Plant health status effects on arbuscular mycorrhizal fungi associated with Lavandula angustifolia and Lavandula intermedia infected by Phytoplasma in France.

We investigated root communities of arbuscular mycorrhizal fungi (AMF) in relation to lavender (Lavandula angustifolia) and lavandin (Lavandula intermedia) health status from organic and conventional fields affected by Phytoplasma infection. The intensity of root mycorrhizal colonization was significantly different between diseased and healthy plants and was higher in the latter regardless of agricultural practice. This difference was more pronounced in lavender. The root AMF diversity was influenced by the plant health status solely in lavender and only under the conventional practice resulting in an increase in the AMF abundance and richness. The plant health status did not influence the distribution of root AMF communities in lavandin unlike its strong impact in lavender in both agricultural practices. Finally, among the most abundant molecular operational taxonomic units (MOTUs), four different MOTUs for each plant species were significantly abundant in the roots of healthy lavender and lavandin in either agricultural practice. Our study demonstrated that the plant health status influences root colonization and can influence the diversity and distribution of root AMF communities. Its effects vary according to plant species, can be modified by agricultural practices and allow plants to establish symbiosis with specific AMF species.

Differences in the fungal communities nursed by two genetic groups of the alpine cushion plant, Silene acaulis.

Foundation plants shape the composition of local biotic communities and abiotic environments, but the impact of a plant's intraspecific variations on these processes is poorly understood. We examined these links in the alpine cushion moss campion (Silene acaulis) on two neighboring mountain ranges in the French Alps. Genotyping of cushion plants revealed two genetic clusters matching known subspecies. The exscapa subspecies was found on both limestone and granite, while the longiscapa one was only found on limestone. Even on similar limestone bedrock, cushion soils from the two S. acaulis subspecies deeply differed in their impact on soil abiotic conditions. They further strikingly differed from each other and from the surrounding bare soils in fungal community composition. Plant genotype variations accounted for a large part of the fungal composition variability in cushion soils, even when considering geography or soil chemistry, and particularly for the dominant molecular operational taxonomic units (MOTUs). Both saprophytic and biotrophic fungal taxa were related to the MOTUs recurrently associated with a single plant genetic cluster. Moreover, the putative phytopathogens were abundant, and within the same genus (Cladosporium) or species (Pyrenopeziza brassicae), MOTUs showing specificity for each plant subspecies were found. Our study highlights the combined influences of bedrock and plant genotype on fungal recruitment into cushion soils and suggests the coexistence of two mechanisms, an indirect selection resulting from the colonization of an engineered soil by free-living saprobes and a direct selection resulting from direct plant-fungi interactions.

Contrasting microbial biogeographical patterns between anthropogenic subalpine grasslands and natural alpine grasslands.

The effect of plant species composition on soil microbial communities was studied at the multiregional level. We compared the soil microbial communities of alpine natural grasslands dominated by Carex curvula and anthropogenic subalpine pastures dominated by Nardus stricta. We conducted paired sampling across the Carpathians and the Alps and used Illumina sequencing to reveal the molecular diversity of soil microbes. We found that bacterial and fungal communities exhibited contrasting regional distributions and that the distribution in each grassland is well discriminated. Beta diversity of microbial communities was much higher in C. curvula grasslands due to a marked regional effect. The composition of grassland-type core microbiomes suggest that C. curvula, and N. stricta to a lesser extent, tend to select a cohort of microbes related to antibiosis/exclusion, pathogenesis and endophytism. We discuss these findings in light of the postglacial history of the studied grasslands, the habitat connectivity and the disturbance regimes. Human-induced disturbance in the subalpine belt of European mountains has led to homogeneous soil microbial communities at large biogeographical scales. Our results confirm the overarching role of the dominant grassland plant species in the distribution of microbial communities and highlight the relevance of biogeographical history.

Autochthonous ascomycetes in depollution of polychlorinated biphenyls contaminated soil and sediment.

We investigated the capacity of a consortium of ascomycetous strains, Doratomyces nanus, Doratomyces purpureofuscus, Doratomyces verrucisporus, Myceliophthora thermophila, Phoma eupyrena and Thermoascus crustaceus in the mycoremediation of historically contaminated soil and sediment by polychlorinated biphenyls (PCBs). Analyses of 15 PCB concentrations in three mesocosms containing soil from which the fungal strains had previously been isolated, revealed significant PCB depletions of 16.9% for the 6 indicator PCBs (i-PCBs) and 18.7% for the total 15 PCBs analyzed after 6months treatment. The degradation rate did not statistically vary whether the soil had been treated with non-inoculated straw or colonized straw or without straw and inoculated with the consortium of the six strains. Concerning the sediment, we evidenced significant depletions of 31.8% for the 6 i-PCBs and 33.3% for the 15 PCB congeners. The PCB depletions affected most of the 15 PCBs analyzed without preference for lower chlorinated congeners. Bioaugmented strains were evidenced in different mesocosms, but their reintroduction, after six months treatment, did not improve the rate of PCB degradation, suggesting that the biodegradation could affect the bioavailable PCB fraction. Our results demonstrate that the ascomycetous strains potentially adapted to PCBs may be propitious to the remediation of PCB contaminated sites.

Effects of organochlorines on microbial diversity and community structure in Phragmites australis rhizosphere.

This study investigated the impacts of an organochlorine (OC, γ-hexachlorocyclohexane and chlorobenzenes) mixture on microbial communities associated to Phragmites australis rhizosphere. Seventy-eight distinct colony morphotypes were isolated, cultivated and analysed by 16S rDNA sequence analysis. Toxicity tests confirmed sensitivity (e.g. Hevizibacter, Acidovorax) or tolerance (e.g. Bacillus, Aeromonas, Pseudomonas, Sphingomonas) of isolates. Rhizosphere analysis by pyrosequencing showed the microbial adaptation induced by OC exposure. Among the most abundant molecular operational taxonomic units, 80 % appeared to be tolerant (55 % opportunist, 25 % unaffected) and 20 % sensitive. P. australis rhizosphere exposed to OCs was dominated by phylotypes related to α-, ß- and γ-Proteobacteria. Specific genera were identified which were previously described as chlorinated organic pollutant degraders: Sphingomonas sp., Pseudomonas sp., Devosia sp. and Sphingobium sp. P. australis could be suitable plants to maintain their rhizosphere active microbial population which can tolerate OCs and potentially improve the OC remediation process in part by biodegradation.

Potential of autochthonous fungal strains isolated from contaminated soils for degradation of polychlorinated biphenyls.

Up to now, most studies on polychlorinated biphenyl (PCB) bioremediation have examined the ability of model fungal strains to biodegrade PCBs. Yet, there is limited information concerning the potential of autochthonous filamentous fungal strains in the biodegradation of PCBs and their possible use in the environmental technologies. In this study, we investigated the capacity of autochthonous fungal strains in the biodegradation of PCBs by isolating 24 taxa from former industrial sites highly contaminated by PCBs. Microscopic and molecular analyses using the internal transcribed spacer (ITS) region revealed that the fungal strains belonged to the phyla Ascomycota (19 strains) and Zygomycota (five strains). The chromatography gas analysis revealed evidence of degradation of seven PCB congeners. With the exception of Circinella muscae which presented no degradation potential, the other fungal strains exhibited a rate of biodegradation ranging from 29 to 85 % after 7 d of incubation in liquid medium. Among these strains, Doratomyces nanus, Doratomyces purpureofuscus, Doratomyces verrucisporus, Myceliophthora thermophila, Phoma eupyrena, and Thermoascus crustaceus showed remarkable degradation ability (>70 %) regardless of the number of chlorine substituents on the biphenyl nucleus and a high tolerance towards PCBs. To our knowledge, this is the first study that demonstrates the ability of PCB degradation by these species and indicates the potential effectiveness of some autochthonous fungal strains in bioremediation systems.

Microbial communities of urban stormwater sediments: the phylogenetic structure of bacterial communities varies with porosity.

This study focuses on the distribution of bacterial and fungal communities within the microstructure of a multi-contaminated sedimentary layer resulting from urban stormwater infiltration. Fractionation was performed on the basis of differential porosity and aggregate grain size, resulting in five fractions: leachable fitting macroporosity, < 10, 10-160, 160-1000 µm fitting aggregates, > 1000 µm. Amounts of both bacterial and fungal biomasses are greater in the < 10 µm and leachable fractions. The aggregates contain numerous bacteria but very low amounts of fungal biomass. Single-strand conformational polymorphism molecular profiles highlighted the differences between bacterial and fungal communities of the leachable fraction and those of the aggregates. Random Sanger sequencing of ssu clones revealed that these differences were mainly because of the presence of Epsilonproteobacteria and Firmicutes in the leachable fractions, while the aggregates contained more Cyanobacteria. The Cyanobacteria phylotypes in the aggregates were dominated by the sequences related to Microcoleus vaginatus while the leachable fractions presented the sequences of chloroplastic origin. Therefore, more than 50% of the phylotypes observed were related to Proteobacteria while 40% were related to Cyanobacteria and Bacteroidetes. Preferential distribution of clades in almost all the phyla or classes detected was observed. This study provides insight into the identities of dominant members of the bacterial communities of urban sediments. Microcoleus vaginatus appeared to predominate in pioneer soils.

Contrasting diversity patterns of crenarchaeal, bacterial and fungal soil communities in an alpine landscape.

BACKGROUND: The advent of molecular techniques in microbial ecology has aroused interest in gaining an understanding about the spatial distribution of regional pools of soil microbes and the main drivers responsible of these spatial patterns. Here, we assessed the distribution of crenarcheal, bacterial and fungal communities in an alpine landscape displaying high turnover in plant species over short distances. Our aim is to determine the relative contribution of plant species composition, environmental conditions, and geographic isolation on microbial community distribution. METHODOLOGY/PRINCIPAL FINDINGS: Eleven types of habitats that best represent the landscape heterogeneity were investigated. Crenarchaeal, bacterial and fungal communities were described by means of Single Strand Conformation Polymorphism. Relationships between microbial beta diversity patterns were examined by using Bray-Curtis dissimilarities and Principal Coordinate Analyses. Distance-based redundancy analyses and variation partitioning were used to estimate the relative contributions of different drivers on microbial beta diversity. Microbial communities tended to be habitat-specific and did not display significant spatial autocorrelation. Microbial beta diversity correlated with soil pH. Fungal beta-diversity was mainly related to soil organic matter. Though the effect of plant species composition was significant for all microbial groups, it was much stronger for Fungi. In contrast, geographic distances did not have any effect on microbial beta diversity. CONCLUSIONS/SIGNIFICANCE: Microbial communities exhibit non-random spatial patterns of diversity in alpine landscapes. Crenarcheal, bacterial and fungal community turnover is high and associated with plant species composition through different set of soil variables, but is not caused by geographical isolation.

Structural stability, microbial biomass and community composition of sediments affected by the hydric dynamics of an urban stormwater infiltration basin. Dynamics of physical and microbial characteristics of stormwater sediment.

The sedimentary layer deposited at the surface of stormwater infiltration basins is highly organic and multicontaminated. It undergoes considerable moisture content fluctuations due to the drying and inundation cycles (called hydric dynamics) of these basins. Little is known about the microflora of the sediments and its dynamics; hence, the purpose of this study is to describe the physicochemical and biological characteristics of the sediments at different hydric statuses of the infiltration basin. Sediments were sampled at five time points following rain events and dry periods. They were characterized by physical (aggregation), chemical (nutrients and heavy metals), and biological (total, bacterial and fungal biomasses, and genotypic fingerprints of total bacterial and fungal communities) parameters. Data were processed using statistical analyses which indicated that heavy metal (1,841 µg/g dry weight (DW)) and organic matter (11%) remained stable through time. By contrast, aggregation, nutrient content (NH4⁺, 53-717 µg/g DW), pH (6.9-7.4), and biological parameters were shown to vary with sediment water content and sediment biomass, and were higher consecutive to stormwater flows into the basin (up to 7 mg C/g DW) than during dry periods (0.6 mg C/g DW). Coinertia analysis revealed that the structure of the bacterial communities is driven by the hydric dynamics of the infiltration basin, although no such trend was found for fungal communities. Hydric dynamics more than rain events appear to be more relevant for explaining variations of aggregation, microbial biomass, and shift in the microbial community composition. We concluded that the hydric dynamics of stormwater infiltration basins greatly affects the structural stability of the sedimentary layer, the biomass of the microbial community living in it and its dynamics. The decrease in aggregation consecutive to rewetting probably enhances access to organic matter (OM), explaining the consecutive release of NH4⁺, the bloom of the microbial biomass, and the change in structure of the bacterial community. These results open new perspectives for basin management since the risk of OM and pollutant transfer to the aquifer is greatly affected by alternating dry and flood periods.

Potentiality of the cox1 gene in the taxonomic resolution of soil fungi.

We explored the potential of the cox1 gene in the species resolution of soil fungi and compared it with the nuclear internal transcribed spacer (ITS) and small subunit (SSU)-rDNA. Conserved primers allowing the amplification of the fungal cox1 gene were designed, and a total of 47 isolates of Zygomycota and Ascomycota were investigated. The analysis revealed a lack of introns in >90% of the isolates. Comparison of the species of each of the six studied genera showed high interspecific sequence polymorphisms. Indeed, the average of nucleotide variations (4.2-11%) according to the genus, due mainly to the nucleotide substitutions, led to the taxonomic resolution of all the species studied regarding both ITS and SSU-rDNA, in which <88% were discriminated. The phylogenetic analysis performed after alignment of the cox1 gene across distant fungal species was in accordance with the well-known taxonomic position of the species studied and no overlap was observed between intra- and interspecific variations. These results clearly demonstrated that the cox1 sequences could provide good molecular markers for the determination of the species composition of environmental samples and constitute an important advance to study soil fungal biodiversity.

Microbial diversity in alpine tundra soils correlates with snow cover dynamics.

The temporal and spatial snow cover dynamics is the primary factor controlling the plant communities' composition and biogeochemical cycles in arctic and alpine tundra. However, the relationships between the distribution of snow and the diversity of soil microbial communities remain largely unexplored. Over a period of 2 years, we monitored soil microbial communities at three sites, including contiguous alpine meadows of late and early snowmelt locations (LSM and ESM, respectively). Bacterial and fungal communities were characterized by using molecular fingerprinting and cloning/sequencing of microbial ribosomal DNA extracted from the soil. Herein, we show that the spatial and temporal distribution of snow strongly correlates with microbial community composition. High seasonal contrast in ESM is associated with marked seasonal shifts for bacterial communities; whereas less contrasted seasons because of long-lasting snowpack in LSM is associated with increased fungal diversity. Finally, our results indicate that, similar to plant communities, microbial communities exhibit important shifts in composition at two extremes of the snow cover gradient. However, winter conditions lead to the convergence of microbial communities independently of snow cover presence. This study provides new insights into the distribution of microbial communities in alpine tundra in relation to snow cover dynamics, and may be helpful in predicting the future of microbial communities and biogeochemical cycles in arctic and alpine tundra in the context of a warmer climate.

Long lasting persistence of Bacillus thuringiensis Subsp. israelensis (Bti) in mosquito natural habitats.

BACKGROUND: The detrimental effects of chemical insecticides on the environment and human health have lead to the call for biological alternatives. Today, one of the most promising solutions is the use of spray formulations based on Bacillus thuringiensis subsp. israelensis (Bti) in insect control programs. As a result, the amounts of Bti spread in the environment are expected to increase worldwide, whilst the common belief that commercial Bti is easily cleared from the ecosystem has not yet been clearly established. METHODOLOGY/MAIN FINDINGS: In this study, we aimed to determine the nature and origin of the high toxicity toward mosquito larvae found in decaying leaf litter collected in several natural mosquito breeding sites in the Rhône-Alpes region. From the toxic fraction of the leaf litter, we isolated B. cereus-like bacteria that were further characterized as B. thuringiensis subsp. israelensis using PCR amplification of specific toxin genes. Immunological analysis of these Bti strains showed that they belong to the H14 group. We finally used amplified length polymorphism (AFLP) markers to show that the strains isolated from the leaf litter were closely related to those present in the commercial insecticide used for field application, and differed from natural worldwide genotypes. CONCLUSIONS/SIGNIFICANCE: Our results raise the issue of the persistence, potential proliferation and environmental accumulation of human-spread Bti in natural mosquito habitats. Such Bti environmental persistence may lengthen the exposure time of insects to this bio-insecticide, thereby increasing the risk of resistance acquisition in target insects, and of a negative impact on non-target insects.

Exonuclease activity of proofreading DNA polymerases is at the origin of artifacts in molecular profiling studies.

CE fingerprint methods are commonly used in microbial ecology. We have previously noticed that the position and number of peaks in CE-SSCP (single-strand conformation polymorphism) profiles depend on the DNA polymerase used in PCR [1]. Here, we studied the fragments produced by Taq polymerase as well as four commercially available proofreading polymerases, using the V3 region of the Escherichia coli rss gene as a marker. PCR products rendered multiple peaks in denaturing CE; Taq polymerase was observed to produce the longest fragments. Incubation of the fragments with T4 DNA polymerase indicated that the 3'-ends of the proofreading polymerase amplicons were recessed, while the Taq amplicon was partially +A tailed. Treatment of the PCR product with proofreading DNA polymerase rendered trimmed fragments. This was due to the 3'-5' exonuclease activity of these enzymes, which is essential for proofreading. The nuclease activity was reduced by increasing the concentration of dNTP. The Platinum Pfx DNA polymerase generated very few artifacts and could produce 85% of blunted PCR products. Nevertheless, despite the higher error rate, we recommend the use of Taq polymerase rather than proofreading in the framework for molecular fingerprint studies. They are more cost-effective and therefore ideally suited for high-throughput analysis; the +A tail artifact rate can be controlled by modifying the PCR primers and the reaction conditions.

CE-SSCP and CE-FLA, simple and high-throughput alternatives for fungal diversity studies.

Fungal communities are key components of soil, but the study of their ecological significance is limited by a lack of appropriated methods. For instance, the assessment of fungi occurrence and spatio-temporal variation in soil requires the analysis of a large number of samples. The molecular signature methods provide a useful tool to monitor these microbial communities and can be easily adapted to capillary electrophoresis (CE) allowing high-throughput studies. Here we assess the suitability of CE-FLA (Fragment Length Polymorphism, denaturing conditions) and CE-SSCP (Single-Stranded Conformation Polymorphism, native conditions) applied to environmental studies since they require a short molecular marker and no post-PCR treatments. We amplified the ITS1 region from 22 fungal strains isolated from an alpine ecosystem and from total genomic DNA of alpine and infiltration basin soils. The CE-FLA and CE-SSCP separated 17 and 15 peaks respectively from a mixture of 19 strains. For the alpine soil-metagenomic DNA, the FLA displayed more peaks than the SSCP and the converse result was found for infiltration basin sediments. We concluded that CE-FLA and CE-SSCP of ITS1 region provided complementary information. In order to improve CE-SSCP sensitivity, we tested its resolution according to migration temperature and found 32 degrees C to be optimal. Because of their simplicity, quickness and reproducibility, we found that these two methods were promising for high-throughput studies of soil fungal communities.

Improvements of polymerase chain reaction and capillary electrophoresis single-strand conformation polymorphism methods in microbial ecology: toward a high-throughput method for microbial diversity studies in soil.

The molecular signature of bacteria from soil ecosystems is an important tool for studying microbial ecology and biogeography. However, a high-throughput technology is needed for such studies. In this article, we tested the suitability of available methods ranging from soil DNA extraction to capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) for high-throughput studies. Our results showed that the extraction method does not dramatically influence CE-SSCP profiles, and that DNA extraction of a 0.25 g soil sample is sufficient to observe overall bacterial diversity in soil matrices. The V3 region of the 16S rRNA gene was amplified by PCR, and the extension time was found to be critical. We have also found that proofreading DNA polymerases generate a better signal in CE-SSCP profiles. Experiments performed with different soil matrices revealed the repeatability, efficiency, and consistency of CE-SSCP. Studies on PCR and CE-SSCP using single-species genomic DNA as a matrix showed that several ribotypes may migrate at the same position, and also that single species can produce double peaks. Thus, the extrapolation between number of peaks and number of species remains difficult. Additionally, peak detection is limited by the analysis software. We conclude that the presented method, including CE-SSCP and the analyzing step, is a simple and effective technique to obtain the molecular signature of a given soil sample.

The capsular polysaccharide biosynthesis of Streptococcus pneumoniae serotype 8: functional identification of the glycosyltransferase WciS (Cap8H).

CPS (capsular polysaccharide) is a major virulence factor in Streptococcus pneumoniae. Biosynthesis of CPS RU (repeat unit) proceeds by sequential transfer of sugar residues from the appropriate sugar donor to an activated lipid carrier by committed GTs (glycosyltransferases). While the nucleotide sequence of many cps loci is already known, the real substrate specificity of the hypothetical GTs, as well as the sequence of sugar addition is unclear. In the present paper, we report the biochemical characterization of one alpha-galactosyltransferase, WciS (Cap8H), a member of GT family 4. This enzyme is implicated in the tetrasaccharide RU biosynthetic pathway of Strep. pneumoniae CPS 8 ([-->4)-alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->4)-beta-D-Glcp-(1-->]n). Expression of WciS-His6 in Escherichia coli BL21 (DE3) strains or BL21 (DE3)/DeltagalU strain resulted in synthesis of a 39 kDa membrane-associated protein identified by N-terminal sequencing and recognized by anti-His6-tag antibody. This protein was capable of adding a galactose residue cellobiuronic acid [beta-D-GlcAp-(1-->4)-D-Glcp]-pyrophosphate-polyprenol from UDP-Gal. The newly added galactose residue is removed by alpha-galactosidase, indicating that WciS is a retaining GT. Our results suggest that WciS catalyses the addition of the third sugar residue of the CPS 8 RU. The recombinant WciS-His6 was solubilized and purified as a soluble multimer, opening the way for structural studies.

The glucosyl-1-phosphate transferase WchA (Cap8E) primes the capsular polysaccharide repeat unit biosynthesis of Streptococcus pneumoniae serotype 8.

The gene wchA (cap8E) belongs to the cps8 locus that is involved in biosynthesis of the capsular polysaccharide (CPS) repeat unit (RU) of the virulent Streptococcus pneumoniae serotype 8. We report here the biochemical characterization of the membrane-associated protein WchA (Cap8E), overexpressed in Escherichia coli BL21(DE3)/pLysS. Our results demonstrate that the recombinant enzyme transfers in vitro a glucosyl-1-phosphate from UDP-glucose to an endogenous phosphoryl-polyprenol, thereby priming the RU biosynthetic pathway of S. pneumoniae CPS 8. We also show that the C-terminal half of WchA is the glycosyltransferase domain as observed for the galactosyl-1-phosphate transferase WbaP from Salmonella enterica, previously described to prime the first step of O-antigen biosynthesis. These results demonstrate that WchA plays a prominent function in the capsule biosynthesis and explain the key role it occupies in the pneumococcal capsule variation.

Production of recombinant xenotransplantation antigen in Escherichia coli.

The synthesis of sufficient amounts of oligosaccharides is the bottleneck for the study of their biological function and their possible use as drug. As an alternative for chemical synthesis, we propose to use Escherichia coli as a "living factory." We have addressed the production of the Galp alpha(1-3)Galp beta(1-4)GlcNAc epitope, the major porcine antigen responsible for xenograft rejection. An E. coli strain was generated which simultaneously expresses NodC (to provide the chitin-pentaose acceptor), beta(1-4) galactosyltransferase LgtB, and bovine alpha(1-3) galactosyltransferase GstA. This strain produced 0.68 g/L of the heptasaccharide Galp alpha(1-3)Galp beta(1-4)(GlcNAc)(5), which harbours the xenoantigen at its non-reducing end, establishing the feasibility of this approach.

Glycogen synthase: towards a minimum catalytic unit?

Classically, alpha-1,4-glucan synthases have been divided into two families, animal/fungal glycogen synthases (GS) and bacterial/plant starch synthases (G(S)S), according to differences in sequence, sugar donor specificity and regulatory mechanisms. Detailed sequence analysis, predicted secondary structure comparison and threading analysis show that these two families are structurally related and that some domains of GSs were acquired to meet regulatory requirements. Archaeal G(S)S present structural and functional features that are conserved in one, the other or both families. Therefore, they are the link between GS and G(S)S and harbor the minimal sequence and structural features that constitute the minimum catalytic unit of the alpha-1,4-glucan synthase superfamily.