A rationale for chemical defense mixtures in spruce oleoresin: most monoterpenes are highly toxic to either bark beetles or to their symbiotic fungi, but not both

doi:10.21203/rs.3.rs-4919445/v1

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A rationale for chemical defense mixtures in spruce oleoresin: most monoterpenes are highly toxic to either bark beetles or to their symbiotic fungi, but not both

https://doi.org/10.21203/rs.3.rs-4919445/v1

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1. Chemical defenses, such as the monoterpenes of conifer oleoresin frequently occur as complex blends of many components, but the underlying ecological reasons for these mixtures are not yet known. Several theories attempt to explain the existence of chemical defense mixtures in plants. However, due to limited empirical evidence, it is unclear which theories might best apply.

2. Here, we tested the bioactivity of 12 individual Norway spruce monoterpenes and their mixtures to adult Eurasian spruce bark beetles, Ips typographus, and three major symbiotic fungi in the vapor phase using survival and growth bioassays. Next, we evaluated whether spruce trees could alter their monoterpene profile in response to fungal infection.

3. Individual monoterpenes had generally opposite effects on bark beetles compared to symbiotic fungi. The compounds that were most toxic to beetles were the least inhibitory to fungal growth and vice versa. The least abundant monoterpenes had the strongest bioactivity against both beetles and fungi, while the most abundant monoterpenes showed intermediate bioactivity. Among the symbiotic fungi tested, one (Grosmannia penicillata) exhibited high tolerance to monoterpenes, and its growth was even stimulated by the monoterpenes most toxic to the beetle. Interestingly, spruce bark responded to G. penicillata inoculation by accumulating higher concentrations of specifically fungistatic monoterpenes.

4. Our results support the interaction diversity hypothesis, which posits that defense mixtures are maintained in plants because the individual components target different attackers. The monoterpene identity and concentration together determine the effectiveness of these compounds against herbivores and pathogens.

Behavioral Ecology

Forestry

Chemical Biology

Chemical mixtures

Norway spruce

spruce bark beetle

ophiostomatoid fungi

monoterpene toxicity

inverse toxicity

interaction diversity.

Plant chemical defenses are characterized by the vast number of compounds produced, their extraordinary structural diversity and their variable distribution among taxa, organs and developmental stages. Moreover, individual defense compounds frequently occur in complex mixtures. The pyrrolizidine alkaloids of comfrey flowers (Stegemann et al., 2019), the cardenolides of milkweed leaves (Züst et al., 2019) and the monoterpenes of conifer oleoresin (Cates 1996) are all present as multi-component blends. Yet researchers have only rarely investigated the ecological factors that maintain mixtures (Gershenzon et al., 2012; Richards et al., 2016; Whitehead et al., 2021).

Several theories aim to explain the existence of defense mixtures in plants. Among the most commonly cited, the synergy hypothesis predicts that the biological activity of blends can be more potent than those of individual compounds (Richards et al., 2016). On the other hand, the screening hypothesis proposes that most components of mixtures are not biologically active, but large numbers of different structures are produced to ensure that at least some will be active (Jones et al., 1997). The interaction diversity hypothesis posits that mixtures are maintained since plants interact with multiple organisms and different defenses are required to target different attackers (Berenbaum & Zangerl, 1996). Researchers have seldom conducted appropriate bioassays of mixtures and their components to determine which of these hypotheses might best apply.

Conifers are among the oldest lineages of woody plants and often dominate boreal and temperate forest ecosystems. The remarkable success of conifers may be partly due to their complex defenses against natural enemies, particularly terpenoid oleoresins, formed both constitutively and after herbivore or pathogen attack (Franceschi et al., 2005; Trapp & Croteau, 2001). Oleoresins were identified to be present in early gymnosperms ca. 320 Mya, representing the oldest known chemical defense of land plants (Bray & Anderson, 2009; Lange, 2015). Oleoresins are mixtures dominated by terpenes of varying carbon length, comprising volatile monoterpenes (C₁₀), non-volatile diterpenes (C₂₀), and a smaller portion of volatile sesquiterpenes (C₁₅) (Celedon & Bohlmann 2019). Norway spruce contains over 20 different monoterpenes, which vary in composition and concentration among constitutive and inducible oleoresins (Krokene, 2015; Schiebe et al., 2012).

Monoterpenes may have multiple modes of biological activity against conifer enemies. They serve as toxins by killing insects and inhibiting microbial growth either in the vapor phase or upon contact with the fluid resin (Chiu et al., 2017; Groot, 1972; Klepzig et al., 1996; Reid et al., 2017). Oleoresin also physically deters attackers by forming a viscous solution, which is forcefully released upon wounding or damage by herbivores (Cabrita, 2018; Phillips & Croteau, 1999). Upon exposure to air, the monoterpenes evaporate and the diterpenes solidify, leading to the entrapment of insects and sealing of wounds. Thus, oleoresins may act in more than one way. Yet, we have little information on why they contain such a multi-component mixture of monoterpenes.

One of the major current threats to conifers world-wide are the increasing outbreaks of bark beetles, amplified by climate change, which have had large ecological and economic consequences (Huang et al., 2020; Seidl et al., 2014). In Eurasia, the spruce bark beetle Ips typographus attacks living Norway spruce (Picea abies) trees. These beetles feed and breed in the phloem of spruce bark and introduce symbiotic fungi into the phloem, ultimately causing the tree’s death (Netherer et al., 2021). Pioneer adult males of I. typographus select suitable hosts and emit aggregation pheromones to attract conspecifics. Once the tree defense is overcome, females join males, lay eggs in tunnels beneath the bark and inoculate ophiostomatoid fungi (Ascomycota: Ophiostomales and Microascales). These fungi may facilitate the development of larvae and immature adults by providing nutrition, detoxifying plant defense or inhibiting the growth of beetle pathogens (Kandasamy et al., 2019).

Throughout their life cycle, all stages of bark beetles encounter monoterpenes, but the role of these compounds is not always clear as they function both as attractants and defenses depending on concentration and identity (Raffa, 2014; Seybold et al., 2006). Bark beetles may use host monoterpenes as kairomones to locate suitable hosts and to synthesize aggregation pheromones. For instance, I. typographus oxidizes α-pinene from Norway spruce into cis-verbenol, which, together with de novo-produced 2-methyl-3-buten-2-ol, attracts conspecific beetles (Ramakrishnan et al., 2022; Rudinsky et al., 1971). The fungal symbionts of I. typographus also metabolize host monoterpenes into distinct oxygenated derivatives, which may aid in successful beetle colonization (Kandasamy et al., 2023). In other bark beetle-fungus systems, monoterpenes have been previously reported to be toxic to bark beetles and inhibit symbiotic fungi (Cates, 1996; Raffa & Berryman, 1986). However, the individual roles of monoterpenes in the mixture are still uncertain for most conifer bark beetles and especially for their fungal symbionts.

In this study, we examined the bioactivity of the major and some minor Norway spruce monoterpenes against a major native enemy complex of the tree- the bark beetle Ips typographus, and its symbiotic fungi using bioassays to measure survival and growth. A few prior studies in other bark beetle systems have tested the insecticidal and fungistatic activity of the major monoterpenes (Chiu et al., 2017; Reid et al., 2017), but these have focused on the dominant compounds. Here, we determined the insecticidal and fungistatic activity of 12 individual monoterpenes and two mixtures formulated to represent the natural blend of constitutive and inducible monoterpenes in the oleoresin. In addition, we investigated whether Norway spruce alters its monoterpene profile in response to fungal infection. Our findings indicate that the three most abundant oleoresin monoterpenes had intermediate effects on beetles and fungi. However, of the other mixture components, monoterpenes with the greatest toxicity to bark beetles were least inhibitory to fungi, and vice versa. These results best align with the predictions of the interaction diversity hypothesis to explain the existence of defense mixtures.

Bark beetle rearing

For all the bioassays, bark beetles were reared in logs, collected and sex determined as previously described (Kandasamy et al., 2019, 2023). The initial population originated from naturally attacked trees near Jena, Germany.

Effect of vapor phase spruce monoterpenes on Ips typographus survival

To assess the toxicity of monoterpenes to adult beetles, we exposed them to individual spruce monoterpenes and mixtures in the vapor phase (Table S1). The composition of monoterpene mixtures mimicking constitutive and induced resin was chosen according to an earlier report (Schiebe et al., 2012). The bioassay was performed according to the method described by Chiu et al., (2017) with slight modifications. Briefly, 23 mm Whatman circular filter papers (No. 3) were affixed to the inner side of the screw cap of 20 mL scintillation glass vials. A single beetle was placed at the bottom of each vial before pure monoterpenes were applied to the filter paper. The weight of experimental beetles ranged from 10–13 mg. Glass vials were then secured tightly with caps and were kept in darkness at room temperature for 24 hours and checked for mortality. Beetles were considered dead if they did not respond after rubbing gently with soft forceps. The concentrations used for the assay were 1, 10, 50, 100, 200, 500, 750, and 1000 µL of monoterpenes /L of vial headspace. Blank filter papers were used as a control. For each concentration of each compound and the control, 20 replicate beetles (10 females and 10 males) were used.

Given the varying vapor pressures of monoterpenes tested (Fig. 1), some trials at high concentrations were conducted at monoterpene pressures over saturation. In these assays, the test compound remained partially in the liquid state after application on the filter paper and may have also condensed on the walls of the vial. Thus, we may have tested both vapor phase and contact toxicity with beetles and fungi. However, since we used monoterpene doses covering the natural range of concentrations found in Norway spruce bark, this should mirror the situation in bark beetle galleries.

Effect of vapor phase spruce monoterpenes on growth of symbiotic fungi

The growth of three major fungal symbionts of I. typographus- Grosmannia penicillata, Endoconidiophora polonica, and Ophiostoma bicolor (Table S2) was assessed in the presence of individual monoterpenes and their mixtures. Briefly, 35 mL of 4% PDA was poured into a 90 mm Petri dish (total volume 75 mL) and a fungal plug was taken from the outer edge of an actively growing 4-day-old mycelium using a no. 3 cork borer (1 cm diameter) and placed at the center of agar with mycelium facing the agar. Different volumes of monoterpenes were applied to a filter paper (23 mm no. 3 Whatman) that was secured underneath the Petri dish lid using double-sided sticky tape. After the addition of monoterpenes, the Petri dishes were immediately sealed first with a layer of polyvinyl chloride (PVC) electrical tape and then with a layer of Parafilm. The controls used filter papers without monoterpenes. Each compound was tested with each fungus at variable concentrations ranging from 31.25 to 10000 µL of monoterpenes/L of Petri dish headspace in five replicates. After 4 days post-inoculation, Petri dishes were photographed, and images were analyzed using ImageJ software to determine the surface area of mycelial growth in mm². Experiments were conducted multiple times with controls included in each round. Fungal growth inhibition was expressed as IC₅₀, the concentration inhibiting 50% of control growth.

Fungal inoculation of spruce logs

Five logs approximately 50 cm long were cut from a 40-year-old spruce tree and inoculated with different I. typographus symbiotic fungi on the next day. Fungi were first grown on PDA at 25°C for 5 d. Two rings were drawn along the circumference of each log 15 cm away from the cut ends. Four equidistant inoculations were made along the circumference of each ring, and the bark was replaced with agar plugs containing fungal mycelium or agar plugs only using a cork borer. The holes were made alternating between the two rings to prevent the overlapping of the fungal lesions. The fungi G. penicillata, E. polonica, and O. bicolor were used for inoculating logs and a PDA plug alone without fungus was used as control. A sample was collected (pre-control) from each log before fungal infection, flash frozen in liquid nitrogen and stored at -80°C. Five logs were then incubated for 14 d under the conditions used for rearing beetles. After 10 d, bark from each log was peeled around the fungal inoculations, and necrotic lesions caused by fungal infections and wounding on bark were marked on transparent sheets. Immediately necrotic phloem samples from each treatment were collected. Additional samples were collected from the uninfected part (post-control) of the logs after 14 d incubation period of the logs to estimate any change in the bark chemistry due to experimental conditions. All bark samples were ground to powder using a cryo-vibratory micro mill and then stored at -80°C until analysis.

Extraction of monoterpenes from spruce bark and their quantification using GC-FID/MS

Approximately 100 mg of frozen bark powder was placed in a pre-weighed 4 mL glass vial placed on dry ice and 1 ml of methyl tert-butyl ether spiked with 50 µg/ml of nonyl acetate (internal standard) was added to the vial. For extractions, the vials were shaken gently using a rotary shaker for 24 h. Then 0.4 ml of 0.1 M (NH₄)₂CO₃ (pH 8.0) was added to each vial and vortexed for 10 s. The samples were then centrifuged at 3320 g for 10 min. After freezing at -80°C for 2–3 h to precipitate the debris, the supernatant was transferred to clean 2 ml amber glass vials with the help of 200 µl glass capillaries attached to a micropipette. Samples were stored at -20°C until analyzed. The eluted volatile samples were subjected to GC-MS analysis for identification and GC-FID analysis for quantification using the protocol described in (Kandasamy et al., 2023)

Data analysis

All statistical analyses were performed using GraphPad Prism version 8.4 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com). The dose-response model (non-linear regression) was used to test the effect of monoterpenes on the mortality of beetles and the growth response of symbiotic fungi. The log-transformed monoterpene concentrations and the corresponding normalized beetle mortality or fungal growth inhibition data were used to determine the LC₅₀ (Lethal concentration for killing 50% of beetles) and IC₅₀ (Inhibitory concentration to reduce fungal growth by 50%), 95% confidence intervals, Hill’s slope and χ² (goodness of fit) values separately for each monoterpene. To analyze the differences among the LC₅₀ or IC₅₀ of monoterpenes, a one-way ANOVA was performed followed by Tukey’s multiple comparisons on the log LC₅₀ and log IC₅₀ values. For the statistical difference between male and female beetles, an unpaired t-test was performed with Welch’s correction.

Lesion area and the concentrations of individual, total monoterpenes and oxygenated monoterpenes in spruce logs inoculated with different fungi were subjected to ANOVA followed by Tukey’s test for normally distributed data. For data that did not follow a Gaussian distribution, a Kruskal-Wallis test followed by a Bonferroni pairwise comparison was used. Principal component analysis was performed in Metaboanalyst 5.0 software (Chong et al., 2019) using 23 compounds, including monoterpene hydrocarbons and oxygenated monoterpenes quantified from fungus-infected and uninfected spruce bark. The data were auto-scaled (mean-centered and divided by the square root of the standard deviation of each variable) before being subjected to PCA analysis. Spearman correlation was performed to test the relationship between the fungal lesion area and the monoterpenes that were identified within the lesion using auto-scaled data and with a significance level of 0.05.

Norway spruce monoterpenes are not equally toxic to adult I. typographus

To evaluate the toxicity of Norway spruce monoterpenes (Fig. 1) to adult bark beetles, insects were exposed to individual compounds or mixtures in the vapor phase for 24 h, and concentrations resulting in 50% mortality (LC₅₀) were calculated. For the tested compounds, these values ranged from ~ 71 µL of monoterpene/L of headspace for myrcene to 2475 µL of monoterpene/L of headspace for (-)-bornyl acetate, a 35-fold quantitative difference in toxicity (ANOVA, F_13,90 = 636.1, P < 0.001) (Fig. 2A). Myrcene and 1,8-cineole were the most toxic among tested compounds, followed by both enantiomers of limonene and 3-carene. The enantiomers of α-pinene differed in their toxicity to beetles by 2-fold with (+)-α-pinene being more toxic than (-)-α-pinene (ANOVA, Tukey’s test, P < 0.05). However, the enantiomers of limonene did not differ in toxicity. The three most abundant monoterpenes in Norway spruce oleoresin, (+)-a-, (-)-a- and (-)-b-pinene, were of intermediate toxicity to I. typographus, while some of the least abundant monoterpenes (-)-terpinen-4-ol, (-)-bornyl acetate, and terpinolene were the least toxic (Fig. 2A). The monoterpene blend induced by jasmonate treatment was slightly more toxic compared to the constitutive blend, but neither blend was as toxic as the individual compounds, myrcene, 1,8-cineole, (+)-limonene and 3-carene. Yet the LC₅₀ values of the blends were lower than those of (-)-α-pinene, (+)-α-pinene, and (-)-β-pinene, which constitute the major proportion (ca. 85%) of the blends (Table S2). Among sexes, there was strong evidence that female beetles were more susceptible to some monoterpenes, such as 1,8-cineole, (±)-3-carene, p-cymene, (+)-α-pinene, (-)-β-pinene, terpinolene, and (-)-bornyl acetate compared to males (Table S4). All LC₅₀ values were within natural concentration ranges of monoterpenes in Norway spruce bark assuming that all of the monoterpenes in a given sample of tissue are in the volatile phase.

Monoterpenes with the highest toxicity to I. typographus were the least inhibitory to symbiotic fungi and vice versa

Like I. typographus, the symbiotic fungi carried by these beetles are also exposed to monoterpenes when inoculated into the bark during a bark beetle attack. Therefore, we calculated the inhibitory concentration (IC₅₀) of monoterpenes to three fungal associates of this beetle. Overall, there is a 35-fold difference between the least and the most fungistatic compounds against the three fungi (Table S5-S7). The IC₅₀ of monoterpenes on the fungi generally showed a trend inverse to their effect on beetles (Fig. 2B). For example, the most toxic compounds to the spruce bark beetle, (-)-limonene and myrcene, were the least inhibitory to all three fungi, while the least toxic monoterpenes to the beetle, such as (-)-bornyl acetate, terpinolene, and (-)-terpinen-4-ol, were the most fungistatic. The growth of the three fungi varied in response to monoterpenes (Tables S5-S7). Grosmannia penicillata was less susceptible to fungistatic oxygenated monoterpenes and more tolerant to insecticidal monoterpenes compared to O. bicolor and E. polonica. The monoterpene blend induced by jasmonate treatment inhibited E. polonica and O. bicolor more than the constitutive monoterpene blend. In contrast, the constitutive blend was more inhibitory to G. penicillata growth than the induced blend. Among the three fungi, E. polonica was the least tolerant to most monoterpenes and was more susceptible to the constitutive and induced blends than the other fungi (Fig. 2B). Intriguingly, comparing the IC₅₀ values for the enantiomers of α-pinene revealed a 20-fold difference in the growth inhibition of E. polonica; (+)-α-pinene was the least fungistatic compound to E. polonica compared to (-)-α-pinene, which was the most fungistatic among the tested compounds (Fig. 2B, Table S6).

Monoterpenes with toxicity to I. typographus promote the growth of the symbiotic fungus G. penicillata

Since it was observed that the lowest concentrations of monoterpenes may even have stimulated the growth of some of the symbiotic fungi, we quantified the effects of individual monoterpenes on the growth rate of fungi. Surprisingly, the five monoterpenes most toxic to I. typographus promoted the growth of the dominant fungal symbiont, G. penicillata (Fig. 3). Both enantiomers of limonene strongly promoted the growth of G. penicillata at all tested concentrations, compared to its growth on terpene-free agar. Similarly, the other monoterpenes most toxic to I. typographus, 3-carene, myrcene, and 1,8-cineole, promoted the growth of G. penicillata at least at the lower concentrations tested. Turning to the other fungal symbionts, the G. penicillata growth-promoting monoterpenes, (+), and (-)-limonene, myrcene, and 3-carene, inhibited the growth of E. polonica and O. bicolor at all tested concentrations. None of the terpenes tested promoted O. bicolor and E. polonica growth, but rather inhibited both fungi at all tested concentrations (Fig. 3).

Lesions from symbiotic fungi in the bark contain high concentrations of fungistatic monoterpenes

To determine which monoterpenes are produced by spruce trees in response to infection by the symbiotic fungi, we quantified the lesion area and monoterpene profile of bark after fungal infection (Fig. 4) (Table S8). Among the symbiotic fungi, G. penicillata induced a larger lesion area in bark compared to the lesion areas of the other fungi and the lesion caused by the mock wounded control (ANOVA, F_(3,35)=134.6, P < 0.00, Tukey’s test)(Fig. 4A). PCA analysis based on total monoterpenes identified from the lesions showed that the highly virulent G. penicillata induced a larger variation in monoterpene profile compared to the less virulent fungi and the controls. PCA components 1 and 2 successfully explained 87.1% of the variation in monoterpene profile, and the monoterpene profile of G. penicillata clustered separately from monoterpene profiles of other fungi and the mock wounded control, which clustered closely together (Fig. 4B). It was apparent from the PCA loadings plot that monoterpenes previously shown to have high anti-fungal activity (Fig. 2B), such as terpinen-4-ol, terpinolene, and (-)-bornyl acetate contribute much to the variation in the monoterpene profile of fungus-infected bark. On the contrary, monoterpenes highly toxic to I. typographus, such as α- and β-pinene, myrcene, limonene, and 3-carene did not make large contributions to the variation in the total monoterpene profile of fungus-induced lesions (Fig. 4C). Interestingly, fungistatic compounds such as terpinen-4-ol (Kruskal-Wallis, Z = 33.8, P < 0.001, Bonferroni’s test) and (-)-bornyl acetate (ANOVA, F_(4,29) = 14.2, P < 0.001, Tukey’s test) (Fig. 4D,E) increased in G. penicillata-infected tissues compared to controls. However, there was little or no increase in the concentrations of monoterpenes toxic to I. typographus in fungal lesions compared to controls (Table S7). The increase in fungistatic monoterpenes upon fungal infection was also correlated with fungal lesion area (Fig. 4F).

The inverse toxicity of monoterpenes to bark beetles and their symbiotic fungi supports the interaction diversity hypothesis to explain the existence of mixtures of chemical defenses

Despite the presence of complex, multi-component mixtures of chemical defense compounds in many plant species, there is still much discussion about the underlying causes of this phenomenon. Several plausible explanations for mixtures have been put forth over the years, including the interaction diversity hypothesis, the synergy hypothesis and the screening hypothesis. Our investigation of the mixture of monoterpenes found in Norway spruce trunk resin showed best support for the interaction diversity hypothesis, which predicts that the different components of a mixture are needed for their activity against different enemies. The top three most effective monoterpene defenses against the spruce bark beetle based on vapor phase toxicity (myrcene, 1,8-cineole, (+)-limonene), proved to be completely different from the three monoterpenes most effective at inhibiting the growth of the spruce bark beetle symbiotic fungi in the vapor phase ((-)-bornyl acetate, terpinolene, (-)-terpinen-4-ol) (Fig. 1). As bark beetles and their associated fungi invade the tree at the same time, it is essential that defenses are present that are effective against both types of attackers. Norway spruce seems to have solved this problem by deploying a mixture containing some compounds with strong activity against one of the two attackers but not necessarily against the other. Meanwhile, the three major monoterpene constituents of Norway spruce oleoresin (+)-a-, (-)-a-, and (-)-b-pinene, (each making up 20–35% of the total monoterpene fraction) exhibit intermediate toxicity to I. typographus and intermediate inhibitory activity to some but not all of the symbiotic fungi. Thus, the strategy for constructing the mixture appears to involve smaller amounts of compounds that are potent defenses towards one group of enemies or the other, with the bulk of the oleoresin composed of compounds with intermediate activities against both groups.

The same strategy may be used by other conifers as well. The monoterpene chemotypes of the lodgepole pine that most inhibited the growth of the mountain pine beetle were found to be least inhibitory to the major symbiotic fungus of this insect, and vice versa (Ullah et al., 2021). Among other classes of chemical defenses, a well-designed study on the phenolics of apple fruits demonstrated that the individual compounds tested also seemed to be most effective against different insect herbivores and fungal pathogens (Whitehead et al., 2021). Additionally, the individual components of glucosinolate hydrolysis product mixtures found in the Brassicaceae appear to be most effect against different plant enemies (Gershenzon et al., 2012; Lankau, 2007).

Our results do not support the synergy hypothesis, that mixtures are inherently more active than individual compounds against a single enemy. We compared the biological activity of the naturally occurring, constitutive mixture of spruce oleoresin monoterpenes, as well as the mixture induced by jasmonate treatment, to the activity of individual compounds. While the two mixtures did provide some of the more active tests against bark beetles and their symbiotic fungi, at least four individual compounds (of the 12 tested) were more toxic to bark beetles than either of the mixtures, and four different compounds were more inhibitory to fungal growth than either of the mixtures. Other studies on mixtures have found little support for the synergistic effects of monoterpenes against the mountain pine beetle (Reid et al., 2017) and phenolics against diverse insect herbivores and fungi (Whitehead et al. 2021), but there are convincing counter examples (Berenbaum & Zangerl, 1996; Richards et al., 2016).

Although no synergy may exist among mixtures of Norway spruce monoterpenes, we did not check for the synergy of monoterpenes with the other major constituents of the trunk oleoresin, sesquiterpenes and diterpene resin acids, in influencing the toxicity or growth inhibiting properties of mixtures. The different size classes of oleoresin terpenoids might also synergize to control the physical properties of the resin, such as viscosity, stickiness to insects, and evaporation rate, which could also have significance in defense against insect or fungal invaders.

Our results also do not favor the screening hypothesis, whose main assumption is that biological activity is rare among the arsenal of potential chemical defenses produced by plants. We found that all of the monoterpene components of Norway spruce trunk oleoresin tested were active against either the spruce bark beetle or their symbiotic fungi. Another explanation for the presence of mixtures is that these are consequences of a biosynthetic machinery that just happens to make multiple products. The last step in the formation of monoterpene hydrocarbons is catalyzed by enzymes called monoterpene synthases, which often make multiple products from a single substrate (Degenhardt et al., 2009). While some spruce monoterpene synthases are indeed multiple product-forming enzymes, it appears that Norway spruce employs at least eight separate enzymes to make its twelve major products, based on those characterized to date (Keeling et al., 2011; Martin et al., 2004) of which at least five are single-product enzymes. Thus, multi-product enzymes make only a small contribution to the mixture of monoterpenes in Norway spruce oleoresin.

Monoterpenes are toxic to I. typographus bark beetles

The individual monoterpenes of the mixture from Norway spruce trunk resin are variably toxic to I. typographus with myrcene, 1,8-cineole and (+)-limonene being the most poisonous in the vapor phase (Fig. 1). Myrcene was described as a contact toxin to I. typographus in an earlier study (Everaerts et al., 1988), while 1,8-cineole inhibited the mass attack of I. typographus by reducing its sensitivity to aggregation pheromones, possible a consequence of its toxicity (Andersson et al., 2010). For other bark beetle species, limonene (chirality unspecified) has been cited in the older literature as one of the most toxic of all conifer oleoresin monoterpenes (Cates, 1996; Seybold et al., 2006). (-)-Limonene was the most toxic monoterpene in the vapor phase to Dendroctonus ponderosae (Chiu et al., 2017), and this insect was twice as sensitive to (-)-limonene as I. typographus, based on the LC₅₀ value. However, both species exhibit similar sensitivity to myrcene. While the results of this and previous studies have shown clear hierarchies of monoterpene toxicity, it is difficult to know if our bioassays in air-tight containers accurately reflect the conditions in the native gallery system of I. typographus. In galleries, monoterpenes from oleoresins may evaporate quickly due to ventilation. However, pioneer bark beetles might come into prolonged contact with monoterpene vapors from fresh oleoresins in their initial tunneling and nuptial chambers. Higher levels of toxic monoterpenes may aid in tree resistance to bark beetles at early stages of attack. In fact, Norway spruce that survived I. typographus attack contained higher amounts of 1,8-cineole and limonene than trees that succumbed to beetle attack (Schiebe et al., 2012; Zhao et al., 2011). During attack, monoterpenes may act as toxins or as deterrent cues indicating reduced host susceptibility.

Spruce monoterpenes that most inhibited growth of bark beetle symbiotic fungi are least toxic to beetles, but accumulate in response to fungal invasion

The monoterpenes most consistently fungistatic to all three symbiotic fungi tested, (-)-bornyl acetate, terpinolene and terpinen-4-ol, were among the least toxic to I. typographus. Two of these compounds are oxygenated, in keeping with previous reports that oxygenated monoterpenes tend to be more fungistatic than monoterpene hydrocarbons (Achotegui-Castells et al., 2016; Kusumoto et al., 2014; Marei et al., 2012). Interestingly, infection of Norway spruce bark with the symbiotic fungus G. penicillata alone (without beetles) led to an enrichment of resin with oxygenated monoterpenes. For example, the concentration of terpinen-4-ol in G. penicillata lesions was 34-fold higher than in wounded (no fungus) controls, and the strong fungistatic compounds, (-)-bornyl acetate and terpinolene, showed a 10-fold higher concentration in G. penicillata lesions. Terpinen-4-ol may be produced by the fungus as a catabolite of α- and β-pinene, as well as being a component of induced host tree oleoresin (Kandasamy et al., 2023). These findings suggest that spruce bark recognizes the infection of bark beetle fungal symbionts and responds by elevating fungistatic compounds to restrict further advancement of fungi.

Some spruce monoterpenes even promote the growth of the symbiotic fungi

Several Norway spruce oleoresin monoterpenes that were toxic to I. typographus not only failed to inhibit the growth of the symbiotic fungi, but even promoted fungal growth. This was especially true for G. penicillata, which exhibited pronounced tolerance to induced monoterpene mixtures and other spruce defense compounds in a previous study (Zhao et al., 2018). The stimulation of G. penicillata growth by monoterpenes may allow this species to out-compete other fungi in terpene-rich bark, and so better deliver its benefits to its bark beetle partners, such as nutritional supplementation (Six, 2012; Zaman et al., 2023). Fungal growth, despite the presence of high concentrations of monoterpenes may result from direct detoxification reactions (Lah et al., 2013; Wang et al., 2014) or excretion via an efflux transporter (Wang et al., 2013), as described for the congeneric G. clavigera. The stimulation of fungal growth by monoterpenes may be due, not only to the ability of G. penicillata to metabolize monoterpenes to less toxic derivatives or excrete them, but also to their use of the metabolites as carbon sources for central metabolism (Cale et al., 2016; DiGuistini et al., 2011; Wang et al., 2014).

Our study on monoterpene mixtures in Norway spruce oleoresin showed that the three most abundant components have intermediate toxicity towards two major classes of enemies- bark beetles and their symbiotic fungi. However, among the next nine components, the most poisonous monoterpenes to bark beetles are completely different from those that most inhibit the growth of symbiotic fungi. Of the theories proposed for the existence of mixtures of chemical defenses in plants, these findings best support the interaction diversity hypothesis, which predicts that individual components of mixtures are most effective against different biotic attackers. Hence the large variety of antagonists that plants face in nature may have selected for their production of chemical defense mixtures.

Conflict of interest

All authors declare that they have no conflicts of interest

Authorship contributions

DK, RZ, JG, and AH developed the initial conceptual framework. RZ and DK developed methods for insect and fungal bioassays. RZ, DK, and AJ collected data. JG provided access to resources and equipment. DK and JG supervised the work. RZ and DK conducted data analyses. DK, JG, and RZ wrote the first draft of the manuscript. All authors contributed to the revisions.

Acknowledgments

We thank Bettina Raguschke and Emily Puckett for their assistance in the laboratory, ThüringenForst for providing spruce trees to maintain the laboratory bark beetle culture, and the Max Planck Society for funding this project.

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The authors declare no competing interests.

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A rationale for chemical defense mixtures in spruce oleoresin: most monoterpenes are highly toxic to either bark beetles or to their symbiotic fungi, but not both

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Abstract

Figures

Introduction

Materials and methods

Bark beetle rearing

Effect of vapor phase spruce monoterpenes on growth of symbiotic fungi

Fungal inoculation of spruce logs

Extraction of monoterpenes from spruce bark and their quantification using GC-FID/MS

Data analysis

Results

Lesions from symbiotic fungi in the bark contain high concentrations of fungistatic monoterpenes

Discussion

Some spruce monoterpenes even promote the growth of the symbiotic fungi

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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