Quantifying the immediate response of the soil microbial community to different grazing intensities on irrigated pastures.

Grazing is known to affect soil microbial communities, nutrient cycling, and forage quantity and quality over time. However, a paucity of information exists for the immediate changes in the soil physicochemical and microbial environment in response to different grazing strategies. Soil microbes drive nutrient cycling and are involved in plant-soil-microbe relationships, making them potentially vulnerable to plant-driven changes in the soil environment caused by grazing. To test the hypothesis that variable grazing intensities modulate immediate effects on the soil microbial community, we conducted a grazing trial of three management approaches; high-intensity, short-duration grazing (HDG), low-intensity, medium-duration grazing (LDG), and no grazing (NG). Soil and vegetation samples were collected before grazing and 24 hours, 1 week, and 4 weeks after HDG grazing ended. Soil labile carbon (C) and nitrogen (N) pools, vegetation biomass, and soil microbial diversity and functional traits were determined, including extracellular enzymatic assays and high-throughput sequencing of the bacterial 16S rRNA and fungal ITS2 regions. We found that labile soil C and inorganic N increased following the LDG grazing while C-cycling extracellular enzymatic activities increased in response to HDG grazing but both total extracellular enzymatic activity profiles and soil abiotic profiles were mostly affected by temporal fluxes. The soil fungal community composition was strongly affected by the interaction of sampling time and grazing treatment, while the soil bacterial community composition was largely affected by sampling time with a lesser impact from grazing treatment. We identified several key fungal taxa that may influence immediate responses to grazing and modulate plant-soil-microbe interactions. There was strong evidence of temporal influences on soil biogeochemical variables and the soil microbiome, even within our narrow sampling scheme. Our results indicate that the soil ecosystem is dynamic and responsive to different grazing strategies within very short time scales, showing the need for further research to understand plant-soil-microbe interactions and how these feedback mechanisms can inform sustainable land management.

Fungal community structure and function shifts with atmospheric nitrogen deposition.

Fungal decomposition of soil organic matter depends on soil nitrogen (N) availability. This ecosystem process is being jeopardized by changes in N inputs that have resulted from a tripling of atmospheric N deposition in the last century. Soil fungi are impacted by atmospheric N deposition due to higher N availability, as soils are acidified, or as micronutrients become increasingly limiting. Fungal communities that persist with chronic N deposition may be enriched with traits that enable them to tolerate environmental stress, which may trade-off with traits enabling organic matter decomposition. We hypothesized that fungal communities would respond to N deposition by shifting community composition and functional gene abundances toward those that tolerate stress but are weak decomposers. We sampled soils at seven eastern US hardwood forests where ambient N deposition varied from 3.2 to 12.6 kg N ha-1  year-1 , five of which also have experimental plots where atmospheric N deposition was simulated through fertilizer application treatments (25-50 kg N ha-1  year-1 ). Fungal community and functional responses to fertilizer varied across the ambient N deposition gradient. Fungal biomass and richness increased with simulated N deposition at sites with low ambient deposition and decreased at sites with high ambient deposition. Fungal functional genes involved in hydrolysis of organic matter increased with ambient N deposition while genes involved in oxidation of organic matter decreased. One of four genes involved in generalized abiotic stress tolerance increased with ambient N deposition. In summary, we found that the divergent response to simulated N deposition depended on ambient N deposition levels. Fungal biomass, richness, and oxidative enzyme potential were reduced by N deposition where ambient N deposition was high suggesting fungal communities were pushed beyond an environmental stress threshold. Fungal community structure and function responses to N enrichment depended on ambient N deposition at a regional scale.

Structural and Functional Dynamics of Soil Microbes following Spruce Beetle Infestation.

As the range of bark beetles expands into new forests and woodlands, the need to understand their effects on multiple trophic levels becomes increasingly important. To date, much attention has been paid to the aboveground processes affected by bark beetle infestation, with a focus on photoautotrophs and ecosystem level processes. However, indirect effects of bark beetle on belowground processes, especially the structure and function of soil microbiota remains largely a black box. Our study examined the impacts of bark beetle-induced tree mortality on soil microbial community structure and function using high-throughput sequencing of the soil bacterial and fungal communities and measurements of extracellular enzyme activities. The results suggest bark beetle infestation affected edaphic conditions through increased soil water content, pH, electrical conductivity, and carbon/nitrogen ratio and altered bulk and rhizosphere soil microbial community structure and function. Finally, increased enzymatic activity suggests heightened microbial decomposition following bark beetle infestation. With this increase in enzymatic activity, nutrients trapped in organic substrates may become accessible to seedlings and potentially alter the trajectory of forest regeneration. Our results indicate the need for incorporation of microbial processes into ecosystem level models.IMPORTANCE Belowground impacts of bark beetle infestation have not been explored as thoroughly as their aboveground counterparts. In order to accurately model impacts of bark beetle-induced tree mortality on carbon and nutrient cycling and forest regeneration, the intricacies of soil microbial communities must be examined. In this study, we investigated the structure and function of soil bacterial and fungal communities following bark beetle infestation. Our results show bark beetle infestation to impact soil conditions, as well as soil microbial community structure and function.

Role of the gut microbiome in mediating standard metabolic rate after dietary shifts in the viviparous cockroach, Diploptera punctata.

Diet may be a significant determinant of insect gut microbiome composition. However, the extent to which dietary shifts shape both the composition and relevant functions of insect gut microbiomes, and ultimately impact host energy balance (i.e. metabolic phenotype), is not well understood. We investigated the impacts of diet switching on Diploptera punctata females maintained on a dog food (DF) diet relative to those fed a comparatively sub-optimal cellulose-amended dog food (CADF) diet for 4 weeks. After this period, dietary shift resulted in a significantly higher average mass-specific standard metabolic rate (SMR) in CADF-fed females compared with DF-fed females. We also uncovered significant 13C-enrichment in DF-fed insect samples relative to CADF-fed insect samples and lowered bacterial essential amino acid (EAA) provisioning in CADF-fed samples. Differences in SMR and EAA provisioning were not accompanied by significant differences in overall microbiome composition between the two groups. However, cellulolytic and nitrogen-fixing bacterial families dominant in wild omnivorous cockroaches and wood-feeding termites were significantly enriched in CADF-fed females than in DF-fed females, at the end of the study. We propose that these changes in microbiome composition after dietary shifts are associated with changes in EAA provisioning and possibly SMR. Further studies are needed to comprehensively understand the relative importance of gut microbial functions among the complexity of factors known to underscore SMR responses in insects under varying dietary conditions.

Species associations overwhelm abiotic conditions to dictate the structure and function of wood-decay fungal communities.

Environmental conditions exert strong controls on the activity of saprotrophic microbes, yet abiotic factors often fail to adequately predict wood decomposition rates across broad spatial scales. Given that species interactions can have significant positive and negative effects on wood-decay fungal activity, one possibility is that biotic processes serve as the primary controls on community function, with abiotic controls emerging only after species associations are accounted for. Here we explore this hypothesis in a factorial field warming- and nitrogen-addition experiment by examining relationships among wood decomposition rates, fungal activity, and fungal community structure. We show that functional outcomes and community structure are largely unrelated to abiotic conditions, with microsite and plot-level abiotic variables explaining at most 19% of the total variability in decomposition and fungal activity, and 2% of the variability in richness and evenness. In contrast, taxonomic richness, evenness, and species associations (i.e., co-occurrence patterns) exhibited strong relationships with community function, accounting for 52% of the variation in decomposition rates and 73% in fungal activity. A greater proportion of positive vs. negative species associations in a community was linked to strong declines in decomposition rates and richness. Evenness emerged as a key mediator between richness and function, with highly even communities exhibiting a positive richness-function relationship and uneven communities exhibiting a negative or null response. These results suggest that community-assembly processes and species interactions are important controls on the function of wood-decay fungal communities, ultimately overwhelming substantial differences in abiotic conditions.

Biotic interactions mediate soil microbial feedbacks to climate change.

Decomposition of organic material by soil microbes generates an annual global release of 50-75 Pg carbon to the atmosphere, ∼7.5-9 times that of anthropogenic emissions worldwide. This process is sensitive to global change factors, which can drive carbon cycle-climate feedbacks with the potential to enhance atmospheric warming. Although the effects of interacting global change factors on soil microbial activity have been a widespread ecological focus, the regulatory effects of interspecific interactions are rarely considered in climate feedback studies. We explore the potential of soil animals to mediate microbial responses to warming and nitrogen enrichment within a long-term, field-based global change study. The combination of global change factors alleviated the bottom-up limitations on fungal growth, stimulating enzyme production and decomposition rates in the absence of soil animals. However, increased fungal biomass also stimulated consumption rates by soil invertebrates, restoring microbial process rates to levels observed under ambient conditions. Our results support the contemporary theory that top-down control in soil food webs is apparent only in the absence of bottom-up limitation. As such, when global change factors alleviate the bottom-up limitations on microbial activity, top-down control becomes an increasingly important regulatory force with the capacity to dampen the strength of positive carbon cycle-climate feedbacks.

Fungi exposed to chronic nitrogen enrichment are less able to decay leaf litter.

Saprotrophic fungi are the primary decomposers of plant litter in temperate forests, and their activity is critical for carbon (C) and nitrogen (N) cycling. Simulated atmospheric N deposition is associated with reduced fungal biomass, shifts in fungal community structure, slowed litter decay, and soil C accumulation. Although rarely studied, N deposition may also result in novel selective pressures on fungi, affecting evolutionary trajectories. To directly test if long-term N enrichment reshapes fungal responses to N, we isolated decomposer fungi from a long-term (28 yr) N-addition experiment and used a common garden approach to compare growth rates and decay abilities of isolates from control and N-amended plots. Both growth and decay were significantly altered by long-term exposure to N enrichment. Changes in growth rates were idiosyncratic, as different species grew either more quickly or more slowly after exposure to N, but litter decay by N isolates was consistent and generally lower compared to control isolates of the same species, a response not readily reversed when N isolates were grown in control (low N) environments. Changes in fungal responses accompany and perhaps drive previously observed N-induced shifts in fungal diversity, community composition, and litter decay dynamics.

Long-Term Warming Alters Carbohydrate Degradation Potential in Temperate Forest Soils.

As Earth's climate warms, soil carbon pools and the microbial communities that process them may change, altering the way in which carbon is recycled in soil. In this study, we used a combination of metagenomics and bacterial cultivation to evaluate the hypothesis that experimentally raising soil temperatures by 5°C for 5, 8, or 20 years increased the potential for temperate forest soil microbial communities to degrade carbohydrates. Warming decreased the proportion of carbohydrate-degrading genes in the organic horizon derived from eukaryotes and increased the fraction of genes in the mineral soil associated with Actinobacteria in all studies. Genes associated with carbohydrate degradation increased in the organic horizon after 5 years of warming but had decreased in the organic horizon after warming the soil continuously for 20 years. However, a greater proportion of the 295 bacteria from 6 phyla (10 classes, 14 orders, and 34 families) isolated from heated plots in the 20-year experiment were able to depolymerize cellulose and xylan than bacterial isolates from control soils. Together, these findings indicate that the enrichment of bacteria capable of degrading carbohydrates could be important for accelerated carbon cycling in a warmer world. IMPORTANCE: The massive carbon stocks currently held in soils have been built up over millennia, and while numerous lines of evidence indicate that climate change will accelerate the processing of this carbon, it is unclear whether the genetic repertoire of the microbes responsible for this elevated activity will also change. In this study, we showed that bacteria isolated from plots subject to 20 years of 5°C of warming were more likely to depolymerize the plant polymers xylan and cellulose, but that carbohydrate degradation capacity is not uniformly enriched by warming treatment in the metagenomes of soil microbial communities. This study illustrates the utility of combining culture-dependent and culture-independent surveys of microbial communities to improve our understanding of the role changing microbial communities may play in soil carbon cycling under climate change.

Extensive trans-specific polymorphism at the mating type locus of the root decay fungus Heterobasidion.

Incompatibility systems in which individuals bearing identical alleles reject each other favor the maintenance of a diversity of alleles. Mushroom mating type loci (MAT) encode for dozens or hundreds of incompatibility alleles whose loss from the population is greatly restricted through negative frequency selection, leading to a system of alleles with highly divergent sequences. Here, we use DNA sequences of homeodomain (HD) encoding genes at the MAT locus of five closely related species of the root rot basidiomycete Heterobasidion annosum sensu lato to show that the extended coalescence time of MAT alleles greatly predates speciation in the group, contrasting loci outside of MAT that show allele divergences largely consistent with the species phylogeny with those of MAT, which show rampant trans-species polymorphism. We observe a roughly 6-fold greater genealogical depth and polymorphism of MAT compared with non-MAT that argues for the maintenance of balanced polymorphism for a minimum duration of 24 My based on a molecular-clock calibrated species phylogeny. As with other basidiomycete HD genes, balancing selection appears to be concentrated at the specificity-determining region in the N-terminus of the protein based on identification of codons under selection and the absence of recombination within the region. However, the elevated polymorphism extends into the nonspecificity determining regions as well as a neighboring non-MAT gene, the mitochondrial intermediate peptidase (MIP). In doing so, increased divergence should decrease recombination among alleles and as a by-product create incompatibilities in the functional domains not involved in allele recognition but in regulating sexual development.

Fungal functioning in a pine forest: evidence from a ¹5N-labeled global change experiment.

• We used natural and tracer nitrogen (N) isotopes in a Pinus taeda free air CO2 enrichment (FACE) experiment to investigate functioning of ectomycorrhizal and saprotrophic fungi in N cycling. • Fungal sporocarps were sampled in 2004 (natural abundance and (15) N tracer) and 2010 (tracer) and δ(15)N patterns were compared against litter and soil pools. • Ectomycorrhizal fungi with hydrophobic ectomycorrhizas (e.g. Cortinarius and Tricholoma) acquired N from the Oea horizon or deeper. Taxa with hydrophilic ectomycorrhizas acquired N from the Oi horizon (Russula and Lactarius) or deeper (Laccaria, Inocybe, and Amanita). (15)N enrichment patterns for Cortinarius and Amanita in 2010 did not correspond to any measured bulk pool, suggesting that a persistent pool of active organic N supplied these two taxa. Saprotrophic fungi could be separated into those colonizing pine cones (Baeospora), wood, litter (Oi), and soil (Ramariopsis), with δ(15)N of taxa reflecting substrate differences. (15)N enrichment between sources and sporocarps varied across taxa and contributed to δ(15)N patterns. • Natural abundance and (15)N tracers proved useful for tracking N from different depths into fungal taxa, generally corresponded to literature estimates of fungal activity within soil profiles, and provided new insights into interpreting natural abundance δ(15)N patterns.

Soil microbiome analysis supports claims of ineffectiveness of Pseudomonas fluorescens D7 as a biocontrol agent of Bromus tectorum.

IMPORTANCE: Cheatgrass is one of North America's most problematic invasive species. Invasion by this annual grass alters ecosystem structure and function and has proven very challenging to remove with traditional approaches. Commercially available bioherbicides, like P. fluorescens D7, are applied with the goal of providing lasting control from a single application. However, experimental results suggest that this bioherbicide has limited efficacy under field conditions. Potential explanations for variable efficacy include a failure of this bioherbicide to establish in the soil microbiome. However, to our knowledge, no data exist to support or refute this hypothesis. Here, we use a deep-sequencing approach to better understand the effects of this bioherbicide on the soil microbiome and screen for P. fluorescens at 18 months post-application.

Comparative Analysis of Core Microbiome Assignments: Implications for Ecological Synthesis.

The concept of a core microbiome has been broadly used to refer to the consistent presence of a set of taxa across multiple samples within a given habitat. The assignment of taxa to core microbiomes can be performed by several methods based on the abundance and occupancy (i.e., detection across samples) of individual taxa. These approaches have led to methodological inconsistencies, with direct implications for ecological interpretation. Here, we reviewed a set of methods most commonly used to infer core microbiomes in divergent systems. We applied these methods using large data sets and analyzed simulations to determine their accuracy in core microbiome assignments. Our results show that core taxa assignments vary significantly across methods and data set types, with occupancy-based methods most accurately defining true core membership. We also found the ability of these methods to accurately capture core assignments to be contingent on the distribution of taxon abundance and occupancy in the data set. Finally, we provide specific recommendations for further studies using core taxa assignments and discuss the need for unifying methodical approaches toward data processing to advance ecological synthesis. IMPORTANCE Different methods are commonly used to assign core microbiome membership, leading to methodological inconsistencies across studies. In this study, we review a set of the most commonly used core microbiome assignment methods and compare their core assignments using both simulated and empirical data. We report inconsistent classifications from commonly applied core microbiome assignment methods. Furthermore, we demonstrate the implication that variable core assignments may have on downstream ecological interpretations. Although we still lack a standardized approach to core taxa assignments, our study provides a direction to properly test core assignment methods and offers advances in model parameterization and method choice across distinct data types.

Insight into trade-off between wood decay and parasitism from the genome of a fungal forest pathogen.

Parasitism and saprotrophic wood decay are two fungal strategies fundamental for succession and nutrient cycling in forest ecosystems. An opportunity to assess the trade-off between these strategies is provided by the forest pathogen and wood decayer Heterobasidion annosum sensu lato. We report the annotated genome sequence and transcript profiling, as well as the quantitative trait loci mapping, of one member of the species complex: H. irregulare. Quantitative trait loci critical for pathogenicity, and rich in transposable elements, orphan and secreted genes, were identified. A wide range of cellulose-degrading enzymes are expressed during wood decay. By contrast, pathogenic interaction between H. irregulare and pine engages fewer carbohydrate-active enzymes, but involves an increase in pectinolytic enzymes, transcription modules for oxidative stress and secondary metabolite production. Our results show a trade-off in terms of constrained carbohydrate decomposition and membrane transport capacity during interaction with living hosts. Our findings establish that saprotrophic wood decay and necrotrophic parasitism involve two distinct, yet overlapping, processes.

A single mating-type locus composed of homeodomain genes promotes nuclear migration and heterokaryosis in the white-rot fungus Phanerochaete chrysosporium.

The white-rot basidiomycete fungus Phanerochaete chrysosporium (Agaricomycetes) is a model species that produces potent wood-degrading enzymes. The mating system of the species has been difficult to characterize due to its cryptic fruiting habit and lack of clamp connections in the heterokaryotic phase. By exploiting the draft genome sequence, we reevaluated the mating system of P. chrysosporium by studying the inheritance and segregation of putative mating-type gene homologues, the homeodomain transcription factor genes (MAT-A) and the pheromone receptors (MAT-B). A pattern of mating incompatibility and fructification consistent with a bipolar system with a single MAT locus was observed, but the rejection response was much weaker than that seen in other agaricomycete species, leading to stable heterokaryons with identical MAT alleles. The homeodomain genes appear to comprise the single MAT locus because they are heterozygous in wild strains and hyperpolymorphic at the DNA sequence level and promote aspects of sexual reproduction, such as nuclear migration, heterokaryon stability, and basidiospore formation. The pheromone receptor loci that might constitute a MAT-B locus, as in many other Agaricomycetes, are not linked to the MAT-A locus and display low levels of polymorphism. This observation is inconsistent with a bipolar mating system that includes pheromones and pheromone receptors as mating-type determinants. The partial uncoupling of nuclear migration and mating incompatibility in this species may be predicted to lead to parasexual recombination and may have contributed to the homothallic behavior observed in previous studies.

Simulated nitrogen deposition affects community structure of arbuscular mycorrhizal fungi in northern hardwood forests.

Our previous investigation found elevated nitrogen deposition caused declines in abundance of arbuscular mycorrhizal fungi (AMF) associated with forest trees, but little is known about how nitrogen affects the AMF community composition and structure within forest ecosystems. We hypothesized that N deposition would lead to significant changes in the AMF community structure. We studied the diversity and community structure of AMF in northern hardwood forests after more than 12 years of simulated nitrogen deposition. We performed molecular analyses on maple (Acer spp.) roots targeting the 18S rDNA region using the fungal-specific primers AM1 and NS31. PCR products were cloned and identified using restriction fragment length polymorphism (RFLP) and sequencing. N addition significantly altered the AMF community structure, and Glomus group A dominated the AMF community. Some Glomus operational taxonomic units (OTUs) responded negatively to N inputs, whereas other Glomus OTUs and an Acaulospora OTU responded positively to N inputs. The observed effect on community structure implies that AMF species associated with maples differ in their response to elevated nitrogen. Given that functional diversity exists among AMF species and that N deposition has been shown to select less beneficial fungi in some ecosystems, this change in community structure could have implications for the functioning of this type of ecosystem.

Diversity begets diversity in competition for space.

Competition can profoundly affect biodiversity patterns by determining whether similar species are likely to coexist. When species compete directly for space, competitive ability differences should theoretically promote trait and phylogenetic clustering, provided that niche differences are otherwise minimal. Yet many sessile communities exhibit high biodiversity despite minimal reliance on niche differentiation. A potential explanation is that intransitive competition ('rock-paper-scissors' competition) not only promotes species richness but also fosters coexistence among highly dissimilar species with different competitive strategies. Here, we test this hypothesis using a combination of empirical and analytical approaches. In an experimental system comprising 37 wood-decay basidiomycete fungi grown in nutrient-rich agar media, pairwise displacement was maximized when species had widely different competitive traits and divergent evolutionary histories. However, when these interactions were embedded in models of species-rich communities, high levels of intransitivity ultimately overwhelmed the pairwise relationships, allowing the weakest and most dissimilar species to survive. In line with theoretical expectations, these multispecies assemblages exhibited reduced functional and phylogenetic diversity, yet the smallest losses were likewise observed in species-rich communities. By demonstrating that species richness can act as a self-reinforcing buffer against competitive exclusion, these results contribute to our understanding of how biodiversity is maintained in natural systems.

Long-term forest soil warming alters microbial communities in temperate forest soils.

Soil microbes are major drivers of soil carbon cycling, yet we lack an understanding of how climate warming will affect microbial communities. Three ongoing field studies at the Harvard Forest Long-term Ecological Research (LTER) site (Petersham, MA) have warmed soils 5°C above ambient temperatures for 5, 8, and 20 years. We used this chronosequence to test the hypothesis that soil microbial communities have changed in response to chronic warming. Bacterial community composition was studied using Illumina sequencing of the 16S ribosomal RNA gene, and bacterial and fungal abundance were assessed using quantitative PCR. Only the 20-year warmed site exhibited significant change in bacterial community structure in the organic soil horizon, with no significant changes in the mineral soil. The dominant taxa, abundant at 0.1% or greater, represented 0.3% of the richness but nearly 50% of the observations (sequences). Individual members of the Actinobacteria, Alphaproteobacteria and Acidobacteria showed strong warming responses, with one Actinomycete decreasing from 4.5 to 1% relative abundance with warming. Ribosomal RNA copy number can obfuscate community profiles, but is also correlated with maximum growth rate or trophic strategy among bacteria. Ribosomal RNA copy number correction did not affect community profiles, but rRNA copy number was significantly decreased in warming plots compared to controls. Increased bacterial evenness, shifting beta diversity, decreased fungal abundance and increased abundance of bacteria with low rRNA operon copy number, including Alphaproteobacteria and Acidobacteria, together suggest that more or alternative niche space is being created over the course of long-term warming.

Decline of arbuscular mycorrhizal fungi in northern hardwood forests exposed to chronic nitrogen additions.

Arbuscular mycorrhizal (AM) fungi are important below-ground carbon (C) sinks that can be sensitive to increased nitrogen (N) availability. The abundance of AM fungi (AMF) was estimated in maple (Acer spp.) fine roots following more than a decade of experimental additions of N designed to simulate chronic atmospheric N deposition. Abundance of AMF was measured by staining and ocular estimation, as well as by analyzing for the AMF indicator fatty acid 16:1omega5c in phospholipid (biomass indicator) and neutral lipid (lipid storage indicator) fractions. Arbuscular mycorrhizal fungal biomass, storage structures and lipid storage declined in response to N addition measured by both methods. This pattern was found when AM response was characterized as colonization intensity, on an areal basis and in proportion to maple above-ground biomass. The phospholipid fraction of the fatty acid 16:1omega5c was positively correlated with total AMF colonization and the neutral lipid fraction with vesicle colonization. Decreased AMF abundance with simulated N deposition suggests reduced C allocation to these fungi or a direct soil N-mediated decline. The fatty acid (phospholipid and neutral lipid fractions) 16:1omega5c was found to be a good indicator for AMF active biomass and stored energy, respectively.