Development of a multigenic metabarcoding protocol for deciphering the diversity of plant-associated Archaea. [144]

Soil and plant microbiota are playing an eminent role in shaping both natural ecosystems and agricultural production systems. It is now widely admitted that bacterial communities show a great diversity in both the plant rhizosphere and the plant endosphere. These microbial communities affect the fitness of the hosts and further play important roles in C and N cycles. While bacterial communities have been widely studied, much less is known about archaeal communities and the role they play within the rhizosphere and the endosphere. The objective of our study was to develop a multigenic metabarcoding approach to accurately describe the diversity of archaeal communities interacting with rice paddies in the Rhône delta region. This pilot agro-ecosystem was selected because it is annually subjected to several abiotic stresses (water stress associated to rice paddies flooding, soil salinity, high production of methane, etc.) that are likely to enhance archaeal growth. We used archaeal specific primers within the universal 16S rRNA gene and the mcrA gene (encoding the methyl-coenzyme M reductase subunit A of methanogens). An evaluation of the efficiency of the primers was carried out on mock cultures and plant samples. Two rice-compartments during both rice paddies flooding and drying periods were collected and analyzed. This study is likely to be a first step towards better describing rice-associated archaeal communities and understanding the dynamics of these communities in relation with abiotic constraints of rice paddies in Camargue

Influence of soil properties on archaeal diversity and distribution in the McMurdo Dry Valleys, Antarctica

Archaea are the least understood members of the microbial community in Antarctic mineral soils. Although their occurrence in Antarctic coastal soils has been previously documented, little is known about their distribution in soils across the McMurdo Dry Valleys, Victoria Land. In this study, terminal-restriction fragment length polymorphism (t-RFLP) analysis and 454 pyrosequencing were coupled with a detailed analysis of soil physicochemical properties to characterize archaeal diversity and identify environmental factors that might shape and maintain archaeal communities in soils of the three southern most McMurdo Dry Valleys (Garwood, Marshall, and Miers Valley). Archaea were successfully detected in all inland and coastal mineral soils tested, revealing a low overall richness (mean of six operational taxonomic units [OTUs] per sample site). However, OTU richness was higher in some soils and this higher richness was positively correlated with soil water content, indicating water as a main driver of archaeal community richness. In total, 18 archaeal OTUs were detected, predominately Thaumarchaeota affiliated with Marine Group 1.1b (> 80% of all archaeal sequences recovered). Less abundant OTUs (2% of all archaeal sequences) were loosely related to members of the phylum Euryarchaeota. This is the first comprehensive study showing a widespread presence and distribution of Archaea in inland Antarctic soils

Ecological and Genomic Attributes of Novel Bacterial Taxa That Thrive in Subsurface Soil Horizons.

While most bacterial and archaeal taxa living in surface soils remain undescribed, this problem is exacerbated in deeper soils, owing to the unique oligotrophic conditions found in the subsurface. Additionally, previous studies of soil microbiomes have focused almost exclusively on surface soils, even though the microbes living in deeper soils also play critical roles in a wide range of biogeochemical processes. We examined soils collected from 20 distinct profiles across the United States to characterize the bacterial and archaeal communities that live in subsurface soils and to determine whether there are consistent changes in soil microbial communities with depth across a wide range of soil and environmental conditions. We found that bacterial and archaeal diversity generally decreased with depth, as did the degree of similarity of microbial communities to those found in surface horizons. We observed five phyla that consistently increased in relative abundance with depth across our soil profiles: Chloroflexi, Nitrospirae, Euryarchaeota, and candidate phyla GAL15 and Dormibacteraeota (formerly AD3). Leveraging the unusually high abundance of Dormibacteraeota at depth, we assembled genomes representative of this candidate phylum and identified traits that are likely to be beneficial in low-nutrient environments, including the synthesis and storage of carbohydrates, the potential to use carbon monoxide (CO) as a supplemental energy source, and the ability to form spores. Together these attributes likely allow members of the candidate phylum Dormibacteraeota to flourish in deeper soils and provide insight into the survival and growth strategies employed by the microbes that thrive in oligotrophic soil environments.IMPORTANCE Soil profiles are rarely homogeneous. Resource availability and microbial abundances typically decrease with soil depth, but microbes found in deeper horizons are still important components of terrestrial ecosystems. By studying 20 soil profiles across the United States, we documented consistent changes in soil bacterial and archaeal communities with depth. Deeper soils harbored communities distinct from those of the more commonly studied surface horizons. Most notably, we found that the candidate phylum Dormibacteraeota (formerly AD3) was often dominant in subsurface soils, and we used genomes from uncultivated members of this group to identify why these taxa are able to thrive in such resource-limited environments. Simply digging deeper into soil can reveal a surprising number of novel microbes with unique adaptations to oligotrophic subsurface conditions

Development of a multigenic metabarcoding of prokaryotic microbiota: application to Bacteria and Archaea of agronomical interest encountered in the rice rhizosphere of Camargue

It is our hypothesis that soil and plant microbiota are playing an eminent role in shaping both natural ecosystems and agricultural production systems. It is now widely admitted that bacterial communities show a great diversity in the rhizosphere and in the endosphere as endophytes. These microbial communities affect the fitness of both the hosts and the microbes and further play important roles in C and N cycles. Much less is known about the role played (if any) by archaea within both the plant rhizosphere and endosphere. Interestingly, a recent study has revealed a relatively high archaeal concentration in internal plant tissues (Müller et al., 2015). The objective of our study is to develop a multigenic metabarcoding approach to accurately describe the biodiversity of relevant microbial communities interacting with rice paddies in the Rhône delta region. This pilot agro-ecosystem was selected because it is annually subjected to several abiotic stresses (water stress associated to rice paddies flooding, soil salinity, etc.) that are likely to enhance archaeal growth. We will retain universal prokaryotic primers and archaeal specific primers within the 16S rRNA gene, two housekeeping genes, gyrB(encoding the ß subunit of the DNA gyrase) and rpoB (encoding the ß subunit of the RNA polymerase) for bacteria, and the thermosome TF55 gene (encoding a type II chaperonin) for archaea. Primers evaluation will be conducted on mock cultures and by sampling rice plants during both rice paddies flooding and drying periods. This study is likely to be a first step towards better understanding the dynamics of Camargue rice paddies microbial communities in relation with abiotic constraints

PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3–V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions

Terminal restriction fragment length polymorphism is an “old school” reliable technique for swift microbial community screening in anaerobic digestion

The microbial community in anaerobic digestion has been analysed through microbial fingerprinting techniques, such as terminal restriction fragment length polymorphism (TRFLP), for decades. In the last decade, high-throughput 16S rRNA gene amplicon sequencing has replaced these techniques, but the time-consuming and complex nature of high-throughput techniques is a potential bottleneck for full-scale anaerobic digestion application, when monitoring community dynamics. Here, the bacterial and archaeal TRFLP profiles were compared with 16S rRNA gene amplicon profiles (Illumina platform) of 25 full-scale anaerobic digestion plants. The α-diversity analysis revealed a higher richness based on Illumina data, compared with the TRFLP data. This coincided with a clear difference in community organisation, Pareto distribution, and co-occurrence network statistics, i.e., betweenness centrality and normalised degree. The β-diversity analysis showed a similar clustering profile for the Illumina, bacterial TRFLP and archaeal TRFLP data, based on different distance measures and independent of phylogenetic identification, with pH and temperature as the two key operational parameters determining microbial community composition. The combined knowledge of temporal dynamics and projected clustering in the β-diversity profile, based on the TRFLP data, distinctly showed that TRFLP is a reliable technique for swift microbial community dynamics screening in full-scale anaerobic digestion plants

Agricultural management and plant selection interactively affect rhizosphere microbial community structure and nitrogen cycling.

BACKGROUND:Rhizosphere microbial communities are key regulators of plant performance, yet few studies have assessed the impact of different management approaches on the rhizosphere microbiomes of major crops. Rhizosphere microbial communities are shaped by interactions between agricultural management and host selection processes, but studies often consider these factors individually rather than in combination. We tested the impacts of management (M) and rhizosphere effects (R) on microbial community structure and co-occurrence networks of maize roots collected from long-term conventionally and organically managed maize-tomato agroecosystems. We also explored the interaction between these factors (M × R) and how it impacts rhizosphere microbial diversity and composition, differential abundance, indicator taxa, co-occurrence network structure, and microbial nitrogen-cycling processes. RESULTS:Host selection processes moderate the influence of agricultural management on rhizosphere microbial communities, although bacteria and fungi respond differently to plant selection and agricultural management. We found that plants recruit management-system-specific taxa and shift N-cycling pathways in the rhizosphere, distinguishing this soil compartment from bulk soil. Rhizosphere microbiomes from conventional and organic systems were more similar in diversity and network structure than communities from their respective bulk soils, and community composition was affected by both M and R effects. In contrast, fungal community composition was affected only by management, and network structure only by plant selection. Quantification of six nitrogen-cycling genes (nifH, amoA [bacterial and archaeal], nirK, nrfA, and nosZ) revealed that only nosZ abundance was affected by management and was higher in the organic system. CONCLUSIONS:Plant selection interacts with conventional and organic management practices to shape rhizosphere microbial community composition, co-occurrence patterns, and at least one nitrogen-cycling process. Reframing research priorities to better understand adaptive plant-microbe feedbacks and include roots as a significant moderating influence of management outcomes could help guide plant-oriented strategies to improve productivity and agroecosystem sustainability

Primer selection impacts specific population abundances but not community dynamics in a monthly time-series 16S rRNA gene amplicon analysis of coastal marine bacterioplankton.

Primers targeting the 16S small subunit ribosomal RNA marker gene, used to characterize bacterial and archaeal communities, have recently been re-evaluated for marine planktonic habitats. To investigate whether primer selection affects the ecological interpretation of bacterioplankton populations and community dynamics, amplicon sequencing with four primer sets targeting several hypervariable regions of the 16S rRNA gene was conducted on both mock communities constructed from cloned 16S rRNA genes and a time-series of DNA samples from the temperate coastal Santa Barbara Channel. Ecological interpretations of community structure (delineation of depth and seasonality, correlations with environmental factors) were similar across primer sets, while population dynamics varied. We observed substantial differences in relative abundances of taxa known to be poorly resolved by some primer sets, such as Thaumarchaeota and SAR11, and unexpected taxa including Roseobacter clades. Though the magnitude of relative abundances of common OTUs differed between primer sets, the relative abundances of the OTUs were nonetheless strongly correlated. We do not endorse one primer set but rather enumerate strengths and weaknesses to facilitate selection appropriate to a system or experimental goal. While 16S rRNA gene primer bias suggests caution in assessing quantitative population dynamics, community dynamics appear robust across studies using different primers

Geobacter, Anaeromyxobacter and Anaerolineae populations are enriched on anodes of root exudate-driven microbial fuel cells in rice field soil

Plant-based sediment microbial fuel cells (PMFCs) couple the oxidation of root exudates in living rice plants to current production. We analysed the composition of the microbial community on anodes from PMFC with natural rice field soil as substratum for rice by analysing 16S rRNA as an indicator of microbial activity and diversity. Terminal restriction fragment length polymorphism (TRFLP) analysis indicated that the active bacterial community on anodes from PMFCs differed strongly compared with controls. Moreover, clones related to Deltaproteobacteria and Chloroflexi were highly abundant (49% and 21%, respectively) on PMFCs anodes. Geobacter (19%), Anaeromyxobacter (15%) and Anaerolineae (17%) populations were predominant on anodes with natural rice field soil and differed strongly from those previously detected with potting soil. In open circuit (OC) control PMFCs, not allowing electron transfer, Deltaproteobacteria (33%), Betaproteobacteria (20%), Chloroflexi (12%), Alphaproteobacteria (10%) and Firmicutes (10%) were detected. The presence of an electron accepting anode also had a strong influence on methanogenic archaea. Hydrogenotrophic methanogens were more active on PMFC (21%) than on OC controls (10%), whereas acetoclastic Methanosaetaceae were more active on OC controls (31%) compared with PMFCs (9%). In conclusion, electron accepting anodes and rice root exudates selected for distinct potential anode-reducing microbial populations in rice soil inoculated PMFC

Effect of Ethanol on Microbial Community Structure and Function During Natural Attenuation of Benzene, Toluene, and \u3cem\u3eo\u3c/em\u3e-Xylene in a Sulfate-reducing Aquifer

Ethanol (EtOH) is a commonly used fuel oxygenate in reformulated gasoline and is an alternative fuel and fuel supplement. Effects of EtOH release on aquifer microbial ecology and geochemistry have not been well characterized in situ. We performed a controlled field release of petroleum constituents (benzene (B), toluene (T), o-xylene (o-X) at ∼1–3 mg/L each) with and without EtOH (∼500 mg/L). Mixed linear modeling (MLM) assessed effects on the microbial ecology of a naturally sulfidic aquifer and how the microbial community affected B, T, and o-X plume lengths and aquifer geochemistry. Changes in microbial community structure were determined by quantitative polymerase chain reaction (qPCR) targeting Bacteria, Archaea, and sulfate reducing bacteria (SRB); SRB were enumerated using a novel qPCR method targeting the adenosine-5′-phosphosulfate reductase gene. Bacterial and SRB densities increased with and without EtOH-amendment (1−8 orders of magnitude). Significant increases in Archaeal species richness; Archaeal cell densities (3–6 orders of magnitude); B, T, and o-X plume lengths; depletion of sulfate; and induction of methanogenic conditions were only observed with EtOH-amendment. MLM supported the conclusion that EtOH-amendment altered microbial community structure and function, which in turn lowered the aquifer redox state and led to a reduction in bioattenuation rates of B, T, and o-X