Complementary roles of two classes of defense chemicals in white spruce against spruce budworm.

MAIN CONCLUSION: Monoterpenes and phenolics play distinct roles in defending white spruce trees from insect defoliators. Monoterpenes contribute to the toxicity of the foliage, deterring herbivory, whereas phenolics impede budworm growth. This study demonstrates the complex interplay between monoterpenes and phenolics and their collective influence on the defense strategy of white spruce trees against a common insect defoliator. Long-lived coniferous trees display considerable variations in their defensive chemistry. The impact of these defense phenotype variations on insect herbivores of the same species remains to be thoroughly studied, mainly due to challenges in replicating the comprehensive defense profiles of trees under controlled conditions. This study methodically examined the defensive properties of foliar monoterpenes and phenolics across 80 distinct white spruce families. These families were subsequently grouped into two chemotypes based on their foliar monoterpene concentrations. To understand the separate and combined effects of these classes on tree defenses to the eastern spruce budworm, we conducted feeding experiments using actual defense profiles from representative families. Specifically, we assessed budworm response when exposed to substrates amended with phenolics alone or monoterpenes. Our findings indicate that the ratios and amounts of monoterpenes and phenolics present in the white spruce foliage influence the survival of spruce budworms. Phenotypes associated with complete larval mortality exhibited elevated ratios (ranging from 0.4 to 0.6) and concentrations (ranging from 1143 to 1796 ng mg-1) of monoterpenes. Conversely, families characterized by higher phenolic ratios (ranging from 0.62 to 0.77) and lower monoterpene concentrations (ranging from 419 to 985 ng mg-1) were less lethal to the spruce budworm. Both classes of defense compounds contribute significantly to the overall defensive capabilities of white spruce trees. Monoterpenes appear critical in determining the general toxicity of foliage, while phenolics play a role in slowing budworm development, thereby underscoring their collective importance in white spruce defenses.

A Pine in Distress: How Infection by Different Pathogenic Fungi Affect Lodgepole Pine Chemical Defenses.

In North America, lodgepole pine is frequently subjected to attacks by various biotic agents that compromise its ability to defend against subsequent attacks by insect herbivores. We investigated whether infections of lodgepole pine by different pathogenic fungal species have varying effects on its defense chemistry. We selected two common pathogens, Atropellis canker, Atropellis piniphila, and western gall rust, Endocronartium harknessii, affecting mature lodgepole pine trees in western Canada. We also included three ophiostomatoid fungi Grosmannia clavigera, Ophiostoma montium, and Leptographium longiclavatum associated with the mountain pine beetle (Dendroctonus ponderosae), because they are commonly used to investigate induced defenses of host trees of bark beetles. We collected phloem samples from lodgepole pines infected with the rust or the canker and healthy lodgepole pines in the same stand. We also inoculated mature lodgepole pines with the three fungal symbionts and collected phloem samples 2 weeks later when the defense chemistry was at its highest level. Different fungal species differentially altered the terpene chemistry of lodgepole pine trees. E. harknessii and the fungal symbionts altered the terpene chemistry in a similar pattern while trees responded to the infection by the A. piniphila differently. Our study highlights the importance of considering specific biotic stress agents in tree susceptibility or resistance to the subsequent attacks by insect herbivores, such as mountain pine beetle.

Decoupling of height growth and drought or pest resistance tradeoffs is revealed through multiple common-garden experiments of lodgepole pine.

The environment could alter growth and resistance tradeoffs in plants by affecting the ratio of resource allocation to various competing traits. Yet, how and why functional tradeoffs change over time and space is poorly understood particularly in long-lived conifer species. By establishing four common-garden test sites for five lodgepole pine populations in western Canada, combined with genomic sequencing, we revealed the decoupling pattern and genetic underpinnings of tradeoffs between height growth, drought resistance based on δ13C and dendrochronology, and metrics of pest resistance based on pest suitability ratings. Height and δ13C correlation displayed a gradient change in magnitude and/or direction along warm-to-cold test sites. All cold test sites across populations showed a positive height and δ13C relationship. However, we did not observe such a clinal correlation pattern between height or δ13C and pest suitability. Further, we found that the study populations exhibiting functional tradeoffs or synergies to various degrees in test sites were driven by non-adaptive evolutionary processes rather than adaptive evolution or plasticity. Finally, we found positive genetic relationships between height and drought or pest resistance metrics and probed five loci showing potential genetic tradeoffs between northernmost and the other populations. Our findings have implications for deciphering the ecological, evolutionary, and genetic bases of the decoupling of functional tradeoffs due to environmental change.

Pest defences under weak selection exert a limited influence on the evolution of height growth and drought avoidance in marginal pine populations.

While droughts, intensified by climate change, have been affecting forests worldwide, pest epidemics are a major source of uncertainty for assessing drought impacts on forest trees. Thus far, little information has documented the adaptability and evolvability of traits related to drought and pests simultaneously. We conducted common-garden experiments to investigate how several phenotypic traits (i.e. height growth, drought avoidance based on water-use efficiency inferred from δ13C and pest resistance based on defence traits) interact in five mature lodgepole pine populations established in four progeny trials in western Canada. The relevance of interpopulation variation in climate sensitivity highlighted that seed-source warm populations had greater adaptive capability than cold populations. In test sites, warming generated taller trees with higher δ13C and increased the evolutionary potential of height growth and δ13C across populations. We found, however, no pronounced gradient in defences and their evolutionary potential along populations or test sites. Response to selection was weak in defences across test sites, but high for height growth particularly at warm test sites. Response to the selection of δ13C varied depending on its selective strength relative to height growth. We conclude that warming could promote the adaptability and evolvability of growth response and drought avoidance with a limited evolutionary influence from pest (biotic) pressures.

Multiple-trait analyses improved the accuracy of genomic prediction and the power of genome-wide association of productivity and climate change-adaptive traits in lodgepole pine.

BACKGROUND: Genomic prediction (GP) and genome-wide association (GWA) analyses are currently being employed to accelerate breeding cycles and to identify alleles or genomic regions of complex traits in forest trees species. Here, 1490 interior lodgepole pine (Pinus contorta Dougl. ex. Loud. var. latifolia Engelm) trees from four open-pollinated progeny trials were genotyped with 25,099 SNPs, and phenotyped for 15 growth, wood quality, pest resistance, drought tolerance, and defense chemical (monoterpenes) traits. The main objectives of this study were to: (1) identify genetic markers associated with these traits and determine their genetic architecture, and to compare the marker detected by single- (ST) and multiple-trait (MT) GWA models; (2) evaluate and compare the accuracy and control of bias of the genomic predictions for these traits underlying different ST and MT parametric and non-parametric GP methods. GWA, ST and MT analyses were compared using a linear transformation of genomic breeding values from the respective genomic best linear unbiased prediction (GBLUP) model. GP, ST and MT parametric and non-parametric (Reproducing Kernel Hilbert Spaces, RKHS) models were compared in terms of prediction accuracy (PA) and control of bias. RESULTS: MT-GWA analyses identified more significant associations than ST. Some SNPs showed potential pleiotropic effects. Averaging across traits, PA from the studied ST-GP models did not differ significantly from each other, with generally a slight superiority of the RKHS method. MT-GP models showed significantly higher PA (and lower bias) than the ST models, being generally the PA (bias) of the RKHS approach significantly higher (lower) than the GBLUP. CONCLUSIONS: The power of GWA and the accuracy of GP were improved when MT models were used in this lodgepole pine population. Given the number of GP and GWA models fitted and the traits assessed across four progeny trials, this work has produced the most comprehensive empirical genomic study across any lodgepole pine population to date.

Integrating genomic information and productivity and climate-adaptability traits into a regional white spruce breeding program.

Tree improvement programs often focus on improving productivity-related traits; however, under present climate change scenarios, climate change-related (adaptive) traits should also be incorporated into such programs. Therefore, quantifying the genetic variation and correlations among productivity and adaptability traits, and the importance of genotype by environment interactions, including defense compounds involved in biotic and abiotic resistance, is essential for selecting parents for the production of resilient and sustainable forests. Here, we estimated quantitative genetic parameters for 15 growth, wood quality, drought resilience, and monoterpene traits for Picea glauca (Moench) Voss (white spruce). We sampled 1,540 trees from three open-pollinated progeny trials, genotyped with 467,224 SNP markers using genotyping-by-sequencing (GBS). We used the pedigree and SNP information to calculate, respectively, the average numerator and genomic relationship matrices, and univariate and multivariate individual-tree models to obtain estimates of (co)variance components. With few site-specific exceptions, all traits examined were under genetic control. Overall, higher heritability estimates were derived from the genomic- than their counterpart pedigree-based relationship matrix. Selection for height, generally, improved diameter and water use efficiency, but decreased wood density, microfibril angle, and drought resistance. Genome-based correlations between traits reaffirmed the pedigree-based correlations for most trait pairs. High and positive genetic correlations between sites were observed (average 0.68), except for those pairs involving the highest elevation, warmer, and moister site, specifically for growth and microfibril angle. These results illustrate the advantage of using genomic information jointly with productivity and adaptability traits, and defense compounds to enhance tree breeding selection for changing climate.

Combined drought and bark beetle attacks deplete non-structural carbohydrates and promote death of mature pine trees.

How carbohydrate reserves in conifers respond to drought and bark beetle attacks are poorly understood. We investigated changes in carbohydrate reserves and carbon-dependent diterpene defences in ponderosa pine trees that were experimentally subjected to two levels of drought stress (via root trenching) and two types of biotic challenge treatments (pheromone-induced bark beetle attacks or inoculations with crushed beetles that include beetle-associated fungi) for two consecutive years. Our results showed that trenching did not influence carbohydrates, whereas both biotic challenges reduced amounts of starch and sugars of trees. However, only the combined trenched-bark beetle attacked trees depleted carbohydrates and died during the first year of attacks. While live trees contained higher carbohydrates than dying trees, amounts of constitutive and induced diterpenes produced did not vary between live and beetle-attacked dying trees, respectively. Based on these results we propose that reallocation of carbohydrates to diterpenes during the early stages of beetle attacks is limited in drought-stricken trees, and that the combination of biotic and abiotic stress leads to tree death. The process of tree death is subsequently aggravated by beetle girdling of phloem, occlusion of vascular tissue by bark beetle-vectored fungi, and potential exploitation of host carbohydrates by bark beetle symbionts as nutrients.

Production of complementary defense metabolites reflects a co-evolutionary arms race between a host plant and a mutualistic bark beetle-fungal complex.

Intra-specific variation in conifers has been extensively studied with respect to defense against herbivores and pathogens. While studies have shown the ability of individual or specific mixtures of compounds to influence insects and microbes, research testing biologically relevant mixtures of defense compounds reflecting intra-specific variation amongst tree populations to enemy complexes is needed. We characterized the variations in lodgepole pine monoterpenes from a progeny trial in western Canada and grouped trees in four clusters using their monoterpene profiles. We then selected 11 representative families across four clusters and amended their entire monoterpene profiles (with the exception of ß-phellandrene) in media to determine how representative families affect the performance of the mountain pine beetle or its fungal symbiont. We placed adult beetles or inoculated fungus on the amended media and measured beetle performance and fungal growth as a proxy to host suitability. We found that different clusters or families differentially influenced beetle or fungal responses. However, monoterpene profiles of trees suitable to the beetle or the fungus were dissimilar. These outcomes reflect a co-evolutionary arms-race between the host and the bark beetle-fungus complex, which has resulted in the production of complementary defense metabolites among different pine populations to enhance tree survival.

Selection of entomopathogenic fungus Beauveria bassiana (Deuteromycotina: Hyphomycetes) for the biocontrol of Dendroctonus ponderosae (Coleoptera: Curculionidae, Scolytinae) in Western Canada.

The mountain pine beetle, Dendroctonus ponderosae, has infested over ~16 Mha of pine forests in British Columbia killing >50% of mature lodgepole pine, Pinus contorta, trees in affected stands. At present, it is functionally an invasive species in Alberta, killing and reproducing in evolutionarily naïve populations of lodgepole pine (P. contorta), novel jack pine (P. banksiana), and their hybrids. The entomopathogenic fungus Beauveria bassiana has shown some potential as a biocontrol agent of several bark beetle species. In this study, nine isolates of B. bassiana were examined for insect virulence characteristics, including conidiation rate, pigmentation, and infection rate in laboratory-reared D. ponderosae, to assess for their potential as biocontrol agents. The strains were categorized into three phenotypic groups based on pigmentation, conidial density, and myceliation rate. Virulence screening utilizing insect-based agar medium (D. ponderosae and European honeybee Apis mellifera carcasses) revealed no difference in selection of fungal growth. However, infection studies on D. ponderosae and A. mellifera showed contrasting results. In vivo A. mellifera infection model revealed ~5% mortality, representing the natural death rate of the hive population, whereas laboratory-reared D. ponderosae showed 100% mortality and mycosis. The LT50 (median lethal time 50) ranges from 2 to 5 ± 0.33 days, and LT100 ranges from 4 to 6 ± 0.5 days. We discuss the selective advantages of the three phenotypic groups in terms of virulence, pigmentation, conidial abundance, and tolerance to abiotic factors like UV and host tree monoterpenes. These results can further provide insights into the development of several phenotypically diverse B. bassiana strains in controlling the spread of the invasive D. ponderosae in Western Canada. KEY POINTS: • Three B. bassiana morphotype groups have been demonstrated to kill D. ponderosae. • A range of effective lethal times (LT50 and LT100) was established against D. ponderosae. • Variable tolerance to UV light and pine monoterpenes were observed in B. bassiana.

Nitrogen and Ergosterol Concentrations Varied in Live Jack Pine Phloem Following Inoculations With Fungal Associates of Mountain Pine Beetle.

Bark beetles form symbiotic associations with multiple species of fungi that supplement their metabolic needs. However, the relative contributions of each symbiont to the nutrition of bark beetles have been largely unexplored. Thus, we evaluated the ability of three fungal symbionts of mountain pine beetle to concentrate nitrogen and produce ergosterol while infecting phloem of a novel host jack pine. Ergosterol was used as proxy to determine the fungal biomass (hyphal density) in the current study. We inoculated 80 trees in two forest stands with one of the three fungal species or a non-fungal (control) agar. Six weeks later, we collected phloem from the necrotic lesions induced by the fungi, uninfected tissues adjacent to lesions, and non-inoculated control trees. We found that nutritional contributions varied with fungal species. Nitrogen in lesions was higher in trees inoculated with Ophiostoma montium or control trees, relative to Grosmannia clavigera or Leptographium longiclavatum. Furthermore, concentrations of ergosterol were higher in O. montium lesions compared to other tissues or treatments. These results suggest that O. montium differs from G. clavigera and L. longiclavatum in terms of acquiring nitrogen from host tissues and producing ergosterol.

Pathophysiological responses of pine defensive metabolites largely lack differences between pine species but vary with eliciting ophiostomatoid fungal species.

Phytopathogenic ophiostomatoid fungi are common associates of bark beetles and contribute to beetle-associated mortality of trees. Mountain pine beetle outbreaks in Canada are facilitating novel associations between its vectored fungi (Grosmannia clavigera, Leptographium longiclavatum and Ophiostoma montium) and jack pine. How the induced defense-related metabolite responses of jack and lodgepole pines vary in response to the fungi is unknown. Understanding this variation is important to clarifying pine susceptibility to and the physiological impacts of infection. We used a comparative metabolite profiling approach to investigate the defense-related signaling, carbon utilization/mobilization, and synthesis responses of both pines to the fungi. Both pine species largely exhibited similar metabolite responses to the fungi. The magnitude of pine metabolite responses positively reflected pathogen virulence. Our findings indicate that pines can recognize and metabolomically respond to novel pathogens, likely due to signals common between the novel fungi and fungi coevolved with the pine. Thus, jack pine is likely as susceptible as lodgepole pine to infections by each of the MPB-vectored fungi. Furthermore, the magnitude of the metabolite responses of both pines varied by the eliciting fungal species, with the most virulent pathogen causing the greatest reduction in carbohydrates and the highest accumulation of defensive terpenes.

Anatomical defences against bark beetles relate to degree of historical exposure between species and are allocated independently of chemical defences within trees.

Conifers possess chemical and anatomical defences against tree-killing bark beetles that feed in their phloem. Resins accumulating at attack sites can delay and entomb beetles while toxins reach lethal levels. Trees with high concentrations of metabolites active against bark beetle-microbial complexes, and more extensive resin ducts, achieve greater survival. It is unknown if and how conifers integrate chemical and anatomical components of defence or how these capabilities vary with historical exposure. We compared linkages between phloem chemistry and tree ring anatomy of two mountain pine beetle hosts. Lodgepole pine, a mid-elevation species, has had extensive, continual contact with this herbivore, whereas high-elevation whitebark pines have historically had intermittent exposure that is increasing with warming climate. Lodgepole pine had more and larger resin ducts. In both species, anatomical defences were positively related to tree growth and nutrients. Within-tree constitutive and induced concentrations of compounds bioactive against bark beetles and symbionts were largely unrelated to resin duct abundance and size. Fewer anatomical defences in the semi-naïve compared with the continually exposed host concurs with directional differences in chemical defences. Partially uncoupling chemical and morphological antiherbivore traits may enable trees to confront beetles with more diverse defence permutations that interact to resist attack.

Dwarf mistletoe infection in jack pine alters growth-defense relationships.

Trees utilize a combination of chemical and anatomical defenses against a myriad of attacking organisms. However, persistent pathogen infection that alters resource acquisition may impact growth and defense relationships, which could have consequences for tree resistance. We characterized systemic chemical and anatomical changes in jack pine (Pinus banksiana) in response to infection by the parasitic plant dwarf mistletoe (Arceuthobium americanum) and identified how the growth-defense relationship is altered due to infection severity. Our study found that the growth and defense relationship in jack pine was altered due to infection and that chemical defenses in the phloem received a relatively higher priority than radial growth and anatomical defenses. Chemical defenses in the phloem had a non-linear relationship with infection severity with increasing concentrations of monoterpenes in trees with moderate infection and decreasing concentrations at high infection. In contrast, both radial growth and vertical resin duct production decreased with increasing infection severity. While constitutive resin duct counts and many monoterpene compound concentrations were positively correlated, this relationship was not maintained in infected trees. Furthermore, radial growth and basal area increment was positively correlated with resin duct production and monoterpene concentration in non-infected trees but had fewer relationships in severely infected trees. We conclude that while both chemical and anatomical defenses may be used as indicators for potential resistance to biotic stress in pines, changes in resource allocation patterns between these defenses after infection will likely have consequences on tree resistance to subsequent biotic attacks.

Drought stress leads to systemic induced susceptibility to a nectrotrophic fungus associated with mountain pine beetle in Pinus banksiana seedlings.

Conifers have complex defense responses to initial attacks by insects and pathogens that can have cascading effects on success of subsequent colonizers. However, drought can affect a plant's ability to respond to biotic agents by potentially altering the resources needed for the energetically costly production of induced defense chemicals. We investigated the impact of reduced water on induced chemical defenses of jack pine (Pinus banksiana) seedlings from initial attack by biotic agents and resistance to subsequent challenge inoculation with a pathogenic fungal associate of mountain pine beetle (Dendroctonus ponderosae), Grosmannia clavigera. Applications of phytohormones (methyl salicylate and methyl jasmonate) and G. clavigera were used for initial induction of defenses. Monoterpene concentrations varied with initial induction from fungal and phytohormone application while watering treatment had no effect. Seedlings treated with G. clavigera and methyl jasmonate had the greatest monoterpene concentrations compared to the control and methyl salicylate-treated seedlings. However, the monoterpene response to the challenge inoculation varied with watering treatments, not with prior induction treatments, with lower monoterpene concentrations in fungal lesions on seedlings in the low to moderate watering treatments compared to normal watering treatment. Furthermore, prior induction from phytohormones resulted in systemic cross-induction of resistance to G. clavigera under normal watering treatment but susceptibility under low watering treatment. Seedlings stressed by low water conditions, which also had lower stomatal conductance than seedlings in the normal watering treatment, likely allocated resources to initial defense response but were left unable to acquire further resources for subsequent responses. Our results demonstrate that drought can affect interactions among tree-infesting organisms through systemic cross-induction of susceptibility.

Rapid monoterpene induction promotes the susceptibility of a novel host pine to mountain pine beetle colonization but not to beetle-vectored fungi.

Chemical induction can drive tree susceptibility to and host range expansions of attacking insects and fungi. Recently, mountain pine beetle (Dendroctonus ponderosae Hopkins; MPB) has expanded its host range from its historic host lodgepole pine (Pinus contorta var. latifolia Douglas ex Loudon) to jack pine (Pinus banksiana Lamb) in western Canada. Beetle success in jack pine forests likely depends upon the suitability of tree chemistry to MPB and its symbiotic phytopathogenic fungi. In particular, how rapid induced defenses of jack pine affect MPB colonization and the beetle's symbionts is unknown. In the field, we characterized and compared differences in rapid induced phloem monoterpenes between lodgepole and jack pines in response to various densities of Grosmannia clavigera (Robinson-Jeffery and Davidson)-a MPB symbiotic fungus used to simulate beetle attack-inoculations. Overall, lodgepole pine had higher limonene and myrcene, but lower α-pinene, concentrations than jack pine. However, myrcene concentrations in jack pine increased with inoculation density, while that in lodgepole pine did not respond to density treatments. We compared the growth and reproduction of MPB's symbiotic fungi, G. clavigera, Ophiostoma montium (Rumford) von Arx and Leptographium longiclavatum Lee, Kim and Breuil, grown on media amended with myrcene, α-pinene and limonene at concentrations reflecting two induction levels from each pine species. Myrcene and α-pinene amendments inhibited the growth but stimulated the reproduction of G. clavigera, whereas limonene stimulated its growth while inhibiting its reproduction. However, the growth and reproduction of the other fungi were generally stimulated by monoterpene amendments. Overall, our results suggest that jack pine rapid induction could promote MPB aggregation due to high levels of α-pinene (pheromone precursor), a positive feedback of myrcene (pheromone synergist) and low levels of limonene (resistance). Jack pine is likely as susceptible to MPB-vectored fungi as lodgepole pine, indicating that jack pine induction will likely not adversely affect symbiont activities enough to inhibit the invasion of MPB into jack pine forests.

A Native Parasitic Plant Systemically Induces Resistance in Jack Pine to a Fungal Symbiont of Invasive Mountain Pine Beetle.

Conifer trees resist pest and pathogen attacks by complex defense responses involving different classes of defense compounds. However, it is unknown whether prior infection by biotrophic pathogens can lead to subsequent resistance to necrotrophic pathogens in conifers. We used the infection of jack pine, Pinus banksiana, by a common biotrophic pathogen dwarf mistletoe, Arceuthobium americanum, to investigate induced resistance to a necrotrophic fungus, Grosmannia clavigera, associated with the mountain pine beetle, Dendroctonus ponderosae. Dwarf mistletoe infection had a non-linear, systemic effect on monoterpene production, with increasing concentrations at moderate infection levels and decreasing concentrations at high infection levels. Inoculation with G. clavigera resulted in 33 times higher monoterpene concentrations and half the level of phenolics in the necrotic lesions compared to uninoculated control trees. Monoterpene production following dwarf mistletoe infection seemed to result in systemic induced resistance, as trees with moderate disease severity were most resistant to G. clavigera, as evident from shorter lesion lengths. Furthermore, trees with moderate disease severity had the highest systemic but lowest local induction of α-pinene after G. clavigera inoculation, suggesting a possible tradeoff between systemically- and locally-induced defenses. The opposing effects to inoculation by G. clavigera on monoterpene and phenolic levels may indicate the potential for biosynthetic tradeoffs by the tree between these two major defense classes. Our results demonstrate that interactions between a biotrophic parasitic plant and a necrotrophic fungus may impact mountain pine beetle establishment in novel jack pine forests through systemic effects mediated by the coordination of jack pine defense chemicals.

Defence syndromes in lodgepole - whitebark pine ecosystems relate to degree of historical exposure to mountain pine beetles.

Warming climate is allowing tree-killing bark beetles to expand their ranges and access naïve and semi-naïve conifers. Conifers respond to attack using complex mixtures of chemical defences that can impede beetle success, but beetles exploit some compounds for host location and communication. Outcomes of changing relationships will depend on concentrations and compositions of multiple host compounds, which are largely unknown. We analysed constitutive and induced chemistries of Dendroctonus ponderosae's primary historical host, Pinus contorta, and Pinus albicaulis, a high-elevation species whose encounters with this beetle are transitioning from intermittent to continuous. We quantified multiple classes of terpenes, phenolics, carbohydrates and minerals. Pinus contorta had higher constitutive allocation to, and generally stronger inducibility of, compounds that resist these beetle-fungal complexes. Pinus albicaulis contained higher proportions of specific monoterpenes that enhance pheromone communication, and lower induction of pheromone inhibitors. Induced P. contorta increased insecticidal and fungicidal compounds simultaneously, whereas P. albicaulis responses against these agents were inverse. Induced terpene accumulation was accompanied by decreased non-structural carbohydrates, primarily sugars, in P. contorta, but not P. albicaulis, which contained primarily starches. These results show some host species with continuous exposure to bark beetles have more thoroughly integrated defence syndromes than less-continuously exposed host species.

Weathering the storm: how lodgepole pine trees survive mountain pine beetle outbreaks.

Recent mountain pine beetle outbreaks in western North America killed millions of lodgepole pine trees, leaving few survivors. However, the mechanism underlying the ability of trees to survive bark beetle outbreaks is unknown, but likely involve phytochemicals such as monoterpenes and fatty acids that can drive beetle aggregation and colonization on their hosts. Thus, we conducted a field survey of beetle-resistant lodgepole pine (Pinus contorta) trees to retrospectively deduce whether these phytochemicals underlie their survival by comparing their chemistry to that of non-attacked trees in the same stands. We also compared beetle attack characteristics between resistant and beetle-killed trees. Beetle-killed trees had more beetle attacks and longer ovipositional galleries than resistant trees, which also lacked the larval establishment found in beetle-killed trees. Resistant trees contained high amounts of toxic and attraction-inhibitive compounds and low amounts of pheromone-precursor and synergist compounds. During beetle host aggregation and colonization, these compounds likely served three critical roles in tree survival. First, low amounts of pheromone-precursor (α-pinene) and synergist (mycrene, terpinolene) compounds reduced or prevented beetles from attracting conspecifics to residual trees. Second, high amounts of 4-allyanisole further inhibited beetle attraction to its pheromone. Finally, high amounts of toxic limonene, 3-carene, 4-allyanisole, α-linolenic acid, and linoleic acid inhibited beetle gallery establishment and oviposition. We conclude that the variation of chemotypic expression of local plant populations can have profound ecological consequences including survival during insect outbreaks.

Water-deficit and fungal infection can differentially affect the production of different classes of defense compounds in two host pines of mountain pine beetle.

Bark beetles are important agents of tree mortality in conifer forests and their interaction with trees is influenced by host defense chemicals, such as monoterpenes and phenolics. Since mountain pine beetle (Dendroctonus ponderosae Hopkins) has expanded its host range from lodgepole pine (Pinus contorta Doug. ex Loud. (var. latifolia Engelm.))-dominated forests to the novel jack pine (Pinus banksiana Lamb.) forests in western Canada, studies investigating the jack pine suitability as a host for this beetle have exclusively focused on monoterpenes, and whether phenolics affect jack pine suitability to mountain pine beetle and its symbiotic fungus Grosmannia clavigera is unknown. We investigated the phenolic and monoterpene composition in phloem and foliage of jack and lodgepole pines, and their subsequent change in response to water deficit and G. clavigera inoculation treatments. In lodgepole pine phloem, water deficit treatment inhibited the accumulation of both the total and richness of phenolics, but had no effect on total monoterpene production or richness. Fungal infection also inhibited the total phenolic production and had no effect on phenolic or monoterpene richness, but increased total monoterpene synthesis by 71%. In jack pine phloem, water deficit treatment reduced phenolic production, but had no effect on phenolic or monoterpene richness or total monoterpenes. Fungal infection did not affect phenolic or monoterpene production. Lesions of both species contained lower phenolics but higher monoterpenes than non-infected phloem in the same tree. In both species, richness of monoterpenes and phenolics was greater in non-infected phloem than in lesions. We conclude that monoterpenes seem to be a critical component of induced defenses against G. clavigera in both jack and lodgepole pines; however, a lack of increased monoterpene response to fungal infection is an important evolutionary factor defining jack pine suitability to the mountain pine beetle invasion in western Canada.

Fungal Volatiles Can Act as Carbon Sources and Semiochemicals to Mediate Interspecific Interactions Among Bark Beetle-Associated Fungal Symbionts.

Mountain pine beetle (Dendroctonus ponderosae) has killed millions of hectares of pine forests in western North America. Beetle success is dependent upon a community of symbiotic fungi comprised of Grosmannia clavigera, Ophiostoma montium, and Leptographium longiclavatum. Factors regulating the dynamics of this community during pine infection are largely unknown. However, fungal volatile organic compounds (FVOCs) help shape fungal interactions in model and agricultural systems and thus may be important drivers of interactions among bark beetle-associated fungi. We investigated whether FVOCs can mediate interspecific interactions among mountain pine beetle's fungal symbionts by affecting fungal growth and reproduction. Headspace volatiles were collected and identified to determine species-specific volatile profiles. Interspecific effects of volatiles on fungal growth and conidia production were assessed by pairing physically-separated fungal cultures grown either on a carbon-poor or -rich substrate, inside a shared-headspace environment. Fungal VOC profiles differed by species and influenced the growth and/or conidia production of the other species. Further, our results showed that FVOCs can be used as carbon sources for fungi developing on carbon-poor substrates. This is the first report demonstrating that FVOCs can drive interactions among bark beetle fungal symbionts, and thus are important factors in beetle attack success.