The diversity of soil microbial communities matters when legumes face drought.

The cultivation of legumes shows promise for the development of sustainable agriculture, but yield instability remains one of the main obstacles for its adoption. Here, we tested whether the yield stability (i.e., resistance and resilience) of pea plants subjected to drought could be enhanced by soil microbial diversity. We used a dilution approach to manipulate the microbial diversity, with a genotype approach to distinguish the effect of symbionts from that of microbial diversity as a whole. We investigated the physiology of plants in response to drought when grown on a soil containing high or low level of microbial diversity. Plants grown under high microbial diversity displayed higher productivity and greater resilience after drought. Yield losses were mitigated by 15% on average in the presence of high soil microbial diversity at sowing. Our study provides proof of concept that the soil microbial community as a whole plays a key role for yield stability after drought even in plant species living in relationships with microbial symbionts. These results emphasize the need to restore soil biodiversity for sustainable crop management and climate change adaptation.

The interaction of soil phototrophs and fungi with pH and their impact on soil CO2, CO18O and OCS exchange.

The stable oxygen isotope composition of atmospheric CO2 and the mixing ratio of carbonyl sulphide (OCS) are potential tracers of biospheric CO2 fluxes at large scales. However, the use of these tracers hinges on our ability to understand and better predict the activity of the enzyme carbonic anhydrase (CA) in different soil microbial groups, including phototrophs. Because different classes of the CA family (α, ß and γ) may have different affinities to CO2 and OCS and their expression should also vary between different microbial groups, differences in the community structure could impact the 'community-integrated' CA activity differently for CO2 and OCS. Four soils of different pH were incubated in the dark or with a diurnal cycle for forty days to vary the abundance of native phototrophs. Fluxes of CO2, CO18O and OCS were measured to estimate CA activity alongside the abundance of bacteria, fungi and phototrophs. The abundance of soil phototrophs increased most at higher soil pH. In the light, the strength of the soil CO2 sink and the CA-driven CO2-H2O isotopic exchange rates correlated with phototrophs abundance. OCS uptake rates were attributed to fungi whose abundance was positively enhanced in alkaline soils but only in the presence of increased phototrophs. Our findings demonstrate that soil-atmosphere CO2, OCS and CO18O fluxes are strongly regulated by the microbial community structure in response to changes in soil pH and light availability and supports the idea that different members of the microbial community express different classes of CA, with different affinities to CO2 and OCS.

Correction: Mapping and predictive variations of soil bacterial richness across France.

[This corrects the article DOI: 10.1371/journal.pone.0186766.].

µgreen-db: a reference database for the 23S rRNA gene of eukaryotic plastids and cyanobacteria.

Studying the ecology of photosynthetic microeukaryotes and prokaryotic cyanobacterial communities requires molecular tools to complement morphological observations. These tools rely on specific genetic markers and require the development of specialised databases to achieve taxonomic assignment. We set up a reference database, called µgreen-db, for the 23S rRNA gene. The sequences were retrieved from generalist (NCBI, SILVA) or Comparative RNA Web (CRW) databases, in addition to a more original approach involving recursive BLAST searches to obtain the best possible sequence recovery. At present, µgreen-db includes 2,326 23S rRNA sequences belonging to both eukaryotes and prokaryotes encompassing 442 unique genera and 736 species of photosynthetic microeukaryotes, cyanobacteria and non-vascular land plants based on the NCBI and AlgaeBase taxonomy. When PR2/SILVA taxonomy is used instead, µgreen-db contains 2,217 sequences (399 unique genera and 696 unique species). Using µgreen-db, we were able to assign 96% of the sequences of the V domain of the 23S rRNA gene obtained by metabarcoding after amplification from soil DNA at the genus level, highlighting good coverage of the database. µgreen-db is accessible at http://microgreen-23sdatabase.ea.inra.fr.

Reduced microbial diversity induces larger volatile organic compound emissions from soils.

Microorganisms in soil are known to be a source and a sink of volatile organic compounds (VOCs). The role of the microbial VOCs on soil ecosystem regulation has been increasingly demonstrated in the recent years. Nevertheless, little is known about the influence of the microbial soil community structure and diversity on VOC emissions. This novel study analyzed the effect of reduced microbial diversity in soil on VOC emissions. We found that reduced levels of microbial diversity in soil increased VOC emissions from soils, while the number of different VOCs emitted decreased. Furthermore, we found that Proteobacteria, Bacteroidetes and fungi phyla were positively correlated to VOC emissions, and other prokaryotic phyla were either negatively correlated or very slightly positively correlated to VOCs emissions. Our interpretation is that Proteobacteria, Bacteroidetes and fungi were VOC producers while the other prokaryotic phyla were consumers. Finally, we discussed the possible role of VOCs as mediators of microbial interactions in soil.

Tillage intensity and pasture in rotation effectively shape soil microbial communities at a landscape scale.

Soil microorganisms are essential to agroecosystem functioning and services. Yet, we still lack information on which farming practices can effectively shape the soil microbial communities. The aim of this study was to identify the farming practices, which are most effective at positively or negatively modifying bacterial and fungal diversity while considering the soil environmental variation at a landscape scale. A long-term research study catchment (12 km2 ) representative of intensive mixed farming (livestock and crop) in Western Europe was investigated using a regular grid for soil sampling (n = 186). Farming systems on this landscape scale were described in terms of crop rotation, use of fertilizer, soil tillage, pesticides treatments, and liming. Molecular microbial biomass was estimated by soil DNA recovery. Bacterial and fungal communities were analyzed by 16S and 18S rRNA gene pyrosequencing. Microbial biomass was significantly stimulated by the presence of pasture during the crop rotation since temporary and permanent pastures, as compared to annual crops, increased the soil microbial biomass by +23% and +93% respectively. While soil properties (mainly pH) explained much of the variation in bacterial diversity, soil tillage seemed to be the most influential among the farming practices. A 2.4% increase in bacterial richness was observed along our gradient of soil tillage intensity. In contrast, farming practices were the predominant drivers of fungal diversity, which was mainly determined by the presence of pastures during the crop rotation. Compared to annual crops, temporary and permanent pastures increased soil fungal richness by +10% and +14.5%, respectively. Altogether, our landscape-scale investigation allows the identification of farming practices that can effectively shape the soil microbial abundance and diversity, with the goal to improve agricultural soil management and soil ecological integrity.

The dynamics of soil bacterial community structure in response to yearly repeated agricultural copper treatments.

The annual dynamics of soil bacterial community structure, including early, dose-dependent and transient modifications, was observed consecutively at different levels of copper contamination (high: 48 kg Cu ha(-1), low: 16 kg Cu ha(-1)) repeated yearly over a three-year field experiment. Repeated low-level Cu contamination led to an increase in community stability to metal stress without a long-term shift in the population structure, whereas repeated high-level Cu contamination induced a novel and stable bacterial community structure. Furthermore, field experimentation highlighted that episodic climatic stress can modulate copper impact by enhancing community stability.

Protein and DNA fingerprinting of a soil bacterial community inoculated into three different sterile soils.

The functional and genetic structures of a soil bacterial community were characterized after inoculation into three different sterile soils using a protein and DNA fingerprinting method, respectively. Principal component analysis (PCA) of profiles revealed that, depending on soil characteristics, bacterial communities with similar genetic structures harbored different functional structures and thus could potentially be of differing ecological significance for soil functioning. Co-inertia analysis between protein fingerprinting data and the corresponding sets of soil physicochemical characteristics demonstrated the correlation between the functional structure of the bacterial community and soil parameters, with pH, clay and CaCO(3) contents being the most discriminating factors.

Biogeography of soil bacteria and archaea across France.

Over the last two decades, a considerable effort has been made to decipher the biogeography of soil microbial communities as a whole, from small to broad scales. In contrast, few studies have focused on the taxonomic groups constituting these communities; thus, our knowledge of their ecological attributes and the drivers determining their composition and distribution is limited. We applied a pyrosequencing approach targeting 16S ribosomal RNA (rRNA) genes in soil DNA to a set of 2173 soil samples from France to reach a comprehensive understanding of the spatial distribution of bacteria and archaea and to identify the ecological processes and environmental drivers involved. Taxonomic assignment of the soil 16S rRNA sequences indicated the presence of 32 bacterial phyla or subphyla and 3 archaeal phyla. Twenty of these 35 phyla were cosmopolitan and abundant, with heterogeneous spatial distributions structured in patches ranging from a 43- to 260-km radius. The hierarchy of the main environmental drivers of phyla distribution was soil pH > land management > soil texture > soil nutrients > climate. At a lower taxonomic level, 47 dominant genera belonging to 12 phyla aggregated 62.1% of the sequences. We also showed that the phylum-level distribution can be determined largely by the distribution of the dominant genus or, alternatively, reflect the combined distribution of all of the phylum members. Together, our study demonstrated that soil bacteria and archaea present highly diverse biogeographical patterns on a nationwide scale and that studies based on intensive and systematic sampling on a wide spatial scale provide a promising contribution for elucidating soil biodiversity determinism.

Fingerprinting and diversity of bacterial copA genes in response to soil types, soil organic status and copper contamination.

A molecular fingerprinting assay was developed to assess the diversity of copA genes, one of the genetic determinants involved in bacterial resistance to copper. Consensus primers of the copA genes were deduced from an alignment of sequences from proteobacterial strains. A PCR detection procedure was optimized for bacterial strains and allowed the description of a novel copA genetic determinant in Pseudomonas fluorescens. The copA DNA fingerprinting procedure was optimized for DNA directly extracted from soils differing in their physico-chemical characteristics and in their organic status (SOS). Particular copA genetic structures were obtained for each studied soil and a coinertia analysis with soil physico-chemical characteristics revealed the strong influence of pH, soil texture and the quality of soil organic matter. The molecular phylogeny of copA gene confirmed that specific copA genes clusters are specific for each SOS. Furthermore, this study demonstrates that this approach was sensitive to short-term responses of copA gene diversity to copper additions to soil samples, suggesting that community adaptation is preferentially controlled by the diversity of the innate copA genes rather than by the bioavailability of the metal.

Mapping and predictive variations of soil bacterial richness across France.

Although numerous studies have demonstrated the key role of bacterial diversity in soil functions and ecosystem services, little is known about the variations and determinants of such diversity on a nationwide scale. The overall objectives of this study were i) to describe the bacterial taxonomic richness variations across France, ii) to identify the ecological processes (i.e. selection by the environment and dispersal limitation) influencing this distribution, and iii) to develop a statistical predictive model of soil bacterial richness. We used the French Soil Quality Monitoring Network (RMQS), which covers all of France with 2,173 sites. The soil bacterial richness (i.e. OTU number) was determined by pyrosequencing 16S rRNA genes and related to the soil characteristics, climatic conditions, geomorphology, land use and space. Mapping of bacterial richness revealed a heterogeneous spatial distribution, structured into patches of about 111km, where the main drivers were the soil physico-chemical properties (18% of explained variance), the spatial descriptors (5.25%, 1.89% and 1.02% for the fine, medium and coarse scales, respectively), and the land use (1.4%). Based on these drivers, a predictive model was developed, which allows a good prediction of the bacterial richness (R2adj of 0.56) and provides a reference value for a given pedoclimatic condition.

Correction: Mapping and predictive variations of soil bacterial richness across France.

[This corrects the article DOI: 10.1371/journal.pone.0186766.].

Field and microcosm experiments to evaluate the effects of agricultural Cu treatment on the density and genetic structure of microbial communities in two different soils.

The effects of Cu amendment on indigenous soil microorganisms were investigated in two soils, a calcareous silty clay (Ep) and a sandy soil (Au), by means of a 1-year field experiment and a two-month microcosm incubation. Cu was added as 'Bordeaux mixture' [CuSO(4), Ca(OH)(2)] at the standard rate used in viticulture (B1=16 kg Cu kg(-1) soil) and at a higher level of contamination (B3=48 kg Cu ha(-1) soil). More extractable Cu was observed in sandy soil (Au) than in silty soil (Ep). Furthermore, total Cu and Cu-EDTA declined with time in Au soil, whereas they remained stable in Ep soil. Quantitative modifications of the microflora were assessed by C-biomass measurements and qualitative modifications were assessed by the characterization of the genetic structure of bacterial and fungal communities from DNA directly extracted from the soil, using B- and F-ARISA (bacterial and fungal automated ribosomal intergenic spacer analysis). In the field study, no significant modifications were observed in C-biomass whereas microcosm incubation showed a decrease in B3 contamination only. ARISA fingerprinting showed slight but significant modifications of bacterial and fungal communities in field and microcosm incubation. These modifications were transient in all cases, suggesting a short-term effect of Cu stress. Microcosm experiments detected the microbial community modifications with greater precision in the short-term, while field experiments showed that the biological effects of Cu contamination may be overcome or hidden by pedo-climatic variations.

Contrasting spatial patterns and ecological attributes of soil bacterial and archaeal taxa across a landscape.

Even though recent studies have clarified the influence and hierarchy of environmental filters on bacterial community structure, those constraining bacterial populations variations remain unclear. In consequence, our ability to understand to ecological attributes of soil bacteria and to predict microbial community response to environmental stress is therefore limited. Here, we characterized the bacterial community composition and the various bacterial taxonomic groups constituting the community across an agricultural landscape of 12 km(2) , by using a 215 × 215 m systematic grid representing 278 sites to precisely decipher their spatial distribution and drivers at this scale. The bacterial and Archaeal community composition was characterized by applying 16S rRNA gene pyrosequencing directly to soil DNA from samples. Geostatistics tools were used to reveal the heterogeneous distribution of bacterial composition at this scale. Soil physical parameters and land management explained a significant amount of variation, suggesting that environmental selection is the major process shaping bacterial composition. All taxa systematically displayed also a heterogeneous and particular distribution patterns. Different relative influences of soil characteristics, land use and space were observed, depending on the taxa, implying that selection and spatial processes might be differentially but not exclusively involved for each bacterial phylum. Soil pH was a major factor determining the distribution of most of the bacterial taxa and especially the most important factor explaining the spatial patterns of α-Proteobacteria and Planctomycetes. Soil texture, organic carbon content and quality were more specific to a few number of taxa (e.g., ß-Proteobacteria and Chlorobi). Land management also influenced the distribution of bacterial taxa across the landscape and revealed different type of response to cropping intensity (positive, negative, neutral or hump-backed relationships) according to phyla. Altogether, this study provided valuable clues about the ecological behavior of soil bacterial and archaeal taxa at an agricultural landscape scale and could be useful for developing sustainable strategies of land management.

Mapping and determinism of soil microbial community distribution across an agricultural landscape.

Despite the relevance of landscape, regarding the spatial patterning of microbial communities and the relative influence of environmental parameters versus human activities, few investigations have been conducted at this scale. Here, we used a systematic grid to characterize the distribution of soil microbial communities at 278 sites across a monitored agricultural landscape of 13 km². Molecular microbial biomass was estimated by soil DNA recovery and bacterial diversity by 16S rRNA gene pyrosequencing. Geostatistics provided the first maps of microbial community at this scale and revealed a heterogeneous but spatially structured distribution of microbial biomass and diversity with patches of several hundreds of meters. Variance partitioning revealed that both microbial abundance and bacterial diversity distribution were highly dependent of soil properties and land use (total variance explained ranged between 55% and 78%). Microbial biomass and bacterial richness distributions were mainly explained by soil pH and texture whereas bacterial evenness distribution was mainly related to land management. Bacterial diversity (richness, evenness, and Shannon index) was positively influenced by cropping intensity and especially by soil tillage, resulting in spots of low microbial diversity in soils under forest management. Spatial descriptors also explained a small but significant portion of the microbial distribution suggesting that landscape configuration also shapes microbial biomass and bacterial diversity.

Microbial diversity and structure are drivers of the biological barrier effect against Listeria monocytogenes in soil.

Understanding the ecology of pathogenic organisms is important in order to monitor their transmission in the environment and the related health hazards. We investigated the relationship between soil microbial diversity and the barrier effect against Listeria monocytogenes invasion. By using a dilution-to-extinction approach, we analysed the consequence of eroding microbial diversity on L. monocytogenes population dynamics under standardised conditions of abiotic parameters and microbial abundance in soil microcosms. We demonstrated that highly diverse soil microbial communities act as a biological barrier against L. monocytogenes invasion and that phylogenetic composition of the community also has to be considered. This suggests that erosion of diversity may have damaging effects regarding circulation of pathogenic microorganisms in the environment.

Evaluation of the ISO standard 11063 DNA extraction procedure for assessing soil microbial abundance and community structure.

Soil DNA extraction has become a critical step in describing microbial biodiversity. Historically, ascertaining overarching microbial ecological theories has been hindered as independent studies have used numerous custom and commercial DNA extraction procedures. For that reason, a standardized soil DNA extraction method (ISO-11063) was previously published. However, although this ISO method is suited for molecular tools such as quantitative PCR and community fingerprinting techniques, it has only been optimized for examining soil bacteria. Therefore, the aim of this study was to assess an appropriate soil DNA extraction procedure for examining bacterial, archaeal and fungal diversity in soils of contrasting land-use and physico-chemical properties. Three different procedures were tested: the ISO-11063 standard; a custom procedure (GnS-GII); and a modified ISO procedure (ISOm) which includes a different mechanical lysis step (a FastPrep ®-24 lysis step instead of the recommended bead-beating). The efficacy of each method was first assessed by estimating microbial biomass through total DNA quantification. Then, the abundances and community structure of bacteria, archaea and fungi were determined using real-time PCR and terminal restriction fragment length polymorphism approaches. Results showed that DNA yield was improved with the GnS-GII and ISOm procedures, and fungal community patterns were found to be strongly dependent on the extraction method. The main methodological factor responsible for differences between extraction procedure efficiencies was found to be the soil homogenization step. For integrative studies which aim to examine bacteria, archaea and fungi simultaneously, the ISOm procedure results in higher DNA recovery and better represents microbial communities.

Molecular biomass and MetaTaxogenomic assessment of soil microbial communities as influenced by soil DNA extraction procedure.

Three soil DNA extraction procedures (homemade protocols and commercial kit) varying in their practicability were applied to contrasting soils to evaluate their efficiency in recovering: (i) soil DNA and (ii) bacterial diversity estimated by 16S rDNA pyrosequencing. Significant differences in DNA yield were systematically observed between tested procedures. For certain soils, 10 times more DNA was recovered with one protocol than with the others. About 15,000 sequences of 16S rDNA were obtained for each sample which were clustered to draw rarefaction curves. These curves, as well as the PCA ordination of community composition based on OTU clustering, did not reveal any significant difference between procedures. Nevertheless, significant differences between procedures were highlighted by the taxonomic identification of sequences obtained at the phylum to genus levels. Depending on the soil, differences in the number of genera detected ranged from 1% to 26% between the most and least efficient procedures, mainly due to a poorer capacity to recover populations belonging to Actinobacteria, Firmicutes or Crenarchaeota. This study enabled us to rank the relative efficiencies of protocols for their recovery of soil molecular microbial biomass and bacterial diversity and to help choosing an appropriate soil DNA extraction procedure adapted to novel sequencing technologies.

Copper dynamics and impact on microbial communities in soils of variable organic status.

The effect of soil organic status on copper impact was investigated by means of a microcosm study carried out on a vineyard soil that had been amended with varying types of organic matter during a previous long-term field experiment. Soil microcosms were contaminated at 250 mg Cu kg(-1) and incubated for 35 days. Copper distribution and dynamics were assessed in the solid matrix by a sequential extraction procedure and in the soil solution by measuring total and free exchangeable copper concentrations. Copper bioavailability was also measured with a whole-cell biosensor. Modifications of microbial communities were assessed by means of biomass-C measurements and characterization of genetic structure using ARISA (automated-ribosomal-intergenic-spacer-analysis). The results showed that copper distribution, speciation, and bioavailability are strongly different between organically amended and nonamended soils. Surprisingly, in solution, bioavailable copper correlated with total copper but not with free copper. Similarly the observed differential copper impact on micro-organisms suggested that organic matter controlled copper toxicity. Bacterial-ARISA modifications also correlated with the estimated metal bioavailability and corresponded to the enrichment of the Actinobacteria. Contrarily, biomass-C and fungal-ARISA measurements did not relate trivially to copper speciation and bioavailability, suggesting that the specific composition of the indigenous-soil communities controls its sensitivity to this metal.

Dynamics and identification of soil microbial populations actively assimilating carbon from 13C-labelled wheat residue as estimated by DNA- and RNA-SIP techniques.

This work is the first report on the use of DNA-, RNA-SIP approaches to elucidate the dynamics and the diversity of bacterial populations actively assimilating C derived from plant residues labelled at more than 90% (13)C. Wheat-residues, were incorporated and incubated into soil microcosms for 28 days. At the end of the incubation time, no more than 55% of the total CO(2) released was (13)C-labelled, suggesting the occurrence of an important priming effect process. After 7 days, more than 30% of the whole DNA extracted were labelled, allowing an efficient separation of labelled from unlabelled DNA using density gradient centrifugation. The genetic structure of bacterial community, assessed by Automated Ribosomal Intergenic Spacer Analysis technique, was deduced from the (13)C- and (12)C-fractions of control and enriched conditions, over the time course of the experiment. Dynamics showed that wheat residues directly induced a rapid and durable stimulation of fresh organic matter (FOM) degrading populations ((13)C), while specific soil organic matter (SOM) degrading populations ((12)C) seemed to be indirectly stimulated only at the early time point (t7d). After 14 days of incubations, 16S rRNA clone libraries were elaborated on (12)C- and (13)C-RNA extracted from enriched microcosms, as well as (12)C-RNA extracted from control condition. Stimulation of the beta- and gamma-subgroups of proteobacteria, where numerous populations were previously described as r-strategists or copiotrophic organisms, was recorded in the (13)C-fraction. In the mean time, several phyla like Actinobacteria, Cyanobacteria, Candidate, Gemmatimonadetes and Planctomycetes were only present in (12)C fractions. Surprisingly, several sequences affiliated to species characterized as oligotrophic organisms were retrieved in both types of fraction. Trophic relationships between soil bacteria involved in FOM and SOM degradation were discussed on the basis of different hypotheses of Fontaine and colleagues (2003) concerning the mechanisms of the priming effect induction.