Publications by authors named "Christophe Mougel"

38 Publications

Influential Insider: , an Intracellular Symbiont, Manipulates Bacterial Diversity in Its Insect Host.

Microorganisms 2021 Jun 16;9(6). Epub 2021 Jun 16.

Institut de Génétique, Environnement et Protection des Plantes (IGEPP), INRAE, Agrocampus Ouest, Université de Rennes, F-35000 Rennes, France.

Facultative intracellular symbionts like the α-proteobacteria influence their insect host phenotype but little is known about how much they affect their host microbiota. Here, we quantified the impact of infection on the bacterial community of the cabbage root fly by comparing the microbiota of -free and infected adult flies of both sexes. We used high-throughput DNA sequencing (Illumina MiSeq, 16S rRNA, V5-V7 region) and performed a community and a network analysis. In both sexes, infection significantly decreased the diversity of bacterial communities and modified their structure and composition by reducing abundance in some taxa but increasing it in others. Infection by was negatively correlated to 8 bacteria genera ( was the most impacted), and positively correlated to and . We suggest that might antagonize for being entomopathogenic (and potentially intracellular), but would favor and because they might protect the host against chemical plant defenses. Although they might seem prisoners in a cell, endocellular symbionts can impact the whole microbiota of their host, hence its extended phenotype, which provides them with a way to interact with the outside world.
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http://dx.doi.org/10.3390/microorganisms9061313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8234596PMC
June 2021

MinYS: mine your symbiont by targeted genome assembly in symbiotic communities.

NAR Genom Bioinform 2020 Sep 3;2(3):lqaa047. Epub 2020 Jul 3.

Univ. Rennes, Inria, CNRS, IRISA, F-35000 Rennes, France.

Most metazoans are associated with symbionts. Characterizing the effect of a particular symbiont often requires getting access to its genome, which is usually done by sequencing the whole community. We present MinYS, a targeted assembly approach to assemble a particular genome of interest from such metagenomic data. First, taking advantage of a reference genome, a subset of the reads is assembled into a set of backbone contigs. Then, this draft assembly is completed using the whole metagenomic readset in a manner. The resulting assembly is output as a genome graph, enabling different strains with potential structural variants coexisting in the sample to be distinguished. MinYS was applied to 50 pea aphid resequencing samples, with variable diversity in symbiont communities, in order to recover the genome sequence of its obligatory bacterial symbiont, . It was able to return high-quality assemblies (one contig assembly in 90% of the samples), even when using increasingly distant reference genomes, and to retrieve large structural variations in the samples. Because of its targeted essence, it outperformed standard metagenomic assemblers in terms of both time and assembly quality.
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http://dx.doi.org/10.1093/nargab/lqaa047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671366PMC
September 2020

Hatching of Induced by Root Exudates Is Not Influenced by Soil Microbiota Composition.

Front Microbiol 2020 8;11:536932. Epub 2020 Oct 8.

Institut national de recherche pour l'agriculture, l'alimentation et l'environnement (INRAE), UMR1349 IGEPP, Institute of Genetic Environment and Plant Protection, Le Rheu, France.

Plant-parasitic nematodes are among the most harmful pests of cultivated crops causing important economic losses. The ban of chemical nematicides requires the development of alternative agroecological approaches to protect crops against nematodes. For cyst nematodes, egg hatching is stimulated by host plant root exudates. Inducing "suicide hatching" of nematode second-stage juveniles (J2), using root exudates in the absence of the host plant, may constitute an effective and innovative biocontrol method to control cyst nematodes. However, before considering the development of this approach, understanding the effect of soil biotic component on cyst nematode hatching by root exudates is a major issue. The effectiveness of this approach could be modulated by other soil organisms consuming root exudates for growth as soil microbiota, and this must be evaluated. To do that, four different native agricultural soils were selected based on their physicochemical properties and their microbiota composition were characterized by rDNA metabarcoding. To disentangle the effect of microbiota from that of soil on hatching, four recolonized artificial soils were obtained by inoculating a common sterile soil matrix with the microbiota proceeding from each agricultural soil. Each soil was then inoculated with cysts of the potato cyst nematode, , and low or high doses of potato root exudates (PREs) were applied. After 40 days, viable J2 remaining in cysts were counted to determine the efficiency of root exudates to stimulate hatching in different soils. Results showed that (i) when physicochemical and microbiota compositions varied among native soils, the hatching rates remained very high albeit small differences were measured and no dose effect was detected and (ii) when only microbiota composition varied among recolonized soils, the hatching rates were also high at the highest dose of PREs, but a strong dose effect was highlighted. This study shows that abiotic and biotic factors may not compromise the development of methods based on suicide hatching of cyst nematodes, using root exudates, molecules inducing J2 hatch, or trap crops.
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http://dx.doi.org/10.3389/fmicb.2020.536932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578397PMC
October 2020

Soil microbiota influences clubroot disease by modulating Plasmodiophora brassicae and Brassica napus transcriptomes.

Microb Biotechnol 2020 09 19;13(5):1648-1672. Epub 2020 Jul 19.

INRAE, Agrocampus Ouest, Université de Rennes, IGEPP, Le Rheu, F-35650, France.

The contribution of surrounding plant microbiota to disease development has led to the 'pathobiome' concept, which represents the interaction between the pathogen, the host plant and the associated biotic microbial community, resulting or not in plant disease. The aim herein is to understand how the soil microbial environment may influence the functions of a pathogen and its pathogenesis, and the molecular response of the plant to the infection, with a dual-RNAseq transcriptomics approach. We address this question using Brassica napus and Plasmodiophora brassicae, the pathogen responsible for clubroot. A time-course experiment was conducted to study interactions between P. brassicae, two B. napus genotypes and three soils harbouring high, medium or low microbiota diversities and levels of richness. The soil microbial diversity levels had an impact on disease development (symptom levels and pathogen quantity). The P. brassicae and B. napus transcriptional patterns were modulated by these microbial diversities, these modulations being dependent on the host genotype plant and the kinetic time. The functional analysis of gene expressions allowed the identification of pathogen and plant host functions potentially involved in the change of plant disease level, such as pathogenicity-related genes (NUDIX effector) in P. brassicae and plant defence-related genes (glucosinolate metabolism) in B. napus.
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http://dx.doi.org/10.1111/1751-7915.13634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7415369PMC
September 2020

Fast computation of genome-metagenome interaction effects.

Algorithms Mol Biol 2020 1;15:13. Epub 2020 Jul 1.

Université Paris-Saclay, CNRS, Univ Évry, Laboratoire de Mathématiques et Modélisation d'Évry, 91037 Évry-Courcouronnes, France.

Motivation: Association studies have been widely used to search for associations between common genetic variants observations and a given phenotype. However, it is now generally accepted that genes and environment must be examined jointly when estimating phenotypic variance. In this work we consider two types of biological markers: genotypic markers, which characterize an observation in terms of inherited genetic information, and metagenomic marker which are related to the environment. Both types of markers are available in their millions and can be used to characterize any observation uniquely.

Objective: Our focus is on detecting interactions between groups of genetic and metagenomic markers in order to gain a better understanding of the complex relationship between environment and genome in the expression of a given phenotype.

Contributions: We propose a novel approach for efficiently detecting interactions between complementary datasets in a high-dimensional setting with a reduced computational cost. The method, named SICOMORE, reduces the dimension of the search space by selecting a subset of supervariables in the two complementary datasets. These supervariables are given by a weighted group structure defined on sets of variables at different scales. A Lasso selection is then applied on each type of supervariable to obtain a subset of potential interactions that will be explored via linear model testing.

Results: We compare SICOMORE with other approaches in simulations, with varying sample sizes, noise, and numbers of true interactions. SICOMORE exhibits convincing results in terms of recall, as well as competitive performances with respect to running time. The method is also used to detect interaction between genomic markers in and metagenomic markers in its rhizosphere bacterial community.

Software Availability: An R package is available [4], along with its documentation and associated scripts, allowing the reader to reproduce the results presented in the paper.
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http://dx.doi.org/10.1186/s13015-020-00173-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7329492PMC
July 2020

Long-lasting effects of antibiotics on bacterial communities of adult flies.

FEMS Microbiol Ecol 2020 04;96(4)

Agrocampus Ouest, INRAE, Université de Rennes, IGEPP, F-35000 Rennes, France.

Insect symbionts benefit their host and their study requires large spectrum antibiotic use like tetracycline to weaken or suppress symbiotic communities. While antibiotics have a negative impact on insect fitness, little is known about antibiotic effects on insect microbial communities and how long they last. We characterized the bacterial communities of adult cabbage root fly Delia radicum in a Wolbachia-free population and evaluated the effect of tetracycline treatment on these communities over several generations. Three D. radicum generations were used: the first- and second-generation flies either ingested tetracycline or not, while the third-generation flies were untreated but differed with their parents and/or grandparents that had or had not been treated. Fly bacterial communities were sequenced using a 16S rRNA gene. Tetracycline decreased fly bacterial diversity and induced modifications in both bacterial abundance and relative frequencies, still visible on untreated offspring whose parents and/or grandparents had been treated, therefore demonstrating long-lasting transgenerational effects on animal microbiomes after antibiotic treatment. Flies with an antibiotic history shared bacterial genera, potentially tetracycline resistant and heritable. Next, the transmission should be investigated by comparing several insect development stages and plant compartments to assess vertical and horizontal transmissions of D. radicum bacterial communities.
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http://dx.doi.org/10.1093/femsec/fiaa028DOI Listing
April 2020

Temporal dynamics of bacterial and fungal communities during the infection of Brassica rapa roots by the protist Plasmodiophora brassicae.

PLoS One 2019 25;14(2):e0204195. Epub 2019 Feb 25.

IGEPP, INRA, AGROCAMPUS OUEST, Université Rennes, Le Rheu, France.

The temporal dynamics of rhizosphere and root microbiota composition was compared between healthy and infected Chinese cabbage plants by the pathogen Plasmodiophora brassicae. When inoculated with P. brassicae, disease was measured at five sampling dates from early root hair infection to late gall development. The first symptoms of clubroot disease appeared 14 days after inoculation (DAI) and increased drastically between 14 and 35 DAI. The structure of microbial communities associated to rhizosphere soil and root from healthy and inoculated plants was characterized through high-throughput DNA sequencing of bacterial (16S) and fungal (18S) molecular markers and compared at each sampling date. In healthy plants, Proteobacteria and Bacteroidetes bacterial phyla dominated the rhizosphere and root microbiota of Chinese cabbage. Rhizosphere bacterial communities contained higher abundances of Actinobacteria and Firmicutes compared to the roots. Moreover, a drastic shift of fungal communities of healthy plants occurred between the two last sampling dates, especially in plant roots, where most of Ascomycota fungi dominated until they were replaced by a fungus assigned to the Chytridiomycota phylum. Parasitic invasion by P. brassicae disrupted the rhizosphere and root-associated community assembly at a late step during the root secondary cortical infection stage of clubroot disease. At this stage, Flavisolibacter and Streptomyces in the rhizosphere, and Bacillus in the roots, were drastically less abundant upon parasite invasion. Rhizosphere of plants colonized by P. brassicae was significantly more invaded by the Chytridiomycota fungus, which could reflect a mutualistic relationship in this compartment between these two microorganisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0204195PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388920PMC
November 2019

Host-microbiota interactions: from holobiont theory to analysis.

Microbiome 2019 01 11;7(1). Epub 2019 Jan 11.

Muséum National d'Histoire Naturelle, Institut de Systématique, Évolution, Biodiversité, ISYEB-UMR 7205-CNRS, MNHN, UPMC, EPHE, Sorbonne Universités, 57 Rue Cuvier-CP39, F-75005, Paris, France.

In the recent years, the holobiont concept has emerged as a theoretical and experimental framework to study the interactions between hosts and their associated microbial communities in all types of ecosystems. The spread of this concept in many branches of biology results from the fairly recent realization of the ubiquitous nature of host-associated microbes and their central role in host biology, ecology, and evolution. Through this special series "Host-microbiota interactions: from holobiont theory to analysis," we wanted to promote this field of research which has considerable implications for human health, food production, and ecosystem protection. In this preface, we highlight a collection of articles selected for this special issue that show, use, or debate the concept of holobiont to approach taxonomically and ecologically diverse organisms, from humans and plants to sponges and insects. We also identify some theoretical and methodological challenges and propose directions for future research on holobionts.
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http://dx.doi.org/10.1186/s40168-019-0619-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6330386PMC
January 2019

Computational analysis of the Plasmodiophora brassicae genome: mitochondrial sequence description and metabolic pathway database design.

Genomics 2019 12 15;111(6):1629-1640. Epub 2018 Nov 15.

IGEPP, INRA, AGROCAMPUS OUEST, Université Rennes, Domaine de la Motte, Le Rheu F-35653, France.

Plasmodiophora brassicae is an obligate biotrophic pathogenic protist responsible for clubroot, a root gall disease of Brassicaceae species. In addition to the reference genome of the P. brassicae European e3 isolate and the draft genomes of Canadian or Chinese isolates, we present the genome of eH, a second European isolate. Refinement of the annotation of the eH genome led to the identification of the mitochondrial genome sequence, which was found to be bigger than that of Spongospora subterranea, another plant parasitic Plasmodiophorid phylogenetically related to P. brassicae. New pathways were also predicted, such as those for the synthesis of spermidine, a polyamine up-regulated in clubbed regions of roots. A P. brassicae pathway genome database was created to facilitate the functional study of metabolic pathways in transcriptomics approaches. These available tools can help in our understanding of the regulation of P. brassicae metabolism during infection and in response to diverse constraints.
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http://dx.doi.org/10.1016/j.ygeno.2018.11.013DOI Listing
December 2019

Multi-scale characterization of symbiont diversity in the pea aphid complex through metagenomic approaches.

Microbiome 2018 10 10;6(1):181. Epub 2018 Oct 10.

INRA, UMR 1349 INRA/Agrocampus Ouest/Université Rennes 1, Institut de Génétique, Environnement et Protection des Plantes (IGEPP), Le Rheu, France.

Background: Most metazoans are involved in durable relationships with microbes which can take several forms, from mutualism to parasitism. The advances of NGS technologies and bioinformatics tools have opened opportunities to shed light on the diversity of microbial communities and to give some insights into the functions they perform in a broad array of hosts. The pea aphid is a model system for the study of insect-bacteria symbiosis. It is organized in a complex of biotypes, each adapted to specific host plants. It harbors both an obligatory symbiont supplying key nutrients and several facultative symbionts bringing additional functions to the host, such as protection against biotic and abiotic stresses. However, little is known on how the symbiont genomic diversity is structured at different scales: across host biotypes, among individuals of the same biotype, or within individual aphids, which limits our understanding on how these multi-partner symbioses evolve and interact.

Results: We present a framework well adapted to the study of genomic diversity and evolutionary dynamics of the pea aphid holobiont from metagenomic read sets, based on mapping to reference genomes and whole genome variant calling. Our results revealed that the pea aphid microbiota is dominated by a few heritable bacterial symbionts reported in earlier works, with no discovery of new microbial associates. However, we detected a large and heterogeneous genotypic diversity associated with the different symbionts of the pea aphid. Partitioning analysis showed that this fine resolution diversity is distributed across the three considered scales. Phylogenetic analyses highlighted frequent horizontal transfers of facultative symbionts between host lineages, indicative of flexible associations between the pea aphid and its microbiota. However, the evolutionary dynamics of symbiotic associations strongly varied depending on the symbiont, reflecting different histories and possible constraints. In addition, at the intra-host scale, we showed that different symbiont strains may coexist inside the same aphid host.

Conclusions: We present a methodological framework for the detailed analysis of NGS data from microbial communities of moderate complexity and gave major insights into the extent of diversity in pea aphid-symbiont associations and the range of evolutionary trajectories they could take.
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http://dx.doi.org/10.1186/s40168-018-0562-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6180509PMC
October 2018

Can soil microbial diversity influence plant metabolites and life history traits of a rhizophagous insect? A demonstration in oilseed rape.

Insect Sci 2017 Dec 10;24(6):1045-1056. Epub 2017 Jul 10.

IGEPP, Agrocampus Ouest, INRA, Université de Rennes 1, Le Rheu, France.

Interactions between plants and phytophagous insects play an important part in shaping the biochemical composition of plants. Reciprocally plant metabolites can influence major life history traits in these insects and largely contribute to their fitness. Plant rhizospheric microorganisms are an important biotic factor modulating plant metabolites and adaptation to stress. While plant-insects or plant-microorganisms interactions and their consequences on the plant metabolite signature are well-documented, the impact of soil microbial communities on plant defenses against phytophagous insects remains poorly known. In this study, we used oilseed rape (Brassica napus) and the cabbage root fly (Delia radicum) as biological models to tackle this question. Even though D. radicum is a belowground herbivore as a larva, its adult life history traits depend on aboveground signals. We therefore tested whether soil microbial diversity influenced emergence rate and fitness but also fly oviposition behavior, and tried to link possible effects to modifications in leaf and root metabolites. Through a removal-recolonization experiment, 3 soil microbial modalities ("high," "medium," "low") were established and assessed through amplicon sequencing of 16S and 18S ribosomal RNA genes. The "medium" modality in the rhizosphere significantly improved insect development traits. Plant-microorganism interactions were marginally associated to modulations of root metabolites profiles, which could partly explain these results. We highlighted the potential role of plant-microbial interaction in plant defenses against Delia radicum. Rhizospheric microbial communities must be taken into account when analyzing plant defenses against herbivores, being either below or aboveground.
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http://dx.doi.org/10.1111/1744-7917.12478DOI Listing
December 2017

Pseudomonas fluorescens C7R12 type III secretion system impacts mycorrhization of Medicago truncatula and associated microbial communities.

Mycorrhiza 2017 Jan 22;27(1):23-33. Epub 2016 Aug 22.

Agroécologie, AgroSup Dijon, CNRS, INRA, Université Bourgogne Franche-Comté, 21000, Dijon, France.

Type three secretion systems (T3SSs) mediate cell-to-cell interactions between Gram-negative bacteria and eukaryotes. We hypothesized that fluorescent pseudomonads harboring T3SS (T3SS+) would be beneficial to arbuscular mycorrhizal symbiosis because non-pathogenic fluorescent pseudomonads have been previously shown to be much more abundant in mycorrhizal than in non-mycorrhizal roots. We tested this hypothesis by comparing mycorrhization and the associated rhizosphere microbial communities of Medicago truncatula grown in a non-sterile soil inoculated with either the T3SS+ mycorrhiza helper bacterium Pseudomonas fluorescens (C7R12) or a T3SS- mutant of the strain. Results showed that the bacterial secretion system was responsible for the promotion of mycorrhization because root colonization by arbuscular mycorrhizal fungi was not promoted by the T3SS- mutant. The observed T3SS-mediated promotion of mycorrhization was associated with changes in the rhizosphere bacterial communities and the increased occurrence of Claroidoglomeraceae within the intraradical arbuscular mycorrhizal fungi. Furthermore, both pseudomonad strains promoted the host-free growth of a model arbuscular mycorrhizal fungus in vitro, suggesting that T3SS-mediated promotion of mycorrhization occurs during plant-fungal interactions rather than during the pre-symbiotic phase of fungal growth. Taken together, these data provide evidence for the involvement of T3SS in promoting arbuscular mycorrhization by a model fluorescent pseudomonad and suggest the implication of interactions between the bacterium and mycorrhizas.
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http://dx.doi.org/10.1007/s00572-016-0730-3DOI Listing
January 2017

Bacterial Community Diversity Harboured by Interacting Species.

PLoS One 2016 3;11(6):e0155392. Epub 2016 Jun 3.

Université Rennes 1, UMR1349 IGEPP, F-35000, Rennes, France.

All animals are infected by microbial partners that can be passengers or residents and influence many biological traits of their hosts. Even if important factors that structure the composition and abundance of microbial communities within and among host individuals have been recently described, such as diet, developmental stage or phylogeny, few studies have conducted cross-taxonomic comparisons, especially on host species related by trophic relationships. Here, we describe and compare the microbial communities associated with the cabbage root fly Delia radicum and its three major parasitoids: the two staphylinid beetles Aleochara bilineata and A. bipustulata and the hymenopteran parasitoid Trybliographa rapae. For each species, two populations from Western France were sampled and microbial communities were described through culture independent methods (454 pyrosequencing). Each sample harbored at least 59 to 261 different bacterial phylotypes but was strongly dominated by one or two. Microbial communities differed markedly in terms of composition and abundance, being mainly influenced by phylogenetic proximity but also geography to a minor extent. Surprisingly, despite their strong trophic interaction, parasitoids shared a very low proportion of microbial partners with their insect host. Three vertically transmitted symbionts from the genus Wolbachia, Rickettsia, and Spiroplasma were found in this study. Among them, Wolbachia and Spiroplasma were found in both the cabbage fly and at least one of its parasitoids, which could result from horizontal transfers through trophic interactions. Phylogenetic analysis showed that this hypothesis may explain some but not all cases. More work is needed to understand the dynamics of symbiotic associations within trophic network and the effect of these bacterial communities on the fitness of their hosts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155392PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892616PMC
July 2017

Plant traits related to nitrogen uptake influence plant-microbe competition.

Ecology 2015 Aug;96(8):2300-10

Plant species are important drivers of soil microbial communities. However, how plant functional traits are shaping these communities has received less attention though linking plant and microbial traits is crucial for better understanding plant-microbe interactions. Our objective was to determine how plant-microbe interactions were affected by plant traits. Specifically we analyzed how interactions between plant species and microbes involved in nitrogen cycling were affected by plant traits related to 'nitrogen nutrition in interaction with soil nitrogen availability. Eleven plant species, selected along an oligotrophic-nitrophilic gradient, were grown individually in a nitrogen-poor soil with two levels of nitrate availability. Plant traits for both carbon and nitrogen nutrition were measured and the genetic structure and abundance of rhizosphere. microbial communities, in particular the ammonia oxidizer and nitrate reducer guilds, were analyzed. The structure of the bacterial community in the rhizosphere differed significantly between plant species and these differences depended on nitrogen availability. The results suggest that the rate of nitrogen uptake per unit of root biomass and per day is a key plant trait, explaining why the effect of nitrogen availability on the structure of the bacterial community depends on the plant species. We also showed that the abundance of nitrate reducing bacteria always decreased with increasing nitrogen uptake per unit of root biomass per day, indicating that there was competition for nitrate between plants and nitrate reducing bacteria. This study demonstrates that nitrate-reducing microorganisms may be adversely affected by plants with a high nitrogen uptake rate. Our work puts forward the role of traits related to nitrogen in plant-microbe interactions, whereas carbon is commonly considered as the main driver. It also suggests that plant traits related to ecophysiological processes, such as nitrogen uptake rates, are more relevant for understanding plant-microbe interactions than composite traits, such as nitrophily, which are related to a number of ecophysiological processes.
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http://dx.doi.org/10.1890/14-1761.1DOI Listing
August 2015

Soil nitrogen availability and plant genotype modify the nutrition strategies of M. truncatula and the associated rhizosphere microbial communities.

PLoS One 2012 15;7(10):e47096. Epub 2012 Oct 15.

INRA, UMR1347 Agroécologie, Dijon, France.

Plant and soil types are usually considered as the two main drivers of the rhizosphere microbial communities. The aim of this work was to study the effect of both N availability and plant genotype on the plant associated rhizosphere microbial communities, in relation to the nutritional strategies of the plant-microbe interactions, for six contrasted Medicago truncatula genotypes. The plants were provided with two different nutrient solutions varying in their nitrate concentrations (0 mM and 10 mM). First, the influence of both nitrogen availability and Medicago truncatula genotype on the genetic structure of the soil bacterial and fungal communities was determined by DNA fingerprint using Automated Ribosomal Intergenic Spacer Analysis (ARISA). Secondly, the different nutritional strategies of the plant-microbe interactions were evaluated using an ecophysiological framework. We observed that nitrogen availability affected rhizosphere bacterial communities only in presence of the plant. Furthermore, we showed that the influence of nitrogen availability on rhizosphere bacterial communities was dependent on the different genotypes of Medicago truncatula. Finally, the nutritional strategies of the plant varied greatly in response to a modification of nitrogen availability. A new conceptual framework was thus developed to study plant-microbe interactions. This framework led to the identification of three contrasted structural and functional adaptive responses of plant-microbe interactions to nitrogen availability.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047096PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471967PMC
March 2013

Interaction between Medicago truncatula and Pseudomonas fluorescens: evaluation of costs and benefits across an elevated atmospheric CO(2).

PLoS One 2012 21;7(9):e45740. Epub 2012 Sep 21.

INRA, UMR 1347 Agroécologie, Dijon, France.

Soil microorganisms play a key role in both plants nutrition and health. Their relation with plant varies from mutualism to parasitism, according to the balance of costs and benefits for the two partners of the interaction. These interactions involved the liberation of plant organic compounds via rhizodeposition. Modification of atmospheric CO(2) concentration may affect rhizodeposition and as a consequence trophic interactions that bind plants and microorganisms. Positive effect of elevated CO(2) on plants are rather well known but consequences for micoorganisms and their interactions with plants are still poorly understood. A gnotobiotic system has been developed to study the interaction between Medicago truncatula Jemalong J5 and the mutualistic bacteria Pseudomonas fluorescens strain C7R12 under two atmospheric CO(2) concentrations: ambient (365 ppm) versus enriched (750 ppm). Costs and benefits for each partner have been determined over time by measuring plant development and growth, the C and N contents of the various plant parts and the density of the bacteria in rhizosphere compartments. Following the increase in CO(2), there was a beneficial effect of P. fluorescens C7R12 on development, vegetative growth, and C/N content of M. truncatula. Concerning plant reproduction, an early seed production was noticed in presence of the bacterial strain combined with increased atmospheric CO(2) conditions. Paradoxically, this transient increase in seed production was correlated with a decrease in bacterial density in the rhizosphere soil, revealing a cost of increased CO(2) for the bacterial strain. This shift of costs-benefits ratio disappeared later during the plant growth. In conclusion, the increase in CO(2) concentration modifies transiently the cost-benefit balance in favor of the plant. These results may be explained either by a competition between the two partners or a change in bacterial physiology. The ecosystem functioning depends on the stability of many plant-microbe associations that abiotic factors can disrupt.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045740PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3448688PMC
February 2013

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

PLoS One 2012 11;7(9):e44279. Epub 2012 Sep 11.

INRA, UMR1347 Agroécologie, Dijon, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044279PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3439486PMC
March 2013

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

Microb Biotechnol 2012 Jan 12;5(1):135-41. Epub 2011 Oct 12.

INRA-Université de Bourgogne, UMR Microbiologie du Sol et de l'Environnement, CMSE, 17, rue Sully, B.V. 86510, 21065 Dijon Cedex, France.

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.
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http://dx.doi.org/10.1111/j.1751-7915.2011.00307.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3815280PMC
January 2012

Validation and application of a PCR primer set to quantify fungal communities in the soil environment by real-time quantitative PCR.

PLoS One 2011 8;6(9):e24166. Epub 2011 Sep 8.

INRA-Université de Bourgogne, UMR Microbiologie du Sol et de l'Environnement, CMSE, Dijon, France.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0024166PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3169588PMC
March 2012

Soil microbial diversity: Methodological strategy, spatial overview and functional interest.

C R Biol 2011 May 15;334(5-6):403-11. Epub 2011 Feb 15.

UMR microbiologie du sol et de l'environnement, CMSE, INRA, université de Bourgogne, 17, rue Sully, BP 86510, 21065 Dijon cedex, France.

Since the development of industrialization, urbanization and agriculture, soils have been subjected to numerous variations in environmental conditions, which have resulted in modifications of the taxonomic diversity and functioning of the indigenous microbial communities. As a consequence, the functional significance of these losses/modifications of biodiversity, in terms of the capacity of ecosystems to maintain the functions and services on which humanity depends, is now of pivotal importance. In this context, one of the main challenges in soil microbial ecology is to better understand and predict the processes that drive soil microbial diversity and the link between diversity and ecosystem process. This review describes past, present and ongoing conceptual and methodological strategies employed to better assess and understand the distribution and evolution of soil microbial diversity with the aim of increasing our capacity to translate such diversity into soil biological functioning and, more widely, into ecosystem services.
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http://dx.doi.org/10.1016/j.crvi.2010.12.003DOI Listing
May 2011

Fluorescent pseudomonads harboring type III secretion genes are enriched in the mycorrhizosphere of Medicago truncatula.

FEMS Microbiol Ecol 2011 Mar 4;75(3):457-67. Epub 2011 Jan 4.

INRA, Université de Bourgogne, UMR 1229 Microbiologie du Sol et de l'Environnement, CMSE, Dijon, France.

Type III secretion systems (T3SSs) of Gram-negative bacteria mediate direct interactions with eukaryotic cells. Pseudomonas spp. harboring T3SS genes (T3SS+) were previously shown to be more abundant in the rhizosphere than in bulk soil. To discriminate the contribution of roots and associated arbuscular mycorrhizal fungi (AMF) on the enrichment of T3SS+ fluorescent pseudomonads in the rhizosphere of Medicago truncatula, their frequency was assessed among pseudomonads isolated from mycorrhizal and nonmycorrhizal roots and from bulk soil. T3SS genes were identified by PCR targeting a conserved hrcRST DNA fragment. Polymorphism of hrcRST in T3SS+ isolates was assessed by PCR-restriction fragment length polymorphism and sequencing. Genotypic diversity of all pseudomonads isolated, whether or not harboring T3SS, was described by BOX-PCR. T3SS+ pseudomonads were significantly more abundant in mycorrhizal than in nonmycorrhizal roots and in bulk soil, and all were shown to belong to the phylogenetic group of Pseudomonas fluorescens on the basis of 16S rRNA gene identity. Four hrcRST genotypes were described; two only included isolates from mycorrhizal roots. T3SS+ and T3SS- pseudomonads showed different genetic backgrounds as indicated by their different BOX-PCR types. Taken together, these data suggest that T3SSs are implicated in interactions between fluorescent pseudomonads and AM in medic rhizosphere.
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http://dx.doi.org/10.1111/j.1574-6941.2010.01021.xDOI Listing
March 2011

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

Environ Microbiol Rep 2009 Apr 3;1(2):97-9. Epub 2009 Mar 3.

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.

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http://dx.doi.org/10.1111/j.1758-2229.2009.00023.xDOI Listing
April 2009

Bacterial effects on arbuscular mycorrhizal fungi and mycorrhiza development as influenced by the bacteria, fungi, and host plant.

Mycorrhiza 2009 Feb 22;19(2):81-90. Epub 2008 Oct 22.

Università del Piemonte Orientale 'Amedeo Avogadro', I-15100, Alessandria, Italy.

Bacterial strains from mycorrhizal roots (three belonging to Comamonadaceae and one to Oxalobacteraceae) and from non-mycorrhizal roots (two belonging to Comamonadaceae) of Medicago truncatula and two reference strains (Collimonas fungivorans Ter331 and Pseudomonas fluorescens C7R12) were tested for their effect on the in vitro saprophytic growth of Glomus mosseae BEG12 and on its colonization of M. truncatula roots. Only the Oxalobacteraceae strain, isolated from barrel medic mycorrhizal roots, and the reference strain P. fluorescens C7R12 promoted both the saprophytic growth and root colonization of G. mosseae BEG12, indicating that they acted as mycorrhiza helper bacteria. Greatest effects were achieved by P. fluorescens C7R12 and its influence on the saprophytic growth of G. mosseae was compared to that on Gigaspora rosea BEG9 to determine if the bacterial stimulation was fungal specific. This fungal specificity, together with plant specificity, was finally evaluated by comparing bacterial effects on arbuscular mycorrhizal symbiosis when each of the fungal species was inoculated to two different plant species (M. truncatula and Lycopersicon esculentum). The results obtained showed that promotion of saprophytic growth by P. fluorescens C7R12 was expressed in vitro towards G. mosseae but not towards G. rosea. Bacterial promotion of mycorhization was also expressed towards G. mosseae, but not G. rosea, in roots of M. truncatula and L. esculentum. Taken together, results indicated that enhancement of arbuscular mycorrhiza development was only induced by a limited number of bacteria, promotion by the most efficient bacterial strain being fungal and not plant specific.
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http://dx.doi.org/10.1007/s00572-008-0205-2DOI Listing
February 2009

Microdiversity of Burkholderiales associated with mycorrhizal and nonmycorrhizal roots of Medicago truncatula.

FEMS Microbiol Ecol 2008 Aug 28;65(2):180-92. Epub 2008 May 28.

INRA, Université de Bourgogne, UMR1229 Microbiologie du Sol et de l'Environnement, CMSE, BP, Dijon, France.

The genetic diversity of bacterial communities associated with mycorrhizal and nonmycorrhizal roots of Medicago truncatula was characterized by two approaches. Firstly, phylogenetic analysis was performed on 164 partial 16S rRNA gene-intergenic spacer (IGS) sequences from operational taxonomic units previously shown to be preferentially associated with mycorrhizal roots. These sequences were distributed into three branches corresponding to Comamonadaceae, Oxalobacteraceae and Rubrivivax subgroups. Most sequences were obtained from mycorrhizal roots, indicating the preferential association of the corresponding families with mycorrhizal roots. A second phylogenetic analysis was performed on the partial 16S rRNA gene-IGS sequences of 173 isolates among a large collection of isolates, from mycorrhizal and nonmycorrhizal roots, belonging to Comamonadaceae and Oxalobacteraceae on the basis of their positive hybridization with a partial 16S rRNA gene-IGS probe obtained in this study. Sequence analysis confirmed the affiliation of 166 isolates to Comamonadaceae and seven to Oxalobacteraceae. Oxalobacteraceae isolates were more abundant in mycorrhizal (five) than in nonmycorrhizal (two) roots, whereas Comamonadaceae isolates were more abundant in nonmycorrhizal (109) than mycorrhizal roots (57). Further analysis of Comamonadaceae isolates by BOX-PCR showed that the genetic structure of culturable populations belonging to this family differed significantly in mycorrhizal and nonmycorrhizal roots, as indicated by distributions in different BOX types, differences being significantly explained by BOX types only including isolates from mycorrhizal roots. These data are discussed in an ecological context.
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http://dx.doi.org/10.1111/j.1574-6941.2008.00504.xDOI Listing
August 2008

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

Environ Microbiol 2008 Aug 6;10(8):2184-7. Epub 2008 May 6.

INRA, Université de Bourgogne, UMR Microbiologie du Sol et de l'Environnement, CMSE, 17, rue Sully, B.V. 86510, 21065 Dijon Cedex, France.

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.
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http://dx.doi.org/10.1111/j.1462-2920.2008.01641.xDOI Listing
August 2008

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

FEMS Microbiol Ecol 2007 Sep;61(3):424-37

INRA-Université de Bourgogne, UMR Microbiologie du Sol et de l'Environnement, CMSE, Dijon, France.

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.
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http://dx.doi.org/10.1111/j.1574-6941.2007.00365.xDOI Listing
September 2007

Diversity of root-associated fluorescent pseudomonads as affected by ferritin overexpression in tobacco.

Environ Microbiol 2007 Jul;9(7):1724-37

INRA, Université de Bourgogne, UMR1229 Microbiologie du Sol et de l'Environnement, CMSE, 17 rue Sully, BV 86510, F-21034 Dijon cedex, France.

A transgenic tobacco overexpressing ferritin (P6) was recently shown to accumulate more iron than the wild type (WT), leading to a reduced availability of iron in the rhizosphere and shifts in the pseudomonad community. The impact of the transgenic line on the community of fluorescent pseudomonads was assessed. The diversity of 635 isolates from rhizosphere soils, rhizoplane + root tissues, and root tissues of WT and P6, and that of 98 isolates from uncultivated soil was characterized. Their ability to grow under iron stress conditions was assessed by identifying their minimal inhibitory concentrations of 8-hydroxyquinoline for each isolate, pyoverdine diversity by isoelectrofocusing and genotypic diversity by random amplified polymorphism DNA. The antagonistic activity of representative isolates and of some purified pyoverdines against a plant pathogen (Pythium aphanidermatum Op4) was tested in vitro. In overall, isolates taken from P6 tobacco showed a greater ability to grow in iron stress conditions than WT isolates. The antagonism by some of the representative isolates was only expressed under iron stress conditions promoting siderophore synthesis and their pyoverdines appeared to have a specific structure as assessed by mass spectrometry. For other isolates, antagonism was still expressed in the presence of iron, suggesting the involvement of metabolites other than siderophores. Altogether, these data indicate that the transgenic tobacco that over-accumulates iron selected fluorescent pseudomonads, less susceptible to iron depletion and more antagonistic to the tested plant pathogen than those selected by the tobacco WT.
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http://dx.doi.org/10.1111/j.1462-2920.2007.01290.xDOI Listing
July 2007

Metaproteomics: a new approach for studying functional microbial ecology.

Microb Ecol 2007 Apr 13;53(3):486-93. Epub 2007 Mar 13.

UMR Microbiologie et Géochimie des Sols, INRA/Université de Bourgogne, CMSE, BP 86510, 17 rue de Sully, 21065, Dijon Cedex, France.

In the postgenomic era, there is a clear recognition of the limitations of nucleic acid-based methods for getting information on functions expressed by microbial communities in situ. In this context, the large-scale study of proteins expressed by indigenous microbial communities (metaproteome) should provide information to gain insights into the functioning of the microbial component in ecosystems. Characterization of the metaproteome is expected to provide data linking genetic and functional diversity of microbial communities. Studies on the metaproteome together with those on the metagenome and the metatranscriptome will contribute to progress in our knowledge of microbial communities and their contribution in ecosystem functioning. Effectiveness of the metaproteomic approach will be improved as increasing metagenomic information is made available thanks to the environmental sequencing projects currently running. More specifically, analysis of metaproteome in contrasted environmental situations should allow (1) tracking new functional genes and metabolic pathways and (2) identifying proteins preferentially associated with specific stresses. These proteins considered as functional bioindicators should contribute, in the future, to help policy makers in defining strategies for sustainable management of our environment.
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http://dx.doi.org/10.1007/s00248-006-9196-8DOI Listing
April 2007

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

Environ Microbiol 2007 Mar;9(3):752-64

INRA-Université de Bourgogne, UMR Microbiologie et Géochimie des Sols, CMSE, 17, rue Sully, B. V. 86510, 21065 Dijon Cedex, France.

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.
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http://dx.doi.org/10.1111/j.1462-2920.2006.01197.xDOI Listing
March 2007

Identification of cellulolytic bacteria in soil by stable isotope probing.

Environ Microbiol 2007 Mar;9(3):625-34

CEA, DSV, DEVM, Laboratoire d'Ecologie Microbienne de la Rhizosphère et des Environnements extrêmes (LEMiRE), UMR 6191 CNRS, CEA, Aix Marseille-Univ, IFR-E 112, Saint-Paul-Lez-Durance F-13108, France.

Plant residues, mainly made up of cellulose, are the largest fraction of organic carbon material in terrestrial ecosystems. Soil microorganisms are mainly responsible for the transfer of this carbon to the atmosphere, but their contribution is not accurately known. The aim of the present study was to identify bacterial populations that are actively involved in cellulose degradation, using the DNA-stable isotope probing (DNA-SIP) technique. (13)C-cellulose was produced by Acetobacter xylinus and incubated in soil for 7, 14, 30 and 90 days. Total DNA was extracted from the soil, the (13)C-labelled (heavy) and unlabelled (light) DNA fractions were separated by ultracentrifugation, and the structure of active bacterial communities was analysed by bacterial-automated ribosomal intergenic spacer analysis (B-ARISA) and characterized with denaturing gradient gel electrophoresis (DGGE). Cellulose degradation was associated with significant changes in bacterial community structure issued from heavy DNA, leading to the appearance of new bands and increase in relative intensities of other bands until day 30. The majority of bands decreased in relative intensity at day 90. Sequencing and phylogenetic analysis of 10 of these bands in DGGE profiles indicated that most sequences were closely related to sequences from organisms known for their ability to degrade cellulose or to uncultured soil bacteria.
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http://dx.doi.org/10.1111/j.1462-2920.2006.01182.xDOI Listing
March 2007
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