Benefits of symbiotic ectomycorrhizal fungi to plant water relations depend on plant genotype in pinyon pine.

Rhizosphere microbes, such as root-associated fungi, can improve plant access to soil resources, affecting plant health, productivity, and stress tolerance. While mycorrhizal associations are ubiquitous, plant-microbe interactions can be species specific. Here we show that the specificity of the effects of microbial symbionts on plant function can go beyond species level: colonization of roots by ectomycorrhizal fungi (EMF) of the genus Geopora has opposite effects on water uptake, and stomatal control of desiccation in drought tolerant and intolerant genotypes of pinyon pine (Pinus edulis Engelm.). These results demonstrate, for the first time, that microorganisms can have significant and opposite effects on important plant functional traits like stomatal control of desiccation that are associated with differential mortality and growth in nature. They also highlight that appropriate pairing of plant genotypes and microbial associates will be important for mitigating climate change impacts on vegetation.

Seasonal variation and potential roles of dark septate fungi in an arid grassland.

High temperatures and extended drought in temperate and tropical arid ecosystems promote the colonization of diverse microenvironments by dark septate fungi (DSF). These fungi contribute to soil nutrient cycling, soil stabilization, and plant survival, but the roles of individual DSF species, their distributions, and their community diversity are poorly understood. The objective of this study was to evaluate the distribution, seasonal variation, and potential roles of DSF on plant growth. We collected biocrust (lichen-, moss-, and cyanobacterium-dominated biocrusts) soils at different depths and rhizosphere soils from two grasses, Bromus tectorum and Pleuraphis jamesii, in an arid grassland near Moab, Utah, USA. Seasonal variation of DSF was evaluated using culture-based approaches and compared with fungal community profiles from next-generation sequencing (NGS). Culturing showed that DSF were 30% more abundant in biocrusts compared with the focal rhizospheres. The abundance of DSF varied seasonally in belowground samples (rhizosphere and below-biocrust), with a significant increase during the summer months. Pleosporales was the dominant order (35%) in both biocrust and rhizosphere soils out of 817 isolated fungi. Dominant DSF genera in culture included Alternaria, Preussia, Cladosporium, Phoma, and an unknown Pleosporales. Similar results were observed in biocrust and rhizosphere soils NGS. Further, seed germination experiments using dominant taxa were conducted to determine their potential roles on germination and seedling growth using maize as a model plant. Cladosporium and unknown Pleosporales isolates showed plant growth-promoting ability. The variation in abundance of DSF, their differential occurrence in different microenvironments, and their ability to grow in a xerotolerant medium reflect adaptations to summer environmental conditions and to changes in the abundance of organic matter, as well as a potential increase in plant investment in these fungi when heat and drought stresses are more severe.

Resistance, Resilience, and Recovery of Dryland Soil Bacterial Communities Across Multiple Disturbances.

Dryland ecosystems are sensitive to perturbations and generally slow to recover post disturbance. The microorganisms residing in dryland soils are especially important as they contribute to soil structure and nutrient cycling. Disturbance can have particularly strong effects on dryland soil structure and function, yet the natural resistance and recovery of the microbial components of dryland soils has not been well documented. In this study, the recovery of surface soil bacterial communities from multiple physical and environmental disturbances is assessed. Samples were collected from three field sites in the vicinity of Moab, UT, United States, 6 to 7 years after physical and climate disturbance manipulations had been terminated, allowing for the assessment of community recovery. Additionally, samples were collected in a transect that included three habitat patches: the canopy zone soils under the dominant shrubs, the interspace soils that are colonized by biological soil crusts, and edge soils at the plot borders. Field site and habitat patch were significant factors structuring the bacterial communities, illustrating that sites and habitats harbored unique soil microbiomes. Across the different sites and disturbance treatments, there was evidence of significant bacterial community recovery, as bacterial biomass and diversity were not significantly different than control plots. There was, however, a small number of 16S rRNA gene amplicon sequence variants that distinguished particular treatments, suggesting that legacy effects of the disturbances still remained. Taken together, these data suggest that dryland bacterial communities may possess a previously unappreciated potential to recover within years of the original disturbance.

Ectomycorrhizal and Dark Septate Fungal Associations of Pinyon Pine Are Differentially Affected by Experimental Drought and Warming.

Changing climates can cause shifts in temperature and precipitation, resulting in warming and drought in some regions. Although each of these factors has been shown to detrimentally affect forest ecosystems worldwide, information on the impacts of the combined effects of warming and drought is lacking. Forest trees rely on mutualistic root-associated fungi that contribute significantly to plant health and protection against climate stresses. We used a six-year, ecosystem-scale temperature and precipitation manipulation experiment targeted to simulate the climate in 2100 in the Southwestern United States to quantify the effects of drought, warming and combined drought and warming on the root colonization (abundance), species composition and diversity of ectomycorrhizal fungi (EMF), and dark septate fungal endophytes in a widespread woodland tree, pinyon pine (Pinus edulis E.). Our results show that pinyon shoot growth after 6 years of these treatments was reduced more by drought than warming. The combined drought and warming treatment reduced the abundance and diversity of EMF more than either treatment alone. Individual ectomycorrhizal fungal taxa, including the drought tolerant Cenococcum geophilum, were present in all treatments but the combined drought and warming treatment. The combined drought and warming treatment also reduced the abundance of dark septate endophytes (DSE), but did not affect their diversity or species composition. The current year shoot growth of the trees correlated positively with ectomycorrhizal fungal diversity, highlighting the importance of diversity in mutualistic relationships to plant growth. Our results suggest that EMF may be more important than DSE to aboveground growth in P. edulis, but also more susceptible to the negative effects of combined climate stressors.

Keratinophilic fungi: Specialized fungal communities in a desert ecosystem identified using cultured-based and Illumina sequencing approaches.

Soil fungi in desert ecosystems are adapted to harsh environmental conditions such as high soil surface temperatures and limited organic matter and water. Given limited carbon inputs from plant material, heterotrophic fungi likely use unconventional sources of carbon in these systems. A baiting method was used to culture keratinophilic fungi from biocrust and rhizosphere soils in an arid grassland in Utah, USA. Fungi were baited using llama and sheep wool, horsehair, and snakeskin on two media, and pure cultures were identified using ITS and LSU rRNA sequences. One hundred-eighteen fungal colonies were grown, representing a total of 32 Operational Taxonomic Units (OTUs) at 97 % similarity. Cultures were dominated by the phylum Ascomycota (88 %) followed by Mucoromycota (8.6 %) and Basidiomycota (3.4 %). The orders Pleosporales, Eurotiales, Hypocreales, and Sordariales were commonly isolated, with the dominant taxa Alternaria (27 %), Aspergillus (22 %), Fusarium (11 %), and Chaetomium (8%). Thirty percent of the fungi isolated have the capacity to degrade keratin in vitro using a keratin azure assay, with Penicillium showing the highest degradation followed by Geomyces, Alternaria, and Fusarium. Although keratin degraders can be infectious, dermatophytes associated with skin infections were not isolated in culture or detected in Illumina sequencing. Illumina sequencing was used to determine general patterns in seasonal variation and habitat preference of keratinophiles. Alternaria was the most abundant genus with >70 % of the sequences. The combination of Illumina data with culture-dependent approaches facilitated the characterization of a specialized community and confirmed the low abundance of dermatophytes in this arid site.

Genomes and secretomes of Ascomycota fungi reveal diverse functions in plant biomass decomposition and pathogenesis.

BACKGROUND: The dominant fungi in arid grasslands and shrublands are members of the Ascomycota phylum. Ascomycota fungi are important drivers in carbon and nitrogen cycling in arid ecosystems. These fungi play roles in soil stability, plant biomass decomposition, and endophytic interactions with plants. They may also form symbiotic associations with biocrust components or be latent saprotrophs or pathogens that live on plant tissues. However, their functional potential in arid soils, where organic matter, nutrients and water are very low or only periodically available, is poorly characterized. RESULTS: Five Ascomycota fungi were isolated from different soil crust microhabitats and rhizosphere soils around the native bunchgrass Pleuraphis jamesii in an arid grassland near Moab, UT, USA. Putative genera were Coniochaeta, isolated from lichen biocrust, Embellisia from cyanobacteria biocrust, Chaetomium from below lichen biocrust, Phoma from a moss microhabitat, and Aspergillus from the soil. The fungi were grown in replicate cultures on different carbon sources (chitin, native bunchgrass or pine wood) relevant to plant biomass and soil carbon sources. Secretomes produced by the fungi on each substrate were characterized. Results demonstrate that these fungi likely interact with primary producers (biocrust or plants) by secreting a wide range of proteins that facilitate symbiotic associations. Each of the fungal isolates secreted enzymes that degrade plant biomass, small secreted effector proteins, and proteins involved in either beneficial plant interactions or virulence. Aspergillus and Phoma expressed more plant biomass degrading enzymes when grown in grass- and pine-containing cultures than in chitin. Coniochaeta and Embellisia expressed similar numbers of these enzymes under all conditions, while Chaetomium secreted more of these enzymes in grass-containing cultures. CONCLUSIONS: This study of Ascomycota genomes and secretomes provides important insights about the lifestyles and the roles that Ascomycota fungi likely play in arid grassland, ecosystems. However, the exact nature of those interactions, whether any or all of the isolates are true endophytes, latent saprotrophs or opportunistic phytopathogens, will be the topic of future studies.

Presence and distribution of insect-associated and entomopathogenic fungi in a temperate pine forest soil: An integrated approach.

For decades entomopathogenic fungi have garnered interest as possible alternatives to chemical pesticides. However, their ecology outside of agroecosystems demands further study. We assessed the diversity and abundance of entomopathogenic and insect-associated fungi at a loblolly pine forest in North Carolina, USA using culture-dependent and next-generation sequencing libraries. Fungi were isolated using Galleriamellonella larvae, as well as from soil dilutions plated on a selective medium. Isolates were identified using Sanger sequencing of the ITS and LSU rRNA gene regions, and represented 36 OTUs including Metarhizium, Lecanicillium, and Paecilomyces. Additionally, we assessed the chitinolytic potential of isolates and found widespread, variable ability to degrade chitin within and between genera. Phylogenetic analyses resolved several isolates to genus, with some forming clades with other insect-associated taxa, as well as with fungi associated with plant tissues. Saprophytes were widely distributed in soil, while entomopathogens were less abundant and present primarily in the top two cm of the soil. The similarity between culture-dependent and next-generation sequencing results demonstrates that both methods can be used concurrently in this system to study the ecology of entomopathogenic fungi.

Chronic Physical Disturbance Substantially Alters the Response of Biological Soil Crusts to a Wetting Pulse, as Characterized by Metatranscriptomic Sequencing.

Biological soil crusts (biocrusts) are microbial communities that are a feature of arid surface soils worldwide. In drylands where precipitation is pulsed and ephemeral, the ability of biocrust microbiota to rapidly initiate metabolic activity is critical to their survival. Community gene expression was compared after a short duration (1 h) wetting pulse in both intact and soils disturbed by chronic foot trampling. Across the metatranscriptomes the majority of transcripts were cyanobacterial in origin, suggesting that cyanobacteria accounted for the bulk of the transcriptionally active cells. Chronic trampling substantially altered the functional profile of the metatranscriptomes, specifically resulting in a significant decrease in transcripts for nitrogen fixation. Soil depth (biocrust and below crust) was a relatively small factor in differentiating the metatranscriptomes, suggesting that the metabolically active bacteria were similar between shallow soil horizons. The dry samples were consistently enriched for hydrogenase genes, indicating that molecular hydrogen may serve as an energy source for the desiccated soil communities. The water pulse was associated with a restructuring of the metatranscriptome, particularly for the biocrusts. Biocrusts increased transcripts for photosynthesis and carbon fixation, suggesting a rapid resuscitation upon wetting. In contrast, the trampled surface soils showed a much smaller response to wetting, indicating that trampling altered the metabolic response of the community. Finally, several biogeochemical cycling genes in carbon and nitrogen cycling were assessed for their change in abundance due to wetting in the biocrusts. Different transcripts encoding the same gene product did not show a consensus response, with some more abundant in dry or wet biocrusts, highlighting the challenges in relating transcript abundance to biogeochemical cycling rates. These observations demonstrate that metatranscriptome sequencing was able to distinguish alterations in the function of arid soil microbial communities at two varying temporal scales, a long-term ecosystems disturbance through foot trampling, and a short term wetting pulse. Thus, community metatranscriptomes have the potential to inform studies on the response and resilience of biocrusts to various environmental perturbations.

Short-Term Transcriptional Response of Microbial Communities to Nitrogen Fertilization in a Pine Forest Soil.

Numerous studies have examined the long-term effect of experimental nitrogen (N) deposition in terrestrial ecosystems; however, N-specific mechanistic markers are difficult to disentangle from responses to other environmental changes. The strongest picture of N-responsive mechanistic markers is likely to arise from measurements over a short (hours to days) time scale immediately after inorganic N deposition. Therefore, we assessed the short-term (3-day) transcriptional response of microbial communities in two soil strata from a pine forest to a high dose of N fertilization (ca. 1 mg/g of soil material) in laboratory microcosms. We hypothesized that N fertilization would repress the expression of fungal and bacterial genes linked to N mining from plant litter. However, despite N suppression of microbial respiration, the most pronounced differences in functional gene expression were between strata rather than in response to the N addition. Overall, ∼4% of metabolic genes changed in expression with N addition, while three times as many (∼12%) were significantly different across the different soil strata in the microcosms. In particular, we found little evidence of N changing expression levels of metabolic genes associated with complex carbohydrate degradation (CAZymes) or inorganic N utilization. This suggests that direct N repression of microbial functional gene expression is not the principle mechanism for reduced soil respiration immediately after N deposition. Instead, changes in expression with N addition occurred primarily in general cell maintenance areas, for example, in ribosome-related transcripts. Transcriptional changes in functional gene abundance in response to N addition observed in longer-term field studies likely result from changes in microbial composition.IMPORTANCE Ecosystems are receiving increased nitrogen (N) from anthropogenic sources, including fertilizers and emissions from factories and automobiles. High levels of N change ecosystem functioning. For example, high inorganic N decreases the microbial decomposition of plant litter, potentially reducing nutrient recycling for plant growth. Understanding how N regulates microbial decomposition can improve the prediction of ecosystem functioning over extended time scales. We found little support for the conventional view that high N supply represses the expression of genes involved in decomposition or alters the expression of bacterial genes for inorganic N cycling. Instead, our study of pine forest soil 3 days after N addition showed changes in microbial gene expression related to cell maintenance and stress response. This highlights the challenge of establishing predictive links between microbial gene expression levels and measures of ecosystem function.

Bifiguratus adelaidae, gen. et sp. nov., a new member of Mucoromycotina in endophytic and soil-dwelling habitats.

Illumina amplicon sequencing of soil in a temperate pine forest in the southeastern United States detected an abundant, nitrogen (N)-responsive fungal genotype of unknown phylogenetic affiliation. Two isolates with ribosomal sequences consistent with that genotype were subsequently obtained. Examination of records in GenBank revealed that a genetically similar fungus had been isolated previously as an endophyte of moss in a pine forest in the southwestern United States. The three isolates were characterized using morphological, genomic, and multilocus molecular data (18S, internal transcribed spacer [ITS], and 28S rRNA sequences). Phylogenetic and maximum likelihood phylogenomic reconstructions revealed that the taxon represents a novel lineage in Mucoromycotina, only preceded by Calcarisporiella, the earliest diverging lineage in the subphylum. Sequences for the novel taxon are frequently detected in environmental sequencing studies, and it is currently part of UNITE's dynamic list of most wanted fungi. The fungus is dimorphic, grows best at room temperature, and is associated with a wide variety of bacteria. Here, a new monotypic genus, Bifiguratus, is proposed, typified by Bifiguratus adelaidae.

Shared features of cryptic plasmids from environmental and pathogenic Francisella species.

The Francisella genus includes several recognized species, additional potential species, and other representatives that inhabit a range of incredibly diverse ecological niches, but are not closely related to the named species. Francisella species have been obtained from a wide variety of clinical and environmental sources; documented species include highly virulent human and animal pathogens, fish pathogens, opportunistic human pathogens, tick endosymbionts, and free-living isolates inhabiting brackish water. While more than 120 Francisella genomes have been sequenced to date, only a few contain plasmids, and most of these appear to be cryptic, with unknown benefit to the host cell. We have identified several putative cryptic plasmids in the sequenced genomes of three Francisella novicida and F. novicida-like strains (TX07-6608, AZ06-7470, DPG_3A-IS) and two new Francisella species (F. frigiditurris CA97-1460 and F. opportunistica MA06-7296). These plasmids were compared to each other and to previously identified plasmids from other Francisella species. Some of the plasmids encoded functions potentially involved in replication, conjugal transfer and partitioning, environmental survival (transcriptional regulation, signaling, metabolism), and hypothetical proteins with no assignable functions. Genomic and phylogenetic comparisons of these new plasmids to the other known Francisella plasmids revealed some similarities that add to our understanding of the evolutionary relationships among the diverse Francisella species.

Bacterial, fungal, and plant communities exhibit no biomass or compositional response to two years of simulated nitrogen deposition in a semiarid grassland.

Nitrogen (N) deposition affects myriad aspects of terrestrial ecosystem structure and function, and microbial communities may be particularly sensitive to anthropogenic N inputs. However, our understanding of N deposition effects on microbial communities is far from complete, especially for drylands where data are comparatively rare. To address the need for an improved understanding of dryland biological responses to N deposition, we conducted a two-year fertilization experiment in a semiarid grassland on the Colorado Plateau in the southwestern United States. We evaluated effects of varied levels of N inputs on archaeal, bacterial, fungal and chlorophyte community composition within three microhabitats: biological soil crusts (biocrusts), soil below biocrusts, and the plant rhizosphere. Surprisingly, N addition did not affect the community composition or diversity of any of these microbial groups; however, microbial community composition varied significantly among sampling microhabitats. Further, while plant richness, diversity, and cover showed no response to N addition, there were strong linkages between plant properties and microbial community structure. Overall, these findings highlight the potential for some dryland communities to have limited biotic ability to retain augmented N inputs, possibly leading to large N losses to the atmosphere and to aquatic systems.

Polysaccharide Degradation Capability of Actinomycetales Soil Isolates from a Semiarid Grassland of the Colorado Plateau.

Among the bacteria, members of the order Actinomycetales are considered quintessential degraders of complex polysaccharides in soils. However, studies examining complex polysaccharide degradation by Actinomycetales (other than Streptomyces spp.) in soils are limited. Here, we examine the lignocellulolytic and chitinolytic potential of 112 Actinomycetales strains, encompassing 13 families, isolated from a semiarid grassland of the Colorado Plateau in Utah. Members of the Streptomycetaceae, Pseudonocardiaceae, Micromonosporaceae, and Promicromonosporaceae families exhibited robust activity against carboxymethyl cellulose, xylan, chitin, and pectin substrates (except for low/no pectinase activity by the Micromonosporaceae). When incubated in a hydrated mixture of blended Stipa and Hilaria grass biomass over a 5-week period, Streptomyces and Saccharothrix (a member of the Pseudonocardiaceae) isolates produced high levels of extracellular enzyme activity, such as endo- and exocellulase, glucosidase, endo- and exoxylosidase, and arabinofuranosidase. These characteristics make them well suited to degrade the cellulose and hemicellulose components of grass cell walls. On the basis of the polysaccharide degradation profiles of the isolates, relative abundance of Actinomycetales sequences in 16S rRNA gene surveys of Colorado Plateau soils, and analysis of genes coding for polysaccharide-degrading enzymes among 237 Actinomycetales genomes in the CAZy database and 5 genomes from our isolates, we posit that Streptomyces spp. and select members of the Pseudonocardiaceae and Micromonosporaceae likely play an important role in the degradation of hemicellulose, cellulose, and chitin substances in dryland soils.IMPORTANCE Shifts in the relative abundance of Actinomycetales taxa have been observed in soil microbial community surveys during large, manipulated climate change field studies. However, our limited understanding of the ecophysiology of diverse Actinomycetales taxa in soil systems undermines attempts to determine the underlying causes of the population shifts or their impact on carbon cycling in soil. This study combines a systematic analysis of the polysaccharide degradation potential of a diverse collection of Actinomycetales isolates from surface soils of a semiarid grassland with analysis of genomes from five of these isolates and publicly available Actinomycetales genomes for genes encoding polysaccharide-active enzymes. The results address an important gap in knowledge of Actinomycetales ecophysiology-identification of key taxa capable of facilitating lignocellulose degradation in dryland soils. Information from this study will benefit future metagenomic studies related to carbon cycling in dryland soils by providing a baseline linkage of Actinomycetales phylogeny with lignocellulolytic functional potential.

Whole-Genome Relationships among Francisella Bacteria of Diverse Origins Define New Species and Provide Specific Regions for Detection.

Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features-for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. IMPORTANCE: DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics, and environmental surveillance, for which response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisella organisms identified in clinical samples or environmental surveys.

Ribosomal RNA gene detection and targeted culture of novel nitrogen-responsive fungal taxa from temperate pine forest soil.

Soil fungal communities are responsible for carbon and nitrogen (N) cycling. The high complexity of the soil fungal community and the high proportion of taxonomically unidentifiable sequences confound ecological interpretations in field studies because physiological information is lacking for many organisms known only by their rRNA sequences. This situation forces experimental comparisons to be made at broader taxonomic racks where functions become difficult to infer. The objective of this study was to determine OTU (operational taxonomic units) level responses of the soil fungal community to N enrichment in a temperate pine forest experiment and to use the sequencing data to guide culture efforts of novel N-responsive fungal taxa. Replicate samples from four soil horizons (up to 10 cm depth) were obtained from ambient, enriched CO2 and N-fertilization plots. Through a fungal large subunit rRNA gene (LSU) sequencing survey, we identified two novel fungal clades that were abundant in our soil sampling (representing up to 27% of the sequences in some samples) and responsive to changes in soil N. The two N-responsive taxa with no predicted taxonomic association were targeted for isolation and culturing from specific soil samples where their sequences were abundant. Representatives of both OTUs were successfully cultured using a filtration approach. One taxon (OTU6) was most closely related to Saccharomycotina; the second taxon (OTU69) was most closely related to Mucoromycotina. Both taxa likely represent novel species. This study shows how observation of specific OTUs level responses to altered N status in a large rRNA gene field survey provided the impetus to design targeted culture approaches for isolation of novel N-responsive fungal taxa.

Assembly of Active Bacterial and Fungal Communities Along a Natural Environmental Gradient.

Dormancy is thought to promote biodiversity within microbial communities, but how assembly of the active community responds to changes in environmental conditions is unclear. To measure the active and dormant communities of bacteria and fungi colonizing decomposing litter in maple forests, we targeted ribosomal genes and transcripts across a natural environmental gradient. Within bacterial and fungal communities, the active and dormant communities were phylogenetically distinct, but patterns of phylogenetic clustering varied. For bacteria, active communities were significantly more clustered than dormant communities, while the reverse was found for fungi. The proportion of operational taxonomic units (OTUs) classified as active and the degree of phylogenetic clustering of the active bacterial communities declined with increasing pH and decreasing C/N. No significant correlations were found for the fungal community. The opposing pattern of phylogenetic clustering in dormant and active communities and the differential response of active communities to environmental gradients suggest that dormancy differentially structures bacterial and fungal communities.

Fungal identification using a Bayesian classifier and the Warcup training set of internal transcribed spacer sequences.

Fungi are key organisms in many ecological processes and communities. Rapid and low cost surveys of the fungal members of a community can be undertaken by isolating and sequencing a taxonomically informative genomic region, such as the ITS (internal transcribed spacer), from DNA extracted from a metagenomic sample, and then classifying these sequences to determine which organisms are present. This paper announces the availability of the Warcup ITS training set and shows how it can be used with the Ribosomal Database Project (RDP) Bayesian Classifier to rapidly and accurately identify fungi using ITS sequences. The classifications can be down to species level and use conventional literature-based mycological nomenclature and taxonomic assignments.

A new fungal large subunit ribosomal RNA primer for high-throughput sequencing surveys.

The inclusion of phylogenetic metrics in community ecology has provided insights into important ecological processes, particularly when combined with high-throughput sequencing methods; however, these approaches have not been widely used in studies of fungal communities relative to other microbial groups. Two obstacles have been considered: (1) the internal transcribed spacer (ITS) region has limited utility for constructing phylogenies and (2) most PCR primers that target the large subunit (LSU) ribosomal unit generate amplicons that exceed current limits of high-throughput sequencing platforms. We designed and tested a PCR primer (LR22R) to target approximately 300-400 bp region of the D2 hypervariable region of the fungal LSU for use with the Illumina MiSeq platform. Both in silico and empirical analyses showed that the LR22R-LR3 pair captured a broad range of fungal taxonomic groups with a small fraction of non-fungal groups. Phylogenetic placement of publically available LSU D2 sequences showed broad agreement with taxonomic classification. Comparisons of the LSU D2 and the ITS2 ribosomal regions from environmental samples and known communities showed similar discriminatory abilities of the two primer sets. Together, these findings show that the LR22R-LR3 primer pair has utility for phylogenetic analyses of fungal communities using high-throughput sequencing methods.

Soil bacterial and fungal community responses to nitrogen addition across soil depth and microhabitat in an arid shrubland.

Arid shrublands are stressful environments, typified by alkaline soils low in organic matter, with biologically-limiting extremes in water availability, temperature, and UV radiation. The widely-spaced plants and interspace biological soil crusts in these regions provide soil nutrients in a localized fashion, creating a mosaic pattern of plant- or crust-associated microhabitats with distinct nutrient composition. With sporadic and limited rainfall, nutrients are primarily retained in the shallow surface soil, patterning biological activity. We examined soil bacterial and fungal community responses to simulated nitrogen (N) deposition in an arid Larrea tridentata-Ambrosia dumosa field experiment in southern Nevada, USA, using high-throughput sequencing of ribosomal RNA genes. To examine potential interactions among the N application, microhabitat and soil depth, we sampled soils associated with shrub canopies and interspace biological crusts at two soil depths (0-0.5 or 0-10 cm) across the N-amendment gradient (0, 7, and 15 kg ha(-1) yr(-1)). We hypothesized that localized compositional differences in soil microbiota would constrain the impacts of N addition to a microhabitat distribution that would reflect highly localized geochemical conditions and microbial community composition. The richness and community composition of both bacterial and fungal communities differed significantly by microhabitat and with soil depth in each microhabitat. Only bacterial communities exhibited significant responses to the N addition. Community composition correlated with microhabitat and depth differences in soil geochemical features. Given the distinct roles of soil bacteria and fungi in major nutrient cycles, the resilience of fungi and sensitivity of bacteria to N amendments suggests that increased N input predicted for many arid ecosystems could shift nutrient cycling toward pathways driven primarily by fungal communities.