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Roussel-Delif, Ludovic
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Roussel-Delif, Ludovic
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- PublicationAccès libreGenome Sequence of Anoxybacillus geothermalis Strain GSsed3, a Novel Thermophilic Endospore-Forming Species(2015)
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- PublicationAccès librePlant and soil microbe interactions in controlled conditions: rhizosphere protozoa and bacterial community structure(2008)
; Plants influence the soil system by the large proportion of photosynthesized matters translocated to the roots and secreted into the soil. This root exudation provides an abundant energy source for rhizosphere living microorganisms. Plants are also strongly affected, positively and negatively, by the presence of soil microbiota, particularly bacteria, protozoa and fungi. Throughout the experiments conducted in this work, we aimed to better understand the influence of protozoa on plant growth. The first part of this work focused on the development of a microcosm method. Firstly, physical soil sterilization methods (autoclaving (A) gamma-ray irradiation (i) and both successively (AI)) were tested to eliminate the soil microbiota and their resistance form (spores and cysts). Although all sterilization methods tested were efficient to eliminate protozoa, AI was the only efficient method to eliminate aerobic heterotrophic cultivable bacteria without changing the soil pH. However the release of NH4+ in the soil after AI sterilization was higher than for other methods. Secondly, a procedure to re-inoculate the sterilized soil with a complex microbial community without protozoa was developed. The protozoa-free bacterial suspension was obtained from rhizosphere soil by subsequent filtering steps to exclude protozoa. The structure of bacterial communities characterised by 16SrDNA PCR-DGGE in the protozoa-free bacterial suspension was similar to that of the native soil. Diversity (Shannon) and evenness indexes increased with time in the sterile soil inoculated with the protozoa-free bacterial suspension. However the final bacterial community composition after 2 months of incubation in the re-inoculated soil presented a lower diversity as compared to the native soil. The second part of this work focused on the plant-microbiota interactions and on protozoa effects on plant growth. The microcosms developed in the first part of the work were re-inoculated with either sterile water or bacterial protozoa-free suspension or bacterial protozoa-free suspension and Acanthamoeba castellanii or with native soil suspension. The growth of Arabidopsis thaliana was clearly influenced by the inoculum and was particularly increased in presence of protozoa. Plants cultivated in presence of protozoa presented higher nitrogen content in leaves. The effect of leaf clipping (simulating herbivore damage) and nitrogen fertilization on soil microorganisms (bacteria, protozoa and nematodes) associated to the rhizosphere of barley was investigated in a pot experiment. The roots-shoots ratio decreased during the plant growth and was lower in the leaf clipping treatment. The abundance of bacteria was not significantly affected by leaf clipping and was higher in the high nitrogen-treatment. The abundance of bacterial-feeders (i.e. protozoa and nematodes) in the rhizosphere of 2, 4 and 6 weeks old plants was marginally affected by the nitrogen treatment as well as by leaf clipping. The role of protozoa in controlling the structure of bacterial community was investigated in the different experiment. The presence of protozoa did not change significantly the richness (numbers of bands) and the diversity (Shannon index) of the DNA-based DGGE fingerprints. The structure of the “total” bacterial communities was significantly changed in response to the functional group of protozoa (amoeba, ciliates and flagellates) inoculated as compared to the control (bacteria inoculum). The presence of protozoa did not change significantly the richness and the diversity of the RNA-based DGGE fingerprints. The structure of the active bacterial communities was significantly influenced by amoebas. - PublicationAccès libreSphenoderiidae (fam. nov.), a New Clade of Euglyphid Testate Amoebae Characterized by Small, Round Scales Surrounding the Aperture
; Euglyphid testate amoebae are a highly conspicuous group of Cercozoa whose systematics is based mainly on the shape and ultrastructure of the shell. However, only a couple of species have been studied with molecular methods. As a consequence, there are still some genera whose classification remains uncertain. Amongst those are Sphenoderia and Trachelocorythion, two genera with diverging ecological requirements that share a collar composed of small scales around the aperture. We demonstrate here with a molecular and morphological approach that they are closely related, and propose a new family, Sphenoderiidae fam. nov. to group these species. Some species share almost similar morphology in spite of being genetically distantly related (Sphenoderia minuta and S. pseudominuta sp. nov.), underlining the importance of combining ultrastructural and morphological data when describing new species of protists. In addition, we describe here Sphenoderia valdiviana sp. nov., a new species isolated from Southern Chile temperate rainforests. - PublicationAccès libreStage 0 sporulation gene A as a molecular marker to study diversity of endospore-forming Firmicutes
; ; ; In this study, we developed and validated a cultureindependent method for diversity surveys to specifically detect endospore-forming Firmicutes. The global transcription regulator of sporulation (spo0A) was identified as a gene marker for endosporeforming Firmicutes. To enable phylogenetic classification, we designed a set of primers amplifying a 602 bp fragment of spo0A that we evaluated in pure cultures and environmental samples. The amplification was positive for 35 strains from 11 genera, yet negative for strains from Alicyclobacillus and Sulfobacillus. We also evaluated various DNA extraction methods because endospores often result in reduced yields. Our results demonstrate that procedures utilizing increased physical force improve DNA extraction. An optimized DNA extraction method on biomass pre-extracted from the environmental sample source (indirect DNA extraction) followed by amplification with the aforementioned primers for spo0A was then tested in sediments from two different sources. Specifically, we validated our cultureindependent diversity survey methodology on a set of 8338 environmental spo0A sequences obtained from the sediments of Lakes Geneva (Switzerland) and Baikal (Russia). The phylogenetic affiliation of the environmental sequences revealed a substantial number of new clades within endospore-formers. This novel culture-independent approach provides a significant experimental improvement that enables exploration of the diversity of endospore-forming Firmicutes. - PublicationAccès libreQuantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Gene spo0A
; ; ; ; ; Bacterial endospores are highly specialized cellular forms that allow endospore-forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the genera Bacillus, Paenibacillus, Brevibacillus, Geobacillus, Alicyclobacillus, Sulfobacillus, Clostridium, and Desulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception of Sulfobacillus and Desulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 104 cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications.