Nanopore Is Preferable over Illumina for 16S Amplicon Sequencing of the Gut Microbiota When Species-Level Taxonomic Classification, Accurate Estimation of Richness, or Focus on Rare Taxa Is Required.

Nanopore sequencing is a promising technology used for 16S rRNA gene amplicon sequencing as it can provide full-length 16S reads and has a low up-front cost that allows research groups to set up their own sequencing workflows. To assess whether Nanopore with the improved error rate of the Kit 12 chemistry should be adopted as the preferred sequencing technology instead of Illumina for 16S amplicon sequencing of the gut microbiota, we used a mock community and human faecal samples to compare diversity, richness, and species-level community structure, as well as the replicability of the results. Nanopore had less noise, better accuracy with the mock community, a higher proportion of reads from the faecal samples classified to species, and better replicability. The difference between the Nanopore and Illumina results of the faecal bacterial community structure was significant but small compared to the variation between samples. The results show that Nanopore is a better choice for 16S rRNA gene amplicon sequencing when the focus is on species-level taxonomic resolution, the investigation of rare taxa, or an accurate estimation of richness. Illumina 16S sequencing should be reserved for communities with many unknown species, and for studies that require the resolution of amplicon sequence variants.

Hidden heterogeneity and co-occurrence networks of soil prokaryotic communities revealed at the scale of individual soil aggregates.

Sequencing PCR-amplified gene fragments from metagenomic DNA is a widely applied method for studying the diversity and dynamics of soil microbial communities. Typically, DNA is extracted from 0.25 to 1 g of soil. These amounts, however, neglect the heterogeneity of soil present at the scale of soil aggregates and thus ignore a crucial scale for understanding the structure and functionality of soil microbial communities. Here, we show with a nitrogen-depleted agricultural soil the impact of reducing the amount of soil used for DNA extraction from 250 mg to approx. 1 mg to access spatial information on the prokaryotic community structure, as indicated by 16S rRNA gene amplicon analyses. Furthermore, we demonstrate that individual aggregates from the same soil differ in their prokaryotic community compositions. The analysis of 16S rRNA gene amplicon sequences from individual soil aggregates allowed us, in contrast to 250 mg soil samples, to construct a co-occurrence network that provides insight into the structure of microbial associations in the studied soil. Two dense clusters were apparent in the network, one dominated by Thaumarchaeota, known to be capable of ammonium oxidation at low N concentrations, and the other by Acidobacteria subgroup 6, representing an oligotrophic lifestyle to obtain energy from SOC. Overall this study demonstrates that DNA obtained from individual soil aggregates provides new insights into how microbial communities are assembled.

Annual replication is essential in evaluating the response of the soil microbiome to the genetic modification of maize in different biogeographical regions.

The importance of geographic location and annual variation on the detection of differences in the rhizomicrobiome caused by the genetic modification of maize (Bt-maize, event MON810) was evaluated at experimental field sites across Europe including Sweden, Denmark, Slovakia and Spain. DNA of the rhizomicrobiome was collected at the maize flowering stage in three consecutive years and analyzed for the abundance and diversity of PCR-amplified structural genes of Bacteria, Archaea and Fungi, and functional genes for bacterial nitrite reductases (nirS, nirK). The nirK genes were always more abundant than nirS. Maize MON810 did not significantly alter the abundance of any microbial genetic marker, except for sporadically detected differences at individual sites and years. In contrast, annual variation between sites was often significant and variable depending on the targeted markers. Distinct, site-specific microbial communities were detected but the sites in Denmark and Sweden were similar to each other. A significant effect of the genetic modification of the plant on the community structure in the rhizosphere was detected among the nirK denitrifiers at the Slovakian site in only one year. However, most nirK sequences with opposite response were from the same or related source organisms suggesting that the transient differences in community structure did not translate to the functional level. Our results show a lack of effect of the genetic modification of maize on the rhizosphere microbiome that would be stable and consistent over multiple years. This demonstrates the importance of considering annual variability in assessing environmental effects of genetically modified crops.

Contrasting microbial community responses to salinization and straw amendment in a semiarid bare soil and its wheat rhizosphere.

Soil salinization is a major constraint of agriculture in semiarid ecosystems. In this study soil microcosms were applied to evaluate the impact of a lower- and higher-level salinization treatment of a pristine scrubland soil on the abundance of Bacteria, Archaea, and Fungi, and on prokaryotic diversity in bare soil and the rhizosphere of wheat assessed by qPCR and high-throughput sequencing of 16S rRNA gene amplicons. Furthermore, the impact of soil straw amendment as a salt-stress alleviation strategy was studied. While the low-level salinity stimulated plant growth, the seedlings did not survive under the higher-level salinity unless the soil was amended with straw. Without the straw amendment, salinization had only minor effects on the microbial community in bare soil. On the other hand, it decreased prokaryotic diversity in the rhizosphere of wheat, but the straw amendment was effective in mitigating this effect. The straw however, was not a significant nutrient source for the rhizosphere microbiota but more likely acted indirectly by ameliorating the salinity stress on the plant. Members of Proteobacteria, Actinobacteria, and Firmicutes were abundant among the bacteria that reacted to soil salinization and the straw amendment but showed inconsistent responses indicating the large physiological diversity within these phyla.

Impact of land-use change and soil organic carbon quality on microbial diversity in soils across Europe.

Land-use and their change have dramatic consequences for above-ground biodiversity, but their impact on soil microbial communities is poorly understood. In this study, soils from 19 European sites representing conversion of croplands to grasslands or forests and of grasslands to croplands or forests were characterized for microbial abundance and bacterial diversity. The abundance of Bacteria and Fungi but not Archaea responded to land-use change. Site was the major determinant of the soil bacterial community structure, explaining 32% of the variation in 16S rRNA gene diversity. While the quantity of soil organic carbon (SOC) only explained 5% of the variation, SOC when differentiated by its quality could explain 22%. This was similar to the impact of soil pH (21%) and higher than that of land-use type (15%). Croplands had the highest bacterial diversity. Converting croplands to grassland caused an increase of Verrucomicrobia; croplands to forest increased Rhizobiales but decreased Bacteroidetes and Nitrospirae; and grasslands to cropland increased Gemmatimonadetes but decreased Verrucomicrobia and Planctomycetes. Network analysis identified associations between particular SOC fractions and specific bacterial taxa. We conclude that land-use-related effects on soil microorganisms can be consistently observed across a continental scale.

Response of the rhizosphere prokaryotic community of barley (Hordeum vulgare L.) to elevated atmospheric CO2 concentration in open-top chambers.

The effect of elevated atmospheric CO2 concentration [CO2 ] on the diversity and composition of the prokaryotic community inhabiting the rhizosphere of winter barley (Hordeum vulgare L.) was investigated in a field experiment, using open-top chambers. Rhizosphere samples were collected at anthesis (flowering stage) from six chambers with ambient [CO2 ] (approximately 400 ppm) and six chambers with elevated [CO2 ] (700 ppm). The V4 region of the 16S rRNA gene was PCR-amplified from the extracted DNA and sequenced on an Illumina MiSeq instrument. Above-ground plant biomass was not affected by elevated [CO2 ] at anthesis, but plants exposed to elevated [CO2 ] had significantly higher grain yield. The composition of the rhizosphere prokaryotic communities was very similar under ambient and elevated [CO2 ]. The dominant taxa were Bacteroidetes, Actinobacteria, Alpha-, Gamma-, and Betaproteobacteria. Elevated [CO2 ] resulted in lower prokaryotic diversity in the rhizosphere, but did not cause a significant difference in community structure.

The Effect of Root Exudate 7,4'-Dihydroxyflavone and Naringenin on Soil Bacterial Community Structure.

Our goal was to investigate how root exudate flavonoids influence the soil bacterial community structure and to identify members of the community that change their relative abundance in response to flavonoid exudation. Using a model system that approximates flavonoid exudation of Medicago sativa roots, we treated a soil with 7,4'-dihydroxyflavone and naringenin in two separate experiments using three different rates: medium (equivalent to the exudation rate of 7,4'-dihydroxyflavone from M. sativa seedlings), high (10× the medium rate), and low (0.1× the medium rate). Controls received no flavonoid. Soil samples were subjected to ATP assays and 16S rRNA gene amplicon sequencing. The flavonoid treatments caused no significant change in the soil ATP content. With the high 7,4'-dihydroxyflavone treatment rate, operational taxonomic units (OTUs) classified as Acidobacteria subdivision 4 increased in relative abundance compared with the control samples, whereas OTUs classified as Gaiellales, Nocardioidaceae, and Thermomonosporaceae were more prevalent in the control. The naringenin treatments did not cause significant changes in the soil bacterial community structure. Our results suggest that the root exudate flavonoid 7,4'-dihydroxyflavone can interact with a diverse range of soil bacteria and may have other functions in the rhizosphere in addition to nod gene induction in legume-rhizobia symbiosis.

Practical survey on antibiotic-resistant bacterial communities in livestock manure and manure-amended soil.

Through livestock manure fertilization, antibiotics, antibiotic-resistant bacteria and genes are transferred to agricultural soils, resulting in a high prevalence of antibiotic-resistant bacteria in the soil. It is not clear, however, whether a correlation exists between resistant bacterial populations in manure and manure-amended soil. In this work, we demonstrate that the prevalence of cephalexin-, amoxicillin-, kanamycin- and gentamicin-resistant bacteria as well as bacteria simultaneously resistant to all four antibiotics was much higher in manure-amended soils than in manure-free soil. 454-pyrosequencing indicated that the ARB and multiple antibiotic-resistant bacteria (MARB) in swine or chicken manure and manure-amended soil were mainly distributed among Sphingobacterium, Myroides, Enterococcus, Comamonas and unclassified Flavobacteriaceae. The genus Sphingobacterium was highly prevalent among ARB from swine manure and manure-amended soil, and was also the most dominant genus among MARB from chicken manure and manure-amended soil. Other dominant genera among ARB or MARB populations in manure samples, including Myroides, Enterococcus and Comamonas, could not be detected or were detected at very low relative abundance in manure-amended soil. The present study suggests the possibility of transfer of ARBs from livestock manures to soils and persistence of ARB in these environments.