Nonetheless, juvenile seedlings up to 1 year old, even of PWN-resistant species, have been associated with greater susceptibility to PWN (Kuroda, 2008). Therefore, the results obtained in 1-year old trees are in fact encouraging, most likely reflecting PWN-resistance of adult trees. On the other hand, differences between artificial inoculations and natural infections should be considered. In artificial inoculations using young, smaller trees, a massive amount of nematodes is introduced; during a natural infection, mature trees are the primary hosts and the amount of nematodes entering the tree is dependent on the vector. Hence, because the initial nematode load strongly influences the severity of the disease (Aikawa, 2008), one would expect that a massive amount of nematodes inoculated in juvenile seedlings would rapidly overwhelm the defence mechanisms, leading to severe consequences. This did not happen: symptom appearance occurred late (in comparison to the known host Pn. pinaster), despite the presence of live nematodes inside the plants until 28 dai.

Decrease in photosynthetic activity, as well in chlorophyll content, was previously described in PWN-inoculated pine seedlings and is regarded as a symptom of the advanced stage of the disease, induced by water deficiency in leaves (Chen et al., 2005). The current results show that chlorophyll concentration in C. lusitanica PWN-inoculated plants did not differ significantly from the controls, possibly indicating that no deleterious physiological changes occurred due to PWN inoculation (both the inoculation procedure and the action of the nematode itself). However, in P. abies plants, total chlorophyll content decreased in both control and in PWN-inoculated seedlings, compared to initial levels, probably due to the combined effect of the inoculation procedure and the activity of the nematode population. Moreover, 21 dai, total chlorophylls in PWN-inoculated seedlings were significantly lower than in control plants; however, this difference ceased to exist 28 dai. Therefore, 21 dai, the action of the nematode population had deleterious effects in total chlorophyll concentration, but plants were able to recover after that. In Pn. pinaster plants, severe chlorophyll reduction occurred 21 dai, thus indicating that in these plants the disease progressed to the advanced stage (Fukuda, 1997). These results seem to suggest once more that P. abies has some degree of susceptibility to PWN, although it must also have more efficient defence mechanisms than Pn. pinaster against PWN that allow plants to cope more successfully with PWNs.

Induced cell-wall lignification is considered to be a major resistance mechanism against a variety of pathogens (Vance et al., 1980; Hammerschmidt, 1999). During PWD, lignin biosynthesis occurs during the advanced stage as a defensive mechanism against PWN (Kawaguchi, 2006) and has been hypothesized to be a resistance factor to PWN inoculation, possibly because harder tissues are able to restrict PWN spread inside the host (Franco et al., 2011). In the current experiment, significant lignin biosynthesis occurred in P. abies and C. lusitanica PWN-inoculated plants 7 dai (Fig. 5). However, despite this significant increase, the overall pattern of lignin biosynthesis was constant over the experiment, and very similar to either non-inoculated plants or Pn. pinaster. Moreover, Pn. pinaster plants showed no significant alterations in lignin content during the experimental period. Taken together, these results cannot confirm if cell lignification is an important mechanism against PWN progression and reproduction within the plant. Nonetheless, there are several bacterial and fungal species capable of degrading lignin (Lozovaya et al., 2006; Kersten & Cullen, 2007). In fact, Bacillus sp. and Pseudomonas sp., known to be associated with PWN in the infection process of PWD (Roriz et al., 2011), have been shown to be able to degrade lignin (Janshekar & Fiechter, 1982; Bandounas et al., 2011). Thus, the decrease in lignin content in both P. abies and C. lusitanica observed 14 dai (and thereafter) may result from the exudation of ligninolytic substances by the bacteria that colonize the PWNs.

Phenolic substances have been considered responsible for the browning of injured or pathogen-infected plant tissues (Futai, 2003), and PWD may be a result of the production of such compounds (Xu et al., 2000). However, more recently, Kuroda et al. (2011) reported greater production of phenolic metabolites in a more PWD-resistant variety of Pn. densiflora, when compared to a more susceptible line. In the current experiment, no significant differences were found in the amount of total soluble phenolic compounds of control and PWN-inoculated P. abies, C. lusitanica and Pn. pinaster seedlings. However, P. abies and C. lusitanica presented much more constitutive phenolic compound production than Pn. pinaster. These results seem to be in accordance with those obtained by Kuroda et al. (2011), where plants with higher resistance to PWN seem to contain increased levels of phenolics. Nevertheless, although the current results did not show an increased production of total soluble phenolics in PWN-inoculated plants, qualitative alterations in the soluble phenolics could have occurred.

Overall, the results indicate that P. abies and C. lusitanica are not susceptible to PWN. Regardless of the close phylogenetic relationship between P. abies and the genus Pinus, both P. abies and C. lusitanica responded in a resistant manner to the infection, namely a decrease in nematode population and dispersion pattern in stems and lignin biosynthesis. Nevertheless, in P. abies, the nematode population size and extent of dispersion was maintained for a longer period of time after inoculation. Aikawa et al. (2009) detected B. xylophilus in P. abies infested with M. alternatus. However, there is no available detailed information on the nematode reproductive fitness in the plant, and this type of information would be useful to identify whether P. abies could act as a temporary repository for PWN in the field – thus potentially contributing to the prevalence of the disease in natural conditions – and to confirm P. abies as a susceptible host to PWN. Also, it is important to factor in the host potential of both species to the insect vector in the evaluation of disease susceptibility to PWN. Picea spp. are reported as hosts to the insect vectors M. galloprovincialis, M. sutor and M. sartor (Schroeder & Magnusson, 1992; Evans et al., 2004). As for C. lusitanica, it seems to be a non-host for M. galloprovincialis, but this will not guarantee protection against B. xylophilus if the insect vector changes host preference or if C. lusitanica is a host for other Monochamus species (which is not yet proven). Also, these studies need to be confirmed in the field, because in natural infections it is likely that nematodes are inoculated into adult trees in much smaller numbers, and it is possible that the adult trees successfully manage to eliminate the pathogen.

Acknowledgements

The authors would like to thank Fundação para a Ciência e a Tecnologia (FCT) for MNS's initiation to investigation grant (BII/LAB/0016/2009), Autoridade Florestal Nacional (AFN) and Ministério da Agricultura, do Desenvolvimento Rural e das Pescas for funding, and Dr Manuel Mota (Universidade de Évora, Portugal) for providing the nematode strain.

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