Mélanie Lelièvre

Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1 / FF390. This in silico analysis of the specificity of FR1 / FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1 / FF390 for Fungi was validated in vitro by cloning - sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils.

Turnover of soil bacterial diversity driven by wide-scale environmental heterogeneity

Spatial scaling and determinism of the wide-scale distribution of macroorganism diversity has been largely demonstrated over a century. For microorganisms, and especially for soil bacteria, this fundamental question requires more thorough investigation, as little information has been reported to date. Here by applying the taxa-area relationship to the largest spatially explicit soil sampling available in France (2,085 soils, area covered ~5.3 × 10(5) km(2)) and developing an innovative evaluation of the habitat-area relationship, we show that the turnover rate of bacterial diversity in soils on a wide scale is highly significant and strongly correlated with the turnover rate of soil habitat. As the diversity of micro- and macroorganisms appears to be driven by similar processes (dispersal and selection), maintaining diverse and spatially structured habitats is essential for soil biological patrimony and the resulting ecosystem services.

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.

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.

Similar Processes but Different Environmental Filters for Soil Bacterial and Fungal Community Composition Turnover on a Broad Spatial Scale

Spatial scaling of microorganisms has been demonstrated over the last decade. However, the processes and environmental filters shaping soil microbial community structure on a broad spatial scale still need to be refined and ranked. Here, we compared bacterial and fungal community composition turnovers through a biogeographical approach on the same soil sampling design at a broad spatial scale (area range: 13300 to 31000 km2): i) to examine their spatial structuring; ii) to investigate the relative importance of environmental selection and spatial autocorrelation in determining their community composition turnover; and iii) to identify and rank the relevant environmental filters and scales involved in their spatial variations. Molecular fingerprinting of soil bacterial and fungal communities was performed on 413 soils from four French regions of contrasting environmental heterogeneity (Landes<Burgundy≤Brittany<<South-East) using the systematic grid of French Soil Quality Monitoring Network to evaluate the communities’ composition turnovers. The relative importance of processes and filters was assessed by distance-based redundancy analysis. This study demonstrates significant community composition turnover rates for soil bacteria and fungi, which were dependent on the region. Bacterial and fungal community composition turnovers were mainly driven by environmental selection explaining from 10% to 20% of community composition variations, but spatial variables also explained 3% to 9% of total variance. These variables highlighted significant spatial autocorrelation of both communities unexplained by the environmental variables measured and could partly be explained by dispersal limitations. Although the identified filters and their hierarchy were dependent on the region and organism, selection was systematically based on a common group of environmental variables: pH, trophic resources, texture and land use. Spatial autocorrelation was also important at coarse (80 to 120 km radius) and/or medium (40 to 65 km radius) spatial scales, suggesting dispersal limitations at these scales.

Platform GenoSol: a new tool for conserving and exploring soil microbial diversity.

Platform GenoSol: a New Tool for Conserving and Exploring Soil Microbial Diversity S DEQUIEDT , M LELIEVRE , PA MARON, C MOUGEL , N CHEMIDLIN PRÉVOST-BOURÉ, F MORIN , AURORE CECCHETTO , C JOLIVET, D ARROUAYS, P LEMANCEAU , L RANJARD 2 INRA, Université de Bourgogne, UMR Microbiologie du Sol et de l’Environnement, CMSE, 17, rue Sully, B.V. 86510, 21065 Dijon Cedex France Platform GenoSol, INRA, Université de Bourgogne, CMSE, 17, rue Sully, B.V. 86510, 21065 Dijon Cedex France 3 INRA Orléans US 1106, Unité INFOSOL, Avenue de la Pomme de Pin BP 20619 Ardon 45166 Olivet cedex – FRANCE

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.

Soil microbial diversity drives the priming effect along climate gradients: a case study in Madagascar

The priming effect in soil is proposed to be generated by two distinct mechanisms: ‘stoichiometric decomposition’ and/or ‘nutrient mining’ theories. Each mechanism has its own dynamics, involves its own microbial actors, and targets different soil organic matter (SOM) pools. The present study aims to evaluate how climatic parameters drive the intensity of each priming effect generation mechanism via the modification of soil microbial and physicochemical properties. Soils were sampled in the center of Madagascar, along climatic gradients designed to distinguish temperature from rainfall effects. Abiotic and biotic soil descriptors were characterized including bacterial and fungal phylogenetic composition. Potential organic matter mineralization and PE were assessed 7 and 42 days after the beginning of incubation with 13C-enriched wheat straw. Both priming mechanisms were mainly driven by the mean annual temperature but in opposite directions. The priming effect generated by stoichiometric decomposition was fostered under colder climates, because of soil enrichment in less developed organic matter, as well as in fast-growing populations. Conversely, the priming effect generated by nutrient mining was enhanced under warmer climates, probably because of the lack of competition between slow-growing populations mining SOM and fast-growing populations for the energy-rich residue entering the soil. Our study leads to hypotheses about the consequences of climate change on both PE generation mechanisms and associated consequences on soil carbon sequestration.

Improving soil bacterial taxa–area relationships assessment using DNA meta-barcoding

The evaluation of the taxa–area relationship (TAR) with molecular fingerprinting data demonstrated the spatial structuration of soil microorganisms and provided insights into the processes shaping their diversity. The increasing use of massive sequencing technologies in biodiversity investigations has now raised the question of the advantages of such technologies over the fingerprinting approach for elucidation of the determinism of soil microbial community assembly in broad-scale biogeographic studies. Our objectives in this study were to compare DNA fingerprinting and meta-barcoding approaches for evaluating soil bacterial TAR and the determinism of soil bacterial community assembly on a broad scale. This comparison was performed on 392 soil samples from four French geographic regions with different levels of environmental heterogeneity. Both molecular approaches demonstrated a TAR with a significant slope but, because of its more sensitive description of soil bacterial community richness, meta-barcoding provided significantly higher and more accurate estimates of turnover rates. Both approaches were useful in evidencing the processes shaping bacterial diversity variations on a broad scale. When different taxonomic resolutions were considered for meta-barcoding data, they significantly influenced the estimation of turnover rates but not the relative importance of each component process. Altogether, DNA meta-barcoding provides a more accurate evaluation of the TAR and may lead to re-examination of the processes shaping soil bacterial community assembly. This should provide new insights into soil microbial ecology in the context of sustainable use of soil resources.

Miscanthus bioenergy crop stimulates nutrient-cycler bacteria and fungi in wastewater-contaminated agricultural soil

Abstract Wastewater can be recycled in agricultural soil as fertilizer to increase crop yields. However, adding wastewater induces sometimes ecotoxicological issues such as pollution by toxic compounds, which may lead to the loss of arable land. Bioenergy crops such as Miscanthus × giganteus have been tested to rehabilitate polluted soils, but the impact of Miscanthus on soil microbes is unknown. Here, we evaluated the effects of Miscanthus cropping on bacterial and fungal taxonomic composition in a wastewater-contaminated soil using synchronic and diachronic evaluation strategies. A 3-year field experiment close to Paris was set up on an agricultural site irrigated by raw wastewater for more than one century, thus resulting in strong metal and organic contamination. Soil microbial taxonomic composition was characterized by direct analysis of soil DNA using metagenomic tools such as 454 pyrosequencing of ribosomal genes. Our results demonstrate that Miscanthus cropping stimulates specific populations of bacteria such as Rhizobiales, increased by 1.4 in relative abundance, Nistrospira (x1.5), Azospira (x2), and Gemmatimonas (x2), and fungi: Glomeromycota (x3) and Mortierella (x1.5) for fungi. Noteworthy, these microbial genera are known to be strongly involved in plant symbiosis, organic matter mineralization, and nutrient cycling. Overall our findings show that Miscanthus cropping enhances regeneration of soil microbiological functions and services in polluted soil by stimulating populations beneficial for soil fertility and crop production.