Next Article in Journal
Developments on Core Collections of Plant Genetic Resources: Do We Know Enough?
Next Article in Special Issue
Friend or Foe? The Endophytic Fungus Alternaria tenuissima Might Be a Major Latent Pathogen Involved in Ginkgo Leaf Blight
Previous Article in Journal
Spatial Distribution of Secondary Forests by Age Group and Biomass Accumulation in the Brazilian Amazon
Previous Article in Special Issue
Monitoring the Severity of Rubber Tree Infected with Powdery Mildew Based on UAV Multispectral Remote Sensing
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Forests
Volume 14
Issue 5
10.3390/f14050925
forests-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Resin Canal Traits Variation in Pinus spp. with Different Susceptibility to the Pine Wood Nematode

by
Aida Rodríguez-García
1,
Juan A. Martín
2,*,
Luis Gil
2,
María Menéndez-Gutiérrez
3 and
Raquel Díaz
3,*
1
Fundación Cesefor, Área Forestal y Gestión de Recursos Naturales. Pol. Ind. Las Casas, Calle C, Parcela 4, 42005 Soria, Spain
2
Departamento de Sistemas y Recursos Naturales, ETSI Montes, Forestal y del Medio Natural, Universidad Politécnica de Madrid, c. José Antonio Novais 10, 28040 Madrid, Spain
3
Departamento de Silvicultura y Mejora, Centro de Investigación Forestal de Lourizán. C/Iglesia, 19., 36153 Pontevedra, Spain
*
Authors to whom correspondence should be addressed.
Forests 2023, 14(5), 925; https://doi.org/10.3390/f14050925
Submission received: 9 March 2023 / Revised: 4 April 2023 / Accepted: 27 April 2023 / Published: 29 April 2023
(This article belongs to the Special Issue Prevention and Control of Forest Diseases)
Browse Figures
Review Reports Versions Notes

Abstract

:
Different studies have emphasized the influence of resin canal traits in the susceptibility of pine trees to the pine wood nematode (PWN) Bursaphelenchus xylophilus. Resin canals can facilitate the PWN migration through the stem and are involved in the accumulation of volatile terpenes in the xylem in response to the pathogen, inducing tracheid embolisms. In this work, we conducted a PWN inoculation experiment under greenhouse conditions to investigate the anatomical traits of constitutive resin canals among seven Pinus species with different degrees of susceptibility to the PWN: P. canariensis, P. halepensis, P. pinea and P. taeda were grouped into a ‘low-susceptible group’, and P. pinaster, P. radiata, and P. sylvestris were grouped into a ‘high-susceptible group’. The high-susceptible group presented higher xylem radial growth, wider constitutive canals in the cortex, lower frequency of constitutive canals in the cortex, and smaller constitutive canals in the xylem than the low-susceptible group. The size of constitutive cortical canals was positively related to the number of seedlings colonized by the PWN, suggesting that wider canals facilitated migration. The inoculation of the PWN increased the frequency and diminished the mean area of canals in the xylem, and the high-susceptible group showed more frequency of induced xylem canals than the low-susceptible group. Additionally, the high-susceptible group presented larger radial growths in the xylem than the low-susceptible group. These results suggest a role of resin canal traits on Pinus spp. susceptibility to the PWN. Nevertheless, the high interspecific variability found in these traits within each susceptibility group evidences the importance of other factors in the susceptibility to the PWN.

1. Introduction

The pine wood nematode (PWN) Bursaphelenchus xylophilus has been described as a major threat to Eurasian pine forests [1,2]. Native to North America, it causes a severe wilt disease outside of its native region known as pine wilt disease, killing trees of several Pinus species in a matter of weeks or months [3]. The PWN has significantly harmed the pine forests in Asia, impacting various pine species [4]. In Europe, the nematode was first detected in 1999, in the Setubal Peninsula in Portugal [5], and has since spread all over continental Portugal and Madeira [6,7]. The first outbreak in Spain was declared in 2008 in Caceres province [8], and other foci have been declared to date in southern Pontevedra (Galicia) and Salamanca (Extremadura). Although nematode infection is confined to these areas, the disease could spread throughout the Iberian Peninsula reaching the Pyrenees from where it could advance towards France [9]. The PWN is a European Union quarantine pathogen and its predicted economic impact in Europe could reach between 16.3 and 36.0 billion euro during the 2008–2030 period [10]. Although the PWN is pathogenic to many pine species [1,2], differences in susceptibility have been observed among pine species [1,11,12] and among individuals within the same species [13,14]. Initially, the pine wilt disease in Europe was thought to be restricted to P. pinaster [15], but in 2013 the nematode was also detected in decayed P. nigra and P. radiata trees in Coimbra, Portugal [16] and Sancti-Spiritus, Spain [17], respectively.
Conifer trees, particularly within Pinaceae, have a complex and extensive defense system against abiotic [18,19] and biotic stress [20,21] or mechanical wounding [22,23]. In pines, the primary defense mechanism is the production of resin, a complex mixture of volatile monoterpenes and sesquiterpenes, as well as non-volatile diterpene resin acids [24]. Resin is synthetized and stored in cellular structures called resin canals [25,26]. Pine species, which contain a developed network of interconnected axial and radial resin canals in wood and bark [25,26], form the first defense against a perturbation on the constitutive canals [21,25,27]. A second defense reaction is the induced synthesis of resin and new canals, often called traumatic canals [28,29]. Previous studies have reported a link between resin canal traits and tree resistance to insects and pathogens [22,30,31]. In some cases, higher abundance or size of constitutive or traumatic resin ducts are indicators of enhanced biotic resistance in conifers [31,32]. However, in cases where the resin canals are colonized and used as a pathway for pathogen spread, an abundance of these structures can be detrimental for the tree [31].
The PWN is transmitted when adult beetles of the genus Monochamus feed on the phloem of healthy trees or during female oviposition into dying trees, where M. galloprovincialis is the only confirmed PWN vector in Europe [7]. The infection continues with the migration of the PWN through the resin canal system [3,33,34]. The infection induces the production and accumulation of volatile terpenes within the stem, which provokes tracheid embolisms, blocks the ascent of xylem-sap and leads to water deficit in pine trees [35]. Different studies have highlighted the importance of resin canal traits in facilitating PWN migration and reproduction. Traits such as canal size and abundance could affect nematode migration, with cortex and xylem resin canals playing the most relevant role [33,36,37]. A recent study performed on four different pine species showed that wider axial resin canals could facilitate nematode dispersal through the stem, increasing tree susceptibility [12]. Additionally, pine trees susceptible to PWN were reported to have more resin canals than trees capable of restricting nematode migration [38]. Yet, other traits can be also involved in resistance, e.g., the accumulation of phytochemicals toxic for the PWN as an induced defense reaction [11,35,39]. The migration rate of nematodes within a pine tree varies with the nematode species [40,41], nematode isolate [40,42], and the resistance level of the host tree [43].
In the present work, we aim to investigate the association between the susceptibility of seven Pinus species to the PWN and the anatomical traits of their constitutive resin canals. Furthermore, we also aim to elucidate if the infection with the PWN induces the formation of resin canals or a shift in their size. We hypothesize that the most susceptible pine species have wider and more abundant constitutive axial resin canals, facilitating nematode migration through the stem, and that PWN inoculation increases the frequency or the area of axial resin canals in the most susceptible pine species. The studied group was formed by five pine species widely distributed and planted in Europe (P. pinaster, P. pinea, P. sylvestris, P. radiata, and P. halepensis), an American resistant species (P. taeda) and the endemic Canary island pine (P. canariensis).

2. Materials and Methods

2.1. Plant Material and Experimental Design

In 2011, seeds from seven different pine species were sown: P. canariensis, P. halepensis, P. pinaster, P. pinea, P. radiata, P. taeda and P. sylvestris. The inoculation experiment was conducted under greenhouse conditions with a randomized complete block design with seven species, four blocks, and three treatments. To study constitutive anatomical traits, the first treatment consisted of non-inoculated seedlings. To study anatomical traits induced by the PWN, the second and third treatments consisted of seedlings inoculated with sterilized distilled water or with the PWN, respectively. A total of 56 PWN-inoculated seedlings (8 per species), 28 water-inoculated seedlings (4 per species) and 35 controls (5 non-inoculated seedlings per species) were used. The initial height of seedlings ranged from 68.83 ± 2.38 cm for P. sylvestris to 138.08 ± 3.04 cm for P. radiata (Table S1). The mean temperature in the greenhouse was 22.5 °C, ranging from 18 °C to 27 °C owing to oscillations between day and night mean temperatures. Seedlings were watered twice a week by a drip irrigation system.

2.2. Pinewood Nematode Inoculation and Quantification

A detailed characterization of the susceptibility of the seven pine species to the PWN was previously published by Menéndez-Gutiérrez et al. [11], where the inoculation methodology followed in our work is described. In brief, we used a B. xylophilus isolate obtained from a P. pinaster in As Neves (Pontevedra, Spain) in 2010. The isolate was reared on a fungal mat of Botrytis cinerea growing on PDA medium. The inoculum was provided by the Areeiro Phytopathological Institute (Estación Fitopatológica Areeiro, EFA, Pontevedra, Spain). Nematodes were extracted from B. cinerea plates using the Baermann funnel technique [44] the day before inoculation and the inoculation suspension was prepared with sterilized distilled water. Seedlings were inoculated in a greenhouse at Lourizán Forest Research Centre (Pontevedra, Spain) on 18 June 2013. A longitudinal cut was made in the previous year’s growth of the main stem. Then, 300 μL of the suspension containing 600 nematodes (mixed developmental stages) was pipetted onto a strip of bandage previously placed on the wound. Parafilm® was used to prevent tissue desiccation. Water-inoculated seedlings were inoculated with 300 μL of distilled water instead of the nematode suspension.
At 42 days after inoculation (dai), 8 PWN-inoculated and 4 water-inoculated seedlings per species were harvested for nematode quantification. Five out of the 8 PWN-inoculated and the 4 water-inoculated seedlings were used for the measurement of anatomical traits. Additionally, 5 non-inoculated seedlings per species were harvested for constitutive anatomical traits analysis. For nematode quantification, nematodes were extracted from the seedlings using a modified Baermann funnel technique at 42 dai, dividing the seedlings into roots and aerial parts. Nematodes in each part were extracted separately. Dried weight was determined for each seedling fraction to express nematode density (ND) in number of nematodes per gram of dry weight for stem (NDs), roots (NDr) and total seedling (NDt). Furthermore, incidence data, i.e., the percentage of seedlings per species with nematode presence in the stem, roots and total seedling (%Ns, %Nr and %Nt, respectively), was calculated.

2.3. Anatomical and Morphological Traits

To study induced responses to the PWN, one 3-cm-long segment of each inoculated individual was cut 5 cm above the inoculation point. For the study of constitutive traits (non-inoculated seedlings), we harvested one 3-cm-long segment of each seedling at a similar height to the inoculated seedlings.
For light microscopy observation, transverse sections (20–30 µm thick) were cut using a sliding microtome (Leica Microsystems Nussloch GmbH, Nussloch, Germany). The samples contained the cortex, xylem and pith tissues. Sections were stained with a solution of safranin 1% and alcian blue 1% and mounted after dehydrating through the ethanol series. Samples were photographed with a light microscope using a Nikon digital Coolpix 4500 camera and analyzed with WinCELL Pro (version 2004a; Regent Instruments Inc. 2004, Québec, Canada). The following parameters were measured (Figure 1): (i) radial growth variables: cortex (Cg, μm) and xylem (Xg, μm) growths, and (ii) axial canals variables in cortex (C) and xylem (X) tissues: mean size of axial canals in cortex and xylem (adjacent epithelial and parenchyma cells included) (CsC and CsX, respectively; µm2), frequency of axial canals (CfC and CfX; number of canals per mm2) and area of axial canals (adjacent epithelial and parenchyma cells included) per mm2 (CaC and CaX; μm2/mm2). On each preparation, a minimum of 3 random measurements of growth variables and all the axial canal variables in the complete transverse section were taken.
Before inoculation, several dendrometric and morphological traits were measured: initial height (IH, cm), previous year height (PH, cm), height increment (HI = IH-PH, cm), root-collar diameter (D, mm), and the number of main branches (BN).

2.4. Susceptibility Groups

Pine species were grouped according to their susceptibility to the PWN based on the symptomatology and mortality observed by Menéndez-Gutierrez et al. [11]. Two susceptibility groups were defined: (i) low-susceptible group, which includes P. canariensis, P. halepensis, P. pinea and P. taeda, and (ii) high-susceptible group, which comprises P. pinaster, P. radiata and P. sylvestris.

2.5. Statistical Analysis

Constitutive anatomical traits were analyzed by a general linear model (GLM), with susceptibility groups and species (nested to susceptibility group) as fixed factors. Least square means (LSMeans) were estimated when significant differences among susceptibility groups or species were detected (p < 0.05). Prior to the analysis, we log10-transformed all variables with the exception of CsX, Xg and Cg to meet normality and homoscedasticity assumptions. The effect of PWN inoculation was analyzed by a GLM considering susceptibility group, species (nested to susceptibility group) and treatment (PWN inoculation and water inoculation) and their interactions. All factors were considered fixed. Prior to the analyses, we log10-transformed all the cortical variables to meet normality and homoscedasticity assumptions. Posthoc LSD tests were applied to compare mean values within factors when any significant effect was detected (p < 0.05). Correlation analysis was performed by Spearman’s rank correlation coefficient between the percentage of seedlings per species with nematode presence in stem, roots and total seedling (%Ns, %Nr, %Nt), the LSMeans per species of the morphological traits (IH, PH, HI, D and BN) and the LSMeans per species of the constitutive anatomical variables in cortex and xylem (CaC, CfC, CsC, Cg, CaX, CfX, CsX and Xg) (N = 7). All statistical analyses were performed using the SAS 9.2 statistical package (SAS Institute, Inc., Cary, NC, USA).

3. Results

3.1. Constitutive Anatomical Traits

A significant variation was found among the two pine susceptibility groups for Xg, CsC and CfC (p < 0.001), and also for CsX (p < 0.05) (Table 1; Figure S1). The high-susceptible group showed higher Xg and CsC, and lower CfC and CsX than the low-susceptible group. At the species level, all constitutive anatomical traits showed differences among pine species (p < 0.05), except for CaC. Two of the three pine species from the high-susceptible group (P. pinaster and P. radiata) presented higher Xg than those from the low-susceptible group. Furthermore, the high-susceptible species P. radiata and P. sylvestris showed the highest values of CsC and the lowest values of CfC (Table 1; Figure 2 and Figure S1). The high-susceptible P. pinaster also presented high CsC values, but its CfC values were similar to most of the low-susceptible species. The rest of the anatomical traits, except CaC, showed variability among the different pine species, but without any evident association with PWN susceptibility.

3.2. Induced Anatomical Variations in Response to PWN Inoculation

At day 41 after inoculation, only the high-susceptible species showed foliage disease symptoms (Figure S2). The PWN inoculation mainly affected anatomical traits in the xylem (Table 2). Two of the three xylem variables were significantly affected by the treatment: CfX and CaX (p < 0.05). Both variables were significantly higher in PWN-inoculated seedlings than in water-inoculated seedlings (0.58 ± 0.01 vs. 0.54 ± 0.01, and 4.56 ± 0.01 vs. 4.51 ± 0.01, respectively).
CsX and CfX showed a significant susceptibility group × treatment interaction (p < 0.001). Thus, in the high-susceptible group, PWN inoculation resulted in smaller and more frequent xylem canals, compared to the water-inoculated ones. In the low-susceptible group, however, PWN inoculation resulted in wider xylem canals but of similar frequency than in water-inoculated seedlings (Figure 3 and Figure 4). At the species level, a similar trend was observed for the highly susceptible P. pinaster and P. radiata, and for the low-susceptible P. canariensis (Figure 5a,c).
In the cortex, CsC and CaC showed significant species × treatment interactions (p < 0.05) (Table 2). PWN inoculation induced the formation of higher values of CsC in P. pinea (low-susceptible species). Yet, the opposite trend was observed in the also low-susceptible P. halepensis (Figure 5b). Similarly, CaC increased with PWN inoculation in P. pinea (as well as in the highly susceptible P. sylvestris) while it decreased in P. halepensis (Figure 5d).

3.3. Relationships between Anatomical/Dendrometric Traits and Nematode Colonization

The number of nematodes recovered from inoculated seedlings differed significantly among species (chi2 = 13.895; p < 0.0308), ranging from 0 to 1152.68 B. xylophilus individuals per dry gram of wood. The median number of nematodes recovered from the total seedling was higher in the high-susceptible species P. radiata and P. sylvestris than in the low-susceptible P. canariensis, P. halepensis and P. pinea (Table S2). The percentage of seedlings per species with nematode presence in roots (%Nr) was associated to CsC (Table 3; Figure 6a). The percentage of seedlings per species with nematode presence in the stem (%Ns) was significantly correlated with the plant diameter (D) (Table 3; Figure 6b). Furthermore, the percentage of seedlings with nematode presence in both stem and root tissues (%Nt) was significantly correlated with Xg (Table 3). Regarding relations between constitutive anatomical and dendrometric traits, D was significantly correlated with Xg, and the seedling initial height (IH) with CfC (Table 3).

4. Discussion

Our study showed a high interspecific variability in axial resin canal traits among Pinus species. Some of these traits showed marked differences between pine susceptibility groups. However, these differences were not always consistent at the species level. These results generally agree with previous works evidencing that resin canal traits are important for PWN infection success [36,45], but also with other studies suggesting that canal traits do not fully determine the pine species susceptibility [12,37].

4.1. PWN Susceptibility and Constitutive Traits

A relation was observed between the percentage of seedlings with nematode presence, especially in roots, and the radial xylem growth. Possibly, the pine species which allocated more resources to radial growth had limited resources available to differentiation processes, such as constitutive defenses [46,47]. This hypothesis is supported by different studies which focused on the chemical defense system of pine trees [1,11]. Some of them, conducted under greenhouse conditions, showed that P. pinea presents slower growth and higher levels of constitutive defenses than P. pinaster and P. radiata [1]. In our anatomical study, we did not find any clear evidence supporting a higher constitutive defense investment in the low-susceptible tree species. The low-susceptible group presented larger resin canals in the xylem and more resin canals in the cortex than the high-susceptible group, but the total area occupied by resin canals was similar in both groups. Moreover, the high-susceptible species P. sylvestris presented lower radial growth than the other two high-susceptible species (P. pinaster and P. radiata), which is evidence that more complex mechanisms (e.g., chemical composition of defenses, early recognition of the pathogen, or ecophysiological traits) are involved in PWN susceptibility.
The constitutive anatomical differences found between susceptibility groups in the cortex, with the high-susceptible group showing less frequent but wider canals, agree with previous studies [36,37], supporting that cortical canal size is a good predictor of tree susceptibility. Cortical axial resin canals are the main path of PWN migration in seedlings [33,34,45], and wider canals could facilitate this migration. For example, PWN migration was much faster through cortical than xylem axial resin canals in Pinus thunbergii, P. strobus and P. rigida [38], indicating that xylem canals probably have a secondary role in PWN migration. Furthermore, the average and total lumen area of cortical resin canals of a P. thunbergii resistant family were significantly smaller than non-selected (susceptible) P. thunbergii families [36]. These authors suggested that the smaller lumen area of cortical canals caused the restriction in PWN migration in resistant P. thunbergii individuals. Similarly, nematode migration through the branch segments of different P. pinaster provenances was positively correlated with the mean area of the resin canals in the cortex [36]. In agreement with these works, we found a positive correlation at an inter-specific level between the size of cortical resin canals and the percentage of seedlings with PWN presence in roots, supporting the idea that smaller lumen canals in the cortex restrict PWN spread. However, our results do not agree with previous research which showed wider cortical canals in P. pinea than in P. sylvestris [12]. These authors suggested that the PWN could also use pathways other than canals to migrate from the inoculation point, such as phloem, the cambium, tracheids and the pith, as was also suggested by other authors [3,33].
Regarding the xylem variables, our results agree with previous studies showing that susceptible species, such as P. sylvestris, present the smallest axial resin canals in the xylem [12]. In our work, the high-susceptible group of seedlings presented smaller canals in xylem tissues than the low-susceptible group. However, this trait was not correlated with the percentage of seedlings with nematode presence in any examined seedling fraction (root or stem). The size of xylem axial canals does not seem to be a good predictor due to the inconsistency found among pine species of different susceptibility level. Thus, P. sylvestris, a high-susceptible species, showed the smallest canal size but was similar to the low-susceptible species P. halepensis. On the contrary, P. pinea, a low-susceptible species showed wide xylem canals, but P. pinaster, a high-susceptible species, showed canals even wider. In sum, constitutive xylem canals cannot be regarded as a cause of susceptibility.

4.2. Anatomical Changes Induced by PWN Inoculation

Pine species show a complex reaction to different types of biotic and abiotic attacks, which operate at different levels (anatomical, chemical and physiological) and at different times [20,21]. The complexity of this defense system should be taken into account when interpreting our results. The formation of traumatic resin canals in response to an external disturbance is an important component of the pine induced resistance [22,48]. In our work, we observed that PWN inoculation induced an increase in the frequency of canals, and in the total area occupied by canals in the xylem. An increase in the axial canal frequency has been observed in response to insect attack, fungal invasion and/or mechanical wounding in Pinus species [22,28,46,49]. Furthermore, we found a greater increase in xylem canal frequency after PWN-inoculation in the high-susceptible group, mainly in the fast-growing P. pinaster and P. radiata. Species with faster growths and lower investments in constitutive defenses might invest more in inducible defenses, as resin canal formation [50]. The induced formation of xylem canals followed the typical pattern of dense concentric series of traumatic canals. This observation agrees with previous studies showing this type of response after PWN inoculation [51].
In spite that the high-susceptible group formed more xylem canals after PWN inoculation, these canals were smaller than those formed by the low-susceptible group. At the species level this was confirmed by the PWN-infected P. radiata and P. pinaster seedlings which showed a decrease in the xylem canal size together with an increase in canal frequency, while P. canariensis showed a significant increase in the average canal size (but no increase in frequency). The formation of more frequent but smaller canals has been also reported as a defense response in Pinus spp. in previous studies [49,52]. The presence of more abundant but smaller defensive structures could be a good strategy for developing a dense network of canals to optimize the barrier effect against insects, but possibly limits the structural resin synthesis. Yet, this strategy of induced defense is not a general trend in the genus. For instance, more frequent and larger xylem resin canals were found in Pinus ponderosa after bark beetle attack [53]. The formation of a denser network of canals might facilitate PWN migration through the xylem, and perhaps a more widespread release of volatiles in the xylem, which seem to be involved in tracheid embolism [35]. This way, changes induced in xylem canals by the PWN could also be the cause of enhanced susceptibility to PWN. It should be noted that the average size of xylem canals would permit, in most cases, the migration of the PWN through them. Thus, the greatest body width of Portuguese Bursaphelenchus xylophilus is of 22.3 ± 1.8 µm [54], while the average xylem canal width in P. sylvestris (the pine species examined with the smallest xylem canals) was of 46.3 ± 12.7 µm (epithelial and parenchyma cells included), with an average lumen of 28.8 ± 7.5 µm.
In general, the effect of the PWN inoculation in the cortical canal traits was low, as no significant effects of the treatment, and the susceptibility group × treatment interaction were observed. Cortical canals are formed during the primary development of a plant, and stay alive for several years of secondary growth [20]. Several studies showed changes in cortical tissues in response to a wound or an infection, which are described as inducible structural and chemical defenses. However, these responses have not been associated to changes in cortical resin canals traits, but to the ability to provide a physical barrier through the formation of callus tissue that can become lignified, suberized or impregnated with phenolics and terpenes [20,21,55]. This way, even though cortical canals seemed to have an important role in PWN dispersal, they did not experience major changes in their size or frequency in response to PWN inoculation. Although the effect of inoculation on cortical canals was generally low, the effect of inoculation was greater for some pine species than others, as evidenced by the significant pine species × treatment interaction (see Figure 5b,d).
Other traits not evaluated in our study could be behind the susceptibility of pine trees to PWN. For instance, Pimentel et al. [1] found higher concentrations of tannins and phenols in the phloem and cambium of P. pinea compared to P. pinaster and P. radiata. In turn, the phloem of P. sylvestris is scarce in defensive polyphenols but abundant in potentially nutritive compounds such as lipid soluble substances [11]. In Pinus species, cortex tissues contain large amounts of phenolics, sclerenchyma and calcium oxalate crystals. These substances create the main defensive barrier during the early stages of stem growth and probably determine, in a stronger way than the anatomical structure, the different susceptibility degree to nematode attack. Pine species produce different amounts of phenolic compounds [11], and there is also some evidence that relative resistance to pathogens might partly be a function of the type of phenolics, nitrogen, lipid-soluble substances or other defense associated substances [1,11,56,57]. These works highlight that further biochemical and physiological research is needed to fully understand susceptibility factors to the PWN.

5. Conclusions

The high-susceptible group of pine species tended to show wider constitutive cortical canals and more xylem canals after infection than the low-susceptible group. The size of constitutive cortical canals could be regarded as one of the causes of susceptibility, influencing the initial migration rate of the PWN. Constitutive xylem canal traits were not clearly associated with susceptibility, although the changes induced after infection in xylem canals might also affect susceptibility. It can be concluded that the anatomy of axial resin canals probably exerts some influence in the interspecific variation of pine susceptibility to the PWN, at least during the seedling stage or in young twigs. In turn, the high-susceptible species tend to show larger radial growths than the low-susceptible species. Yet, these trends were not always straightforward at the species level, most likely due to the role of other chemical or physiological key defensive traits. The results of controlled greenhouse inoculation experiments should be confirmed in the field, as the defense mechanisms of adult trees could be quite different in outdoors conditions; nevertheless, since pine wilt disease is subjected to strict quarantine measures in Europe, field experiments involving PWN inoculation are not permitted in Spain.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f14050925/s1, Table S1. Height and basal diameter of the experimental seedlings (mean ± SE); Table S2. Median number of nematodes per gram of dry weight recovered from roots (NDr), stem (NDs) and total seedling (NDt) at 42 days after inoculation. Within each column, different letters denote significant differences between pine species (Kruskal-Wallis test, p < 0.05, N = 8); Figure S1. Constitutive anatomical traits showing differences between susceptibility groups: (a) xylem growth, (b) canal size in xylem, (c) canal size in the cortex, and (d) canal frequency in the cortex. Different letters denote significant differences between pine species (p < 0.05). A = P. pinea; C = P. canariensis; H = P. halepensis; T = P. taeda; P = P. pinaster; R = P. radiata and S = P. sylvestris; Figure S2. Changes in foliage symptoms shown by high-susceptible species after 3 and 41 days after inoculation (DAI) with the pine wood nematode. No significant changes were observed in low-susceptible species.

Author Contributions

Conceptualization, R.D., L.G. and A.R.-G.; methodology, A.R.-G., R.D. and M.M.-G.; formal analysis, R.D., A.R.-G., M.M.-G. and J.A.M.; writing—original draft preparation, A.R.-G. and L.G.; writing—review and editing, J.A.M., M.M.-G. and R.D.; supervision, R.D.; project administration, R.D. All authors have read and agreed to the published version of the manuscript.

Funding

This work has been supported by the research projects RTA2011-069-C03-01 and RTA2017-012-C2-01 from the National Institute for Agricultural and Food Research and Technology (INIA) and cofunded by the Plan de Mejora e Innovación Forestal de Galicia (2010–2020) and INDITEX company.

Data Availability Statement

Data available on request.

Acknowledgments

We thank Ricardo Ferradás, Maribel Juncal and Francis Ignacio for their technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Pimentel, C.S.; Firmino, P.N.; Calvão, T.; Ayres, M.P.; Miranda, I.; Pereira, H. Pinewood Nematode Population Growth in Relation to Pine Phloem Chemical Composition. Plant Pathol. 2017, 66, 856–864. [Google Scholar] [CrossRef]
  2. Togashi, K.; Jikumaru, S. Evolutionary Change in a Pine Wilt System Following the Invasion of Japan by the Pinewood Nematode, Bursaphelenchus xylophilus. Ecol. Res. 2007, 22, 862–868. [Google Scholar] [CrossRef]
  3. Kuroda, K. Physiological Incidences Related to Symptom Development and Wilting Mechanism. In Pine Wilt Disease; Springer: Tokyo, Japan, 2008; pp. 204–222. [Google Scholar] [CrossRef]
  4. Mota, M.M.; Futai, K.; Vieira, P. Pine Wilt Disease and the Pinewood Nematode, Bursaphelenchus xylophilus. In Integrated Management of Fruit Crops Nematodes; Ciancio, A., Mukerji, K.G., Eds.; Springer: Berlin/Heidelberg, Germany, 2009; pp. 253–274. [Google Scholar] [CrossRef]
  5. Burgermeister, W.; Sousa, E.; Mota, M.; Penas, A.C.; Bravo, M.A.; Metge, K.; Braasch, H. First Report of Bursaphelenchus xylophilus in Portugal and in Europe. Nematology 1999, 1, 727–734. [Google Scholar] [CrossRef]
  6. Fonseca, L.; Cardoso, J.M.S.; Lopes, A.; Pestana, M.; Abreu, F.; Nunes, N.; Mota, M.; Abrantes, I. The Pinewood Nematode, Bursaphelenchus xylophilus, in Madeira Island. Helminthologia 2012, 49, 96–103. [Google Scholar] [CrossRef]
  7. Naves, P.; Bonifácio, L.; de Sousa, E. The Pine Wood Nematode and Its Local Vectors in the Mediterranean Basin. In Insects and Diseases of Mediterranean Forest Systems; Paine, T.D., Lieutier, F., Eds.; Springer: Berlin/Heidelberg, Germany, 2016; pp. 329–378. ISBN 9783319247441. [Google Scholar]
  8. Robertson, L.; Cobacho Arcos, S.; Escuer, M.; Santiago Merino, R.; Esparrago, G.; Abelleira, A.; Navas, A. Incidence of the Pinewood Nematode Bursaphelenchus Xylophlius Steiner & Buhrer, 1934 (Nickle, 1970) in Spain. Nematology 2011, 13, 755–757. [Google Scholar] [CrossRef]
  9. de la Fuente, B.; Saura, S. Long-Term Projections of the Natural Expansion of the Pine Wood Nematode in the Iberian Peninsula. Forests 2021, 12, 849. [Google Scholar] [CrossRef]
  10. Soliman, T.; Mourits, M.C.M.; van der Werf, W.; Hengeveld, G.M.; Robinet, C.; Lansink, A.G.J.M.O. Framework for Modelling Economic Impacts of Invasive Species, Applied to Pine Wood Nematode in Europe. PLoS ONE 2012, 7, e45505. [Google Scholar] [CrossRef] [PubMed]
  11. Menéndez-Gutiérrez, M.; Alonso, M.; Jiménez, E.; Toval, G.; Mansilla, P.; Abelleira, A.; Abelleira-Sanmartín, A.; Díaz, R. Interspecific Variation of Constitutive Chemical Compounds in Pinus Spp. Xylem and Susceptibility to Pinewood Nematode (Bursaphelenchus xylophilus). Eur. J. Plant Pathol. 2018, 150, 939–953. [Google Scholar] [CrossRef]
  12. Nunes da Silva, M.; Solla, A.; Sampedro, L.; Zas, R.; Vasconcelos, M.W. Susceptibility to the Pinewood Nematode (PWN) of Four Pine Species Involved in Potential Range Expansion across Europe. Tree Physiol. 2015, 35, 987–999. [Google Scholar] [CrossRef]
  13. Matsunaga, K.; Togashi, K. A Simple Method for Discriminating Bursaphelenchus xylophilus and B. mucronatus by Species-Specific Polymerase Chain Reaction Primer Pairs. Nematology 2004, 6, 273–277. [Google Scholar] [CrossRef]
  14. Menéndez-Gutiérrez, M.; Alonso, M.; Toval, G.; Díaz, R. Testing of Selected Pinus pinaster Half-Sib Families for Tolerance to Pinewood Nematode (Bursaphelenchus xylophilus). Forestry 2018, 91, 38–48. [Google Scholar] [CrossRef]
  15. Vicente, C.; Espada, M.; Vieira, P.; Mota, M. Pine Wilt Disease: A Threat to European Forestry. Eur. J. Plant Pathol. 2012, 133, 89–99. [Google Scholar] [CrossRef]
  16. Inácio, M.L.; Nóbrega, F.; Vieira, P.; Bonifácio, L.; Naves, P.; Sousa, E.; Mota, M. First Detection of Bursaphelenchus xylophilus Associated with Pinus nigra in Portugal and in Europe. For. Pathol. 2015, 45, 235–238. [Google Scholar] [CrossRef]
  17. Zamora, P.; Rodríguez, V.; Renedo, F.; Sanz, A.V.; Domínguez, J.C.; Pérez-Escolar, G.; Miranda, J.; Álvarez, B.; González-Casas, A.; Mayor, E.; et al. First Report of Bursaphelenchus xylophilus causing Pine Wilt Disease on Pinus radiata in Spain. Plant Dis. 2015, 99, 1449. [Google Scholar] [CrossRef]
  18. Blanche, C.A.; Lorio, P.L.; Sommers, R.A.; Hodges, J.D.; Nebeker, T.E. Seasonal cambial growth and development of loblolly-pine—Xylem formation, inner bark chemistry, resin ducts, and resin flow. For. Ecol. Manag. 1992, 49, 151–165. [Google Scholar] [CrossRef]
  19. Lombardero, M.J.; Ayers, M.P.; Lorio, P.L., Jr.; Ruel, J.J. Environmental Effects on Constitutive and Indicible Resin Defences of Pinus taeda. Ecol. Lett. 2000, 3, 329–339. [Google Scholar] [CrossRef]
  20. Franceschi, V.R.; Krokene, P.; Christiansen, E.; Krekling, T. Anatomical and Chemical Defenses of Conifer Bark against Bark Beetles and Other Pests. New Phytol. 2005, 167, 353–376. [Google Scholar] [CrossRef] [PubMed]
  21. Lewinsohn, E.; Gijzen, M.; Croteau, R. Defense Mechanisms of Conifers1. Plant Physiol. 1991, 96, 44–49. [Google Scholar] [CrossRef]
  22. Luchi, N.; Ma, R.; Capretti, P.; Bonello, P. Systemic Induction of Traumatic Resin Ducts and Resin Flow in Austrian Pine by Wounding and Inoculation with Sphaeropsis Sapinea and Diplodia Scrobiculata. Planta 2005, 221, 75–84. [Google Scholar] [CrossRef] [PubMed]
  23. Cheniclet, C. Effects of Wounding and Fungus Inoculation on Terpene Producing Systems of Maritime Pine. J. Exp. Bot. 1987, 38, 1557–1572. [Google Scholar] [CrossRef]
  24. Zulak, K.G.; Bohlmann, J. Terpenoid Biosynthesis and Specialized Vascular Cells of Conifer Defense. J. Integr. Plant Biol. 2010, 52, 86–97. [Google Scholar] [CrossRef]
  25. Bannan, M.W. Vertical Resin Ducts in the Secondary Wood of the Abietineae. New Phytol. 1936, 35, 11–46. [Google Scholar] [CrossRef]
  26. Wu, H.; Hu, Z. Comparative Anatomy of Resin Ducts of the Pinaceae. Trees 1997, 11, 135–143. [Google Scholar] [CrossRef]
  27. Nagy, N.E.; Krokene, P.; Solheim, H. Anatomical-Based Defense Responses of Scots Pine (Pinus sylvestris) Stems to Two Fungal Pathogens. Tree Physiol. 2006, 26, 159–167. [Google Scholar] [CrossRef]
  28. Eyles, A.; Bonello, P.; Ganley, R.; Mohammed, C. Induced Resistance to Pests and Pathogens in Trees. New Phytol. 2010, 185, 893–908. [Google Scholar] [CrossRef]
  29. Klutsch, J.G.; Kee, C.X.; Cappa, E.P.; Ratcliffe, B.; Thomas, B.R.; Erbilgin, N. Increment Coring Induced Traumatic Resin Ducts in White Spruce But Not in Lodgepole Pine. Tree-Ring Res. 2020, 76, 54–58. [Google Scholar] [CrossRef]
  30. Krokene, P.; Solheim, H.; Krekling, T.; Christiansen, E. Inducible Anatomical Defense Responses in Norway Spruce Stems and Their Possible Role in Induced Resistance. Tree Physiol. 2003, 23, 191–197. [Google Scholar] [CrossRef]
  31. Vazquez-Gonzalez, C.; Zas, R.; Erbilgin, N.; Ferrenberg, S.; Rozas, V.; Sampedro, L. Resin Ducts as Resistance Traits in Conifers: Linking Dendrochronology and Resin-Based Defences. Tree Physiol. 2020, 40, 1313–1326. [Google Scholar] [CrossRef] [PubMed]
  32. Hood, S.; Sala, A.; Heyerdahl, E.K.; Boutin, M.; Raffa, K.F. Low-Severity Fire Increases Tree Defense against Bark Beetle Attacks. Ecology 2015, 96, 1846–1855. [Google Scholar] [CrossRef]
  33. Son, J.A.; Komatsu, M.; Matsushita, N.; Hogetsu, T. Migration of Pine Wood Nematodes in the Tissues of Pinus thunbergii. J. For. Res. 2010, 15, 186–193. [Google Scholar] [CrossRef]
  34. Ichihara, Y.; Fukuda, K.; Suzuki, K. Early Symptom Development and Histological Changes Associated with Migration of Bursaphelenchus xylophilus in Seedling Tissues of pinus thunbergii. Plant Dis. 2000, 84, 675–680. [Google Scholar] [CrossRef] [PubMed]
  35. Kuroda, K. Mechanism of Cavitation Development in the Pine Wilt Disease. Eur. J. For. Pathol. 1991, 21, 82–89. [Google Scholar] [CrossRef]
  36. Kawaguchi, E. Relationship between the Anatomical Characteristics of Cortical Resin Canals and Migration of Bursaphelenchus xylophilus in Stem Cuttings of Pinus thunbergtt Seedlings. Nihon Ringakkai Shi/J. Jpn. For. Soc. 2006, 88, 240–244. [Google Scholar] [CrossRef]
  37. Zas, R.; Moreira, X.; Ramos, M.; Lima, M.R.M.; Nunes da Silva, M.; Solla, A.; Vasconcelos, M.W.; Sampedro, L. Intraspecific Variation of Anatomical and Chemical Defensive Traits in Maritime Pine (Pinus pinaster) as Factors in Susceptibility to the Pinewood Nematode (Bursaphelenchus xylophilus). Trees-Struct. Funct. 2015, 29, 663–673. [Google Scholar] [CrossRef]
  38. Son, J.A.; Matsushita, N.; Hogetsu, T. Migration of Bursaphelenchus xylophilus in Cortical and Xylem Axial Resin Canals of Resistant Pines. For. Pathol. 2015, 45, 246–253. [Google Scholar] [CrossRef]
  39. Zhang, F.; Kajiwara, J.; Mori, Y.; Ohira, M.; Tsutsumi, Y.; Kondo, R. Metabolites from Resistant and Susceptible Pinus thunbergii after Inoculation with Pine Wood Nematode. Am. J. Plant Sci. 2013, 4, 512–518. [Google Scholar] [CrossRef]
  40. Fukuda, K.; Hogetsu, T.; Suzuki, K. Cavitation and Cytological Changes in Xylem of Pine Seedlings Inoculated with Virulent and Avirulent Isolates of Bursaphelenchus xylophilus and B. mucronatus. J. Jpn. For. Soc. 1992, 74, 289–299. [Google Scholar] [CrossRef]
  41. Mori, Y.; Miyahara, F.; Tsutsumi, Y.; Kondo, R. Relationship between Resistance to Pine Wilt Disease and the Migration or Proliferation of Pine Wood Nematodes. Eur. J. Plant Pathol. 2008, 122, 529–538. [Google Scholar] [CrossRef]
  42. Ichihara, Y.; Fukuda, K.; Suzuki, K. The Effect of Periderm Formation in the Cortex of Pinus thunbergii on Early Invasion by the Pinewood Nematode. For. Pathol. 2000, 30, 141–148. [Google Scholar] [CrossRef]
  43. Menéndez-Gutiérrez, M.; Matsunaga, K.; Togashi, K. Relationship between Pine Wilt-Tolerance Rankings of Pinus thunbergii Trees and the Number of Bursaphelenchus xylophilus Passing through Branch Sections. Nematology 2017, 19, 1083–1093. [Google Scholar] [CrossRef]
  44. Hallman, J.; Viaene, N. PM 7/119 (1) Nematode Extraction. EPPO Bull. 2013, 43, 471–495. [Google Scholar] [CrossRef]
  45. Kuroda, K.; Ohira, M.; Okamura, M.; Fujisawa, Y. Migration and Population Growth of the Pine Wood Nematode (Bursaphelenchus xylophilus) Related to the Symptom Development in the Seedlings of Japanese Black Pine (Pinus thunbergii) Families Selected as Resistant to Pine Wilt. Nihon Ringakkai Shi/J. Jpn. For. Soc. 2007, 89, 241–248. [Google Scholar] [CrossRef]
  46. Bonello, P.; Gordon, T.R.; Herms, D.A.; Wood, D.L.; Erbilgin, N. Nature and Ecological Implications of Pathogen-Induced Systemic Resistance in Conifers: A Novel Hypothesis. Physiol. Mol. Plant Pathol. 2006, 68, 95–104. [Google Scholar] [CrossRef]
  47. Moreira, X.; Abdala-Roberts, L.; Zas, R.; Merlo, E.; Lombardero, M.J.; Sampedro, L.; Mooney, K.A. Masting Behaviour in a Mediterranean Pine Tree Alters Seed Predator Selection on Reproductive Output. Plant Biol. 2016, 18, 973–980. [Google Scholar] [CrossRef] [PubMed]
  48. López-Villamor, A.; Zas, R.; Pérez, A.; Cáceres, Y.; Nunes da Silva, M.; Vasconcelos, M.; Vázquez-González, C.; Sampedro, L.; Solla, A. Traumatic Resin Ducts Induced by Methyl Jasmonate in Pinus Spp. Trees-Struct. Funct. 2021, 35, 557–567. [Google Scholar] [CrossRef]
  49. Rodríguez-García, A.; Martín, J.A.; López, R.; Sanz, A.; Gil, L. Effect of Four Tapping Methods on Anatomical Traits and Resin Yield in Maritime Pine (Pinus pinaster Ait.). Ind. Crops Prod. 2016, 86, 143–154. [Google Scholar] [CrossRef]
  50. Moreira, X.; Mooney, K.A.; Rasmann, S.; Petry, W.K.; Carrillo-Gavilán, A.; Zas, R.; Sampedro, L. Trade-Offs between Constitutive and Induced Defences Drive Geographical and Climatic Clines in Pine Chemical Defences. Ecol. Lett. 2014, 17, 537–546. [Google Scholar] [CrossRef]
  51. Mamiya, Y.; Shoji, T. Pathogenicity of the Pinewood Nematode, Bursaphelenchus xylophilus, to Japanese Larch, Larix Kaempferi, Seedlings. J. Nematol. 2009, 41, 157–162. [Google Scholar]
  52. Ferrenberg, S.; Kane, J.M.; Mitton, J.B. Resin Duct Characteristics Associated with Tree Resistance to Bark Beetles across Lodgepole and Limber Pines. Oecologia 2014, 174, 1283–1292. [Google Scholar] [CrossRef]
  53. Kane, J.M.; Kolb, T.E. Importance of Resin Ducts in Reducing Ponderosa Pine Mortality from Bark Beetle Attack. Oecologia 2010, 164, 601–609. [Google Scholar] [CrossRef]
  54. Fonseca, L.; Vieira, M.C.; Santos, D.; Susana, M.; De, N.; Santos, A.; Curtis, R.H.C.; De, I.M.; Abrantes, O. Morpho-Biometrical Characterisation of Portuguese Bursaphelenchus xylophilus Isolates with Mucronate, Digitate or Round Tailed Females. Phytopathol. Mediterr. 2008, 47, 223–233. [Google Scholar] [CrossRef]
  55. Hudgins, J.W.; Christiansen, E.; Franceschi, V.R. Induction of Anatomically Based Defense Responses in Stems of Diverse Conifers by Methyl Jasmonate: A Phylogenetic Perspective. Tree Physiol. 2004, 24, 251–264. [Google Scholar] [CrossRef] [PubMed]
  56. Bonello, P.; Blodgett, J.T. Pinus nigra-Sphaeropsis Sapinea as a Model Pathosystem to Investigate Local and Systemic Effects of Fungal Infection of Pines. Physiol. Mol. Plant Pathol. 2003, 63, 249–261. [Google Scholar] [CrossRef]
  57. Brignolas, F.; Lacroix, B.; Lieutier, F.; Sauvard, D.; Drouet, A.; Claudot, A.C.; Yart, A.; Berryman, A.A.; Christiansen, E. Induced Responses in Phenolic Metabolism in Two Norway Spruce Clones after Wounding and Inoculations with Ophiostoma Polonicum, a Bark Beetle-Associated Fungus. Plant Physiol. 1995, 109, 821–827. [Google Scholar] [CrossRef] [PubMed]
Forests 14 00925 g001 550
Figure 1. Transverse section of a Pinus halepensis seedling stained with safranin (red) and alcian blue (blue). Parameters measured in each one of the analyzed samples were: (i) radial growth variables; xylem growth (Xg, μm) and cortex growth (Cg, μm), and (ii) axial canals variables; size of axial canals (adjacent epithelial and parenchyma cells included) in cortex (CsC; μm2) and size of axial canals (adjacent epithelial and parenchyma cells included) in xylem (CsX; μm2). Other traits measured in xylem and cortex were the transverse area occupied by canals (μm2/mm2), and the frequency of canals (N/mm2).
Figure 1. Transverse section of a Pinus halepensis seedling stained with safranin (red) and alcian blue (blue). Parameters measured in each one of the analyzed samples were: (i) radial growth variables; xylem growth (Xg, μm) and cortex growth (Cg, μm), and (ii) axial canals variables; size of axial canals (adjacent epithelial and parenchyma cells included) in cortex (CsC; μm2) and size of axial canals (adjacent epithelial and parenchyma cells included) in xylem (CsX; μm2). Other traits measured in xylem and cortex were the transverse area occupied by canals (μm2/mm2), and the frequency of canals (N/mm2).
Forests 14 00925 g001
Forests 14 00925 g002 550
Figure 2. Transverse sections of Pinus sylvestris (a), Pinus canariensis (b), Pinus radiata (c), and Pinus taeda (d) seedlings stained with safranin (red) and alcian blue (blue). The images show the constitutive anatomy of high-susceptible (a,c) and low-susceptible (b,d) pine species to the pine wood nematode. Note axial canals in the cortex (arrowheads) and canals in the xylem (arrows). Scale bars = 500 μm.
Figure 2. Transverse sections of Pinus sylvestris (a), Pinus canariensis (b), Pinus radiata (c), and Pinus taeda (d) seedlings stained with safranin (red) and alcian blue (blue). The images show the constitutive anatomy of high-susceptible (a,c) and low-susceptible (b,d) pine species to the pine wood nematode. Note axial canals in the cortex (arrowheads) and canals in the xylem (arrows). Scale bars = 500 μm.
Forests 14 00925 g002
Forests 14 00925 g003 550
Figure 3. Interaction between the treatment (pine wood nematode and water inoculation) and the susceptibility group (low-susceptible and high-susceptible pine species) in two resin canal traits: (a) xylem canal size (CsX) and (b) xylem canal frequency (CfX). The least square means values (mean ± SE) are shown. Different letters indicate significant differences (p < 0.05) among the studied treatments within each susceptibility group.
Figure 3. Interaction between the treatment (pine wood nematode and water inoculation) and the susceptibility group (low-susceptible and high-susceptible pine species) in two resin canal traits: (a) xylem canal size (CsX) and (b) xylem canal frequency (CfX). The least square means values (mean ± SE) are shown. Different letters indicate significant differences (p < 0.05) among the studied treatments within each susceptibility group.
Forests 14 00925 g003
Forests 14 00925 g004 550
Figure 4. Transverse sections of Pinus canariensis (a,b), Pinus radiata (c,d), and Pinus pinaster (e,f) seedlings 42 days after inoculation with water (a,c,e) or with the pine wood nematode (b,d,f). Note the formation of xylem traumatic resin canals as a response to the pine wood nematode infection (arrowheads). Low-susceptible (LS) and high-susceptible (HS) species to the pine wood nematode are shown. (Scale bar = 500 μm).
Figure 4. Transverse sections of Pinus canariensis (a,b), Pinus radiata (c,d), and Pinus pinaster (e,f) seedlings 42 days after inoculation with water (a,c,e) or with the pine wood nematode (b,d,f). Note the formation of xylem traumatic resin canals as a response to the pine wood nematode infection (arrowheads). Low-susceptible (LS) and high-susceptible (HS) species to the pine wood nematode are shown. (Scale bar = 500 μm).
Forests 14 00925 g004
Forests 14 00925 g005 550
Figure 5. Effect of the treatment (pine wood nematode and water inoculation) on some studied anatomical traits for each pine species: (a) canal size in the xylem, (b) canal size in the cortex, (c) canal frequency in the xylem, and (d) canal area in the cortex. The LS mean values (mean ± SE) are shown. For each species asterisks indicate significant differences between the two treatments at p < 0.001 (***), p < 0.01 (**), p < 0.05 (*). C = P. canariensis; H = P. halepensis; A = P. pinea; T = P. taeda; P = P. pinaster; R = P. radiata and S = P. sylvestris.
Figure 5. Effect of the treatment (pine wood nematode and water inoculation) on some studied anatomical traits for each pine species: (a) canal size in the xylem, (b) canal size in the cortex, (c) canal frequency in the xylem, and (d) canal area in the cortex. The LS mean values (mean ± SE) are shown. For each species asterisks indicate significant differences between the two treatments at p < 0.001 (***), p < 0.01 (**), p < 0.05 (*). C = P. canariensis; H = P. halepensis; A = P. pinea; T = P. taeda; P = P. pinaster; R = P. radiata and S = P. sylvestris.
Forests 14 00925 g005
Forests 14 00925 g006 550
Figure 6. Relationships between two constitutive anatomical traits and the percentage of seedlings with pine wood nematode presence: (a) percentage of seedlings with presence of nematodes in roots (%Nr) vs. cortical canal size (CsC); (b) percentage of seedlings with presence of nematodes in the total seedling (%Nt) vs. seedling root collar diameter (D). C = P. canariensis; H = P. halepensis; A = P. pinea; T = P. taeda; P = P. pinaster; R = P. radiata; S = P. sylvestris. Blue dots = low-susceptible species; orange dots = high-susceptible species.
Figure 6. Relationships between two constitutive anatomical traits and the percentage of seedlings with pine wood nematode presence: (a) percentage of seedlings with presence of nematodes in roots (%Nr) vs. cortical canal size (CsC); (b) percentage of seedlings with presence of nematodes in the total seedling (%Nt) vs. seedling root collar diameter (D). C = P. canariensis; H = P. halepensis; A = P. pinea; T = P. taeda; P = P. pinaster; R = P. radiata; S = P. sylvestris. Blue dots = low-susceptible species; orange dots = high-susceptible species.
Forests 14 00925 g006
Table
Table 1. F-values and least square means (±s.e.) obtained from the general linear model of anatomical variables in xylem and cortex tissues in control seedlings (CsX and CsC: size of axial canals in xylem and cortex, respectively; CfX and CfC: frequency of axial canals in xylem and cortex, respectively; CaX and CaC: area occupied by canals per mm2 in xylem and cortex, respectively; Xg: xylem growth; Cg: cortex growth).
Table 1. F-values and least square means (±s.e.) obtained from the general linear model of anatomical variables in xylem and cortex tissues in control seedlings (CsX and CsC: size of axial canals in xylem and cortex, respectively; CfX and CfC: frequency of axial canals in xylem and cortex, respectively; CaX and CaC: area occupied by canals per mm2 in xylem and cortex, respectively; Xg: xylem growth; Cg: cortex growth).
Anatomical TraitF-ValuesLSMeans
Suscept. GroupSpecies (Group)Susceptibility GroupSpecies (Susceptibility Group)
LowHighLow-SusceptibleHigh-Susceptible
P. pineaP. canariensisP. halepensisP. taedaP. pinasterP. radiataP. sylvestris
Xylem
Xg (µm)50.75 ***6.14 ***2563 ± 74 a3441 ± 163 b2714 ± 48 a2379 ± 170 a2387 ± 141 a2769 ± 139 a3595 ± 129 b3926 ± 152 b2800 ± 267 a
CsX (µm2)4.66 *8.16 ***10,350 ± 488 b9186 ± 619 a10,574 ± 722 cd12,599 ± 763 d8041 ± 185 ab10,185 ± 878 c11,043 ± 595 cd9782 ± 976 bc6735 ± 507 a
CfX (N/mm2)0.089.83 ***3.25 ± 0.253.47 ± 0.422.13 ± 0.14 a2.77 ± 0.42 a4.19 ± 0.43 b3.90 ± 0.27 b2.38 ± 0.19 a2.67 ± 0.51 a5.36 ± 0.53 b
CaX (µm2/mm2)0.892.90 *32,458 ± 217429,724 ± 252222,876 ± 2740 a34,603 ± 4548 b32,704 ± 2502 ab39,647 ± 4282 b26,455 ± 3004 ab26,179 ± 5271 ab36,537 ± 3549 b
Cortex
Cg (µm)1.673.68 *952.44 ± 46.74886.10 ± 32.211117.4 ± 55.13 b1071.1 ± 112.6 b806.91 ± 53.26 a814.40 ± 50.81 a879.09 ± 37.95 ab861.22 ± 77.01 a918.01 ± 55.72 b
CsC (µm2)24.41 ***3.24 *10,739 ± 931 a18,330 ± 1649 b12,176 ± 1541 bc7805 ± 1415 a14,801 ± 1408 cd8171 ± 1299 ab15,784 ± 1617 cd19,102 ± 3567 cd20,104 ± 3265 d
CfC (N/mm2)15.69 ***4.41 **1.14 ± 0.1 b0.77 ± 0.06 a0.87 ± 0.09 ab1.15 ± 0.13 bc0.99 ± 0.23 b1.53 ± 0.16 c1.02 ± 0.11 b0.65 ± 0.07 a0.63 ± 0.02 a
CaC (µm2/mm2)2.991.5510,946 ± 82613,651 ± 130410,267 ± 8078663 ± 165413,616 ± 213111,239 ± 134715,810 ± 176311,930 ± 254913,214 ± 2510
Significance levels: *** = p < 0.001, ** = p < 0.01, * = p < 0.05. Different letters indicate significant differences (p < 0.05) among susceptibility groups or among Pinus species within susceptibility groups.
Table
Table 2. F-values and significance levels obtained from the analysis of variance of anatomical variables. Values represent the effects of susceptibility group (SG; high-susceptible and low-susceptible), species within susceptibility group, and treatment (PWN inoculation and water inoculation) and of their interactions on the studied anatomical traits (canal size, canal frequency and canal area) in xylem and cortical tissues.
Table 2. F-values and significance levels obtained from the analysis of variance of anatomical variables. Values represent the effects of susceptibility group (SG; high-susceptible and low-susceptible), species within susceptibility group, and treatment (PWN inoculation and water inoculation) and of their interactions on the studied anatomical traits (canal size, canal frequency and canal area) in xylem and cortical tissues.
FactorXylemCortex
CsX (µm2)CfX (N/mm2)CaX (µm2/mm2)CsC (µm2)CfC (N/mm2)CaC (µm2/mm2)
SG0.031.234.19 *22.45 ***21.66 ***0.03
Species (SG)26.89 ***34.23 ***11.55 ***11.28 ***7.73 ***2.16
Treatment0.455.84 *5.59 *0.230.280.13
SG × treatment17.15 ***12.48 ***1.520.151.662.19
Species (SG) × treatment5.68 ***1.260.213.29 **0.333.12 *
Significance levels: *** = p < 0.001, ** = p < 0.01, * = p < 0.05.
Table
Table 3. Correlation matrix (Spearman’s rank correlation coefficients) between different anatomical, dendrometric and pine wood nematode incidence variables. Asterisks indicate significant values at p < 0.05 (*), and p < 0.01 (**). CaC and CaX = area occupied by canals in cortex and xylem, respectively; CfC and CfX = frequency of canals in in cortex and xylem, respectively; CsC and CsX = size of canals in cortex and xylem, respectively; Cg and Xg = radial growth of cortex and xylem, respectively; %Nr and %Nt = percentage of seedlings with nematode presence in roots and in total seedling, respectively; D = seedling root collar diameter; PH = seedling height in the previous year; IH = initial plant height; HI = height increment; BN = number of main branches. %Ns (percentage of seedlings with nematode presence in stems) is not included in the Table because it is highly correlated with %Nt.
Table 3. Correlation matrix (Spearman’s rank correlation coefficients) between different anatomical, dendrometric and pine wood nematode incidence variables. Asterisks indicate significant values at p < 0.05 (*), and p < 0.01 (**). CaC and CaX = area occupied by canals in cortex and xylem, respectively; CfC and CfX = frequency of canals in in cortex and xylem, respectively; CsC and CsX = size of canals in cortex and xylem, respectively; Cg and Xg = radial growth of cortex and xylem, respectively; %Nr and %Nt = percentage of seedlings with nematode presence in roots and in total seedling, respectively; D = seedling root collar diameter; PH = seedling height in the previous year; IH = initial plant height; HI = height increment; BN = number of main branches. %Ns (percentage of seedlings with nematode presence in stems) is not included in the Table because it is highly correlated with %Nt.
CaCCaXCfCCfXCsCCsXCgXg%Nr%NtDPHIHHIBN
CaC10.14−0.070.280.54−0.39−0.570.390.230.070.18−0.040.070.070.32
CaX 10.390.79 *−0.17−0.21−0.32−0.21−0.160.26−0.14−0.430.40−0.18−0.43
CfC 1−0.11−0.82 *0.64−0.21−0.43−0.72−0.26−0.140.210.96 **0.36−0.64
CfX 10.21−0.68−0.46−0.18−0.040.11−0.32−0.68−0.07−0.390.11
CsC 1−0.68−0.140.75 *0.85 *0.480.460.00−0.65−0.040.46
CsX 10.46−0.28−0.41−0.330.000.390.560.36−0.64
Cg 1−0.180.20−0.07−0.11−0.14−0.40−0.39−0.14
Xg 10.85 *0.78 *0.93 **0.61−0.200.500.00
%Nr 10.77 *0.700.20−0.590.020.14
%Nt 10.82 *0.37−0.110.26−0.26
D 10.79 *0.070.68−0.32
PH 10.380.89 *−0.36
IH 10.58−0.68
HI 1−0.50
BN 1
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Rodríguez-García, A.; Martín, J.A.; Gil, L.; Menéndez-Gutiérrez, M.; Díaz, R. Resin Canal Traits Variation in Pinus spp. with Different Susceptibility to the Pine Wood Nematode. Forests 2023, 14, 925. https://doi.org/10.3390/f14050925

AMA Style

Rodríguez-García A, Martín JA, Gil L, Menéndez-Gutiérrez M, Díaz R. Resin Canal Traits Variation in Pinus spp. with Different Susceptibility to the Pine Wood Nematode. Forests. 2023; 14(5):925. https://doi.org/10.3390/f14050925

Chicago/Turabian Style

Rodríguez-García, Aida, Juan A. Martín, Luis Gil, María Menéndez-Gutiérrez, and Raquel Díaz. 2023. "Resin Canal Traits Variation in Pinus spp. with Different Susceptibility to the Pine Wood Nematode" Forests 14, no. 5: 925. https://doi.org/10.3390/f14050925

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop