Terpenoids are involved in the expression of systemic-induced resistance in Austrian pine.

Terpenoids are defense metabolites that are induced upon infection or wounding. However, their role in systemic-induced resistance (SIR) is not known. Here, we explored the role of terpenoids in this phenomenon at a very early stage in the interaction between Austrian pine and the tip blight and canker pathogen Diplodia pinea. We induced Austrian pine saplings by either wounding or inoculating the lower stems with D. pinea. The seedlings were then challenged after 12 h, 72 h, or 10 days with D. pinea on the stem 15 cm above the induction. Lesion lengths and terpenoids were quantified at both induction and challenge locations. Key terpenoids were assayed for antifungal activity in in vitro bioassays. SIR increased with time and was correlated with the inducibility of several compounds. α-Pinene and a cluster of ß-pinene, limonene, benzaldehyde, dodecanol, and n-dodecyl acrylate were positively correlated with SIR and were fungistatic in vitro, while other compounds were negatively correlated with SIR and appeared to serve as a carbon source for D. pinea. This study shows that, overall, terpenoids are involved in SIR in this system, but their role is nuanced, depending on the type of induction and time of incubation. We hypothesize that some, such as α-pinene, could serve in SIR signaling.

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.

Unraveling the multifaceted effects of climatic factors on mountain pine beetle and its interaction with fungal symbionts.

Mountain pine beetles (MPBs) pose a substantial threat to North American pine forests, causing extensive tree mortality over large areas. Their tree-killing ability is closely linked to mass aggregation on host trees triggered via pheromones and dependence on their symbiotic fungi. However, the influence of a changing climate on the biology of MPBs and their co-evolved interactions with their fungal symbionts remains uncertain. To investigate this, male and female pairs of beetles were introduced into freshly cut logs from lodgepole pine trees and placed in controlled climate chambers with manipulated environmental conditions, including two levels of CO2 (ambient vs. 1000 ppm), O3 (ambient vs. 100 ppb) and humidity (33% vs. 65%). The beetle-infested logs were left in these chambers for 1 month and then returned to ambient conditions until brood emergence. Emerging broods were collected for further analysis. Additionally, three species of fungal symbionts (Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum) were subjected to the same CO2 , O3 and humidity conditions for 5 days. Lower humidity promoted MPB reproduction and fungal growth. Elevated CO2 accelerated larval growth and emergence while improving brood pheromone production. Elevated O3 had a negative impact on MPB reproduction and brood fitness while improving its immune responses to an entomopathogenic fungus (Beauveria bassiana). It also inhibited fungal growth and reproduction, whereas elevated CO2 had varied (positive or negative) effects on fungal growth and ergosterol (proxy to fungal mass) production depending on the fungal species. Together, these findings suggest that climate change can potentially alter the interactions between MPBs and their fungal symbionts, highlighting the importance of understanding how climate change affects forest pests and their symbiotic relationships to develop effective management strategies in the future.

Long-insert sequence capture detects high copy numbers in a defence-related beta-glucosidase gene ßglu-1 with large variations in white spruce but not Norway spruce.

Conifers are long-lived and slow-evolving, thus requiring effective defences against their fast-evolving insect natural enemies. The copy number variation (CNV) of two key acetophenone biosynthesis genes Ugt5/Ugt5b and ßglu-1 may provide a plausible mechanism underlying the constitutively variable defence in white spruce (Picea glauca) against its primary defoliator, spruce budworm. This study develops a long-insert sequence capture probe set (Picea_hung_p1.0) for quantifying copy number of ßglu-1-like, Ugt5-like genes and single-copy genes on 38 Norway spruce (Picea abies) and 40 P. glauca individuals from eight and nine provenances across Europe and North America respectively. We developed local assemblies (Piabi_c1.0 and Pigla_c.1.0), full-length transcriptomes (PIAB_v1 and PIGL_v1), and gene models to characterise the diversity of ßglu-1 and Ugt5 genes. We observed very large copy numbers of ßglu-1, with up to 381 copies in a single P. glauca individual. We observed among-provenance CNV of ßglu-1 in P. glauca but not P. abies. Ugt5b was predominantly single-copy in both species. This study generates critical hypotheses for testing the emergence and mechanism of extreme CNV, the dosage effect on phenotype, and the varying copy number of genes with the same pathway. We demonstrate new approaches to overcome experimental challenges in genomic research in conifer defences.

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.

Beauveria bassiana exhibits strong virulence against Dendroctonus ponderosae in greenhouse and field experiments.

The mountain pine beetle (MPB) has infested over 16 million hectares of pine forests in western Canada, killing over 50% of mature lodgepole pine, Pinus contorta, in British Columbia alone. There are few tools available to manage irruptive bark beetle populations and to mitigate tree mortality. Beauveria bassiana is an entomopathogenic fungus that causes mortality to several bark beetle species. However, the potential for B. bassiana as a biocontrol agent against pine beetle populations is unknown. We selected three strains of B. bassiana from several culture collections and evaluated their conidial stability under cold storage, in planta (greenhouse, and pine bolts) and in natura (forest stand, pine bolts, and live pines) conditions. The stability assays showed that all fungal strains maintained a minimum effective conidial yield through the assay durations (3-12 weeks). In addition, we adapted a biphasic liquid-solid fermentation approach for the large-scale production of conidial biomass, yielding up to a 100-fold increase in production. In greenhouse virulence assays, the mean lethal time of MPBs was reduced to 3-4 days upon treatment with B. bassiana, where high B. bassiana-associated mycosis was also observed. Furthermore, the application of B. bassiana formulation substantially affected the gallery network of MPBs in bolts in the field, resulting in shorter larval galleries and significantly reduced offspring production. Indeed, high titer treatments reduced the mean larvae per gallery to virtually zero. Together these results demonstrate that B. bassiana may be a viable biocontrol tool to reduce mountain pine beetle populations in pine forests in western Canada. KEY POINTS: • Three B. bassiana strains identified to be stable at various test conditions. • Large-scale conidial biomass production using liquid-solid biphasic fermentation. • Reproductive success of D. ponderosae significantly reduced by B. bassiana formulation.

Bark Beetles Utilize Ophiostomatoid Fungi to Circumvent Host Tree Defenses.

Bark beetles maintain symbiotic associations with a diversity of microbial organisms, including ophiostomatoid fungi. Studies have frequently reported the role of ophiostomatoid fungi in bark beetle biology, but how fungal symbionts interact with host chemical defenses over time is needed. We first investigated how inoculations by three fungal symbionts of mountain pine beetle affect the terpene chemistry of live lodgepole pine trees. We then conducted a complimentary laboratory experiment specifically measuring the host metabolite degradation by fungi and collected the fungal organic volatiles following inoculations with the same fungal species on lodgepole pine logs. In both experiments, we analyzed the infected tissues for their terpene chemistry. Additionally, we conducted an olfactometer assay to determine whether adult beetles respond to the volatile organic chemicals emitted from each of the three fungal species. We found that all fungi upregulated terpenes as early as two weeks after inoculations. Similarly, oxygenated monoterpene concentrations also increased by several folds (only in logs). A large majority of beetles tested showed a strong attraction to two fungal species, whereas the other fungus repelled the beetles. Together this study shows that fungal symbionts can alter host defense chemistry, assist beetles in overcoming metabolite toxicity, and provide possible chemical cues for bark beetle attraction.

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.

Improving lodgepole pine genomic evaluation using spatial correlation structure and SNP selection with single-step GBLUP.

Modeling environmental spatial heterogeneity can improve the efficiency of forest tree genomic evaluation. Furthermore, genotyping costs can be lowered by reducing the number of markers needed. We investigated the impact on variance components, breeding value accuracy, and bias of two phenotypic data adjustments (experimental design and autoregressive spatial models), and a relationship matrix calculated from a subset of markers selected for their ability to infer ancestry. Using a multiple-trait multiple-site single-step Genomic Best Linear Unbiased Prediction (ssGBLUP) approach, four scenarios (2 phenotype adjustments × 2 marker sets) were applied to diameter at breast height (DBH), height (HT), and resistance to western gall rust (WGR) in four open-pollinated progeny trials of lodgepole pine, with 1490 (out of 11,188) trees genotyped with 25,099 SNPs. As a control, we fitted the conventional ABLUP model using pedigree information. The highest heritability estimates were achieved for the ABLUP followed closely by the ssGBLUP with the full marker set and using the spatial phenotype adjustments. The highest predictive ability was obtained by using a reduced marker subset (8000 SNPs) when either the spatial (DBH: 0.429, and WGR: 0.513) or design (HT: 0.467) phenotype corrections were used. No significant difference was detected in prediction bias among the six fitted models, and all values were close to 1 (0.918-1.014). Results demonstrated that selecting informative markers, such as those capturing ancestry, can improve the predictive ability. The use of spatial correlation structure increased traits' heritability and reduced prediction bias, while increases in predictive ability were trait-dependent.

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.

Long-term nitrogen addition does not sustain host tree stem radial growth but doubles the abundance of high-biomass ectomycorrhizal fungi.

Global change has altered nitrogen availability in boreal forest soils. As ectomycorrhizal fungi play critical ecological functions, shifts in their abundance and community composition must be considered in the response of forests to changes in nitrogen availability. Furthermore, ectomycorrhizas are symbiotic, so the response of ectomycorrhizal fungi to nitrogen cannot be understood in isolation of their plant partners. Most previous studies, however, neglect to measure the response of host trees to nitrogen addition simultaneously with that of fungal communities. In addition to being one-sided, most of these studies have also been conducted in coniferous forests. Deciduous and "dual-mycorrhizal" tree species, namely those that form ecto- and arbuscular mycorrhizas, have received little attention despite being widespread in the boreal forest. We applied nitrogen (30 kg ha-1  year-1 ) for 13 years to stands dominated by aspen (Populus tremuloides Michx.) and hypothesized that tree stem radial growth would increase, ectomycorrhizal fungal biomass would decrease, ectomycorrhizal fungal community composition would shift, and the abundance of arbuscular mycorrhizal (AM) fungi would increase. Nitrogen addition initially increased stem radial growth of aspen, but it was not sustained at the time we characterized their mycorrhizas. After 13 years, the abundance of fungi possessing extramatrical hyphae, or "high-biomass" ectomycorrhizas, doubled. No changes occurred in ectomycorrhizal and AM fungal community composition, or in ecto- and AM abundance measured as root colonization. This dual-mycorrhizal tree species did not shift away from ectomycorrhizal fungal dominance with long-term nitrogen input. The unexpected increase in high-biomass ectomycorrhizal fungi with nitrogen addition may be due to increased carbon allocation to their fungal partners by growth-limited trees. Given the focus on conifers in past studies, reconciling results of plant-mycorrhizal fungal relationships in stands of deciduous trees may demand a broader view on the impacts of nitrogen addition on the structure and function of boreal forests.

Assessing the dual-mycorrhizal status of a widespread tree species as a model for studies on stand biogeochemistry.

Viewing plant species by their mycorrhizal type has explained a range of ecosystem processes. However, mycorrhizal type is confounded with plant phylogeny and the environments in which mycorrhizal partners occur. To circumvent these confounding effects, "dual-mycorrhizal" plant species may be potential models for testing the influence of mycorrhizal type on stand biogeochemistry. To assess their use as models, duality in mycorrhizas within a single host species must be confirmed and factors underlying their variation understood. We surveyed roots, soils, and leaves of mature aspen (Populus tremuloides) across 27 stands in western Canada spanning two biomes: boreal forest and parklands. Aspen roots were mostly ectomycorrhizal with sporadic and rare occurrences of arbuscular mycorrhizas. We further tested whether a climate moisture index predicted abundance of ectomycorrhizal roots (number of ectomycorrhizal root tips m-1 root length) surveyed at two depths (0-20 cm and 20-40 cm) and found that ectomycorrhizal root abundance in subsoils (20-40 cm) was positively related to the index. We subsequently examined the relationships between ectomycorrhizal root abundance, leaf traits, and slow and fast pools of soil organic carbon and nitrogen. The ratio of leaf lignin:N, but not its components, increased along with ectomycorrhizal root abundance in subsoils. Soil carbon and nitrogen pools were independent of ectomycorrhizal root abundance. Our results suggest that (1) categorizing aspen as dual-mycorrhizal may overstate the functional importance of arbuscular mycorrhizas in this species and life stage, (2) water availability influences ectomycorrhizal root abundance, and (3) ectomycorrhizal root abundance coincides with leaf quality.

Primary and Secondary Metabolite Profiles of Lodgepole Pine Trees Change with Elevation, but Not with Latitude.

Climate change has a large influence on plant functional and phenotypic traits including plant primary and secondary metabolites. One well-established approach to investigating the variation in plant metabolites involves studying plant populations along elevation and latitude gradients. We considered how two space-for-time climate change gradients (elevation and latitude) influence carbohydrate reserves (soluble sugars, starches) and secondary metabolites (monoterpenes, diterpene resin acids) of lodgepole pine trees in western Canada. We were particularly interested in the relationship of terpenes and carbohydrates with a wide range of tree, site, and climatic factors. We found that only elevation had a strong influence on the expression of both terpenes and carbohydrates of trees. Specifically, as elevation increased, concentrations of monoterpenes and diterpenes generally increased and soluble sugars (glucose, sucrose, total sugars) decreased. In contrast, latitude had no impact on either of terpenes or carbohydrates. Furthermore, we found a positive relationship between concentrations of starch and total terpenes and diterpenes in the elevation study; whereas neither starches nor sugars were correlated to terpenes in the latitude study. Similarly, both terpenes and carbohydrates had a much greater number of significant correlations to site characteristics such as slope, basal area index, and sand basal area, in the elevational than in the latitude study. Overall, these results support the conclusion that both biotic and abiotic factors likely drive the patterns of primary and secondary metabolite profiles of lodgepole pine along geographical gradients. Also, presence of a positive relationship between terpenes and starches suggests an interaction between primary ad secondary metabolites of lodgepole pine trees.

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.

Mutualistic Ophiostomatoid Fungi Equally Benefit from Both a Bark Beetle Pheromone and Host Tree Volatiles as Nutrient Sources.

Interactions between mutualistic bark beetles and ophiostomatoid fungi have received considerable attention in recent years. Studies have shown how volatile organic compounds emitted from mutualist fungi affect the behaviors of several bark beetle species. However, we currently lack sufficient knowledge regarding whether bark beetle pheromones can influence mutualist fungi. Here, we measured growth and biomass of two mutualistic fungi of the mountain pine beetle in response to headspace of a beetle pheromone (trans-verbenol), a blend of host tree volatiles, the combination of both, or control (no volatile source) in vitro experiments consisting of a nitrogen-based medium. The surface area and ergosterol content of the mycelia were used as surrogates for fungal growth and biomass respectively. We found that both growth and biomass of Grosmannia clavigera and Ophiostoma montium were greater in medium exposed to any type of volatile sources than the control. While growth and ergosterol content of G. clavigera were highest in the combination treatment, there were no differences in growth or biomass among the types of volatiles introduced for O. montium. These results suggest that both mutualistic fungi can utilize both bark beetle pheromone and host tree volatiles as nutrient sources. Overall, these results support the on-going studies on the role of volatile organic compounds mediating mutualistic bark beetle-fungi interactions.

Changes in soil fungal community composition depend on functional group and forest disturbance type.

Disturbances have altered community dynamics in boreal forests with unknown consequences for belowground ecological processes. Soil fungi are particularly sensitive to such disturbances; however, the individual response of fungal guilds to different disturbance types is poorly understood. Here, we profiled soil fungal communities in lodgepole pine forests following a bark beetle outbreak, wildfire, clear-cut logging, and salvage-logging. Using Illumina MiSeq to sequence ITS1 and SSU rDNA, we characterized communities of ectomycorrhizal, arbuscular mycorrhizal, saprotrophic, and pathogenic fungi in sites representing each disturbance type paired with intact forests. We also quantified soil fungal biomass by measuring ergosterol. Abiotic disturbances changed the community composition of ectomycorrhizal fungi and shifted the dominance from ectomycorrhizal to saprotrophic fungi compared to intact forests. The disruption of the soil organic layer with disturbances correlated with the decline of ectomycorrhizal and the increase of arbuscular mycorrhizal fungi. Wildfire changed the community composition of pathogenic fungi but did not affect their proportion and diversity. Fungal biomass declined with disturbances that disrupted the forest floor. Our results suggest that the disruption of the forest floor with disturbances, and the changes in C and nutrient dynamics it may promote, structure the fungal community with implications for fungal biomass-C.