Eiko E. Kuramae

Taxonomical and functional microbial community selection in soybean rhizosphere

This study addressed the selection of the rhizospheric microbial community from the bulk soil reservoir under agricultural management of soybean in Amazon forest soils. We used a shotgun metagenomics approach to investigate the taxonomic and functional diversities of microbial communities in the bulk soil and in the rhizosphere of soybean plants and tested the validity of neutral and niche theories to explain the rhizosphere community assembly processes. Our results showed a clear selection at both taxonomic and functional levels operating in the assembly of the soybean rhizosphere community. The taxonomic analysis revealed that the rhizosphere community is a subset of the bulk soil community. Species abundance in rhizosphere fits the log-normal distribution model, which is an indicator of the occurrence of niche-based processes. In addition, the data indicate that the rhizosphere community is selected based on functional cores related to the metabolisms of nitrogen, iron, phosphorus and potassium, which are related to benefits to the plant, such as growth promotion and nutrition. The network analysis including bacterial groups and functions was less complex in rhizosphere, suggesting the specialization of some specific metabolic pathways. We conclude that the assembly of the microbial community in the rhizosphere is based on niche-based processes as a result of the selection power of the plant and other environmental factors.

Soil characteristics more strongly influence soil bacterial communities than land-use type

To gain insight into the factors driving the structure of bacterial communities in soil, we applied real-time PCR, PCR-denaturing gradient gel electrophoreses, and phylogenetic microarray approaches targeting the 16S rRNA gene across a range of different land usages in the Netherlands. We observed that the main differences in the bacterial communities were not related to land-use type, but rather to soil factors. An exception was the bacterial community of pine forest soils (PFS), which was clearly different from all other sites. PFS had lowest bacterial abundance, lowest numbers of operational taxonomic units (OTUs), lowest soil pH, and highest C : N ratios. C : N ratio strongly influenced bacterial community structure and was the main factor separating PFS from other fields. For the sites other than PFS, phosphate was the most important factor explaining the differences in bacterial communities across fields. Firmicutes were the most dominant group in almost all fields, except in PFS and deciduous forest soils (DFS). In PFS, Alphaproteobacteria was most represented, while in DFS, Firmicutes and Gammaproteobacteria were both highly represented. Interestingly, Bacillii and Clostridium OTUs correlated with pH and phosphate, which might explain their high abundance across many of the Dutch soils. Numerous bacterial groups were highly correlated with specific soil factors, suggesting that they might be useful as indicators of soil status.

Acidobacterial community responses to agricultural management of soybean in Amazon forest soils

This study focused on the impact of land-use changes and agricultural management of soybean in Amazon forest soils on the abundance and composition of the acidobacterial community. Quantitative real-time PCR (q-PCR) assays and pyrosequencing of 16S rRNA gene were applied to study the acidobacterial community in bulk soil samples from soybean croplands and adjacent native forests, and mesocosm soil samples from soybean rhizosphere. Based on qPCR measurements, Acidobacteria accounted for 23% in forest soils, 18% in cropland soils, and 14% in soybean rhizosphere of the total bacterial signals. From the 16S rRNA gene sequences of Bacteria domain, the phylum Acidobacteria represented 28% of the sequences from forest soils, 16% from cropland soils, and 17% from soybean rhizosphere. Acidobacteria subgroups 1-8, 10, 11, 13, 17, 18, 22, and 25 were detected with subgroup 1 as dominant among them. Subgroups 4, 6, and 7 were significantly higher in cropland soils than in forest soils, which subgroups responded to decrease in soil aluminum. Subgroups 6 and 7 responded to high content of soil Ca, Mg, Mn, and B. These results showed a differential response of the Acidobacteria subgroups to abiotic soil factors, and open the possibilities to explore acidobacterial subgroups as early-warning bioindicators of agricultural soil management effects in the Amazon area.

Soil-Borne Microbiome: Linking Diversity to Function

Soil microorganisms are sensitive to environment disturbances, and such alterations have consequences on microbial diversity and functions. Our hypothesis is that alpha diversity of microbial communities and functional diversity decrease from undisturbed to disturbed soils, with consequences for functional redundancy in the soil ecosystem. To test this hypothesis, we used soil DNA shotgun metagenomics approach to assess the soil microbiome in a chronosequence of land-use from a native tropical forest, followed by deforestation and cultivation of soybean croplands and pasture in different seasons. Agriculture and pasture soils were among the most diverse and presented higher functional redundancy, which is important to maintain the ecosystem functioning after the forest conversion. On the other hand, the ecosystem equilibrium in forest is maintained based on a lower alpha diversity but higher abundance of microorganisms. Our results indicate that land-use change alters the structure and composition of microbial communities; however, ecosystem functionality is overcome by different strategies based on the abundance and diversity of the communities.

Soil and plant factors driving the community of soil-borne microorganisms across chronosequences of secondary succession of chalk grasslands with a neutral pH.

Although soil pH has been shown to be an important factor driving microbial communities, relatively little is known about the other potentially important factors that shape soil-borne microbial community structure. This study examined plant and microbial communities across a series of neutral pH fields (pH=7.0-7.5) representing a chronosequence of secondary succession after former arable fields were taken out of production. These fields ranged from 17 to >66 years since the time of abandonment, and an adjacent arable field was included as a reference. Hierarchical clustering analysis, nonmetric multidimensional scaling and analysis of similarity of 52 different plant species showed that the plant community composition was significantly different in the different chronosequences, and that plant species richness and diversity increased with time since abandonment. The microbial community structure, as analyzed by phylogenetic microarrays (PhyloChips), was significantly different in arable field and the early succession stage, but no distinct microbial communities were observed for the intermediate and the late succession stages. The most determinant factors in shaping the soil-borne microbial communities were phosphorous and NH(4)(+). Plant community composition and diversity did not have a significant effect on the belowground microbial community structure or diversity.

Impact of long-term N, P, K, and NPK fertilization on the composition and potential functions of the bacterial community in grassland soil

Soil abiotic and biotic interactions govern important ecosystem processes. However, the mechanisms behind these interactions are complex, and the links between specific environmental factors, microbial community structures, and functions are not well understood. Here, we applied DNA shotgun metagenomic techniques to investigate the effect of inorganic fertilizers N, P, K, and NPK on the bacterial community composition and potential functions in grassland soils in a 54-year experiment. Differences in total and available nutrients were found in the treatment soils; interestingly, Al, As, Mg, and Mn contents were variable in N, P, K, and NPK treatments. Bacterial community compositions shifted and Actinobacteria were overrepresented under the four fertilization treatments compared to the control. Redundancy analysis of the soil parameters and the bacterial community profiles showed that Mg, total N, Cd, and Al were linked to community variation. Using correlation analysis, Acidobacteria, Bacteroidetes, and Verrucomicrobia were linked similarly to soil parameters, and Actinobacteria and Proteobacteria were linked separately to different suites of parameters. Surprisingly, we found no fertilizers effect on microbial functional profiles which supports functional redundancy as a mechanism for stabilization of functions during changes in microbial composition. We suggest that functional profiles are more resistant to environmental changes than community compositions in the grassland ecosystem.

Network topology reveals high connectance levels and few key microbial genera within soils

Microbes have a central role in soil global biogeochemical process, yet specific microbe-microbe relationships are largely unknown. Analytical approaches as network analysis may shed new lights in understanding of microbial ecology and environmental microbiology. We investigated the soil bacterial community interactions through cultivation-independent methods in several land uses common in two Brazilian biomes. Using correlation network analysis we identified bacterial genera that presented important microbial associations within the soil community. The associations revealed nonrandomly structured microbial communities and clusters of operational taxonomic units (OTUs) that reflected relevant bacterial relationships. Possible keystone genera were found in each soil. Irrespective of the biome or land use studied only a small portion of OTUs showed positive or negative interaction with other members of the soil bacterial community. The more interactive genera were also more abundant however, within those genera, the abundance was not related to taxon importance as measured by the Betweenness Centrality. Most of the soil bacterial genera were important to the overall connectance of the network, whereas only few genera play a key role as connectors, mainly belonged to phyla Proteobacteria and Actinobacteria. Finally it was observed that each land use presented a different set of keystone genera and that no keystone genus presented a generalized distribution. Taking into account that species interactions could be more important to soil processes than species richness and abundance, especially in complex ecosystems, this approach might represent a step forward in microbial ecology beyond the conventional studies of microbial richness and abundance.

Soil microbiome responses to the short-term effects of Amazonian deforestation.

Slash‐and‐burn clearing of forest typically results in increase in soil nutrient availability. However, the impact of these nutrients on the soil microbiome is not known. Using next generation sequencing of 16S rRNA gene and shotgun metagenomic DNA, we compared the structure and the potential functions of bacterial community in forest soils to deforested soils in the Amazon region and related the differences to soil chemical factors. Deforestation decreased soil organic matter content and factors linked to soil acidity and raised soil pH, base saturation and exchangeable bases. Concomitant to expected changes in soil chemical factors, we observed an increase in the alpha diversity of the bacterial microbiota and relative abundances of putative copiotrophic bacteria such as Actinomycetales and a decrease in the relative abundances of bacterial taxa such as Chlamydiae, Planctomycetes and Verrucomicrobia in the deforested soils. We did not observe an increase in genes related to microbial nutrient metabolism in deforested soils. However, we did observe changes in community functions such as increases in DNA repair, protein processing, modification, degradation and folding functions, and these functions might reflect adaptation to changes in soil characteristics due to forest clear‐cutting and burning. In addition, there were changes in the composition of the bacterial groups associated with metabolism‐related functions. Co‐occurrence microbial network analysis identified distinct phylogenetic patterns for forest and deforested soils and suggested relationships between Planctomycetes and aluminium content, and Actinobacteria and nitrogen sources in Amazon soils. The results support taxonomic and functional adaptations in the soil bacterial community following deforestation. We hypothesize that these microbial adaptations may serve as a buffer to drastic changes in soil fertility after slash‐and‐burning deforestation in the Amazon region.

Microbial secondary succession in a chronosequence of chalk grasslands

Although secondary succession has been studied extensively, we have little knowledge of the succession of soil-borne microbial communities. In this study, we therefore examined the structures of the microbial communities across two separate chronosequences of chalk grasslands in Limburg, the Netherlands, which are at different stages of secondary succession after being abandoned for between 17 and >66 years. Arable fields were also included in the investigation as non-abandoned references. Changes in the soil-borne microbial communities, as determined by phylogenetic microarray and quantitative PCR methodologies, were correlated with the prevailing environmental conditions related to vegetation and soil biochemistry. We observed clear patterns of microbial secondary succession related to soil age, pH and phosphate status, as exemplified by the overrepresentation of Verrucomicrobia, Acidobacteria, Gemmatimonadetes, and α-, δ- and ɛ-Proteobacteria at late successional stages. Moreover, effects of secondary succession versus changes in soil pH could be resolved, with pH significantly altering the trajectory of microbial succession.

Testing Potential Effects of Maize Expressing the Bacillus thuringiensis Cry1Ab Endotoxin (Bt Maize) on Mycorrhizal Fungal Communities via DNA- and RNA-Based Pyrosequencing and Molecular Fingerprinting

ABSTRACT The cultivation of genetically modified (GM) crops has increased significantly over the last decades. However, concerns have been raised that some GM traits may negatively affect beneficial soil biota, such as arbuscular mycorrhizal fungi (AMF), potentially leading to alterations in soil functioning. Here, we test two maize varieties expressing the Bacillus thuringiensis Cry1Ab endotoxin (Bt maize) for their effects on soil AM fungal communities. We target both fungal DNA and RNA, which is new for AM fungi, and we use two strategies as an inclusive and robust way of detecting community differences: (i) 454 pyrosequencing using general fungal rRNA gene-directed primers and (ii) terminal restriction fragment length polymorphism (T-RFLP) profiling using AM fungus-specific markers. Potential GM-induced effects were compared to the normal natural variation of AM fungal communities across 15 different agricultural fields. AM fungi were found to be abundant in the experiment, accounting for 8% and 21% of total recovered DNA- and RNA-derived fungal sequences, respectively, after 104 days of plant growth. RNA- and DNA-based sequence analyses yielded most of the same AM fungal lineages. Our research yielded three major conclusions. First, no consistent differences were detected between AM fungal communities associated with GM plants and non-GM plants. Second, temporal variation in AMF community composition (between two measured time points) was bigger than GM trait-induced variation. Third, natural variation of AMF communities across 15 agricultural fields in The Netherlands, as well as within-field temporal variation, was much higher than GM-induced variation. In conclusion, we found no indication that Bt maize cultivation poses a risk for AMF.