Seasonality and longer-term development generate temporal dynamics in the Populus microbiome.

Temporal variation in community composition is central to our understanding of the assembly and functioning of microbial communities, yet the controls over temporal dynamics for microbiomes of long-lived plants, such as trees, remain unclear. Temporal variation in tree microbiomes could arise primarily from seasonal (i.e., intra-annual) fluctuations in community composition or from longer-term changes across years as host plants age. To test these alternatives, we experimentally isolated temporal variation in plant microbiome composition using a common garden and clonally propagated plants, and we used amplicon sequencing to characterize bacterial/archaeal and fungal communities in the leaf endosphere, root endosphere, and rhizosphere of two Populus spp. over four seasons across two consecutive years. Microbial community composition differed among seasons and years (which accounted for up to 21% of the variation in microbial community composition) and was correlated with seasonal dissimilarity in climatic conditions. However, microbial community dissimilarity was also positively correlated with time, reflecting longer-term compositional shifts as host trees aged. Together, our findings demonstrate that temporal patterns in tree microbiomes arise from both seasonal fluctuations and longer-term changes, which interact to generate unique seasonal patterns each year. In addition to shedding light on two important controls over the assembly of plant microbiomes, our results also suggest future studies of tree microbiomes should account for background temporal dynamics when testing the drivers of spatial patterns in microbial community composition and temporal responses of plant microbiomes to environmental change.IMPORTANCEMicrobiomes are integral to the health of host plants, but we have a limited understanding of the factors that control how the composition of plant microbiomes changes over time. Especially little is known about the microbiome of long-lived trees, relative to annual and non-woody plants. We tested how tree microbiomes changed between seasons and years in poplar (genus Populus), which are widespread and ecologically important tree species that also serve as important biofuel feedstocks. We found the composition of bacterial, archaeal, and fungal communities differed among seasons, but these seasonal differences depended on year. This dependence was driven by longer-term changes in microbial composition as host trees developed across consecutive years. Our findings suggest that temporal variation in tree microbiomes is driven by both seasonal fluctuations and longer-term (i.e., multiyear) development.

Signatures of prescribed fire in the microbial communities of Cornus florida are largely undetectable five months post-fire.

Prescribed burn is a management tool that influences the physical structure and composition of forest plant communities and their associated microorganisms. Plant-associated microorganisms aid in host plant disease tolerance and increase nutrient availability. The effects of prescribed burn on microorganisms associated with native ecologically and economically important tree species, such as Cornus florida L. (flowering dogwood), are not well understood, particularly in aboveground plant tissues (e.g., leaf, stem, and bark tissues). The objective of this study was to use 16S rRNA gene and ITS2 region sequencing to evaluate changes in bacterial and fungal communities of five different flowering dogwood-associated niches (soil, roots, bark, stem, and leaves) five months following a prescribed burn treatment. The alpha- and beta-diversity of root bacterial/archaeal communities differed significantly between prescribed burn and unburned control-treated trees. In these bacterial/archaeal root communities, we also detected a significantly higher relative abundance of sequences identified as Acidothermaceae, a family of thermophilic bacteria. No significant differences were detected between prescribed burn-treated and unburned control trees in bulk soils or bark, stem, or leaf tissues. The findings of our study suggest that prescribed burn does not significantly alter the aboveground plant-associated microbial communities of flowering dogwood trees five months following the prescribed burn application. Further studies are required to better understand the short- and long-term effects of prescribed burns on the microbial communities of forest trees.

Relationships between Sphaerulina musiva Infection and the Populus Microbiome and Metabolome.

Pathogenic fungal infections in plants may, in some cases, lead to downstream systematic impacts on the plant metabolome and microbiome that may either alleviate or exacerbate the effects of the fungal pathogen. While Sphaerulina musiva is a well-characterized fungal pathogen which infects Populus tree species, an important wood fiber and biofuel feedstock, little is known about its systematic effects on the metabolome and microbiome of Populus. Here, we investigated the metabolome of Populus trichocarpa and Populus deltoides leaves and roots and the microbiome of the leaf and root endospheres, phylloplane, and rhizosphere to understand the systematic impacts of S. musiva abundance and infection on Populus species in a common garden field setting. We found that S. musiva is indeed present in both P. deltoides and P. trichocarpa, but S. musiva abundance was not statistically related to stem canker onset. We also found that the leaf and root metabolomes significantly differ between the two Populus species and that certain leaf metabolites, particularly the phenolic glycosides salirepin and salireposide, are diminished in canker-infected P. trichocarpa trees compared to their uninfected counterparts. Furthermore, we found significant associations between the metabolome, S. musiva abundance, and microbiome composition and α-diversity, particularly in P. trichocarpa leaves. Our results show that S. musiva colonizes both resistant and susceptible hosts and that the effects of S. musiva on susceptible trees are not confined to the site of canker infection. IMPORTANCE Poplar (Populus spp.) trees are ecologically and economically important trees throughout North America. However, many western North American poplar plantations are at risk due to the introduction of the nonnative fungal pathogen Sphaerulina musiva, which causes leaf spot and cankers, limiting their production. To better understand the interactions among the pathogen S. musiva, the poplar metabolome, and the poplar microbiome, we collected leaf, root, and rhizosphere samples from poplar trees consisting of 10 genotypes and two species with differential resistance to S. musiva in a common garden experiment. Here, we outline the nuanced relationships between the poplar metabolome, microbiome, and S. musiva, showing that S. musiva may affect poplar trees in tissues distal to the site of infection (i.e., stem). Our research contributes to improving the fundamental understanding of S. musiva and Populus sp. ecology and the utility of a holobiont approach in understanding plant disease.

Ecosystem consequences of introducing plant growth promoting rhizobacteria to managed systems and potential legacy effects.

The rapidly growing industry of crop biostimulants leverages the application of plant growth promoting rhizobacteria (PGPR) to promote plant growth and health. However, introducing nonnative rhizobacteria may impact other aspects of ecosystem functioning and have legacy effects; these potential consequences are largely unexplored. Nontarget consequences of PGPR may include changes in resident microbiomes, nutrient cycling, pollinator services, functioning of other herbivores, disease suppression, and organic matter persistence. Importantly, we lack knowledge of whether these ecosystem effects may manifest in adjacent ecosystems. The introduced PGPR can leave a functional legacy whether they persist in the community or not. Legacy effects include shifts in resident microbiomes and their temporal dynamics, horizontal transfer of genes from the PGPR to resident taxa, and changes in resident functional groups and interaction networks. Ecosystem functions may be affected by legacies PGPR leave following niche construction, such as when PGPR alter soil pH that in turn alters biogeochemical cycling rates. Here, we highlight new research directions to elucidate how introduced PGPR impact resident microbiomes and ecosystem functions and their capacity for legacy effects.

Incorporating concentration-dependent sediment microbial activity into methylmercury production kinetics modeling.

In anoxic environments, anaerobic microorganisms carrying the hgcAB gene cluster can mediate the transformation of inorganic mercury (Hg(II)) to monomethylmercury (MMHg). The kinetics of Hg(II) transformation to MMHg in periphyton from East Fork Poplar Creek (EFPC) in Oak Ridge, TN have previously been modeled using a transient availability model (TAM). The TAM for Hg(II) methylation combines methylation/demethylation kinetics with kinetic expressions for processes that decrease Hg(II) and MMHg availability for methylation and demethylation (multisite sorption of Hg(II) and MMHg, Hg(II) reduction/Hg(0) oxidation). In this study, the TAM is used for the first time to describe MMHg production in sediment. We assessed MMHg production in sediment microcosms using two different sediment types from EFPC: a relatively anoxic, carbon-rich sediment with higher microbial activity (higher CO2 production from sediment) and a relatively oxic, sandy, carbon-poor sediment with lower microbial activity (lower CO2 production from sediment). Based on 16s rRNA sequencing, the overall microbial community structure in the two sediments was retained during the incubations. However, the hgcA containing methanogenic Euryarchaeota communities differed between sediment types and their growth followed different trajectories over the course of incubations, potentially contributing to the distinct patterns of MMHg production observed. The general TAM paradigm performed well in describing MMHg production in the sediments. However, the MMHg production and ancillary data suggested the need to revise the model structure to incorporate terms for concentration-dependent microbial activity over the course of the incubations. We modified the TAM to include Monod-type kinetics for methylation and demethylation and observed an improved fit for the carbon-rich, microbially active sediment. Overall our work shows that the TAM can be applied to describe Hg(II) methylation in sediments and that including expressions accounting for concentration-dependent microbial activity can improve the accuracy of the model description of the data in some cases.

Nitrogen addition alters soil fungal communities, but root fungal communities are resistant to change.

Plants are colonized by numerous microorganisms serving important symbiotic functions that are vital to plant growth and success. Understanding and harnessing these interactions will be useful in both managed and natural ecosystems faced with global change, but it is still unclear how variation in environmental conditions and soils influence the trajectory of these interactions. In this study, we examine how nitrogen addition alters plant-fungal interactions within two species of Populus - Populus deltoides and P. trichocarpa. In this experiment, we manipulated plant host, starting soil (native vs. away for each tree species), and nitrogen addition in a fully factorial replicated design. After ~10 weeks of growth, we destructively harvested the plants and characterized plant growth factors and the soil and root endosphere fungal communities using targeted amplicon sequencing of the ITS2 gene region. Overall, we found nitrogen addition altered plant growth factors, e.g., plant height, chlorophyll density, and plant N content. Interestingly, nitrogen addition resulted in a lower fungal alpha diversity in soils but not plant roots. Further, there was an interactive effect of tree species, soil origin, and nitrogen addition on soil fungal community composition. Starting soils collected from Oregon and West Virginia were dominated by the ectomycorrhizal fungi Inocybe (55.8% relative abundance), but interestingly when P. deltoides was grown in its native West Virginia soil, the roots selected for a high abundance of the arbuscular mycorrhizal fungi, Rhizophagus. These results highlight the importance of soil origin and plant species on establishing plant-fungal interactions.

Cultivating the Bacterial Microbiota of Populus Roots.

The integral role of microbial communities in plant growth and health is now widely recognized, and, increasingly, the constituents of the microbiome are being defined. While phylogenetic surveys have revealed the taxa present in a microbiome and show that this composition can depend on, and respond to, environmental perturbations, the challenge shifts to determining why particular microbes are selected and how they collectively function in concert with their host. In this study, we targeted the isolation of representative bacterial strains from environmental samples of Populus roots using a direct plating approach and compared them to amplicon-based sequencing analysis of root samples. The resulting culture collection contains 3,211 unique isolates representing 10 classes, 18 orders, 45 families, and 120 genera from 6 phyla, based on 16S rRNA gene sequence analysis. The collection accounts for ∼50% of the natural community of plant-associated bacteria as determined by phylogenetic analysis. Additionally, a representative set of 553 had their genomes sequenced to facilitate functional analyses. The top sequence variants in the amplicon data, identified as Pseudomonas, had multiple representatives within the culture collection. We then explore a simplified microbiome, comprised of 10 strains representing abundant taxa from environmental samples, and tested for their ability to reproducibly colonize Populus root tissue. The 10-member simplified community was able to reproducibly colonize on Populus roots after 21 days, with some taxa found in surface-sterilized aboveground tissue. This study presents a comprehensive collection of bacteria isolated from Populus for use in exploring microbial function and community inoculation experiments to understand basic concepts of plant and environmental selection. IMPORTANCE Microbial communities play an integral role in the health and survival of their plant hosts. Many studies have identified key members in these communities and led to the use of synthetic communities for elucidating their function; however, these studies are limited by the available cultured bacterial representatives. Here, we present a bacterial culture collection comprising 3,211 isolates that is representative of the root community of Populus. We then demonstrate the ability to examine underlying microbe-microbe interactions using a synthetic community approach. This culture collection will allow for the greater exploration of the microbial community function through targeted experimentation and manipulation.

Assembly of the Populus Microbiome Is Temporally Dynamic and Determined by Selective and Stochastic Factors.

Recent work shows that the plant microbiome, particularly the initial assembly of this microbiome, influences plant health, survival, and fitness. Here, we characterize the initial assembly of the Populus microbiome across ten genotypes belonging to two poplar species in a common garden using 16S rRNA gene and ITS2 region amplicon sequencing of the leaf endosphere, leaf surface, root endosphere, and rhizosphere. We sampled these microbiomes three times throughout the first growing season and found that the composition of the microbiome changed dramatically over time across all plant-associated habitats and host genotypes. For archaea and bacteria, these changes were dominated by strong homogenizing selection (accounting for 29 to 62% of pairwise comparisons). However, fungal assembly was generally characterized by multiple ecological assembly processes (i.e., a mix of weak selective and dispersal processes). Interestingly, genotype, while a significant moderator of microbiome composition, generally explained less variation than sample date across plant-associated habitats. We defined a set of core genera that accounted for, on average, 36% of the microbiome. The relative abundance of this core community was consistent over time. Additionally, using source tracking modeling, we determined that new microbial taxa colonize from both aboveground and belowground sources, and combined with our ecological assembly null models, we found that both selective and dispersal processes explained the differences between exo- (i.e., leaf surface and rhizosphere) and endospheric microbiomes. Taken together, our results suggest that the initial assembly of the Populus microbiome is time-, genotype-, and habitat-dependent and is moderated by both selective and stochastic factors. IMPORTANCE The initial assembly of the plant microbiome may establish the trajectory of forthcoming microbiome states, which could determine the overall future health of the plant. However, while much is known about the initial microbiome assembly of grasses and agricultural crops, less is known about the initial microbiome of long-lived trees, such as poplar (Populus spp.). Thus, a greater understanding of initial plant microbiome assembly in an ecologically and economically important plant such as Populus is highly desirable. Here, we show that the initial microbiome community composition and assembly in the first growing season of Populus is temporally dynamic and is determined by a combination of both selective and stochastic factors. Our findings could be used to prescribe ecologically informed microbial inoculations and better predict the composition of the Populus microbiome into the future and to better understand its influence on plant health.

Nutrient Exposure Alters Microbial Composition, Structure, and Mercury Methylating Activity in Periphyton in a Contaminated Watershed.

The conversion of mercury (Hg) to monomethylmercury (MMHg) is a critical area of concern in global Hg cycling. Periphyton biofilms may harbor significant amounts of MMHg but little is known about the Hg-methylating potential of the periphyton microbiome. Therefore, we used high-throughput amplicon sequencing of the 16S rRNA gene, ITS2 region, and Hg methylation gene pair (hgcAB) to characterize the archaea/bacteria, fungi, and Hg-methylating microorganisms in periphyton communities grown in a contaminated watershed in East Tennessee (United States). Furthermore, we examined how nutrient amendments (nitrate and/or phosphate) altered periphyton community structure and function. We found that bacterial/archaeal richness in experimental conditions decreased in summer and increased in autumn relative to control treatments, while fungal diversity generally increased in summer and decreased in autumn relative to control treatments. Interestingly, the Hg-methylating communities were dominated by Proteobacteria followed by Candidatus Atribacteria across both seasons. Surprisingly, Hg methylation potential correlated with numerous bacterial families that do not contain hgcAB, suggesting that the overall microbiome structure of periphyton communities influences rates of Hg transformation within these microbial mats. To further explore these complex community interactions, we performed a microbial network analysis and found that the nitrate-amended treatment resulted in the highest number of hub taxa that also corresponded with enhanced Hg methylation potential. This work provides insight into community interactions within the periphyton microbiome that may contribute to Hg cycling and will inform future research that will focus on establishing mixed microbial consortia to uncover mechanisms driving shifts in Hg cycling within periphyton habitats.

Fire alters plant microbiome assembly patterns: integrating the plant and soil microbial response to disturbance.

It is increasingly evident that the plant microbiome is a strong determinant of plant health. While the ability to manipulate the microbiome in plants and ecosystems recovering from disturbance may be useful, our understanding of the plant microbiome in regenerating plant communities is currently limited. Using 16S ribosomal RNA (rRNA) gene and internal transcribed spacer (ITS) region amplicon sequencing, we characterized the leaf, stem, fine root, rhizome, and rhizosphere microbiome of < 1-yr-old aspen saplings and the associated bulk soil after a recent high-intensity prescribed fire across a burn severity gradient. Consistent with previous studies, we found that soil microbiomes are responsive to fire. We extend these findings by showing that certain plant tissue microbiomes also change in response to fire. Differences in soil microbiome compositions could be attributed to soil chemical characteristics, but, generally, plant tissue microbiomes were not related to plant tissue elemental concentrations. Using source tracking modeling, we also show that fire influences the relative dominance of microbial inoculum and the vertical inheritance of the sapling microbiome from the parent tree. Overall, our results demonstrate how fire impacts plant microbiome assembly, diversity, and composition and highlights potential for further research towards increasing plant fitness and ecosystem recovery after fire events.

In Vivo Entombment of Bacteria and Fungi during Calcium Oxalate, Brushite, and Struvite Urolithiasis.

Background: Human kidney stones form via repeated events of mineral precipitation, partial dissolution, and reprecipitation, which are directly analogous to similar processes in other natural and manmade environments, where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Methods: Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, whereas one patient formed each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from seven of these 20 patients (five CaOx, one brushite, and one struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, super-resolution autofluorescence (SRAF), and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1-V3, V3-V5) and internal transcribed spacer (ITS1, ITS2) regions. Results: Bulk-entombed DNA was sequenced from stone fragments in 11 of the 18 patients who formed CaOx stones, and the patients who formed brushite and struvite stones. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Aspergillus niger. Bacterial cells approximately 1 µm in diameter were also optically observed to be entombed and well preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. Conclusions: These results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.

Microbiome Variation Across Two Hemlock Species With Hemlock Woolly Adelgid Infestation.

The hemlock woolly adelgid (Adelges tsugae, HWA), an invasive insect, is devastating native hemlock populations in eastern North America, and management outcomes have so far had limited success. While many plant microbiomes influence and even support plant immune responses to insect herbivory, relatively little is known about the hemlock microbiome and its interactions with pathogens or herbivores such as HWA. Using 16S rRNA and ITS gene amplicon sequencing, we characterized the needle, branch, root, and rhizosphere microbiome of two hemlock species, Tsuga canadensis and T. sieboldii, that displayed low and high levels of HWA populations. We found that both archaeal/bacterial and fungal needle communities, as well as the archaeal/bacterial branch and root communities, varied in composition in both hemlock species relative to HWA population levels. While host species and plant-associated habitats explained a greater proportion of the variance in the microbiome than did HWA population level, high HWA populations were associated with enrichment of 100 likely fungal pathogen sequence variants across the four plant-associated habitats (e.g., needle, branch, root, rhizosphere) compared to trees with lower HWA populations. This work contributes to a growing body of literature linking plant pathogens and pests with the changes in the associated plant microbiome and host health. Furthermore, this work demonstrates the need to further investigate plant microbiome effects across multiple plant tissues to understand their influences on host health.

Plant Hosts Modify Belowground Microbial Community Response to Extreme Drought.

Drought stress negatively impacts microbial activity, but the magnitude of stress responses is likely dependent on a diversity of belowground interactions. Populus trichocarpa individuals and no-plant bulk soils were exposed to extended drought (∼0.03% gravimetric water content [GWC] after 12 days), rewet, and a 12-day "recovery" period to determine the effects of plant presence in mediating soil microbiome stability to water stress. Plant metabolomic analyses indicated that drought exposure increased host investment in C and N metabolic pathways (amino acids, fatty acids, phenolic glycosides) regardless of recovery. Several metabolites positively correlated with root-associated microbial alpha-diversity, but not those of soil communities. Soil bacterial community composition shifted with P. trichocarpa presence and with drought relative to irrigated controls, whereas soil fungal composition shifted only with plant presence. However, root fungal communities strongly shifted with drought, whereas root bacterial communities changed to a lesser degree. The proportion of bacterial water-stress opportunistic operational taxonomic units (OTUs) (enriched counts in drought) was high (∼11%) at the end of drying phases and maintained after rewet and recovery phases in bulk soils, but it declined over time in soils with plants present. For root fungi, opportunistic OTUs were high at the end of recovery in drought treatments (∼17% abundance), although relatively not responsive in soils, particularly planted soils (<0.5% abundance for sensitive or opportunistic). These data indicate that plants modulate soil and root-associated microbial drought responses via tight plant-microbe linkages during extreme drought scenarios, but trajectories after extreme drought vary with plant habitat and microbial functional groups.IMPORTANCE Climate change causes significant alterations in precipitation and temperature regimes that are predicted to become more extreme throughout the next century. Microorganisms are important members within ecosystems, and how they respond to these changing abiotic stressors has large implications for the functioning of ecosystems, the recycling of nutrients, and the health of the aboveground plant community. Drought stress negatively impacts microbial activity, but the magnitude of this stress response may be dependent on above- and belowground interactions. This study demonstrates that beneficial associations between plants and microbes can enhance tolerance to abiotic stress.

One-time nitrogen fertilization shifts switchgrass soil microbiomes within a context of larger spatial and temporal variation.

Soil microbiome responses to short-term nitrogen (N) inputs remain uncertain when compared with previous research that has focused on long-term fertilization responses. Here, we examined soil bacterial/archaeal and fungal communities pre- and post-N fertilization in an 8 year-old switchgrass field, in which twenty-four plots received N fertilization at three levels (0, 100, and 200 kg N ha-1 as NH4NO3) for the first time since planting. Soils were collected at two depths, 0-5 and 5-15 cm, for DNA extraction and amplicon sequencing of 16S rRNA genes and ITS regions for assessment of microbial community composition. Baseline assessments prior to fertilization revealed no significant pre-existing divergence in either bacterial/archaeal or fungal communities across plots. The one-time N fertilizations increased switchgrass yields and tissue N content, and the added N was nearly completely removed from the soil of fertilized plots by the end of the growing season. Both bacterial/archaeal and fungal communities showed large spatial (by depth) and temporal variation (by season) within each plot, accounting for 17 and 12-22% of the variation as calculated from the Sq. root of PERMANOVA tests for bacterial/archaeal and fungal community composition, respectively. While N fertilization effects accounted for only ~4% of overall variation, some specific microbial groups, including the bacterial genus Pseudonocardia and the fungal genus Archaeorhizomyces, were notably repressed by fertilization at 200 kg N ha-1. Bacterial groups varied with both depth in the soil profile and time of sampling, while temporal variability shaped the fungal community more significantly than vertical heterogeneity in the soil. These results suggest that short-term effects of N fertilization are significant but subtle, and other sources of variation will need to be carefully accounted for study designs including multiple intra-annual sampling dates, rather than one-time "snapshot" analyses that are common in the literature. Continued analyses of these trends over time with fertilization and management are needed to understand how these effects may persist or change over time.

Rhizosphere microbiomes diverge among Populus trichocarpa plant-host genotypes and chemotypes, but it depends on soil origin.

BACKGROUND: Plants have developed defense strategies for phytopathogen and herbivore protection via coordinated metabolic mechanisms. Low-molecular weight metabolites produced within plant tissues, such as salicylic acid, represent one such mechanism which likely mediates plant - microbe interactions above and below ground. Salicylic acid is a ubiquitous phytohormone at low levels in most plants, yet are concentrated defense compounds in Populus, likely acting as a selective filter for rhizosphere microbiomes. We propagated twelve Populus trichocarpa genotypes which varied an order of magnitude in salicylic acid (SA)-related secondary metabolites, in contrasting soils from two different origins. After four months of growth, plant properties (leaf growth, chlorophyll content, and net photosynthetic rate) and plant root metabolomics specifically targeting SA metabolites were measured via GC-MS. In addition, rhizosphere microbiome composition was measured via Illumina MiSeq sequencing of 16S and ITS2 rRNA-genes. RESULTS: Soil origin was the primary filter causing divergence in bacterial/archaeal and fungal communities with plant genotype secondarily influential. Both bacterial/archaeal and fungal evenness varied between soil origins and bacterial/archaeal diversity and evenness correlated with at least one SA metabolite (diversity: populin; evenness: total phenolics). The production of individual salicylic acid derivatives that varied by host genotype resulted in compositional differences for bacteria /archaea (tremuloidin) and fungi (salicylic acid) within one soil origin (Clatskanie) whereas soils from Corvallis did not illicit microbial compositional changes due to salicylic acid derivatives. Several dominant bacterial (e.g., Betaproteobacteria, Acidobacteria, Verrucomicrobia, Chloroflexi, Gemmatimonadete, Firmicutes) and one fungal phyla (Mortierellomycota) also correlated with specific SA secondary metabolites; bacterial phyla exhibited more negative interactions (declining abundance with increasing metabolite concentration) than positive interactions. CONCLUSIONS: These results indicate microbial communities diverge most among soil origin. However, within a soil origin, bacterial/archaeal communities are responsive to plant SA production within greenhouse-based rhizosphere microbiomes. Fungal microbiomes are impacted by root SA-metabolites, but overall to a lesser degree within this experimental context. These results suggest plant defense strategies, such as SA and its secondary metabolites, may partially drive patterns of both bacterial/archaeal and fungal taxa-specific colonization and assembly.

Methanogenic Archaea dominate mature heartwood habitats of Eastern Cottonwood (Populus deltoides).

While recent reports demonstrate that the direct emission of methane from living tree trunks may be a significant terrestrial emission source, there has been debate whether tree emissions are due to transport from soils or produced in the wood environment itself. Reports of methanogens from wood of trees were prominent in the literature 40 years ago but have not been revisited with molecular ecology approaches. We examined communities associated with Populus deltoides using rRNA gene sequence analyses and how these vary with tree and wood properties. Our data indicate that wood environments are dominated by anaerobic microbiomes. Methanogens are prominent in heartwood (mean 34% relative abundance) compared to sapwood environments (13%), and dominant operational taxonomic units (OTUs) were classified as the Methanobacterium sp. Members of the Firmicutes phylum comprised 39% of total sequences and were in 42% greater abundance in sapwood over heartwood niches. Tree diameter was the strongest predictor of methanogen abundance, but wood moisture content and pH were also significant predictors of taxon abundance and overall community composition. Unlike microbiomes of the soil, rhizosphere and phyllosphere, wood associated communities are shaped by unique environmental conditions and may be prominent and overlooked sources of methane emissions in temperate forest systems.

Microbial communities respond to experimental warming, but site matters.

Because microorganisms are sensitive to temperature, ongoing global warming is predicted to influence microbial community structure and function. We used large-scale warming experiments established at two sites near the northern and southern boundaries of US eastern deciduous forests to explore how microbial communities and their function respond to warming at sites with differing climatic regimes. Soil microbial community structure and function responded to warming at the southern but not the northern site. However, changes in microbial community structure and function at the southern site did not result in changes in cellulose decomposition rates. While most global change models rest on the assumption that taxa will respond similarly to warming across sites and their ranges, these results suggest that the responses of microorganisms to warming may be mediated by differences across the geographic boundaries of ecosystems.

The effects of insects, nutrients, and plant invasion on community structure and function above-and belowground.

Soil nutrient availability, invasive plants, and insect presence can directly alter ecosystem structure and function, but less is known about how these factors may interact. In this 6-year study in an old-field ecosystem, we manipulated insect abundance (reduced and control), the propagule pressure of an invasive nitrogen-fixing plant (propagules added and control), and soil nutrient availability (nitrogen added, nitrogen reduced and control) in a fully crossed, completely randomized plot design. We found that nutrient amendment and, occasionally, insect abundance interacted with the propagule pressure of an invasive plant to alter above-and belowground structure and function at our site. Not surprisingly, nutrient amendment had a direct effect on aboveground biomass and soil nutrient mineralization. The introduction of invasive nitrogen-fixing plant propagules interacted with nutrient amendment and insect presence to alter soil bacterial abundance and the activity of the microbial community. While the larger-scale, longer-term bulk measurements such as biomass production and nutrient mineralization responded to the direct effects of our treatments, the shorter-term and dynamic microbial communities tended to respond to interactions among our treatments. Our results indicate that soil nutrients, invasive plants, and insect herbivores determine both above-and belowground responses, but whether such effects are independent versus interdependent varies with scale.