Eiko Kuramae

The bacteria that dominate and become enriched in the rhizosphere during continuous cropping are of increasing interest, as they can greatly adapt to the rhizosphere. However, there are still little knowledge about the general composition and function of these bacteria. In this study, we planted tomatoes in three different soils for three planting cycles and used both high-throughput sequencing and culture-dependent workflows. Despite significant differences in bacterial communities from the initial soils, we observed a similar succession in the rhizosphere bacterial community compositions. We identified certain bacteria that were gradually enriched and potentially beneficial, such as Rhizobium and Flavobacterium. However, some other potentially beneficial bacteria, such as Massilia and Lysobacter, were gradually depleted. Additionally, we found that predicted functions related to xenobiotic biodegradation, nutrient metabolism and antibiotic biosynthesis were enriched in different rhizosphere soils. Beijerinckia fluminensis GR2, which was gradually enriched in all tested soils, significantly inhibited the growth of Ralstonia solanacearum and protected the host from infection. Our study provides new insights into the assembly mechanism of gradually enriched bacteria and their role as plant-beneficial microbes that adapt well to the rhizosphere.

Periodic inoculations of soil-beneficial microbes can increase their populations, but they also act as recurring biotic disturbances on the native microbial community. Soil rare and abundant microorganisms disproportionally shape the community diversity and stability. Uncovering their dynamic responses to recurring biotic disturbances and the underlying driving factors helps improve our understanding of the inoculation effects. Here, we imposed temporally recurring biotic disturbances by inoculating soils with phosphate-solubilizing bacteria, nitrogen-fixing bacteria, and the combination of both, with the overall aim of studying the successive responses of bacterial and fungal subcommunities along a rarity index. Our results showed that, in both bacterial and fungal communities, the relatively rare taxa exhibited higher diversity than the abundant taxa, and the relative abundance of rare taxa increased with recurring disturbances. However, the responses of rare and abundant taxa to inoculations were different between bacteria and fungi and were related to time and inoculation frequency. The rarer bacteria and the more abundant fungi explained most of the effects of inoculations on the resident microbial community. About 20 percent of the microbes changed their rarity categories over time, and most of the changes and interactions occurred within the rarer taxa during the first 45 days. Modeling analyses and co-occurrence networks indicated that microbial interactions, soil biochemical factors, and inoculation time drove the shifts of subcommunities. In summary, relatively rare bacteria and relatively abundant fungi play major roles in understanding the impacts of recurring biotic disturbances, while the conditionality of microbial rarity is dependent on both biotic and abiotic factors.

Introducing probiotics to soil is a sustainable way to stimulate the production of plant metabolites. However, the soil-resident microbes may compromise the efficiency of probiotics. To date, it remains challenging to integrate the effects of probiotics on plant performance with soil microbiome changes. Using Cyclocarya paliurus (Batal.) Iljinsk as a model medicinal plant and two types of probiotic consortia combined with organic fertilizer at three levels (low: 0.5, medium: 1.0, and high: 1.5 kg·plant⁻¹), we examined the impacts of three fertilization regimes (O: organic fertilizer, OMF: O coupled with Bacillus megaterium and Pseudomonas fluorescens, OCB: O coupled with Azotobacter chroococcum and Azospirillum brasilense) on plant metabolites and nutrient stoichiometry after three-year applications and identified the key soil microbes relating to the accumulation of plant metabolites via generalized joint attribute model (GJAM) analysis. Our results indicated that the concentration of flavonoids reached 36.9 mg·g⁻¹ in OCB treatment at a low level, and 30.0 mg·g⁻¹ in OMF treatment at a medium level, both were significantly higher than that in O treatment (25.8 mg·g⁻¹ on average). Furthermore, the accumulations of metabolites were associated with plant nutrient acquisition and C: N: P stoichiometry. GJAM analysis showed that higher fertilizer levels restricted the influence of probiotic consortia on the variance of plant-soil-microbe system, with fewer differences observed between fertilizer types. Specific soil microbes were predicted as potential indicators that may assist or impede the effects of probiotics on plant metabolite production. The predictions were further tested in a comparative pot experiment, and the effects of common indicators in both pot and field experiments were consistently associated with probiotics’ addition. This study reveals that the effects of probiotics on plant metabolites are associated with fertilization regimes and soil-indigenous microbes. Identifying microbial indicators will help to understand the probiotics' effects and further improve plant productivity.

Cover crops are a potential pathway for ecological cultivation in agricultural systems. In tropical no-till agricultural systems, the maintenance of residues on the soil surface and the addition of nitrogen (N) benefit the growth and grain yield of cash crops as well as the chemical and physical properties of the soil. However, the effects of these management practices on the soil microbiota are largely unknown. Here, we evaluated the effects of the timing of N application as a pulse disturbance and the growth of different cover crop species before maize in rotation on soil properties, maize productivity, and soil bacterial and fungal community diversity and composition. N fertilizer was applied either on live cover crops (palisade grass or ruzigrass), on cover crop straw just before maize seeding or in the maize V₄ growth stage. Soils previously cultivated with palisade grass established similar microbial communities regardless of N application timing, with increases in total bacteria, total archaea, nutrients, and the C:N ratio. The soil microbial alpha diversity in treatments with palisade grass did not vary with N application timing, whereas the bacterial and fungal diversities in the treatments with ruzigrass decreased when N was applied to live ruzigrass or maize in the V₄ growth stage. We conclude that palisade grass is a more suitable cover crop than ruzigrass, as palisade grass enhanced soil microbial diversity and maize productivity regardless of N application timing. Ruzigrass could be used as an alternative to palisade grass when N is applied during the straw phase. However, considering the entire agricultural system (soil–plant–microbe), ruzigrass is not as efficient as palisade grass in tropical no-till cover crop–maize rotation systems. Palisade grass is a suitable cover crop alternative for enhancing maize productivity, soil chemical properties and nutrient cycling, regardless of the timing of N application. Additionally, this study demonstrates that a holistic approach is valuable for evaluating soil diversity and crop productivity in agricultural systems.

Nitrous oxide (N₂O) production in tropical soils cultivated with sugarcane is associated with ammonia-oxidizing bacteria (AOB) and fungal denitrifiers. However, the taxonomic identities and the community diversities, compositions, and structures of AOB and fungal denitrifiers in these soils are not known. Here, we examined the effects of applying different concentrations of an organic recycled residue (vinasse: regular non-concentrated or 5.8-fold concentrated) on the dynamics of AOB and fungal denitrifier community diversity and composition and greenhouse gas emissions during the sugarcane cycle in two different seasons, rainy and dry. DNA was extracted from soil samples collected at six timepoints to determine the dynamics of amoA-AOB and nirK-fungal community diversity and composition by amplicon sequencing with gene-specific primers. Bacterial and archaeal amoA, fungal and bacterial nirK, bacterial nirS and nosZ, total bacteria (16S rRNA) and total fungi (18S rRNA) were quantified by real-time PCR, and N₂O and CO₂ emissions were measured. The genes amoA-AOB and bacterial nirK clade II correlated with N₂O emissions, followed by fungal nirK. The application of inorganic nitrogen fertilizer combined with organic residue, regardless of concentration, did not affect the diversity and structure of the AOB and fungal denitrifier communities but increased their abundances and N₂O emissions. Nitrosospira sp. was the dominant AOB, while unclassified fungi were the dominant fungal denitrifiers. Furthermore, the community structures of AOB and fungal denitrifiers were affected by season, with dominance of uncultured Nitrosospira and unclassified fungi in the rainy season and the genera Nitrosospira and Chaetomium in the dry season. Nitrosospira, Chaetomium, Talaromyces purpureogenus, and Fusarium seemed to be the main genera governing N₂O production in the studied tropical soils. These results highlight the importance of deciphering the main players in N₂O production and demonstrate the impact of fertilization on soil microbial N functions.

Soil microbial communities are essential components of agroecological ecosystems that influence soil fertility, nutrient turnover, and plant productivity. Metagenomics data are increasingly easy to obtain, but studies of soil metagenomics face three key challenges: (1) accounting for soil physicochemical properties; (2) incorporating untreated controls; and (3) sharing data. Accounting for soil physicochemical properties is crucial for better understanding the changes in soil microbial community composition, mechanisms, and abundance. Untreated controls provide a good baseline to measure changes in soil microbial communities and separate treatment effects from random effects. Sharing data increases reproducibility and enables meta-analyses, which are important for investigating overall effects. To overcome these challenges, we suggest establishing standard guidelines for the design of experiments for studying soil metagenomics. Addressing these challenges will promote a better understanding of soil microbial community composition and function, which we can exploit to enhance soil quality, health, and fertility.

Chemolitho-autotrophic microorganisms like the nitrite-oxidizing Nitrobacter winogradskyi create an environment for heterotrophic microorganisms that profit from the production of organic compounds. It was hypothesized that the assembly of a community of heterotrophic microorganisms around N. winogradskyi depends on the ecosystem from which the heterotrophs are picked. To test this hypothesis, pure cultures of N. winogradskyi were grown in continuously nitrite-fed bioreactors in a mineral medium free of added organic carbon that had been inoculated with diluted sewage sludge or with a suspension from a grassland soil. Samples for chemical and 16S rRNA gene amplicon analyses were taken after each volume change in the bioreactor. At the end of the enrichment runs, samples for shotgun metagenomics were also collected. Already after two volume changes, the transformations in community structure became less dynamic. The enrichment of heterotrophs from both sewage and soil was highly stochastic and yielded different dominant genera in most of the enrichment runs that were independent of the origin of the inoculum. Hence, the hypothesis had to be refuted. Notwithstanding the large variation in taxonomic community structure among the enrichments, the functional compositions of the communities were statistically not different between soil- and sludge-based enrichments. IMPORTANCE In the process of aerobic nitrification, nitrite-oxidizing bacteria together with ammonia-oxidizing microorganisms convert mineral nitrogen from its most reduced appearance, i.e., ammonium, into its most oxidized form, i.e., nitrate. Because the form of mineral nitrogen has large environmental implications, nitrite-oxidizing bacteria such as Nitrobacter winogradskyi play a central role in the global biogeochemical nitrogen cycle. In addition to this central role, the autotrophic nitrite-oxidizing bacteria also play a fundamental role in the global carbon cycle. They form the basis of heterotrophic food webs, in which the assimilated carbon is recycled. Little is known about the heterotrophic microorganisms that participate in these food webs, let alone their assembly in different ecosystems. This study showed that the assembly of microbial food webs by N. winogradskyi was a highly stochastic process and independent of the origin of the heterotrophic microorganisms, but the functional characteristics of the different food webs were similar.

The increase in sequencing capacity has amplified the number of taxonomically unclassified sequences in most databases. The classification of such sequences demands phylogenetic tree construction and comparison to currently classified sequences, a process that demands the processing of large amounts of data and use of several different software. Here, we present PhyloFunDB, a pipeline for extracting, processing, and inferring phylogenetic trees from specific functional genes. The goal of our work is to decrease processing time and facilitate the grouping of sequences that can be used for improved taxonomic classification of functional gene datasets.

Soil microbiota plays fundamental roles in maintaining ecosystem functions and services, including biogeochemical processes and plant productivity. Despite the ubiquity of soil microorganisms from the topsoil to deeper layers, their vertical distribution and contribution to element cycling in subsoils remain poorly understood. Here, nine soil profiles (0 to 135 cm) were collected at the local scale (within 300 km) from two canonical paddy soil types (Fe-accumuli and Hapli stagnic anthrosols), representing redoximorphic and oxidative soil types, respectively. Variations with depth in edaphic characteristics and soil bacterial and diazotrophic community assemblies and their associations with element cycling were explored. The results revealed that nitrogen and iron status were the most distinguishing edaphic characteristics of the two soil types throughout the soil profile. The acidic Fe-accumuli stagnic anthrosols were characterized by lower concentrations of free iron oxides and total iron in topsoil and ammonia in deeper layers compared with the Hapli stagnic anthrosols. The bacterial and diazotrophic community assemblies were mainly shaped by soil depth, followed by soil type. Random forest analysis revealed that nitrogen and iron cycling were strongly correlated in Fe-accumuli stagnic anthrosol, whereas in Hapli soil, available sulfur was the most important variable predicting both nitrogen and iron cycling. The distinctive biogeochemical processes could be explained by the differences in enrichment of microbial taxa between the two soil types. The main discriminant clades were the iron-oxidizing denitrifier Rhodanobacter, Actinobacteria, and diazotrophic taxa (iron-reducing Geobacter, Nitrospirillum, and Burkholderia) in Fe-accumuli stagnic anthrosol and the sulfur-reducing diazotroph Desulfobacca in Hapli stagnic anthrosol. IMPORTANCE Rice paddy ecosystems support nearly half of the global population and harbor remarkably diverse microbiomes and functions in a variety of soil types. Diazotrophs provide significant bioavailable nitrogen in paddy soil, priming nitrogen transformation and other biogeochemical processes. This study provides a novel perspective on the vertical distribution of bacterial and diazotrophic communities in two hydragric anthrosols. Microbiome analysis revealed divergent biogeochemical processes in the two paddy soil types, with a dominance of nitrogen-iron cycling processes in Fe-accumuli stagnic anthrosol and sulfur-nitrogen-iron coupling in Hapli stagnic anthrosol. This study advances our understanding of the multiple significant roles played by soil microorganisms, especially diazotrophs, in biogeochemical element cycles, which have important ecological and biogeochemical ramifications.

BACKGROUND: The assembly of the rhizomicrobiome, i.e., the microbiome in the soil adhering to the root, is influenced by soil conditions. Here, we investigated the core rhizomicrobiome of a wild plant species transplanted to an identical soil type with small differences in chemical factors and the impact of these soil chemistry differences on the core microbiome after long-term cultivation. We sampled three natural reserve populations of wild rice (i.e., in situ) and three populations of transplanted in situ wild rice grown ex situ for more than 40 years to determine the core wild rice rhizomicrobiome.

RESULTS: Generalized joint attribute modeling (GJAM) identified a total of 44 amplicon sequence variants (ASVs) composing the core wild rice rhizomicrobiome, including 35 bacterial ASVs belonging to the phyla Actinobacteria, Chloroflexi, Firmicutes, and Nitrospirae and 9 fungal ASVs belonging to the phyla Ascomycota, Basidiomycota, and Rozellomycota. Nine core bacterial ASVs belonging to the genera Haliangium, Anaeromyxobacter, Bradyrhizobium, and Bacillus were more abundant in the rhizosphere of ex situ wild rice than in the rhizosphere of in situ wild rice. The main ecological functions of the core microbiome were nitrogen fixation, manganese oxidation, aerobic chemoheterotrophy, chemoheterotrophy, and iron respiration, suggesting roles of the core rhizomicrobiome in improving nutrient resource acquisition for rice growth. The function of the core rhizosphere bacterial community was significantly (p < 0.05) shaped by electrical conductivity, total nitrogen, and available phosphorus present in the soil adhering to the roots.

CONCLUSION: We discovered that nitrogen, manganese, iron, and carbon resource acquisition are potential functions of the core rhizomicrobiome of the wild rice Oryza rufipogon. Our findings suggest that further potential utilization of the core rhizomicrobiome should consider the effects of soil properties on the abundances of different genera. Video Abstract.

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 slashand-burning deforestation in the Amazon region.