Choices on sampling, sequencing, and analyzing DNA influence the estimation of community composition of plant fungal symbionts.

Plant root symbionts, namely mycorrhizal fungi, can be characterized using a variety of methods, but most of these rely on DNA. While Sanger sequencing still fulfills particular research objectives, next-generation sequencing currently dominates the field, thus understanding how the two methods differ is important for identifying both opportunities and limitations to characterizing fungal communities. In addition to testing sequencing methods, we also examined how roots and soils may yield different fungal communities and how disturbance may affect those differences. We sequenced DNA from ectomycorrhizal fungi colonizing roots of Pinus banksiana and found that operational taxonomic unit richness was higher, and compositional variance lower, for Illumina MiSeq-sequenced communities compared to Sanger-sequenced communities. We also found that fungal communities associated with roots were distinct in composition compared to those associated with soils and, moreover, that soil-associated fungi were more clustered in composition than those of roots. Finally, we found community dissimilarity between roots and soils was insensitive to disturbance; however, rarefying read counts had a sizeable influence on trends in fungal richness. Although interest in mycorrhizal communities is typically focused on the abiotic and biotic filters sorting fungal species, our study shows that the choice of methods to sample, sequence, and analyze DNA can also influence the estimation of community composition.

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.

Exposure to Fungal Volatiles Can Influence Volatile Emissions From Other Ophiostomatoid Fungi.

Fungal volatile organic compounds (FVOCs) can act as intra- and inter-kingdom communication signals that influence the growth and behaviors of organisms involved in antagonistic or mutualistic relationships with fungi. There is growing evidence suggesting that FVOCs can mediate interactions between organisms within and across different ecological niches. Bark beetles have established mutualistic relationships with ophiostomatoid fungi which can serve as a food source and condition host plant tissues for developing beetle larvae. While the profiles (both composition and concentrations) of volatile emission from ophiostomatoid fungi can be influenced by abiotic factors, whether emissions from a given fungal species can be influenced by those from another is still unknown. Here, we analyzed FVOCs emitted from the two ophiostomatoid fungi, Grosmannia clavigera and Ophiostoma ips, associated with mountain pine beetle and pine engraver beetle, respectively, when each fungus was growing alone or in a shared headspace. We used two isolates of each fungus species. Overall, we detected a total of eight volatiles in both G. clavigera alone or in combination with O. ips including acetoin, ethyl acetate, cis-grandisol, isoamyl alcohol, isobutanol, 2-methyl-1-butanol, phenethyl acetate, and phenethyl alcohol. The profiles of volatiles emitted differed between the two fungal species but not between the two isolates of the same fungus. Six compounds were common between the species, whereas two compounds were detected only when G. clavigera was present. Moreover, the majority of volatiles were detected less frequently and at lower concentrations when the two fungi were grown together in a shared headspace. These results are likely due to reduced volatile emissions from O. ips in the presence of G. clavigera. However, changes in the profiles of fungal volatiles did not correspond with the observed changes in the growth of either species. Overall, these results suggest that the similarities in fungal volatiles among different species of fungi may reflect a common ecological niche and that the differences may correspond to species-specific adaptation to their respective host beetles or genetic factors.

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.

Induced Defenses of a Novel Host Tree Affect the Growth and Interactions of Bark Beetle-Vectored Fungi.

Mountain pine beetle (MPB) has recently expanded its host range to the novel jack pine forests in Alberta. Invasion success of MPB may depend on the outcome of interactions between its symbiotic fungus Grosmannia clavigera and Ophiostoma ips, a fungal associate of a potential competitor Ips pini. However, how the quality of jack pine phloem could influence interactions between the fungi is unknown. We investigated whether introduced concentrations of host nitrogen and monoterpenes affect the growth of and interaction between the fungi. Nitrogen concentrations did not affect the growth rate of either fungus. In the absence of monoterpenes, the presence of O. ips promoted G. clavigera growth. Monoterpenes either promoted or inhibited the growth of both fungi, and altered the outcome of species interactions from facilitation to no-effect. Overall, these results suggest that jack pine phloem quality and the presence of a niche-sharing fungus could influence MPB development.

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.

Effects of Pheromone Dose and Conspecific Density on the Use of Aggregation-Sex Pheromones by the Longhorn Beetle Phymatodes grandis and Sympatric Species (Coleoptera: Cerambycidae).

Many species of longhorn beetles (Coleoptera: Cerambycidae) utilize male-produced aggregation-sex pheromones that attract both sexes. However, the reasons why and the details of how this type of pheromone is used by cerambycids and other coleopteran species that utilize analogous male-produced pheromones remain unclear. Thus, our goals were to test the hypotheses that 1) cerambycids respond to pheromones in a dose-dependent (= release rate-dependent) manner and 2) pheromone emission is density-dependent. If true, these characteristics of pheromone use could suggest that cerambycids utilize an optimal density strategy to limit competition for scarce and ephemeral hosts, i.e., the stressed or dying trees that typically constitute their larval hosts. Attraction of beetles to a range of release rates of two common pheromone components - 2-methylbutanol and 3-hydroxyhexan-2-one - was tested in field trials. Responses, as measured by the number of beetles caught in pheromone-baited traps, increased with release rates for five endemic species, even at the highest rates tested (~1450 µg/h for 2-methylbutanol and ~720 µg/h for 3-hydroxyhexan-2-one). The effect of density of conspecific males on per capita pheromone production was tested by collecting the volatiles produced by individuals, pairs, or groups of three or four male Phymatodes grandis Casey. Frequency of pheromone production was significantly different among the treatment densities, and emission rates of the pheromone (R)-2-methylbutanol decreased with increasing density. These results are discussed in the context of a possible optimal density strategy used by cerambycids, and more broadly, in relation to the use of male-produced aggregation-sex pheromones by other coleopterans. In addition, we report the identification of the pheromones of four of our five test species, specifically, Phymatodes obliquus Casey, Brothylus conspersus LeConte, Brothylus gemmulatus LeConte, and Xylotrechus albonotatus Casey.

Ectomycorrhizal fungal species differentially affect the induced defensive chemistry of lodgepole pine.

Plants interact simultaneously with multiple organisms, including ectomycorrhizal (EM) fungal symbionts which benefit plants by facilitating resource acquisition. Yet, their role in induced plant defenses that rely on the allocation of plant resources has received little attention. We investigated whether EM fungi can affect the induction of defense-related monoterpenes in greenhouse-grown lodgepole pine (Pinus contorta var. latifolia) seedlings, and whether such effects differed between EM fungal species occurring alone or in combination. Fungal interactions on growth media were also assessed to complement the greenhouse study. Our study revealed that the production of certain monoterpenes is influenced by the fungal species colonizing pine roots. Furthermore, pine seedlings did not necessarily benefit from having associations with multiple EM fungi, as we found contrasting effects of single vs. multiple species of fungi on induced monoterpene responses. Finally, monoterpene responses were altered when early-colonizing species inhibited the colonization or development of later-arriving species. We conclude that the presence of EM fungi can impact host susceptibility to insect and pathogen attack, suggesting that seedlings establishing in areas lacking fungi that promote the induction of tree defense chemicals may suffer from increased susceptibility to future pest damage.

Successful Colonization of Lodgepole Pine Trees by Mountain Pine Beetle Increased Monoterpene Production and Exhausted Carbohydrate Reserves.

Lodgepole pine (Pinus contorta) forests have experienced severe mortality from mountain pine beetle (MPB) (Dendroctonus ponderosae Hopkins) in western North America for the last several years. Although the mechanisms by which beetles kill host trees are unclear, they are likely linked to pine defense monoterpenes that are synthesized from carbohydrate reserves. However, how carbohydrates and monoterpenes interact in response to MPB colonization is unknown. Understanding this relationship could help to elucidate how pines succumb to bark beetle attack. We compared concentrations of individual and total monoterpenes and carbohydrates in the phloem of healthy pine trees with those naturally colonized by MPB. Trees attacked by MPB had nearly 300% more monoterpenes and 40% less carbohydrates. Total monoterpene concentrations were most strongly associated with the concentration of sugars in the phloem. These results suggest that bark beetle colonization likely depletes carbohydrate reserves by increasing the production of carbon-rich monoterpenes, and other carbon-based secondary compounds. Bark beetle attacks also reduce water transport causing the disruption of carbon transport between tree foliage and roots, which restricts carbon assimilation. Reduction in carbohydrate reserves likely contributes to tree mortality.

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.

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.

A Blend of Ethanol and (-)-α-Pinene were Highly Attractive to Native Siricid Woodwasps (Siricidae, Siricinae) Infesting Conifers of the Sierra Nevada and the Allegheny Mountains.

Woodwasps in Sirex and related genera are well-represented in North American conifer forests, but the chemical ecology of native woodwasps is limited to a few studies demonstrating their attraction to volatile host tree compounds, primarily monoterpene hydrocarbons and monoterpene alcohols. Thus, we systematically investigated woodwasp-host chemical interactions in California's Sierra Nevada and West Virginia's Allegheny Mountains. We first tested common conifer monoterpene hydrocarbons and found that (-)-α-pinene, (+)-3-carene, and (-)-ß-pinene were the three most attractive compounds. Based on these results and those of earlier studies, we further tested three monoterpene hydrocarbons and four monoterpene alcohols along with ethanol in California: monoterpene hydrocarbons caught 72.3% of all woodwasps. Among monoterpene hydrocarbons, (+)-3-carene was the most attractive followed by (-)-ß-pinene and (-)-α-pinene. Among alcohols, ethanol was the most attractive, catching 41.4% of woodwasps trapped. Subsequent tests were done with fewer selected compounds, including ethanol, 3-carene, and ethanol plus (-)-α-pinene in both Sierra Nevada and Allegheny Mountains. In both locations, ethanol plus (-)-α-pinene caught more woodwasps than other treatments. We discussed the implications of these results for understanding the chemical ecology of native woodwasps and invasive Sirex noctilio in North America. In California, 749 woodwasps were caught, representing five species: Sirex areolatus Cresson, Sirex behrensii Cresson, Sirex cyaneus Fabricius, Sirex longicauda Middlekauff, and Urocerus californicus Norton. In West Virginia 411 woodwasps were caught representing four species: Sirex edwardsii Brullé, Tremex columba Linnaeus, Sirex nigricornis F., and Urocerus cressoni Norton.

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.

Direction of interaction between mountain pine beetle (Dendroctonus ponderosae) and resource-sharing wood-boring beetles depends on plant parasite infection.

Plant pathogens can have cascading consequences on insect herbivores, though whether they alter competition among resource-sharing insect herbivores is unknown. We experimentally tested whether the infection of a plant pathogen, the parasitic plant dwarf mistletoe (Arceuthobium americanum), on jack pine (Pinus banksiana) altered the competitive interactions among two groups of beetles sharing the same resources: wood-boring beetles (Coleoptera: Cerambycidae) and the invasive mountain pine beetle (Dendroctonus ponderosae) (Coleoptera: Curculionidae). We were particularly interested in identifying potential mechanisms governing the direction of interactions (from competition to facilitation) between the two beetle groups. At the lowest and highest disease severity, wood-boring beetles increased their consumption rate relative to feeding levels at moderate severity. The performance (brood production and feeding) of mountain pine beetle was negatively associated with wood-boring beetle feeding and disease severity when they were reared separately. However, when both wood-boring beetles and high severity of plant pathogen infection occurred together, mountain pine beetle escaped from competition and improved its performance (increased brood production and feeding). Species-specific responses to changes in tree defense compounds and quality of resources (available phloem) were likely mechanisms driving this change of interactions between the two beetle groups. This is the first study demonstrating that a parasitic plant can be an important force in mediating competition among resource-sharing subcortical insect herbivores.

Bacteria influence mountain pine beetle brood development through interactions with symbiotic and antagonistic fungi: implications for climate-driven host range expansion.

Bark beetles are associated with diverse communities of symbionts. Although fungi have received significant attention, we know little about how bacteria, and in particular their interactions with fungi, affect bark beetle reproduction. We tested how interactions between four bacterial associates, two symbiotic fungi, and two opportunistic fungi affect performance of mountain pine beetles (Dendroctonus ponderosae) in host tissue. We compared beetle performance in phloem of its historical host, lodgepole pine (Pinus contorta), and its novel host recently accessed through warming climate, jack pine (Pinus banksiana). Overall, beetles produced more larvae, and established longer ovipositional and larval galleries in host tissue predominantly colonized by the symbiotic fungi, Grosmannia clavigera, or Ophiostoma montium than by the opportunistic colonizer Aspergillus and to a lesser extent, Trichoderma. This occurred in both historical and naïve hosts. Impacts of bacteria on beetle reproduction depended on particular fungus-bacterium combinations and host species. Some bacteria, e.g., Pseudomonas sp. D4-22 and Hy4T4 in P. contorta and Pseudomonas sp. Hy4T4 and Stenotrophomonas in P. banksiana, reduced antagonistic effects by Aspergillus and Trichoderma resulting in more larvae and longer ovipositional and larval galleries. These effects were not selective, as bacteria also reduced beneficial effects by symbionts in both host species. Interestingly, Bacillus enhanced antagonistic effects by Aspergillus in both hosts. These results demonstrate that bacteria influence brood development of bark beetles in host tissue. They also suggest that climate-driven range expansion of D. ponderosae through the boreal forest will not be significantly constrained by requirements of, or interactions among, its microbial associates.