Pine defenses against the pitch canker disease are modulated by a native insect newly associated with the invasive fungus

Forest Ecology and Management 437 (2019) 253–262

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Pine defenses against the pitch canker disease are modulated by a native insect newly associated with the invasive fungus

T

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María J. Lombarderoa, , Alejandro Sollab, Matthew P. Ayresc a

Departamento de Producción Vegetal y Proyectos de Ingeniería, Universidad de Santiago de Compostela, 27002 Lugo, Spain Institute for Dehesa Research (INDEHESA), Ingeniería Forestal y del Medio Natural, Universidad de Extremadura, Avenida Virgen del Puerto 2, 10600 Plasencia, Spain c Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA b

1. Introduction Individual plants, especially long lived plants such as trees, are likely to be attacked by more than one organism, often at the same time. For this reason trees are equipped with numerous constitutive as well as inducible structural and chemical defenses against insects and pathogens (Lewinsohn et al., 1991; Pearce, 1996; Larsson, 2002). Conifers may respond to pathogenic infection and insect attacks by increasing physical barriers (Franceschi et al., 2005), phenolics (Klepzig et al., 1995; Franceschi et al., 2000), terpenoids (Erbilgin et al., 2006; Zeneli et al., 2006), resin flow (Ruel et al., 1998; Lombardero et al., 2000; Hudgins and Franceschi, 2004) or defensive proteins (Ekramoddoullah et al., 2000; Smith et al., 2006; Lombardero et al., 2016). The plant hormones salicylic acid (SA) and jasmonic acid (JA) function in the regulation of plant defenses (Jones and Dangl, 2006; Bent and Mackey, 2007) The SA pathway is thought to be effective primarily against biotrophic pathogens and JA against herbivores and necrotrophic pathogens (Glazebrook, 2005; Stout et al., 2006) while hemibiotrophs might trigger both (Ding et al., 2011). Beside these two main pathways other plant hormones may be also involved (RobertSeilaniantz et al., 2011). Plants produce a specific blend of these signaling hormones after pathogen or pest attacks. These signals activate different sets of defense-related genes that determine the defense response against the attacker (Reymond and Farmer, 1998; De Vos et al., 2005). Terpenoids may function in plant defense as repellents or toxins against herbivores or pathogens (Langenheim, 1994), but are also involved in insect attraction (Franceschi et al., 2005) and play a role in indirect defenses attracting parasitoids (Kessler and Baldwin, 2001). Terpenoids are typically derived from one of two biosynthetic pathways: cytosolic mevalonic-acid pathway and the mevalonate-independent pathway (Nagegowda, 2010). Tomicus piniperda (L) (Coleoptera, Curculionidae) is a native insect in Europe. Its capacity for damaging forest productivity has been well documented, especially for northern countries (Bakke, 1968). There are reports of damage to trees in nearly all European countries (e.g.,

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Masutti, 1969; Lombardero et al., 2008; Öhrn et al., 2018) and in the United States (Czokajlo et al., 1997), where it has been accidentally introduced (Haack and Kucera, 1993). Tomicus piniperda beetles cause three types of damage to trees: (1) they excavate galleries within the phloem of the main stem, which affects vascular transportation and can kill the tree; (2) they feed on shoots in the canopy making branches more susceptible to wind breakage leading to multiple-shoot malformations; and (3) they facilitate the introduction of fungi into shoots and stems. Fusarium circinatum Nirenberg and O’Donnell is the ascomycete causing pine pitch canker, which is one of the most destructive fungal diseases of pines worldwide (Wingfield et al., 2008). It has been reported to affect more than 60 pine species as well as Pseudotsuga menziesii (Mirb.) Franco and even some herbaceous plants (Swett and Gordon, 2012; Hernandez-Escribano et al., 2018). Fusarium circinatum enters host tissues through wounds caused by insect feeding, silvicultural activity, or weather-related damages (Gordon, 2006). This fungus was first reported in Spain in 2005 (Landeras et al., 2005) and is now spread throughout northwestern Spain (EFSA, 2010). Berbegal et al. (2013) found two different populations to be present and a separate clonal population has been detected on the Basque country (Iturritxa et al., 2015). The pathogen was considered a necrotroph by Lewis (1973) but Bacon and Yates (2006) considered all Fusarium species to be hemibiotrophs. The fungus is able to produce cell-degrading enzymes and mycotoxins for subsequent penetration inside the host (Leslie and Summerell, 2006; Martin-Rodrigues et al., 2015). Some studies reported T. piniperda as a potential vector of F. circinatum (Romon et al., 2007; Bezos et al., 2015) which could be especially harmful when the insect feeds in shoots of healthy trees. The damage caused by T. piniperda to plantations of native and introduced pines in Europe may increase significantly if it becomes a vector of newly introduced pathogens. However, the extent of damage, and its impact on different pine species, is likely to depend upon tree defenses, which in turn may depend on environmental conditions (Lombardero et al., 2000; Sampedro et al., 2011), and current and previous

Corresponding author at: Departamento de Producción Vegetal y Proyectos de Ingeniería, Universidad de Santiago de Compostela, 27002 Lugo, Spain. E-mail address: [email protected] (M.J. Lombardero).

https://doi.org/10.1016/j.foreco.2019.01.041 Received 26 November 2018; Received in revised form 24 January 2019; Accepted 26 January 2019 0378-1127/ © 2019 Published by Elsevier B.V.

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allow tripartite interactions to occur. Ten conidia is in the range that has been reported to be carried by other bark beetles that are vectors of F. circinatum (Gordon et al., 1998, Erbilgin et al., 2008). At the same time as inoculations with F. circinatum, the mock-inoculated plants (W) were treated with 10 μl of sterile distilled water (Davis et al., 2002) and the true control plants (C) were left untouched. The experiment was carried out in a biosafety greenhouse (Lugo, Spain) during early September (2012), coinciding in time when T. piniperda naturally enters into shoots to overwinter and could introduces the pathogen into healthy trees. Six weeks after the experiment was initiated, we measured the exudation of resin from points of insect attack or simulated attack (W, T and T + F treatments) by carefully removing and weighing the dry resin. Next we measured the lesion length with a caliper after slicing away the outer bark around the inoculation point. Then we harvested the upper shoot of plants and froze them at – 40 °C prior to terpene extraction. We verified that the lesions were the result of infection by F. circinatum by re-isolating it from pieces of stem taken near the inoculation point of a random subset of plants.

interactions with biotic or abiotic factors (Durrant and Dong, 2004; Eyles et al., 2010; Fu and Dong, 2013). Previous exposure of trees to biotic or abiotic stressors might increase susceptibility to pathogens (Milanović et al., 2015) or predispose them to further attacks (Gaylord et al., 2013). On the other hand, previous or coincident damage to plants could reduce susceptibility to pathogens by promoting systemic acquired resistance or induced systemic resistance (priming) (Christiansen et al., 1999; Bonello et al., 2001; Eyles et al., 2010). Indirect interactions among plant enemies may arise when the attack by one enemy alters the shared host plant in a way that affects a second enemy (Stout et al., 2006). In addition to effects on plant defensive systems (secondary metabolism), attacks by pathogens and herbivores can also affect aspects of plant primary metabolism relevant to arthropod and pathogen nutrition, including quantity and quality of nitrogen, concentration of non-structural and structural carbohydrates, and water content (Hatcher, 1995; Lombardero et al., 2012; Milanović et al., 2015). Most investigations of such tripartite interactions have addressed plants that are first affected by one organism and later attacked by a second but there is an increasing number of studies that explore interactions when damaging agents arrive at about the same time to trees (Erbilgin et al., 2009; Eberl et al., 2018; Gallardo et al., 2018). Here we study the interaction of an arthropod, T. piniperda, and a pathogen, F. circinatum, in two pine species, the native Pinus pinaster Ait. and the introduced P. radiata D. Don. The simultaneous occurrence of T. piniperda and F. circinatum may result in altered defense response of the pine species. Therefore the outcome of this interaction may have implications for forest management in areas where both organisms concur.

2.2. Chemical analysis We analyzed nitrogen content of the phloem from a subset of 5 plants of each treatment. The samples were collected with an increment borer, 1 cm in diameter, from the lower part of the stem, far from the inoculation point. Samples were weighed fresh then oven-dried at 60 °C. The dried samples were weighed again to estimate water content, milled to a fine powder, and then submitted to instant oxidation (as 0.1 g tissue samples); the gases released were identified with a conductimeter. Analyses were performed by the analytical unit of the University of Santiago de Compostela (RIAIDT). To analyze concentrations of resins (chiefly di-terpenes) we followed Wainhouse et al. (1998). A 2-cm section of stem just above the inoculation point (upper half of the lesion) was harvested after all the other measurements were taken. For Control and Tomicus treatments, the section was harvested at a similar distance from the top of the tree. One gram of tissue was cut from each sample in very small sections and resin compounds were quantitatively extracted twice with n-hexane (with each extraction including 25 min in an ultrasonic bath). Later the plant material was recovered by filtration, the solvent was evaporated, and the mass of the non-volatile resin residue was determined gravimetrically with a precision scale. The gravimetric determination of non-volatile resin was well correlated with the concentration of diperpenes fraction quantified by gas chromatography in previous work with the same species (Sampedro et al., 2011). Extraction and analysis of the rest of terpenoids followed the methodology of Blanch et al. (2009). A subsection of samples harvested as in the previous paragraph was pulverized with liquid nitrogen. Then, the samples were extracted with ultrapure n- pentane in an ultrasonic bath at 25 °C using dodecane (Merck, no. 1.09658.0005) as the internal standard. The monoterpenes and sesquiterpenes in the extract were analyzed by gas chromatography using a 320 GC/MS/MS (Bruker Corporation) in a DBWAX capillary column (30 m Length, 0.25 mm ID, 0.25 µm film), with He as the carrier gas. Identification of peaks was performed by a comparison of the mass spectra in the single ion 93 m/z with the NIST library and with known standards (eleven monoterpenes and three sesquiterpenes, from Fluka Chemie AG, Buchs, Switzerland and Sigma-Aldrich). Calibration curves for quantification were prepared with commercial standards of 14 compounds frequently cited in samples of both species: α-pinene, β-pinene, β-myrcene, δ-3-carene, αterpinene, limonene, α-terpinolene, γ-terpinene, camphene, linalool, sabinene, trans-caryophyllene, geranyl acetate and α-humulene. We quantified diterpenes in a different subset of fresh phloem tissue from each tree following Wainhouse et al. (1998) and Sampedro et al. (2011). All terpene concentrations were expressed as mg/g phloem dry mass.

2. Materials and methods 2.1. Experiment overview and plant inoculation We selected 200 seedlings (2 years old), 100 of P. pinaster and 100 of P. radiata that were established with a certified mixed seedlot from a seed orchard. Plants were growing in pots with the same history of irrigation and fertilization. We measured plant height and stem diameter at the root collar. We counted the number of branches of the crown to estimate crown size. One hundred plants of each species were randomly assigned to one of five different treatments (n = 20 plants by treatment): Tomicus (T), Fusarium (F), Tomicus + Fusarium (T + F), mock treatment with sterilized water (W), and control (C). The Tomicus treatment (T) was carried out by caging a male and female of T. piniperda inside a mesh enclosure around the top of the plant allowing them to bore into the upper shoot. Tomicus piniperda adults were collected from the field by locating and opening shoots under attack. The timing and location of our insect collections ensured that we were working with T. piniperda rather than T. destruens (Gallego et al., 2004). Each attacked shoot typically contained one male and one female. The pairing was maintained for the study. Once in the lab, the body surface of each insect was cleaned of fungi by gently washing the insect with sterilized water and modified White’s solution (1 g HgCl2/l H2O) (Kopper et al., 2003). For the Fusarium treatments (F and T + F), F. circinatum inoculum was prepared by subculturing the fungus on Potato Dextrose Agar (PDA) before transfer to a sterile flask containing potato dextrose broth (PDB). The flask was held in the dark and shaking for 7 days. Then the suspension was filtered and adjusted to 1,000 spores/ ml water. Ten microliters of inoculum solution was inoculated into the shoot of experimental plants at the point of T. piniperda entrance for plants in the T + F treatment or, in the F treatment, at the same height with a sterile punch. Fungal inoculum was applied right after insects entered the plant to mimic the situation when T. piniperda functions as a vector. Our inoculation dosage (∼10 conidia) was lower than normally used in resistance tests (700-1000 conidia/tree; Vivas et al., 2012) because our intention was to avoid rapid plant mortality and 254

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terpinene, limonene, α-terpinolene, γ–terpinene, linalool, and sabinene. Two monoterpenes and all three sesquiterpenes were significantly higher in P. pinaster: β-myrcene, camphene, trans-caryophyllene, geranyl acetate and α-humulene. Finally δ-3-carene and α-pinene were similar in both species (Table 1, control treatment). Diterpenes increased in concentration after treatment (F4,190 = 17.30, p < 0.0001) and there was also a species × treatment interaction (F4,190 = 18.93, p < 0.0001; Fig. 1). Diterpenes increased in P. pinaster following attacks by T. piniperda, both for T and T + F treatments (Fig. 1a). Pinus radiata had higher diterpene concentration with exposure to F. circinatum, also in both treatments (F and T + F; Fig. 1d). There was also an increase in diterpene concentration when T. piniperda was alone (T) compared with control treatments (Fig. 1d) but the increase was significantly less than with F. circinatum alone (F1,95 = 20.24, p < 0.0001). There was a similar pattern in monoterpenes. There was an effect of treatment (F4,190 = 28.53, p < 0.0001) that differed between species (species × treatment interaction: F4,118 = 14.08, p < 0.0001; Fig. 1). Monoterpenes were also higher in P. pinaster with exposure to T. piniperda and higher in P. radiata with exposure to F. circinatum (Fig. 1b). There were also increases in monoterpenes in P. pinaster after F. circinatum inoculations (F) and in P. radiata after T. piniperda attack (T) but these increases were not significant compared with controls or mock controls (Fig. 1b,e). Sesquiterpenes followed a different pattern. There were also differences between species after exposure to T. piniperda or F. circinatum (F4,190 = 358.06, p < 0.0001; Fig. 1), but there were no differences among T, F, or T + F in the concentrations of sesquiterpenes. There was a species × treatment interaction (F4,190 = 4.40, p = 0.002; Fig. 1). Sesquiterpens increased significantly only in T + F in P. pinaster compared with the control treatments (F1,95 = 4.29, p = 0.003; Fig. 1c) and there were no significant treatment effects in P. radiata (Fig. 1f) although there was a modest increased in sesquiterpenes after F. circinatum inoculation (F). Analyzed individually, almost all compounds varied among treatments (Table 1). Sabinene appeared in P. pinaster samples only after exposure to T. piniperda. Most compounds (9 of 14) increased significantly in P. pinaster with exposure to T. piniperda (T and T + F; Table 1): α-pinene, β-pinene, β-myrcene, limonene, α-terpinolene, camphene, linalool, trans-caryophyllene and α-humulene. And most compounds (10 of 14) increased significantly in P. radiata after inoculation with F. circinatum (F; Table 1): α-pinene, β-pinene, β-myrcene, α-terpinene, α-terpinolene, camphene, linalool, and sabinene.

2.3. Statistical analyses Terpene concentrations and N contents were analyzed with a twoway ANOVA that included species (P. pinaster and P. radiata), treatment (T, F, T + F, W and C), and their interactions. Because the tree species differed greatly in their baseline terpene concentrations and sometimes responded differently to treatments, we also analyzed each species separately with a model that included treatment only and conducted a priori contrasts to compare individual treatments against the control. Tests of specific terms in the statistical model were reported whenever there were significant effects; for brevity, statistical details were not reported for all non-significant effects. Occasionally, peaks from the chromatography could not be reliably resolved, and occasional samples were lost for other reasons; consequently the error degrees of freedom varied slightly depending on the response variable. Terpene concentrations were square root transformed to normalize the distributions and reduce heteroscedasticity. Analyses of terpenes included principal component analysis (PCA) to reduce dimensionality. All analyses were performed with the statistical package JMP (SAS Institute Inc.). 3. Results Six week after the inoculation, no plants had died; 27% of P. radiata and 4% P. pinaster showed some symptoms of declining health with some lateral branches dead, but there was no detectable effect of treatment on this decline. 3.1. Terpene chemistry P. pinaster and P. radiata were different in their terpene chemistry (Table 1). Diterpenes (Fig. 1a and d) and monoterpenes (Fig. 1b and e) were significantly higher in P. radiata compared with P. pinaster (F1,190 = 17.30, p < 0.0001 and F1,190 = 25.19, p < 0.0001 for species effect on diterpenes and monoterpenes, respectively; Fig. 1). This was chiefly a result of differences in induced response between species because there were no differences between species in concentrations of monoterpenes or diterpenes in control plants (i.e., constitutive defenses; Fig. 1). Sesquiterpenes, however, were higher in P. pinaster (Fig. 1c) than P. radiata (Fig. 1f) for both constitutive (F1,38 = 108,72, p < 0.0001; Fig. 1) and inducible (F1,190 = 358.06, p < 0.0001) defenses (Fig. 1). In control plants, constitutive levels of seven monoterpenes were significantly higher in P. radiata than in P. pinaster: β-pinene, α-

Table 1 Concentrations of monoterpenes, diterpenes, and sesquiterpenes in the phloem of P. pinaster and P. radiata in each of five treatments (T + F = Tomicus and Fusarium together). Values that differ significantly from controls (p < 0.05) are in bold (n.d. = nondetectable). Means are from untransformed data. Pinus pinaster (mg/g)

Pinus radiata (mg/g)

Control

Water

Fusarium

Tomicus

T+F

Control

Water

Fusarium

Tomicus

T+F

Monoterpenes α-Pinene β-Pinene β-Myrcene δ-3-Carene α-Terpinene Limonene α-Terpinolene γ-Terpinene Camphene Linalool Sabinene Diterpenes

8,319 5,487 1,004 0,562 0,004 0,520 0,083 0,003 0,292 n.d. n.d. 48,445

7,325 6,838 1,336 n.d. n.d. 0,858 0,035 n.d. 0,338 0,003 n.d. 43,319

11,037 9,138 1,429 0,809 0,005 0,550 0,097 n.d. 0,441 n.d. n.d. 45,316

20,726 22,711 2,267 0,131 0,002 0,983 0,100 n.d. 1,532 0,126 0,004 92,312

26,709 20,227 1,695 0,771 0,003 0,998 0,129 0,006 1,209 0,062 0,004 89,180

6,842 8,002 0,592 0,632 0,023 2,969 0,386 0,040 0,152 0,028 0,025 45,481

6,660 7,235 0,601 0,177 0,009 6,170 0,214 0,003 0,149 0,059 0,009 44,373

41,876 55,636 1,807 0,503 0,053 12,937 0,686 0,114 0,834 0,100 0,077 152,574

11,647 17,074 0,716 0,475 0,023 3,765 0,432 0,050 0,235 0,095 0,027 77,136

38,054 49,807 1,256 0,188 0,042 4018 0,569 0,068 0,668 0,074 0,040 139,603

Sesquiterpenes Trans-Caryophyllene Geranyl Acetate α-Humulene

0,471 0,006 0,104

0,367 0,003 0,077

0,651 0,005 0,144

0,964 0,005 0,193

1,211 0,002 0,233

0,088 n.d. 0,009

0,034 n.d. 0,007

0,068 n.d. 0,012

0,045 n.d. 0,002

n.d. n.d. 0,005

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Fig. 1. Concentrations (mg/g) of total monoterpenes, diterpenes, and sesquiterpenes in the phloem of P. pinaster (a, b, and c, respectively) and P. radiata (d, e, and f, respectively) under five treatments: control, mock treatment with water, attack by the bark beetle Tomicus piniperda, inoculation with the fungus Fusarium circinatum, and beetle attack + fungal inoculation. Asterisks indicate significant treatment differences relative to control. Means ± SE.

pine species between the amounts of any class of terpenes with N content, water content, or the dry mass of phloem.

However, increases in two of these compounds were negated when T. piniperda attacks were combined with F. circinatum (T + F): limonene and γ–terpinene (Table 1). In P. radiata there were only 2 of 14 compounds that increased after T. piniperda attack alone (β-pinene and linalool) and no compounds increased significantly in P. pinaster after exposure to F. circinatum alone. Principal component analyses (PCA) reinforced that terpenes responded to the treatments differently in P. pinaster and P. radiata. Correlations among the 14 terpenoids analyzed were mainly positive for both P. pinaster and P. radiata (Table 2). Consequently, the first principal components axis (PC-1) had positive loadings for both species (Table 3) and was interpretable as reflecting terpene concentrations in general. PC-2 showed no differences among treatments. For both P. pinaster and P. radiata there was an effect of treatment on overall terpene concentrations (PC-1) that matched patterns from analyses of individual compounds. For P. pinaster, there was a significant increase of terpenes in any treatments that included T. piniperda (T and T + F; F4,95 = 17.49, P < 0.0001; Fig. 2), but no effects with exposure to F. circinatum alone (F). For P. pinaster, there were also significant but relatively modest treatment effects on PC-2 (F4,95 = 3.40, P = 0.01). For P. radiata, there was an increase of terpenoids in treatments with F. circinatum (F and T + F) on PC-1 (F4,95 = 18.93; P < 0.0001; Fig. 2), effects were significantly higher in F than T + F, and treatment with T. piniperda alone (T) did not differ from the mock treatment (W) or control trees (C). Differences in PC-2 were also significant in P. radiata (F4,95 = 2.88, P = 0.03) with high positive loadings for treatment C and lower for T while there was negative loadings for W, F and T + F treatments. Diterpenes were positively correlated with monoterpenes and sesquiterpenes in P. pinaster, and with monoterpenes, but not sesquiterpenes, in P. radiata (Table 4). There were no correlations in either

3.2. Lesions Lesion length and diameter were highly correlated (r2 = 0.91) and showed the same statistical patterns. Lesion length was significantly longer in P. radiata (F1,183 = 91.02, p < 0.0001 for species effect; Fig. 3). Lesions in P. pinaster were small and there were no differences in size compared with the mock treatment (Fig. 3). However lesions were conspicuous in P. radiata and varied strongly among treatments (F4,90 = 196.20, p < 0.0001; Fig. 3), with longer lesions in trees inoculated with F. circinatum alone (Fig. 3). Lesion length was also significantly longer in P. radiata inoculated with T. piniperda and F. circinatum (T + F) compared with the mock treatment (F2,52 = 16.91, p < 0.0001; Fig. 3). Lesion length in P. pinaster was independent of total terpene concentrations (Table 4, Fig. 4), but in P. radiata was positive correlated with the concentration of monoterpenes and diterpenes but not with sesquiterpenes (Table 4, Fig. 4). From analysis of individual compounds, lesion length in P. pinaster was negatively correlated with the concentration of linalool, and in P. radiata was positive correlated with α–pinene, β-pinene, myrcene, camphene and limonene. Lesion length was positively correlated with plant diameter in P. pinaster and with crown size in P. radiata (Table 4). There were no significant correlations in P. pinaster or P. radiata between lesion length and N content, dry mass of phloem or water content. 3.3. Other plant traits External resin flow from wounds was higher in P. pinaster than in P. 256

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Table 2 Matrix of Pearson correlation coefficients among terpene concentrations and lesion size in the phloem of P. pinaster (upper) and P. radiata (lower). N = 95–100 trees per species. Significant correlations (p < 0.05) are in bold. Correlations were calculated from square-root transformed data. Tables S2 and S3 in supplemental materials show correlation matrices for each treatment group in each pine species. P. pinaster A. δ-3-Carene B. α-Pinene C. α-Terpinene D. α-Terpinolene E. β-Myrcene F. β-Pinene G. Camphene H. Trans-Caryophyllene I. γ-Terpinene J. Geranyl Acetate K. α-Humulene L. Limonene M. Linalool N. Diterpenes O. Lesion size P. radiata A. δ-3-Carene B. α-Pinene C. α-Terpinene D. α-Terpinolene E. β-Myrcene F. β-Pinene G. Camphene H. γ-Terpinene I. α-Humulene J. Limonene K. Linalool L. Diterpenes M. Lesion size

A

B

C

D

E

F

G

H

I

J

K

L

M

N

1 −0,03 0,88 0,82 0,13 0,06 −0,04 0,15 0,58 0,03 0,13 −0,01 −0,10 0,12 0,12

1 0,02 0,36 0,63 0,63 0,78 0,63 0,09 0,10 0,55 0,57 0,55 0,48 0,02

1 0,82 0,22 0,18 0,07 0,13 0,37 −0,02 0,08 0,06 0,00 0,07 0,00

1 0,44 0,45 0,42 0,39 0,50 0,05 0,31 0,37 0,27 0,37 0,02

1 0,56 0,62 0,46 0,25 0,24 0,38 0,46 0,48 0,27 0,01

1 0,80 0,52 0,17 −0,08 0,41 0,51 0,69 0,45 −0,02

1 0,62 0,08 −0,03 0,48 0,51 0,77 0,51 −0,09

1 0,33 0,06 0,92 0,38 0,32 0,49 0,05

1 0,02 0,29 0,05 0,03 0,31 0,14

1 0,12 0,18 −0,04 −0,01 −0,09

1 0,29 0,20 0,37 0,04

1 0,36 0,31 0,03

1 0,46 −0,21

1 −0,09

B

C

D

E

F

G

H

I

J

K

L

1 0,58 0,48 0,77 0,87 0,98 0,54 0,01 0,31 0,44 0,68 0,43

1 0,85 0,60 0,48 0,59 0,77 0,08 0,28 0,31 0,43 0,16

1 0,52 0,43 0,49 0,77 0,04 0,17 0,24 0,39 0,07

1 0,82 0,82 0,62 0,08 0,58 0,51 0,64 0,44

1 0,90 0,57 0,00 0,41 0,48 0,76 0,52

1 0,56 0,06 0,37 0,45 0,72 0,45

1 0,12 0,21 0,29 0,54 0,18

1 0,23 −0,03 0,06 0,17

1 0,32 0,22 0,37

1 0,33 0,16

1 0,49

A 1 0,10 0,25 0,22 0,22 0,13 0,14 0,29 0,13 0,23 0,16 0,15 −0,03

39.2 ± 0.6% for P. radiata and P. pinaster respectively). There was no effect of treatment on phloem water content. The percent nitrogen in phloem was unaffected by treatment but was a bit higher in P. pinaster than P. radiata (F1,70 = 14.19, p = 0.003; mean ± SE = 0.52 ± 0.01 vs. 0.47 ± 0.01%, respectively).

Table 3 Loadings of individual terpenes onto the 1st and 2nd axes of principal component analyses of P. pinaster and P. radiata. Pinus pinaster

Pinus radiata

PC-1

PC-2

PC-1

PC-2

δ-3-Carene α-Pinene α-Terpinene α-Terpinolene β-Myrcene β-Pinene Camphene Trans-Caryophyllene γ-Terpinene Geranyl Acetate α-Humulene Limonene Linalool Diterpenes

0,125 0,341 0,144 0,290 0,303 0,339 0,359 0,329 0,161 0,029 0,282 0,257 0,264 0,246

0,562 −0,191 0,507 0,390 −0,039 −0,115 −0,198 −0,041 0,345 0,002 −0,033 −0,124 −0,214 −0,040

0,112 0,356 0,312 0,282 0,364 0,360 0,370

0,425 −0,251 0,386 0,435 −0,095 −0,285 −0,230

0,313

0,347

0,055 0,198 0,221 0,308

0,287 −0,015 −0,189 −0,190

Percent variance

40,7

19,5

47,0

10,4

3.4. Indirect effects of the insects on pathogen Lesion size did not differ between T and T + F in P. pinaster, but in P. radiata was significantly shorter when T. piniperda was combined with F. circinatum than when F. circinatum was alone (F1,35 = 7.46, p = 0.01; Fig. 3). In P. pinaster, there was no signal of differences between T and T + F treatments in mono-, di- or sesquiterpene concentration (Fig. 1, left), but in P. radiata there was a tendency for reduced concentrations of terpenes in T + F treatments compared to F alone: nonsignificant reductions of 13%, 7% and 50%, respectively for mono-, di- or sesquiterpenes (Fig. 1, right), and a significant reduction in PC-1 (Fig. 2). The analysis of individual compounds in P. pinaster showed that linalool was significantly higher in P. pinaster when T. piniperda was alone vs. combined with F. circinatum (F1,38 = 4.88, P = 0.03) but no other compounds differed between treatments. For P. radiata, limonene was significantly lower when T. piniperda was also present (T + F) compared to when F. circinatum was alone (F) (F1,38 = 10.57, P = 0.002). Other terpenes also tended to be lower in T + F vs. F but not significantly so. Resin flow was significantly higher in P. pinaster after T. piniperda attack (T) compared with both the insect and fungus together (T vs. T + F; F1,38 = 20.9, p < 0.0001). In contrast, the resin flow in P. radiata was significantly lower when exposed to both the insect and fungus (T + F) versus F. circinatum was alone (F) (F1,35 = 7.05, p = 0.01).

radiata (F1,183 = 23.58, p < 0.0001; mean ± SE = 49 ± 5 vs. 11 ± 5 mg for P. pinaster and P. radiata respectively; main effect of treatment: F4,183 = 20.21, p < 0.0001; species × treatment interaction: F4,183 = 13.92, p < 0.0001). Resin flow was negatively correlated with lesion length in P. pinaster while being positively (but nonsignificantly) correlated in P. radiata (Table 4). Resin flow was positive correlated with the concentration of monoterpenes and diterpenes in both pine species (Table 4). There were no significant correlations for either species between resin flow and N content, water content, or the dry mass of phloem for any of the two pine species. There were no significant differences in phloem dry mass between species or treatments. Water content was significantly higher in P. radiata (F1,185 = 9.98, p = 0.002; mean ± SE = 41.7 ± 0.6 vs. 257

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Fig. 2. Amount and composition of terpenes in the phloem of P. pinaster and P. radiata (as reflected by 1st axis of Principal Components Analysis) under five treatments: control, mock treatment with water, attack by the bark beetle Tomicus piniperda, inoculation with the fungus Fusarium circinatum, beetle attack + fungal inoculation. Asterisks indicate significant treatment differences relative to control. Means ± SE. Details on PCA in Table 3.

4. Discussion The selection of plant species when establishing new planted forests influences subsequent damage caused by pests and pathogens due to the different susceptibility of the trees species. Fusarium circinatum is one of the most aggressive fungus attacking pine species worldwide and P. radiata is especially susceptible to the pitch canker disease (Wingfield et al., 2008). However, interactions among non-native species within newly formed communities may change the outcome of interactions, making it difficult to generalize. Fusarium circinatum usually enters adult trees when suitable fresh wounds are available for infection (Dwinell et al., 1985; Gordon et al., 2001). The presence of T. piniperda in stands affected by F. circinatum might increase infections because the insect uses the shoots of healthy trees to overwinter or for maturation feeding, providing the fresh wounds for the fungus to enter. Furthermore, T. piniperda is considered a plausible vector of the disease (Bezos et al., 2015). Therefore T. piniperda not only act as wounding agents when they bore overwintering

Fig. 3. Length of lesions formed in the phloem of P. pinaster and P. radiata under mock treatment with water, inoculation with the fungus Fusarium circinatum, and beetle attack + fungal inoculation. There were no lesions in true controls or Tomicus treatments. Means ± SE.

Table 4 Correlation matrix among plant traits for P. pinaster and P. radiata (100 trees per species). Significant correlations (p < 0.05) are in bold. P. pinaster

A

B

C

D

A. Plant diameter (cm) B. Plant height (cm) C. Crown size (total branches) D. Monoterpenes (mg/g) E. Sesquiterpenes (mg/g) F. Diterpenes (mg/g) G. Lesion Length (mm) H. Resin flow (mg)

1 0,37 0,09 −0,07 −0,15 −0,11 0,23 0,06

1 −0,32 −0,01 −0,04 0,09 0,07 −0,05

1 −0,09 −0,16 0,00 −0,16 0,18

1 0,54 0,51 0,01 0,33

P. radiata A. Plant diameter (cm) B. Plant height (cm) C. Crown size (total branches) D. Monoterpenes (mg/g) E. Sesquiterpenes (mg/g) F. Diterpenes (mg/g) G. Lesion Length (mm) H. Resin flow (mg)

A 1 0,52 0,21 0,09 −0,05 0,19 0,01 0,09

B

C

1 0,07 0,15 0,10 0,13 0,10 −0,01

1 0,19 −0,03 0,05 0,25 −0,18

258

E

F

G

1 0,37 0,03 0,04

1 −0,08 0,26

1 −0,26

D

E

F

G

1 0,03 0,74 0,50 0,25

1 0,06 0,17 −0,16

1 0,49 0,37

1 0,16

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were associated with the suppression of SA induction. Ding et al. (2011) reported that infection of wheat by Fusarium graminearum induced both pathways, with SA appearing to function in limiting fungal establishment and JA in limiting tissue damage after establishment. We identify three hypotheses for the detrimental effect of T. piniperda on F. circinatum. Hypothesis 1: The insect induced the JA pathway and the resulting expression of secondary metabolites limited fungal establishment and growth. Consistent with this, T. piniperda induced upregulation of β-pinene, linalool, and diterpenes (Table 1), and βpinene at least is known to inhibit germination of F. circinatum spores (Slinski et al., 2015). JA products in general have been reported to deter growth of Fusarium after establishment (Ding et al., 2011). Hypothesis 1 is consistent with the systemic induced resistance described by Christiansen et al. (1999) and demonstrated in previous studies of P. radiata and F. circinatum (Bonello et al., 2001; Gordon et al., 2011). The hypothesis is weakened by our result that inducible increases in terpenes were less when F. circinatum inoculation was combined with T. piniperda attacks, but this could be related to the timing and order of activation of the SA and JA defense pathways (Makandar et al., 2010; Ding et al., 2011). In nature, the wounds caused by insects may occur simultaneously to inoculations, but there must be a delay of hours to days before the pathogen is colonizes plant tissues. Hypothesis 2: The immediate release of pre-formed resin at the point of insect attack interfered with the germination and initial establishment of fungal spores (Erbilgin et al., 2009; Kim et al., 2010). A problem with this hypothesis is that mechanical wounds to trees (the other source of potential infection points for the fungus) also induce resin flow, and yet infection risks from F. circinatum are higher in fresh wounds (with freshly released resin) compared with old wounds (Sakamoto and Gordon, 2006). Hypothesis 3: plant responses to the insect interfered with the ability of F. circinatum to benefit from traumatic resin ducts and their associated epithelial cells that are substrate for the growth of fungi (MartinRodrigues et al., 2015). Hypothesis 3 differs from 1 and 2 with regard to the role of terpenes in the biology of F. circinatum. In hypotheses 1 and 2, terpenes are viewed as defenses against the fungus, which is in accord with conventional wisdom for conifers (Cheniclet, 1987; Lieutier et al., 1993; Phillips and Croteau, 1999; Schmidt et al., 2005; Bonello et al., 2008). In the alternative possibility (H3), the benefits of increased epithelial cells for fungal development are hypothesized to outweigh the detriments of increased terpenes that are products of the newly formed resin ducts (Martin-Rodrigues et al., 2015). The plausibility of hypothesis 3 is enhanced by the observation that F. circinatum, at least once established in the plant tissue, is surprisingly tolerant of terpene-rich environments – as reflected in the common name “pitch canker”. Our results add to evidence of interactive effects on inducible plant defenses of plant-feeding insects and a plant pathogen with which they are associated. A case similar to ours is that defoliation of Pinus nigra Arnold by the pine sawfly Neodiprion sertifer Geoffory results in less tissue damage from the fungus Sphaeropsis sapinea (Eyles et al., 2007). Examples that work in the opposite direction – where prior fungal infection interferes with induced responses against herbivores – include: the infection of Pinus ponderosa by Heterobasidion annosum deters feeding by the bark beetle Ips paraconfusus Lanier (McNee et al., 2003); the infection of Populus nigra L. by leaf rust limits the increase in volatile emissions following folivory (Eberl et al., 2018); and the infection of Quercus ilex by Phytophthora reduces upregulation of tannins following leaf damage (Gallardo et al., 2018). Broader examples of interacting effects between herbivores and plant pathogens include Hatcher (1995), Hatcher and Paul (2000), Rostás et al. (2003), Thaler et al. (2010). It seems increasingly likely that the order of attack is of general importance because the initial attacker can compromise (or prime) a plant’s induced responses to secondary challengers. Such temporally structured dynamics can have general consequences for the structure and function of green food webs (Poelman et al., 2008). Understanding plant-mediated interactions among pests and

Fig. 4. Relations between lesion length and terpene concentrations in P. radiata. Regressions were significant for diterpenes (p < 0.0001) and monoterpenes (p < 0.0001), but not sesquiterpenes (p = 0.09).

or feeding galleries, but also can transfer the fungi directly to healthy trees. Thus, the co-occurence of T. piniperda and F. circinatum could be expected to increase the pitch canker disease incidence. However, our results show that in the case of P. radiata, tree responses to the attacking insect interact with the establishment and growth of the fungi within the plant. Lesion size, the most common indicator of fungal growth within the plant tissue, was significantly smaller on trees treated with T. piniperda and F. circinatum (T + F) compared with trees inoculated only with F. circinatum (F). Plant defenses against herbivores and pathogens are mediated by a network of interacting signal transduction pathways. Salicylic Acid (SA) and Jasmonic Acid (JA) are prominent plant hormones regulating the production of downstream resistance molecules. It is thought that the SA pathway is primarily induced by and effective in mediating resistance against biotrophic pathogens while the JA pathway is primarily induced by and effective in mediating resistance against herbivores and necrotrophic pathogens (Glazebrook, 2005; Stout et al., 2006). If attacks by the insect are followed by pathogen infection it could be thought that vectoring would activate first the JA pathway over the SA pathway. Our results showed that attacks by T. piniperda reduced the growth of F. circinatum in P. radiata (Fig. 3). There have been clear demonstrations of antagonism between the SA and JA pathways (Glazebrook, 2005; Thaler et al., 2012; Hou et al., 2013; Proietti et al., 2013), but it is difficult to know how frequent are the exceptions; Thaler et al. (2012) identified at least seven cases where JA responses 259

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pathogens has practical value by helping to anticipate and understand the properties of new species assemblages in plant production systems. In the case of our study system, it appears that when the newly introduced fungal pathogen is introduced into pine trees by the native bark beetle T. piniperda, it tends to be less damaging than when the fungus enters a wound not caused by insects. This is a fortunate result from the perspective of forest management because there was a possibility that T. piniperda would diminish plant defenses against the new pathogen and therefore amplify the risks of forest damage. As it turned out, physical wounds not caused by T. piniperda seem to be a more dangerous route for establishment of F. circinatum in host trees than introduction by the bark beetle. A practical conclusion is that limiting mechanical wounding to trees during management activities will reduce risks of infection within the stand. Bezos et al. (2012) found a significant relationship between pruning and incidence of the disease in northern Spain. Our results have further relevance to management by adding information about the relative susceptibility of the two most widely propagated pine species in Spain. P. radiata is considered more susceptible to F. circinatum than the native P. pinaster (Iturritxa et al., 2015) and this was supported in our studies by the larger lesions in P. radiata (Fig. 3). However, there have been reports of successful attacks of F. circinatum on P. pinaster in nurseries and forests (Landeras et al., 2005; Iturritxa et al., 2015). In our study, 100% of lesions in P. pinaster were shorter than 18 mm in length, which has been suggested as an approximate threshold for resistance (Gordon et al., 1998). Better understanding of pine defenses against F. circinatum, and genetic variation in the defense responses, would contribute to the development of safe, effective, and sustainable tactics for managing damage from F. circinatum to pine forests in Spain and elsewhere. There is also a need to study how T. piniperda influences the extent and severity of damage of F. circinatum in the field. Although our results indicate that T. piniperda is not as dangerous for fungal establishment as might have been expected, the beetles can still act as vectors and wounding agents. It would be relatively easy and informative to compare the incidence and spread of fungal infections in stands of P. radiata and P. pinaster with low and high abundance of T. piniperda. This knowledge can be constructively combined with existing tactics for predicting, monitoring, and managing the abundance of T. piniperda (Veteli et al., 2006; Bezos et al., 2017).

21, 481–488. https://doi.org/10.5424/fs/2012213-02262. Bezos, D., Martinez-Alvarez, P., Diez, J.J., Fernandez, M.M., 2015. The pine shoot beetle Tomicus piniperda as a plausible vector of Fusarium circinatum in northern Spain. Ann. For. Sci. 72, 1079–1088. https://doi.org/10.1007/s13595-015-0515-4. Bezos, D., Martinez-Alvarez, P., Fernandez, M., Diez, J.J., 2017. Epidemiology and management of pine pitch canker disease in Europe – a review. Balt. For. 23, 279–293. Blanch, J., Peñuelas, J., Sardans, J., Llusia, J., 2009. Drought, warming and soil fertilization effects on leaf volatile terpene concentrations in Pinus halepensis and Quercus ilex. Acta Physiol. Plantarum 31, 207–218. https://doi.org/10.1007/s11738-0080221-z. Bonello, P., Capretti, P., Luchi, N., Martini, V., Michelozzi, M., 2008. Systemic effects of Heterobasidion annosum s.s. infection on severity of Diplodia pinea tip blight and terpenoid metabolism in Italian stone pine (Pinus pinea). Tree Physiol. 28, 1653–1660. https://doi.org/10.1093/treephys/28.11.1653. Bonello, P., Gordon, T.R., Storer, A.J., 2001. Systemic induced resistance in Monterey pine. For. Pathol. 31, 99–106. https://doi.org/10.1046/j.1439-0329.2001.00230.x. Cheniclet, C., 1987. Effects of wounding and fungus inoculation on terpene producing systems of Maritime pine. J. Exp. Bot. 38, 1557–1572. https://doi.org/10.1093/jxb/ 38.9.1557. Christiansen, E., Krokene, P., Berryman, A.A., Franceschi, V.R., Krekling, T., Lieutier, F., Lonneborg, A., Solheim, H., 1999. Mechanical injury and fungal infection induce acquired resistance in Norway spruce. Tree Physiol. 19, 399–403. https://doi.org/10. 1093/treephys/19.6.399. Czokajlo, D., Wink, R.A., Warren, J.C., Teale, S.A., 1997. Growth reduction of Scots pine, Pinus sylvestris, caused by the larger pine shoot beetle, Tomicus piniperda (Coleoptera, Scolytidae), in New York State. Can. J. Forest Res. 27, 1394–1397. https://doi.org/ 10.1139/x97-111. Davis, J.M., Wu, H.G., Cooke, J.E.K., Reed, J.M., Luce, K.S., Michler, C.H., 2002. Pathogen challenge, Salicylic acid, and Jasmonic acid regulate expression of chitinase gene homologs in pine. Mol. Plant-Microbe Interact. 15, 380–387. https://doi.org/ 10.1094/MPMI.2002.15.4.380. De Vos, M., Van Oosten, V.R., Van Poecke, R.M.P., Van Pelt, J.A., Pozo, M.J., Mueller, M.J., Buchala, A.J., Metraux, J.P., Van Loon, L.C., Dicke, M., Pieterse, C.M.J., 2005. Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol. Plant Microbe. In. 18 (9), 923–937. https://doi.org/10.1094/MPMI-180923. Ding, L., Xu, H., Yi, H., Yang, L., Kong, Z., Zhang, L., Xue, S., Jia, H., Ma, Z., 2011. Resistance to hemi-biotrophic Fusarium graminearum infection is associated with coordinated and ordered expression of diverse defense signaling pathways. PLoS One 6, e19008. https://doi.org/10.1371/journal.pone.0019008. Durrant, W.E., Dong, X., 2004. Systemic acquired resistance. Annu. Rev. Phytopathol. 42, 185–209. https://doi.org/10.1146/annurev.phyto.42.040803.140421. Dwinell, L.D., Barrows-Broaddus, J.B., Kuhlman, E.G., 1985. Pitch canker – a disease complex of Southern pines. Plant Dis. 69, 270–276. https://doi.org/10.1094/PD-69270. Eberl, F., Hammerbacher, A., Gershenzon, J., Unsicker, S.B., 2018. Leaf rust infection reduces herbivore-induced volatile emission in black poplar and attracts a generalist herbivore. New Phytol. 760–772. https://doi.org/10.1111/nph.14565. Ekramoddoullah, A.K.M., Yu, X.S., Sturrock, R., Zamani, A., Taylor, D., 2000. Detection and seasonal expression pattern of a pathogenesis-related protein (PR-10) in Douglasfir (Pseudotsuga menziesii) tissues. Physiol. Plantarum 110, 240–247. https://doi.org/ 10.1034/j.1399-3054.2000.110214.x. EFSA Panel on Plant Health (PLH), 2010. Risk assessment of Gibberella circinata for the EU territory and identification and evaluation of risk management options. EFSA J. 8, 1620. Erbilgin, N., Ritokova, G., Gordon, T.R., Wood, D.L., Storer, A.J., 2008. Temporal variation in contamination of pine engraver beetles with Fusarium circinatum in native Monterey pine forests in California. Plant Pathol. 57, 1103–1108. https://doi.org/10. 1111/j.1365-3059.2008.01887.x. Erbilgin, N., Gordon, T.R., Wood, D.L., Storer, A.J., 2009. Bark beetle-mediated fungal infections of susceptible trees induce resistance to subsequent infections in a dose dependent manner. Agricult. For. Entomol. 11, 255–263. https://doi.org/10.1111/j. 1461-9563.2009.00435.x. Erbilgin, N., Krokene, P., Christiansen, E., Zeneli, G., Gershenzon, J., 2006. Exogenous application of methyl jasmonate elicits defenses in Norway spruce (Picea abies) and reduces host colonization by the bark beetle Ips typographus. Oecologia 148, 426–436. https://doi.org/10.1007/s00442-006-0394-3. Eyles, A., Bonello, P., Ganley, R., Mohammed, C., 2010. Induced resistance to pests and pathogens in trees. New Phytol. 185, 893–908. https://doi.org/10.1111/j.14698137.2009.03127.x. Eyles, A., Chorbadjian, R., Wallis, C., Hansen, R., Cipollini, D., Herms, D., Bonello, P., 2007. Cross-induction of systemic induced resistance between an insect and a fungal pathogen in Austrian pine over a fertility gradient. Oecologia 153, 365–374. https:// doi.org/10.1007/s00442-007-0741-z. Franceschi, V.R., Krokene, P., Christiansen, E., Krekling, T., 2005. Anatomical and chemical defenses of conifer bark against bark beetles and other pests. New Phytol. 167, 353–375. https://doi.org/10.1111/j.1469-8137.2005.01436.x. Franceschi, V.R., Krokene, P., Krekling, T., Christiansen, E., 2000. Phloem parenchyma cells are involved in local and distant defense responses to fungal inoculation or barkbeetle attack in Norway spruce (Pinaceae). Am. J. Bot. 87, 314–326. https://doi.org/ 10.2307/2656627. Fu, Z.Q., Dong, X., 2013. Systemic acquired resistance: turning local infection into global defense. Annu. Rev. Plant Biol. 6464, 839–863. https://doi.org/10.1146/annurevarplant-042811-105606. Gallardo, A., Morcuende, D., Solla, A., Moreno, G., Pulido, F., Quesada, A., 2018.

Acknowledgements Research was funded by Xunta de Galicia, Grant: 10MRU291017PR. Additional support was provided by US Forest Service International Programs. Thanks to Javier Pereira-Espinel for assistance with greenhouse and laboratory work. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foreco.2019.01.041. References Bacon, C.W., Yates, I.E., 2006. Endophitic root colonization by Fusarium species: histology, plant interactions, and toxicity. In: Schulz, B., Boyle, C.J.V., Sieber, T.N. (Eds.), Micriobial Root Endophytes. Springer, Berlin, pp. 133–152. Bakke, A., 1968. Ecological studies on bark beetles (Coleoptera: Scolytidae) associated with Scots pine (Pinus sylvestris L.) in Norway with particular reference to influence of temperature. Meddelelser fra det Norske Skogforsoksvesen 21, 447–602. Bent, A.F., Mackey, D., 2007. Elicitors, effectors, and R genes: the new paradigm and a lifetime supply of questions. Annu. Rev. Phytopathol. 45, 399–436. https://doi.org/ 10.1146/annurev.phyto.45.062806.094427. Berbegal, M., Perez-Sierra, A., Armengol, J., Gruenwald, N.J., 2013. Evidence for multiple introductions and clonality in Spanish populations of Fusarium circinatum. Phytopathology 103, 851–861. https://doi.org/10.1094/PHYTO-11-12-0281-R. Bezos, D., Lomba, J.M., Martinez-Alvarez, P., Fernandez, M., Diez, J.J., 2012. Effects of pruning in Monterrey pine plantations affected by Fusarium circinatum. Forest Syst.

260

Forest Ecology and Management 437 (2019) 253–262

M.J. Lombardero et al.

Lombardero, M.J., Alonso-Rodríguez, M., Roca-Posada, E.P., 2012. Tree insects and pathogens display opposite tendencies to attack native vs. non-native pines. For. Ecol. Manage. 281, 121–129. https://doi.org/10.1016/j.foreco.2012.06.036. Lombardero, M.J., Ayres, M.P., Bonello, P., Cipollini, D., Herms, D.A., 2016. Effects of defoliation and site quality on growth and defenses of Pinus pinaster and P. radiata. For. Ecol. Manage. 382, 39–50. https://doi.org/10.1016/j.foreco.2016.10.003. Lombardero, M.J., Ayres, M.P., Lorio, P.L., Ruel, J.J., 2000. Environmental effects on constitutive and inducible resin defences of Pinus taeda. Ecol. Lett. 3, 329–339. https://doi.org/10.1046/j.1461-0248.2000.00163.x. Lombardero, M.J., Vázquez-Mejuto, P.y.M., Ayres, P., 2008. Role of plant enemies in the forestry of indigenous vs. nonindigenous pines. Ecol. Appl. 18 (5), 1171–1181. https://doi.org/10.1890/07-1048.1. Makandar, R., Nalam, V., Chaturvedi, R., Jeannotte, R., Sparks, A.A., Shah, J., 2010. Involvement of salicylate and jasmonate signaling pathways in Arabidopsis interaction with Fusarium graminearum. Mol. Plant. Microbe Interact. 23, 861–870. https:// doi.org/10.1094/MPMI-23-7-0861. Martin-Rodrigues, N., Sanchez-Zabala, J., Salcedo, I., Majada, J., Gonzalez-Murua, C., Dunabeitia, M.K., 2015. New insights into Radiata pine seedling root infection by Fusarium circinatum. Plant Pathol. 64, 1336–1348. https://doi.org/10.1111/ppa. 12376. Masutti, L., 1969. Pine stands of the Adriatic coast and Blastophagus piniperda: a bad combination. Monti e Boschi 20, 15–27. McNee, W.R., Bonello, P., Wood, D.L., Storer, A.J., Gordon, T.R., 2003. Feeding response of Ips paraconfusus to phloem and phloem metabolites of Heterobasidion annosuminoculated ponderosa pine, Pinus ponderosa. J. Chem. Ecol. 29, 1183–1202. https:// doi.org/10.1023/A:1023885724220. Milanović, S., Lazarević, J., Karadžić, D., Milenković, I., Jankovský, L., Vuleta, A., Solla, A., 2015. Belowground infections of the invasive Phytophthora plurivora pathogen enhance the suitability of red oak leaves to the generalist herbivore Lymantria dispar. Ecol. Entomol. 40, 479–482. https://doi.org/10.1111/een.12193. Nagegowda, D.A., 2010. Plant volatile terpenoid metabolism: Biosynthetic genes, transcriptional regulation and subcellular compartmentation. FEBS Lett. 584, 2965–2973. https://doi.org/10.1016/j.febslet.2010.05.045. Öhrn, P., Björklund, N., Långström, B., 2018. Occurrence, performance and shoot damage of Tomicus piniperda in pine stands in southern Sweden after storm-felling. J. Appl. Entomol. 142, 854–862. https://doi.org/10.1111/jen.12533. Pearce, R.B., 1996. Antimicrobial defenses in the wood of living trees. New Phytol. 132, 203–233. https://doi.org/10.1111/j.1469-8137.1996.tb01842.x. Phillips, M.A., Croteau, R.B., 1999. Resin-based defenses in conifers. Trends Plant Sci. 4, 184–190. https://doi.org/10.1016/S1360-1385(99)01401-6. Poelman, E.H., van Loon, Joop, J.A., Dicke, M., 2008. Consequences of variation in plant defense for biodiversity at higher trophic levels. Trends Plant Sci. 13, 534–541. https://doi.org/10.1016/j.tplants.2008.08.003. Proietti, S., Bertini, L., Timperio, A.M., Zolla, L., Caporale, C., Caruso, C., 2013. Crosstalk between salicylic acid and jasmonate in Arabidopsis investigated by an integrated proteomic and transcriptomic approach. Mol. BioSyst. 9, 1169–1187. https://doi. org/10.1039/c3mb25569g. Reymond, P., Farmer, E.E., 1998. Jasmonate and salicylate as global signals for defense gene expression. Curr. Opin. Plant. Biol. 1 (5), 404–411. https://doi.org/10.1016/ S1369-5266(98)80264-1. Robert-Seilaniantz, A., Grant, M., Jones, J.D.G., 2011. Hormone crosstalk in plant disease and defense: more than just jasmonate-salicylate antagonism. Annu. Rev. Phytopathol. 4949, 317–343. https://doi.org/10.1146/annurev-phyto-073009114447. Romon, P., Iturrondobeitia, J.C., Gibson, K., Lindgren, B.S., Goldarazena, A., 2007. Quantitative association of bark beetles with pitch canker fungus and effects of verbenone on their semiochemical communication in Monterey pine forests in Northern Spain. Environ. Entomol. 36, 743–750. https://doi.org/10.1093/ee/36.4. 743. Rostás, M., Simon, M., Hilker, M., 2003. Ecological cross-effects of induced plant responses towards herbivores and phytopathogenic fungi. Basic Appl. Ecol. 4, 43–62. https://doi.org/10.1078/1439-1791-00132. Ruel, J.J., Ayres, M.P., Lorio, P.L., 1998. Loblolly pine responds to mechanical wounding with increased resin flow. Can. J. Forest Res. 28, 596–602. https://doi.org/10.1139/ x98-030. Sakamoto, J.M., Gordon, T.R., 2006. Factors influencing infection of mechanical wounds by Fusarium circinatum on Monterey pines (Pinus radiata). Plant Path. 55, 130–136. https://doi.org/10.1111/j.1365-3059.2005.01310.x. Sampedro, L., Moreira, X., Zas, R., 2011. Costs of constitutive and herbivore-induced chemical defenses in pine trees emerge only under low nutrient availability. J. Ecol. 99, 818–827. https://doi.org/10.1111/j.1365-2745.2011.01814.x. Schmidt, D.D., Voelckel, C., Hartl, M., Schmidt, S., Baldwin, I.T., 2005. Specificity in ecological interactions. Attack from the same lepidopteran herbivore results in species-specific transcriptional responses in two solanaceous host plants. Plant Physiol. 138, 1763–1773. https://doi.org/10.1104/pp.105.061192. Slinski, S.L., Zakharov, F., Gordon, T.R., 2015. the effect of resin and monoterpenes on spore germination and growth in Fusarium circinatum. Phytopathology 105, 119–125. https://doi.org/10.1094/PHYTO-02-14-0027-R. Smith Jr., M.F., Gautam, J.K., Black, S.G., Ernst, P.B., 2006. Microbial-induced regulation of syndecan expression: important host defense mechanism or an opportunity for pathogens? Sci. World J. 6, 442–445. https://doi.org/10.1100/tsw.2006.84. Stout, M.J., Thaler, J.S., Thomma, B.P.H.J., 2006. Plant-mediated interactions between pathogenic microorganisms and herbivorous arthropods. Annu. Rev. Entomol. 51, 663–689. https://doi.org/10.1146/annurev.ento.51.110104.151117. Swett, C.L., Gordon, T.R., 2012. First report of grass species (Poaceae) as naturally occurring hosts of the pine pathogen Gibberella circinata. Plant Dis. 96, 908. https://doi.

Regulation by biotic stress of tannins biosynthesis in Quercus ilex: crosstalk between defoliation and Phytophthora cinnamomi infection. Physiol Plant. https://doi.org/10. 1111/ppl.12848. Gallego, D., Cánovas, F., Esteve, M.A., Galián, J., 2004. Descriptive biogeography of Tomicus (Coleoptera: Scolytidae) species in Spain. J. Biogeog. 31, 2011–2024. https://doi.org/10.1111/j.1365-2699.2004.01131.x. Gaylord, M.L., Kolb, T.E., Pockman, W.T., Plaut, J.A., Yepez, E.A., Macalady, A.K., Pangle, R.E., McDowell, N.G., 2013. Drought predisposes pinon–juniper woodlands to insect attacks and mortality. New Phytol. 198, 567–578. https://doi.org/10.1111/ nph.12174. Glazebrook, J., 2005. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205–227. https://doi.org/10.1146/ annurev.phyto.43.040204.135923. Gordon, T.R., 2006. Pitch canker disease of pines. Phytopathology 96, 657–659. https:// doi.org/10.1094/PHYTO-96-0657. Gordon, T.R., Kirkpatrick, S.C., Aegerter, B.J., Fisher, A.J., Storer, A.J., Wood, D.L., 2011. Evidence for the occurrence of induced resistance to pitch canker, caused by Gibberella circinata (anamorph Fusarium circinatum), in populations of Pinus radiata. For. Pathol. 41, 227–232. https://doi.org/10.1111/j.1439-0329.2010.00678.x. Gordon, T.R., Storer, A.J., Wood, D.L., 2001. The pitch canker epidemic in California. Plant Dis. 85, 1128–1139. https://doi.org/10.1094/PDIS.2001.85.11.1128. Gordon, T.R., Wikler, K.R., Clark, S.L., Okamoto, D., Storer, A.J., Bonello, P., 1998. Resistance to pitch canker disease, caused by Fusarium subglutinans f.sp. pini, in Monterey pine (Pinus radiata). Plant Pathol. 47, 706–711. Haack, R.A., Kucera, D., 1993. New introduction: common pine shoot beetle, Tomicus piniperda (L.). USDA Forest Service, Northeastern Area, Pest Alert NA-TP-05-93. USDA Forest service, Washington, D.C., USA. Hatcher, P.E., 1995. Three-way interactions between plant pathogenic fungi, herbivorous insects and their host plants. Biol. Rev. 70, 639–694. https://doi.org/10.1111/j. 1469-185X.1995.tb01655.x. Hatcher, P.E., Paul, N.D., 2000. Beetle grazing reduces natural infection of Rumex obtusifolius by fungal pathogens. New Phytol. 146, 325–333. https://doi.org/10.1046/j. 1469-8137.2000.00646.x. Hernandez-Escribano, L., Iturritxa, E., Elvira-Recuenco, M., Berbegal, M., Campos, J.A., Renobales, G., García, I., Raposo, R., 2018. Herbaceous plants in the understory of a pitch canker-affected Pinus radiata plantation are endophytically infected with Fusarium circinatum. Fungal Ecol. 32, 65–71. https://doi.org/10.1016/j.funeco.2017. 12.001. Hou, X., Ding, L., Yu, H., 2013. Crosstalk between GA and JA signaling mediates plant growth and defense. Plant Cell Rep. 32, 1067–1074. https://doi.org/10.1007/ s00299-013-1423-4. Hudgins, J.W., Franceschi, V.R., 2004. Methyl jasmonate-induced ethylene production is responsible for conifer phloem defense responses and reprogramming of stem cambial zone for traumatic resin duct formation. Plant Physiol. 135, 2134–2149. https:// doi.org/10.1104/pp.103.037929. Iturritxa, E., Mesanza, N., Brenning, A., 2015. Spatial analysis of the risk of major forest diseases in Monterey pine plantations. Plant Pathol. 64, 880–889. https://doi.org/10. 1111/ppa.12328. Jones, J.D.G., Dangl, J.L., 2006. The plant immune system. Nature 444, 323–329. https:// doi.org/10.1038/nature05286. Kessler, A., Baldwin, I.T., 2001. Defensive function of herbivore-induced plant volatile emissions in nature. Science 291, 2141–2144. https://doi.org/10.1126/science.291. 5511.2141. Kim, K.W., Lee, I.J., Kim, C.S., Eom, I., Choi, J., Lee, D.K., Park, E.W., 2010. Resin flow, symptom development, and lignin biosynthesis of two pine species in response to wounding and inoculation with Fusarium circinatum. Plant Pathol. J. 26, 394–401. https://doi.org/10.5423/PPJ.2010.26.4.394. Klepzig, K.D., Kruger, E.L., Smalley, E.B., Raffa, K.F., 1995. Effects of biotic and abiotic stress on induced accumulation of terpenes and phenolics in red pines inoculated with bark beetle-vectored fungus. J. Chem. Ecol. 21, 601–626. https://doi.org/10. 1007/BF02033704. Kopper, B.J., Klepzig, K.D., Raffa, K.F., 2003. Effectiveness of modified White's solution at removing ascomycetes associated with the bark beetle Ips pini. For. Pathol. 33, 237–240. https://doi.org/10.1046/j.1439-0329.2003.00327.x. Landeras, E., Garcia, P., Fernandez, Y., Brana, M., Fernandez-Alonso, O., Mendez-Lodos, S., Perez-Sierra, A., Leon, M., Abad-Campos, P., Berbegal, M., Beltran, R., GarciaJimenez, J., Armengol, J., 2005. Outbreak of pitch canker caused by Fusarium circinatum on Pinus spp. in Northern Spain. Plant Dis 89, 1015. https://doi.org/10.1094/ PD-89-1015A. Langenheim, J.H., 1994. Higher-plant terpenoids – a phytocentric overview of their ecological roles. J. Chem. Ecol. 20, 1223–1280. https://doi.org/10.1007/ BF02059809. Larsson, S., 2002. Resistance of trees to insects—an overview of mechanisms and interactions. In: Wagner, M.R., Clancy, K.M., Lieutier, F., Paine, T.D. (Eds.), Mechanisms and Deployment of Resistance in Trees to Insects. Kluwer, Dordrecht, pp. 1–29. Leslie, J.F., Summerell, B.A., 2006. The Fusarium Laboratory Manual. Blackwell Publishing, Iowa, USA, pp. 388. Lewis, D.H., 1973. Concepts in fungal nutrition and the origin of biotrophy. Biol. Rev. 48, 261–278. https://doi.org/10.1111/j.1469-185X.1973.tb00982.x. Lewinsohn, E., Gijzen, M., Croteau, R., 1991. Defense mechanisms of conifers. Differences in constitutive and wound-induced monoterepene biosynthesis among species. Plant Physiol. 96, 44–49. https://doi.org/10.1104/pp.96.1.44. Lieutier, F., Garcia, J., Romary, P., Yart, A., Jactel, H., Sauvard, D., 1993. Inter-tree variability in the induced defense reaction of Scots pine to single inoculations by Ophiostoma brunneo-ciliatum, a bark-beetle-associated fungus. For. Ecol. Manage. 59, 257–270. https://doi.org/10.1016/0378-1127(93)90006-9.

261

Forest Ecology and Management 437 (2019) 253–262

M.J. Lombardero et al.

Wainhouse, D., Ashburner, R., Ward, E., Rose, J., 1998. The effect of variation in light and nitrogen on growth and defence in young Sitka Spruce. Funct. Ecol. 12, 561–572. https://doi.org/10.1046/j.1365-2435.1998.00232.x. Wingfield, M.J., Hammerbacher, A., Ganley, R.J., Steenkamp, E.T., Gordon, T.R., Wingfield, B.D., Coutinho, T.A., 2008. Pitch canker caused by Fusarium circinatum – a growing threat to pine plantations and forests worldwide. Australas. Plant Pathol. 37, 319–334. https://doi.org/10.1071/AP08036. Zeneli, G., Krokene, P., Christiansen, E., Krekling, T., Gershenzon, J., 2006. Methyl jasmonate treatment of mature Norway spruce (Picea abies) trees increases the accumulation of terpenoid resin components and protects against infection by Ceratocystis polonica, a bark beetle-associated fungus. Tree Physiol. 26, 977–988. https://doi.org/ 10.1093/treephys/26.8.977.

org/10.1094/PDIS-02-12-0136-PDN. Thaler, J.S., Agrawal, A.A., Halitschke, R., 2010. Salicylate-mediated interactions between pathogens and herbivores. Ecology 91, 1075–1082. https://doi.org/10.1890/ 08-2347.1. Thaler, J.S., Humphrey, P.T., Whiteman, N.K., 2012. Evolution of jasmonate and salicylate signal crosstalk. Trends Plant Sci. 17, 260–270. https://doi.org/10.1016/j. tplants.2012.02.010. Veteli, T.O., Koricheva, J., Niemela, P., Kellomaki, S., 2006. Effects of forest management on the abundance of insect pests on Scots pine. For. Ecol. Manage. 231, 214–217. https://doi.org/10.1016/j.foreco.2006.05.048. Vivas, M., Zas, R., Solla, A., 2012. Screening of Maritime pine (Pinus pinaster) for resistance to Fusarium circinatum, the causal agent of Pitch Canker disease. Forestry 85, 185–192.

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