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1.
Environ Sci Technol ; 58(16): 7056-7065, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38608141

RESUMO

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.


Assuntos
Óxido Nitroso , Óxido Nitroso/metabolismo , Bactérias/metabolismo , Oxirredutases/metabolismo , Desnitrificação
2.
Proc Natl Acad Sci U S A ; 118(25)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34161254

RESUMO

In this study, a suite of complementary environmental geochemical analyses, including NMR and gas chromatography-mass spectrometry (GC-MS) analyses of central metabolites, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of secondary metabolites, and lipidomics, was used to investigate the influence of organic matter (OM) quality on the heterotrophic microbial mechanisms controlling peatland CO2, CH4, and CO2:CH4 porewater production ratios in response to climate warming. Our investigations leverage the Spruce and Peatland Responses under Changing Environments (SPRUCE) experiment, where air and peat warming were combined in a whole-ecosystem warming treatment. We hypothesized that warming would enhance the production of plant-derived metabolites, resulting in increased labile OM inputs to the surface peat, thereby enhancing microbial activity and greenhouse gas production. Because shallow peat is most susceptible to enhanced warming, increases in labile OM inputs to the surface, in particular, are likely to result in significant changes to CO2 and CH4 dynamics and methanogenic pathways. In support of this hypothesis, significant correlations were observed between metabolites and temperature consistent with increased availability of labile substrates, which may stimulate more rapid turnover of microbial proteins. An increase in the abundance of methanogenic genes in response to the increase in the abundance of labile substrates was accompanied by a shift toward acetoclastic and methylotrophic methanogenesis. Our results suggest that as peatland vegetation trends toward increasing vascular plant cover with warming, we can expect a concomitant shift toward increasingly methanogenic conditions and amplified climate-peatland feedbacks.


Assuntos
Ecossistema , Metaboloma , Picea/metabolismo , Solo/química , Dióxido de Carbono/análise , Ciclotrons , Cromatografia Gasosa-Espectrometria de Massas , Íons , Isótopos/análise , Lipídeos/análise , Espectroscopia de Ressonância Magnética , Metagenômica , Metano/análise , Análise Multivariada , Ácidos Nucleicos/genética , Oxirredução , Análise de Componente Principal , Proteômica , RNA Ribossômico 16S/genética , Água
3.
Am J Psychother ; 76(3): 124-127, 2023 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36794443

RESUMO

OBJECTIVE: Brief dynamic interpersonal therapy (DIT) is an evidence-based psychodynamic intervention for depression offered by the U.K. National Health Service and previously studied in the context of a U.S. Department of Veterans Affairs medical center. This study assessed the clinical value of DIT in primary care for veterans with general medical conditions. METHODS: The authors examined outcome data of veterans (N=30; all but one had ≥1 comorbid general medical conditions) referred to DIT from primary care. RESULTS: Veterans who began treatment with clinically elevated depression or anxiety experienced a 42% reduction in symptom severity, as measured by the nine-item Patient Health Questionnaire or by the seven-item Generalized Anxiety Disorder questionnaire, respectively, representing large effect sizes. CONCLUSIONS: Significant decreases in depression and anxiety symptoms suggest the utility of DIT for veterans with comorbid general medical conditions. DIT's dynamically informed framework may improve patients' help seeking, which is relevant for patients experiencing comorbid medical conditions.


Assuntos
Veteranos , Estados Unidos/epidemiologia , Humanos , Medicina Estatal , United States Department of Veterans Affairs , Transtornos de Ansiedade/diagnóstico , Transtornos de Ansiedade/epidemiologia , Transtornos de Ansiedade/terapia , Atenção Primária à Saúde
4.
New Phytol ; 230(6): 2433-2446, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33525047

RESUMO

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.


Assuntos
Microbiota , Solo , Bactérias/genética , Raízes de Plantas , RNA Ribossômico 16S/genética , Rizosfera , Microbiologia do Solo
5.
Microb Ecol ; 80(3): 593-602, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32388577

RESUMO

Peatlands are important players in climate change-biosphere feedbacks via long-term net carbon (C) accumulation in soil organic matter and as potential net C sources including the potent greenhouse gas methane (CH4). Interactions of climate, site-hydrology, plant community, and groundwater chemical factors influence peatland development and functioning, including C dioxide (CO2) and CH4 fluxes, but the role of microbial community composition is not well understood. To assess microbial functional and taxonomic dissimilarities, we used high throughput sequencing of the small subunit ribosomal DNA (SSU rDNA) to determine bacterial and archaeal community composition in soils from twenty North American peatlands. Targeted DNA metabarcoding showed that although Proteobacteria, Acidobacteria, and Actinobacteria were the dominant phyla on average, intermediate and rich fens hosted greater diversity and taxonomic richness, as well as an array of candidate phyla when compared with acidic and nutrient-poor poor fens and bogs. Moreover, pH was revealed to be the strongest predictor of microbial community structure across sites. Predictive metagenome content (PICRUSt) showed increases in specific genes, such as purine/pyrimidine and amino-acid metabolism in mid-latitude peatlands from 38 to 45° N, suggesting a shift toward utilization of microbial biomass over utilization of initial plant biomass in these microbial communities. Overall, there appears to be noticeable differences in community structure between peatland classes, as well as differences in microbial metabolic activity between latitudes. These findings are in line with a predicted increase in the decomposition and accelerated C turnover, and suggest that peatlands north of 37° latitude may be particularly vulnerable to climate change.


Assuntos
Archaea/isolamento & purificação , Bactérias/isolamento & purificação , Clima , Microbiota , Áreas Alagadas , Ontário , Microbiologia do Solo , Estados Unidos
6.
Mol Plant Microbe Interact ; 32(7): 853-864, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30699306

RESUMO

Mortierella and Ilyonectria genera include common species of soil fungi that are frequently detected as root endophytes in many plants, including Populus spp. However, the ecological roles of these and other endophytic fungi with respect to plant growth and function are still not well understood. The functional ecology of two key taxa from the P. trichocarpa rhizobiome, M. elongata PMI93 and I. europaea PMI82, was studied by coupling forest soil bioassays with environmental metatranscriptomics. Using soil bioassay experiments amended with fungal inoculants, M. elongata was observed to promote the growth of P. trichocarpa. This response was cultivar independent. In contrast, I. europaea had no visible effect on P. trichocarpa growth. Metatranscriptomic studies revealed that these fungi impacted rhizophytic and endophytic activities in P. trichocarpa and induced shifts in soil and root microbial communities. Differential expression of core genes in P. trichocarpa roots was observed in response to both fungal species. Expression of P. trichocarpa genes for lipid signaling and nutrient uptake were upregulated, and expression of genes associated with gibberellin signaling were altered in plants inoculated with M. elongata, but not I. europaea. Upregulation of genes for growth promotion, downregulation of genes for several leucine-rich repeat receptor kinases, and alteration of expression of genes associated with plant defense responses (e.g., jasmonic acid, salicylic acid, and ethylene signal pathways) also suggest that M. elongata manipulates plant defenses while promoting plant growth.


Assuntos
Endófitos , Fungos , Regulação da Expressão Gênica de Plantas , Populus , Biodiversidade , Endófitos/fisiologia , Fungos/fisiologia , Fenótipo , Raízes de Plantas/microbiologia , Populus/microbiologia , Rizosfera
7.
New Phytol ; 222(1): 115-121, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29978909

RESUMO

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.


Assuntos
Archaea/metabolismo , Ecossistema , Metano/metabolismo , Populus/microbiologia , Madeira/microbiologia , Bactérias/crescimento & desenvolvimento , Biodiversidade , Microbiota , Análise de Componente Principal
8.
New Phytol ; 222(1): 18-28, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30394559

RESUMO

Tree stems from wetland, floodplain and upland forests can produce and emit methane (CH4 ). Tree CH4 stem emissions have high spatial and temporal variability, but there is no consensus on the biophysical mechanisms that drive stem CH4 production and emissions. Here, we summarize up to 30 opportunities and challenges for stem CH4 emissions research, which, when addressed, will improve estimates of the magnitudes, patterns and drivers of CH4 emissions and trace their potential origin. We identified the need: (1) for both long-term, high-frequency measurements of stem CH4 emissions to understand the fine-scale processes, alongside rapid large-scale measurements designed to understand the variability across individuals, species and ecosystems; (2) to identify microorganisms and biogeochemical pathways associated with CH4 production; and (3) to develop a mechanistic model including passive and active transport of CH4 from the soil-tree-atmosphere continuum. Addressing these challenges will help to constrain the magnitudes and patterns of CH4 emissions, and allow for the integration of pathways and mechanisms of CH4 production and emissions into process-based models. These advances will facilitate the upscaling of stem CH4 emissions to the ecosystem level and quantify the role of stem CH4 emissions for the local to global CH4 budget.


Assuntos
Ciclo do Carbono , Metano/metabolismo , Caules de Planta/metabolismo , Árvores/metabolismo , Modelos Biológicos , Água
9.
Glob Chang Biol ; 25(4): 1514-1528, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30659721

RESUMO

We examined the hypothesis that climate-driven evolution of plant traits will influence associated soil microbiomes and ecosystem function across the landscape. Using a foundation tree species, Populus angustifolia, observational and common garden approaches, and a base population genetic collection that spans 17 river systems in the western United States, from AZ to MT, we show that (a) as mean annual temperature (MAT) increases, genetic and phenotypic variation for bud break phenology decline; (b) soil microbiomes, soil nitrogen (N), and soil carbon (C) vary in response to MAT and conditioning by trees; and (c) with losses of genetic variation due to warming, population-level regulation of community and ecosystem functions strengthen. These results demonstrate a relationship between the potential evolutionary response of populations and subsequent shifts in ecosystem function along a large temperature gradient.

10.
Appl Environ Microbiol ; 84(1)2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29079614

RESUMO

Numerous studies have shown that the continuous increase of atmosphere CO2 concentrations may have profound effects on the forest ecosystem and its functions. However, little is known about the response of belowground soil microbial communities under elevated atmospheric CO2 (eCO2) at different soil depth profiles in forest ecosystems. Here, we examined soil microbial communities at two soil depths (0 to 5 cm and 5 to 15 cm) after a 10-year eCO2 exposure using a high-throughput functional gene microarray (GeoChip). The results showed that eCO2 significantly shifted the compositions, including phylogenetic and functional gene structures, of soil microbial communities at both soil depths. Key functional genes, including those involved in carbon degradation and fixation, methane metabolism, denitrification, ammonification, and nitrogen fixation, were stimulated under eCO2 at both soil depths, although the stimulation effect of eCO2 on these functional markers was greater at the soil depth of 0 to 5 cm than of 5 to 15 cm. Moreover, a canonical correspondence analysis suggested that NO3-N, total nitrogen (TN), total carbon (TC), and leaf litter were significantly correlated with the composition of the whole microbial community. This study revealed a positive feedback of eCO2 in forest soil microbial communities, which may provide new insight for a further understanding of forest ecosystem responses to global CO2 increases.IMPORTANCE The concentration of atmospheric carbon dioxide (CO2) has continuously been increasing since the industrial revolution. Understanding the response of soil microbial communities to elevated atmospheric CO2 (eCO2) is important for predicting the contribution of the forest ecosystem to global atmospheric change. This study analyzed the effect of eCO2 on microbial communities at two soil depths (0 to 5 cm and 5 to 15 cm) in a forest ecosystem. Our findings suggest that the compositional and functional structures of microbial communities shifted under eCO2 at both soil depths. More functional genes involved in carbon, nitrogen, and phosphorus cycling were stimulated under eCO2 at the soil depth of 0 to 5 cm than at the depth of 5 to 15 cm.


Assuntos
Dióxido de Carbono/metabolismo , Florestas , Microbiota , Microbiologia do Solo , Microbiota/genética , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , Tennessee
11.
Glob Chang Biol ; 24(8): 3452-3461, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29645398

RESUMO

Long-term elevated nitrogen (N) input from anthropogenic sources may cause soil acidification and decrease crop yield, yet the response of the belowground microbial community to long-term N input alone or in combination with phosphorus (P) and potassium (K) is poorly understood. We explored the effect of long-term N and NPK fertilization on soil bacterial diversity and community composition using meta-analysis of a global dataset. Nitrogen fertilization decreased soil pH, and increased soil organic carbon (C) and available N contents. Bacterial taxonomic diversity was decreased by N fertilization alone, but was increased by NPK fertilization. The effect of N fertilization on bacterial diversity varied with soil texture and water management, but was independent of crop type or N application rate. Changes in bacterial diversity were positively related to both soil pH and organic C content under N fertilization alone, but only to soil organic C under NPK fertilization. Microbial biomass C decreased with decreasing bacterial diversity under long-term N fertilization. Nitrogen fertilization increased the relative abundance of Proteobacteria and Actinobacteria, but reduced the abundance of Acidobacteria, consistent with the general life history strategy theory for bacteria. The positive correlation between N application rate and the relative abundance of Actinobacteria indicates that increased N availability favored the growth of Actinobacteria. This first global analysis of long-term N and NPK fertilization that differentially affects bacterial diversity and community composition provides a reference for nutrient management strategies for maintaining belowground microbial diversity in agro-ecosystems worldwide.


Assuntos
Agricultura , Ecossistema , Fertilizantes/análise , Microbiota , Microbiologia do Solo , Actinobacteria , Nitrogênio/análise , Fósforo/análise , Potássio/análise , Proteobactérias
12.
Appl Environ Microbiol ; 83(17)2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28667112

RESUMO

Microbial N2 fixation (diazotrophy) represents an important nitrogen source to oligotrophic peatland ecosystems, which are important sinks for atmospheric CO2 and are susceptible to the changing climate. The objectives of this study were (i) to determine the active microbial group and type of nitrogenase mediating diazotrophy in an ombrotrophic Sphagnum-dominated peat bog (the S1 peat bog, Marcell Experimental Forest, Minnesota, USA); and (ii) to determine the effect of environmental parameters (light, O2, CO2, and CH4) on potential rates of diazotrophy measured by acetylene (C2H2) reduction and 15N2 incorporation. A molecular analysis of metabolically active microbial communities suggested that diazotrophy in surface peat was primarily mediated by Alphaproteobacteria (Bradyrhizobiaceae and Beijerinckiaceae). Despite higher concentrations of dissolved vanadium ([V] 11 nM) than molybdenum ([Mo] 3 nM) in surface peat, a combination of metagenomic, amplicon sequencing, and activity measurements indicated that Mo-containing nitrogenases dominate over the V-containing form. Acetylene reduction was only detected in surface peat exposed to light, with the highest rates observed in peat collected from hollows with the highest water contents. Incorporation of 15N2 was suppressed 90% by O2 and 55% by C2H2 and was unaffected by CH4 and CO2 amendments. These results suggest that peatland diazotrophy is mediated by a combination of C2H2-sensitive and C2H2-insensitive microbes that are more active at low concentrations of O2 and show similar activity at high and low concentrations of CH4 IMPORTANCE Previous studies indicate that diazotrophy provides an important nitrogen source and is linked to methanotrophy in Sphagnum-dominated peatlands. However, the environmental controls and enzymatic pathways of peatland diazotrophy, as well as the metabolically active microbial populations that catalyze this process, remain in question. Our findings indicate that oxygen levels and photosynthetic activity override low nutrient availability in limiting diazotrophy and that members of the Alphaproteobacteria (Rhizobiales) catalyze this process at the bog surface using the molybdenum-based form of the nitrogenase enzyme.

13.
New Phytol ; 235(5): 2127, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35781272
14.
Environ Sci Technol ; 51(7): 3609-3620, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28300407

RESUMO

To further understand the diversity and dynamics of SRB in response to substrate amendment, we sequenced genes coding for the dissimilatory sulfite reductase (dsrA) in groundwater samples collected after an emulsified vegetable oil (EVO) amendment, which sustained U(VI)-reducing conditions for one year in a fast-flowing aquifer. EVO amendment significantly altered the composition of groundwater SRB communities. Sequences having no closely related-described species dominated (80%) the indigenous SRB communities in nonamended wells. After EVO amendment, Desulfococcus, Desulfobacterium, and Desulfovibrio, known for long-chain-fatty-acid, short-chain-fatty-acid and H2 oxidation and U(VI) reduction, became dominant accounting for 7 ± 2%, 21 ± 8%, and 55 ± 8% of the SRB communities, respectively. Succession of these SRB at different bioactivity stages based on redox substrates/products (acetate, SO4-2, U(VI), NO3-, Fe(II), and Mn(II)) was observed. Desulfovibrio and Desulfococcus dominated SRB communities at 4-31 days, whereas Desulfobacterium became dominant at 80-140 days. By the end of the experiment (day 269), the abundance of these SRB decreased but the overall diversity of groundwater SRB was still higher than non-EVO controls. Up to 62% of the SRB community changes could be explained by groundwater geochemical variables, including those redox substrates/products. A significant (P < 0.001) correlation was observed between groundwater U(VI) concentrations and Desulfovibrio abundance. Our results showed that the members of SRB and their dynamics were correlated significantly with slow EVO biodegradation, electron donor production and maintenance of U(VI)-reducing conditions in the aquifer.


Assuntos
Água Subterrânea/química , Urânio/metabolismo , Bactérias/metabolismo , Biodegradação Ambiental , Oxirredução , Sulfatos/química , Óxidos de Enxofre
15.
Mycologia ; 109(1): 18-26, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28402786

RESUMO

Among fungi isolated from healthy root mycobiomes of Populus, we discovered a new endorrhizal fungal species belonging to the rust lineage Pucciniomycotina, described here as Atractiella rhizophila. We characterized this species by transmission electron microscopy (TEM), phylogenetic analysis, and plant bioassay experiments. Phylogenetic sequence analysis of isolates and available environmental and reference sequences indicates that this new species, A. rhizophila, has a broad geographic and host range. Atractiella rhizophila appears to be present in North America, Australia, Asia, and Africa and is associated with trees, orchids, and other agriculturally important species, including soybean, corn, and rice. Despite the large geographic and host range of this species sampling, A. rhizophila appears to have exceptionally low sequence variation within nuclear rDNA markers examined. With inoculation studies, we demonstrate that A. rhizophila is nonpathogenic, asymptomatically colonizes plant roots, and appears to foster plant growth and elevated photosynthesis rates.


Assuntos
Basidiomycota/classificação , Basidiomycota/isolamento & purificação , Endófitos/classificação , Endófitos/isolamento & purificação , Raízes de Plantas/microbiologia , Populus/microbiologia , África , Ásia , Austrália , Basidiomycota/ultraestrutura , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , DNA Ribossômico/genética , DNA Espaçador Ribossômico/química , DNA Espaçador Ribossômico/genética , Endófitos/ultraestrutura , Microscopia Eletrônica de Transmissão , América do Norte , Filogeografia , Análise de Sequência de DNA
16.
Environ Microbiol ; 18(1): 205-18, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26177312

RESUMO

Discerning network interactions among different species/populations in microbial communities has evoked substantial interests in recent years, but little information is available about temporal dynamics of microbial network interactions in response to environmental perturbations. Here, we modified the random matrix theory-based network approach to discern network succession in groundwater microbial communities in response to emulsified vegetable oil (EVO) amendment for uranium bioremediation. Groundwater microbial communities from one control and seven monitor wells were analysed with a functional gene array (GeoChip 3.0), and functional molecular ecological networks (fMENs) at different time points were reconstructed. Our results showed that the network interactions were dramatically altered by EVO amendment. Dynamic and resilient succession was evident: fairly simple at the initial stage (Day 0), increasingly complex at the middle period (Days 4, 17, 31), most complex at Day 80, and then decreasingly complex at a later stage (140-269 days). Unlike previous studies in other habitats, negative interactions predominated in a time-series fMEN, suggesting strong competition among different microbial species in the groundwater systems after EVO injection. Particularly, several keystone sulfate-reducing bacteria showed strong negative interactions with their network neighbours. These results provide mechanistic understanding of the decreased phylogenetic diversity during environmental perturbations.


Assuntos
Bactérias/metabolismo , Biodegradação Ambiental , Água Subterrânea/microbiologia , Óleos de Plantas/metabolismo , Urânio/metabolismo , Bactérias/genética , Ecossistema , Microbiota/genética , Filogenia
17.
Appl Environ Microbiol ; 82(10): 2919-2928, 2016 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-26969694

RESUMO

UNLABELLED: Members of the Fungi convert nitrate (NO3 (-)) and nitrite (NO2 (-)) to gaseous nitrous oxide (N2O) (denitrification), but the fungal contributions to N loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations, and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N2O from added NO3 (-) or NO2 (-) in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, 151 of which produced N2O from NO2 (-) Novel PCR primers targeting the p450nor gene, which encodes the nitric oxide (NO) reductase responsible for N2O production in fungi, yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54 to 98% amino acid identity with reference P450nor sequences within the phylum Ascomycota and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N2O from NO2 (-), whereas nirK (encoding the NO-forming NO2 (-) reductase) was amplified in only 13 to 74% of the N2O-forming isolates using two separate nirK primer sets. Collectively, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation. IMPORTANCE: A comprehensive understanding of the microbiota controlling soil N loss and greenhouse gas (N2O) emissions is crucial for sustainable agricultural practices and addressing climate change concerns. We report the design and application of a novel PCR primer set targeting fungal p450nor, a biomarker for fungal N2O production, and demonstrate the utility of the new approach to assess fungal denitrification potential in fungal isolates and agricultural soils. These new PCR primers may find application in a variety of biomes to assess the fungal contributions to N loss and N2O emissions.


Assuntos
Proteínas Fúngicas/genética , Fungos/enzimologia , Metagenoma , Oxirredutases/genética , Microbiologia do Solo , DNA Fúngico/genética , Proteínas Fúngicas/análise , Fungos/classificação , Fungos/isolamento & purificação , Variação Genética , Meio-Oeste dos Estados Unidos , Nitratos/metabolismo , Nitritos/metabolismo , Óxido Nitroso/metabolismo , Oxirredução , Oxirredutases/análise , Reação em Cadeia da Polimerase , Análise de Sequência de DNA
18.
Appl Environ Microbiol ; 82(18): 5698-708, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27422831

RESUMO

UNLABELLED: Bacterial endophytes that colonize Populus trees contribute to nutrient acquisition, prime immunity responses, and directly or indirectly increase both above- and below-ground biomasses. Endophytes are embedded within plant material, so physical separation and isolation are difficult tasks. Application of culture-independent methods, such as metagenome or bacterial transcriptome sequencing, has been limited due to the predominance of DNA from the plant biomass. Here, we describe a modified differential and density gradient centrifugation-based protocol for the separation of endophytic bacteria from Populus roots. This protocol achieved substantial reduction in contaminating plant DNA, allowed enrichment of endophytic bacteria away from the plant material, and enabled single-cell genomics analysis. Four single-cell genomes were selected for whole-genome amplification based on their rarity in the microbiome (potentially uncultured taxa) as well as their inferred abilities to form associations with plants. Bioinformatics analyses, including assembly, contamination removal, and completeness estimation, were performed to obtain single-amplified genomes (SAGs) of organisms from the phyla Armatimonadetes, Verrucomicrobia, and Planctomycetes, which were unrepresented in our previous cultivation efforts. Comparative genomic analysis revealed unique characteristics of each SAG that could facilitate future cultivation efforts for these bacteria. IMPORTANCE: Plant roots harbor a diverse collection of microbes that live within host tissues. To gain a comprehensive understanding of microbial adaptations to this endophytic lifestyle from strains that cannot be cultivated, it is necessary to separate bacterial cells from the predominance of plant tissue. This study provides a valuable approach for the separation and isolation of endophytic bacteria from plant root tissue. Isolated live bacteria provide material for microbiome sequencing, single-cell genomics, and analyses of genomes of uncultured bacteria to provide genomics information that will facilitate future cultivation attempts.


Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Endófitos/classificação , Endófitos/isolamento & purificação , Raízes de Plantas/microbiologia , Populus/microbiologia , Bactérias/genética , Centrifugação com Gradiente de Concentração/métodos , Biologia Computacional , Endófitos/genética , Metagenômica , Análise de Sequência de DNA , Análise de Célula Única/métodos
19.
Appl Environ Microbiol ; 82(1): 375-83, 2016 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-26519390

RESUMO

The Pseudomonas genus contains a metabolically versatile group of organisms that are known to occupy numerous ecological niches, including the rhizosphere and endosphere of many plants. Their diversity influences the phylogenetic diversity and heterogeneity of these communities. On the basis of average amino acid identity, comparative genome analysis of >1,000 Pseudomonas genomes, including 21 Pseudomonas strains isolated from the roots of native Populus deltoides (eastern cottonwood) trees resulted in consistent and robust genomic clusters with phylogenetic homogeneity. All Pseudomonas aeruginosa genomes clustered together, and these were clearly distinct from other Pseudomonas species groups on the basis of pangenome and core genome analyses. In contrast, the genomes of Pseudomonas fluorescens were organized into 20 distinct genomic clusters, representing enormous diversity and heterogeneity. Most of our 21 Populus-associated isolates formed three distinct subgroups within the major P. fluorescens group, supported by pathway profile analysis, while two isolates were more closely related to Pseudomonas chlororaphis and Pseudomonas putida. Genes specific to Populus-associated subgroups were identified. Genes specific to subgroup 1 include several sensory systems that act in two-component signal transduction, a TonB-dependent receptor, and a phosphorelay sensor. Genes specific to subgroup 2 contain hypothetical genes, and genes specific to subgroup 3 were annotated with hydrolase activity. This study justifies the need to sequence multiple isolates, especially from P. fluorescens, which displays the most genetic variation, in order to study functional capabilities from a pangenomic perspective. This information will prove useful when choosing Pseudomonas strains for use to promote growth and increase disease resistance in plants.


Assuntos
Variação Genética , Genoma Bacteriano , Populus/microbiologia , Pseudomonas/classificação , Pseudomonas/genética , Hibridização Genômica Comparativa , Filogenia , Raízes de Plantas/microbiologia , Pseudomonas/isolamento & purificação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas fluorescens/classificação , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/isolamento & purificação , Pseudomonas putida/genética , Pseudomonas putida/isolamento & purificação , Rizosfera , Análise de Sequência de DNA
20.
Glob Chang Biol ; 22(2): 957-64, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26414247

RESUMO

Although elevated CO2 (eCO2 ) significantly affects the α-diversity, composition, function, interaction and dynamics of soil microbial communities at the local scale, little is known about eCO2 impacts on the geographic distribution of micro-organisms regionally or globally. Here, we examined the ß-diversity of 110 soil microbial communities across six free air CO2 enrichment (FACE) experimental sites using a high-throughput functional gene array. The ß-diversity of soil microbial communities was significantly (P < 0.05) correlated with geographic distance under both CO2 conditions, but declined significantly (P < 0.05) faster at eCO2 with a slope of -0.0250 than at ambient CO2 (aCO2 ) with a slope of -0.0231 although it varied within each individual site, indicating that the spatial turnover rate of soil microbial communities was accelerated under eCO2 at a larger geographic scale (e.g. regionally). Both distance and soil properties significantly (P < 0.05) contributed to the observed microbial ß-diversity. This study provides new hypotheses for further understanding their assembly mechanisms that may be especially important as global CO2 continues to increase.


Assuntos
Dióxido de Carbono/farmacologia , Microbiologia do Solo , Bactérias/efeitos dos fármacos , Bactérias/genética , DNA Bacteriano/análise
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