Samuel Dequiedt

Determinants of the distribution of nitrogen-cycling microbial communities at the landscape scale

Little information is available regarding the landscape-scale distribution of microbial communities and its environmental determinants. However, a landscape perspective is needed to understand the relative importance of local and regional factors and land management for the microbial communities and the ecosystem services they provide. In the most comprehensive analysis of spatial patterns of microbial communities to date, we investigated the distribution of functional microbial communities involved in N-cycling and of the total bacterial and crenarchaeal communities over 107 sites in Burgundy, a 31 500 km2 region of France, using a 16 × 16 km2 sampling grid. At each sampling site, the abundance of total bacteria, crenarchaea, nitrate reducers, denitrifiers- and ammonia oxidizers were estimated by quantitative PCR and 42 soil physico-chemical properties were measured. The relative contributions of land use, spatial distance, climatic conditions, time, and soil physico-chemical properties to the spatial distribution of the different communities were analyzed by canonical variation partitioning. Our results indicate that 43–85% of the spatial variation in community abundances could be explained by the measured environmental parameters, with soil chemical properties (mostly pH) being the main driver. We found spatial autocorrelation up to 739 km and used geostatistical modelling to generate predictive maps of the distribution of microbial communities at the landscape scale. The present study highlights the potential of a spatially explicit approach for microbial ecology to identify the overarching factors driving the spatial heterogeneity of microbial communities even at the landscape scale.

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.

Biogeography of soil microbial communities: a review and a description of the ongoing french national initiative

Microbial biogeography is the study of the distribution of microbial diversity on large scales of space and time. This science aims at understanding biodiversity regulation and its link with ecosystem biological functioning, goods and services such as maintenance of productivity, of soil and atmospheric quality, and of soil health. Although the initial concept dates from the early 20th century (Beijerinck (1913) De infusies en de ontdekking der backterien, in: Jaarboek van de Knoniklijke Akademie van Wetenschappen, Muller, Amsterdam), only recently have an increasing number of studies have investigated the biogeographical patterns of soil microbial diversity. A such delay is due to the constraints of the microbial models, the need to develop relevant molecular and bioinformatic tools to assess microbial diversity, and the non-availability of an adequate sampling strategy. Consequently, the conclusions from microbial ecology studies have rarely been generally applicable and even the fundamental power-laws differ because the taxa-area relationship and the influence of global and distal parameters on the spatial distribution of microbial communities have not been examined. In this article we define and discuss the scientific, technical and operational limits and outcomes resulting from soil microbial biogeography together with the technical and logistical feasibility. The main results are that microbial communities are not stochastically distributed on a wide scale and that biogeographical patterns are more influenced by local parameters such as soil type and land use than by distal ones, e.g. climate and geomorphology, contrary to plants and animals. We then present the European soil biological survey network, focusing on the French national initiative and the „ECOMIC-RMQS” project. The objective of the ECOMIC-RMQS project is to characterise the density and diversity of bacterial communities in all soils in the RMQS library in order to assess, for the first time, not only microbial biogeography across the whole of France but also the impact of land use on soil biodiversity (Réseau de Mesures de la Qualité des Sols = French Soil Quality Monitoring Network, 2200 soils covering all the French territory with a systematic grid of sampling). The scientific, technical and logistical outputs are examined with a view to the future prospects needed to develop this scientific domain and its applications in sustainable land use.

Response of soil bacterial community structure to successive perturbations of different types and intensities

In soil, genetic structure modifications of indigenous bacterial community consecutively to a severe stress (mercury contamination) were delayed when the community was pre-exposed to various minor perturbations (heat, copper and atrazine). Such minor perturbations induced transitory community structure modifications leading to an increase of community stability towards a severe mercury stress. These results illustrated well the short-term pre-adaptation process for bacterial community hypothesizing that community submitted to perturbations become more resistant to withstand another stress.

Meta-barcoded evaluation of the ISO standard 11063 DNA extraction procedure to characterize soil bacterial and fungal community diversity and composition

This study was designed to assess the influence of three soil DNA extraction procedures, namely the International Organization for Standardization (ISO‐11063, GnS‐GII and modified ISO procedure (ISOm), on the taxonomic diversity and composition of soil bacterial and fungal communities. The efficacy of each soil DNA extraction method was assessed on five soils, differing in their physico‐chemical characteristics and land use. A meta‐barcoded pyrosequencing approach targeting 16S and 18S rRNA genes was applied to characterize soil microbial communities. We first observed that the GnS‐GII introduced some heterogeneity in bacterial composition between replicates. Then, although no major difference was observed between extraction procedures for soil bacterial diversity, we saw that the number of fungal genera could be underestimated by the ISO‐11063. In particular, this procedure underestimated the detection in several soils of the genera Cryptococcus, Pseudallescheria, Hypocrea and Plectosphaerella, which are of ecological interest. Based on these results, we recommend using the ISOm method for studies focusing on both the bacterial and fungal communities. Indeed, the ISOm procedure provides a better evaluation of bacterial and fungal communities and is limited to the modification of the mechanical lysis step of the existing ISO‐11063 standard.

Land Use History Shifts In Situ Fungal and Bacterial Successions following Wheat Straw Input into the Soil

Soil microbial communities undergo rapid shifts following modifications in environmental conditions. Although microbial diversity changes may alter soil functioning, the in situ temporal dynamics of microbial diversity is poorly documented. Here, we investigated the response of fungal and bacterial diversity to wheat straw input in a 12-months field experiment and explored whether this response depended on the soil management history (grassland vs. cropland). Seasonal climatic fluctuations had no effect on the diversity of soil communities. Contrastingly fungi and bacteria responded strongly to wheat regardless of the soil history. After straw incorporation, diversity decreased due to the temporary dominance of a subset of copiotrophic populations. While fungi responded as quickly as bacteria, the resilience of fungal diversity lasted much longer, indicating that the relative involvement of each community might change as decomposition progressed. Soil history did not affect the response patterns, but determined the identity of some of the populations stimulated. Most strikingly, the bacteria Burkholderia, Lysobacter and fungi Rhizopus, Fusarium were selectively stimulated. Given the ecological importance of these microbial groups as decomposers and/or plant pathogens, such regulation of the composition of microbial successions by soil history may have important consequences in terms of soil carbon turnover and crop health.

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.