As key components for sustaining terrestrial ecosystem functions, soil bacterial communities make it vital to clarify their variation patterns and driving mechanisms at regional and continental scales. Here, 16S rRNA gene sequencing was selected to analyze soil bacterial communities in 162 Chinese natural forest plots across different eco-geographical divisions. At the regional/continent scale, variations in bacterial community spatial patterns were revealed by analyzing the shaping of community composition by environmental heterogeneity. From the taxonomic perspective, niche differentiation of dominant phyla driven by environmental changes was also explored. Results showed that distinct climatic zones and arid-humid regions select for unique genera. At the regional scale, solar radiation and above-ground biomass were the primary drivers of community composition in the cold temperate zone; pH, soil organic carbon, and total phosphorus acted as regulatory factors in the temperate zone; while mean annual air temperature and mean annual ground temperature emerged as dominant drivers in the subtropical zone; mean annual air temperature, mean annual ground temperature, sand content, soil water content, and above-ground biomass collectively shaped the composition in humid regions, whereas solar radiation was the main driver in arid/semi-arid regions. At the continental scale, pH primarily affects the community composition along the north-south gradient, while soil water content and above-ground biomass drove that along the west-east gradient. Moreover, dominant bacterial phyla respond to environmental factors in complex ways at the regional scale; but environmental filtering shapes their continental distribution, which may in turn affect the spatial distribution of entire bacterial communities continentally. Overall, our study clarifies the scale-dependent and taxa-dependent responses of soil bacterial communities in Chinese natural forests to environmental factors, which not only establishes a foundation for predicting the evolutionary trends of forest soil microecosystems under future climate change but also provides references for formulating adaptive forest management plans based on ecological process mechanisms.

Plants can enhance their resistance to pathogens by regulating their associated microbial communities, which contributes to the ecological adaptability and potential application of afforestation species. However, microbial interactions in the rhizosphere and roots, and their implications for pathogen suppression in afforestation systems, remain unclear. In this study, we investigated the structure and potential functions of bacterial and fungal communities in roots, rhizosphere soil, and bulk soil of major afforestation species in northeastern China, Birch, Larch, and Poplar, using amplicon sequencing, with a particular focus on associations among functional microbial groups. In rhizosphere soils, the relative abundance of ectomycorrhizal (EcM) fungi was negatively correlated with that of pathogenic fungi in Birch and Larch forests. In roots, the relative abundance of saprotrophic fungi was negatively correlated with that of pathogenic fungi across all tree species. In contrast, major bacterial genera showed no consistent associations with pathogen abundance across compartments. Additionally, Larch forests showed a greater influx of bacterial and fungal taxa from the rhizosphere into the roots, yet root pathogen abundance remained comparatively low. These results suggest that negative associations between pathogenic fungi and EcM fungi in the rhizosphere, together with compartment specific microbial filtering and negative associations with saprotrophic fungi within roots, may contribute to reduced abundance of pathogenic fungi particularly in Larch. These results highlight the potential role of host-associated microbial community structure in mediating pathogen pressure and enhancing the ecological adaptability of afforestation species.

Soil bacterial communities exhibit biogeographic patterns along environmental gradients, yet why some environmental factors contribute more strongly to community turnover than others remains poorly understood. Here, we tested whether this variation can be explained by the phylogenetic depth at which bacterial responses to each environmental factor are conserved. Across 40 forest sites in Japan spanning multiple soil and climatic gradients, environmental factors whose bacterial responses were conserved at deeper phylogenetic levels contributed more strongly to bacterial community turnover. We further asked whether phylogenetic clades that share similar environmental responses represent ecologically meaningful units for understanding bacterial community responses. Using soil pH as a focal test case, we found that response-defined clades improved prediction of taxon-level abundance shifts and community-level compositional shifts compared with models that treated taxa as independent units. Together, these findings show that the phylogenetic depth of bacterial environmental responses links trait conservation, community turnover and soil bacterial biogeographic patterns.

Natural restoration is an effective approach for restoring degraded ecosystems, yet the successional patterns and assembly mechanisms of aboveground (litter layer) and belowground (topsoil) microbial communities remain poorly understood. We applied the niche conservatism framework to investigate niche partitioning, successional patterns and community assembly processes of microbial communities in the litter and topsoil layers during long-term vegetation restoration in southwestern China. The results showed that, during vegetation succession, the potential source communities of microbial communities in the litter layer gradually shifted from being dominated by the topsoil to being dominated by the litter. Fungal communities had a significantly higher proportion of external immigrants (> 80%) than bacteria (> 40%) and archaea (< 20%). During succession, bacterial and fungal communities in the litter and topsoil layers underwent niche differentiation, displaying a divergent succession pattern, while archaeal communities showed niche overlap, following a convergent pattern driven by stochastic processes. Additionally, the dispersal rate (m) and β-diversity turnover rate (slope) of bacterial and fungal species in the litter were significantly lower than in the topsoil, with community assembly being more influenced by deterministic processes in the litter. This study reveals that higher habitat specialisation in the litter imposes stronger filtering effects on the colonisation of most microbial groups, particularly fungal communities, highlighting the role of strategy differentiation in shaping microbial communities.

Ammonium release (ammonification) and uptake (immobilization) by soil microbial communities are fundamental processes of forest nitrogen (N) cycling, representing major N fluxes that influence plant productivity and ecosystem N retention. However, because these processes involve diverse metabolic pathways distributed across many taxa, they are difficult to evaluate using gene- or taxon-specific approaches, and it remains unclear how microbial community structure governs the patterns of these processes. In this study, we examined how the abundance, taxonomic composition, richness, and metabolic capabilities of microbial communities regulate ammonium-related N cycling processes across a wide range of forests in Japan, using rRNA gene sequencing and quantification, shotgun metagenomics, and ¹⁵N tracer assays. Across the full gradients of soil pH and N content, microbial abundance was primarily correlated with the absolute rates of N cycling processes, while taxonomic composition and richness were more strongly correlated with N allocation-that is, the balance among ammonium release, ammonium uptake, and subsequent nitrification. Soils with higher pH supported taxonomic compositions linked to enhanced ammonium release and nitrification, whereas lower-pH soils hosted compositions associated with greater ammonium uptake and retention. Notably, the regulatory influence of taxonomic composition on N allocation was pronounced within the higher-pH range but diminished within the lower-pH range. Despite this environmental dependency, N allocation by soil microbial communities was ultimately constrained by their overall metabolic capabilities. In higher-pH soils, microbial communities were enriched in metabolic functions related to nutrient acquisition and respiratory N transformations, supporting increased ammonium release and N mobility. By contrast, microbial communities in lower-pH soils were enriched in stress-adaptive functions, which promoted ammonium retention and limited N transformations-thereby diminishing the regulatory influence in N cycling. Together, our findings provide a mechanistic understanding of how microbial community structure and metabolic capabilities regulate ammonium-related N cycling processes across forests under varying environmental conditions.

There is growing awareness of the need for regenerative practices in the fight against biodiversity loss and climate change. Yet, we lack a mechanistic understanding of how microbial community composition and functioning are likely to change alongside transition from high-density tillage to large-scale vegetation restoration. Here, we investigated the functional dynamics of microbial communities following a complete vegetation successional chronosequence in a subtropical zone, Southwestern China, using shotgun metagenomics approaches. The contents of total soil phosphorus (P), available P, litter P, and microbial biomass P decreased significantly during vegetation succession, indicating that P is the most critical limiting nutrient. The abundance of genes related to P-uptake and transport, inorganic P-solubilization, organic P-mineralization, and P-starvation response regulation significantly increased with successional time, indicating an increased microbial "mining" for P under P limitation. Multi-analysis demonstrated microbial P limitation strongly inhibits carbon (C) catabolism potential, resulting in a significant decrease in carbohydrate-active enzyme family gene abundances. Nevertheless, over successional time, microorganisms increased investment in genes involved in degradation-resistant compounds (lignin and its aromatic compounds) to acquire P resources in the litter. Our study provides functional gene-level insights into how P limitation during vegetation succession in subtropical regions inhibits soil microbial C metabolic processes, thereby advancing our understanding of belowground C cycling and microbial metabolic feedback during forest restoration.

Background:The composition of the human gut microbiome varies tremendously among individuals, making the effects of dietary or treatment interventions difficult to detect and characterize. The consumption of fiber is important for gut health, yet the specific effects of increased fiber intake on the gut microbiome vary across studies. The variation in study outcomes might be due to inter-individual (or inter-population) variation or to the details of the interventions including the types of fiber, length of study, size of cohort, and molecular approaches. Thus, to identify consistent fiber-induced responses in the gut microbiome of healthy individuals, we re-analyzed 16S rRNA sequencing data from 21 dietary fiber interventions from 12 human studies, which included 2564 fecal samples from 538 subjects across all interventions. Results:Short-term increases in dietary fiber consumption resulted in highly consistent gut microbiome responses across studies. Increased fiber consumption explained an average of 1.5% of compositional variation (versus 82% of variation attributed to the individual), reduced alpha diversity, and resulted in phylogenetically conserved responses in relative abundances among bacterial taxa. Additionally, we identified bacterial clades, at approximately the genus level, that were highly consistent in their response (increasing or decreasing in their relative abundance) to dietary fiber interventions across the studies. Conclusions:Our study is an example of the power of synthesizing and reanalyzing microbiome data from many intervention studies. Despite high inter-individual variation of the composition of the human gut microbiome, dietary fiber interventions cause a consistent response both in the degree of change as well as the particular taxa that respond to increased fiber.

Soil burial processes create large pools of soil organic carbon that have been sequestered in the deep for long periods of time. This buried organic carbon is generally thought to be stable and resistant to decomposition, consisting of the remains of past plants and soil microbes. As a result, there is a lack of research on the role of microbiomes in the carbon transformation processes that may occur in the deep buried soil layers. In this study, we characterized the microbiomes and their metabolic potentials in organic soil layers buried following the volcanic eruption approximately 300 and 1,640 years ago, using bacterial and archaeal 16S rRNA gene sequencing. Both buried layers had comparable or even larger carbon and nitrogen contents and a microbial population as abundant and diverse as that of the surface layer. However, the organic soil layer buried 1,640 years ago showed a distinct microbiome from the surface layer, with a higher proportion of chemolithotrophic taxa, such as methanogens and NH3-oxidizing archaea. Additionally, this buried layer had a higher metabolic potential for fixing CO2 and synthesizing organic matter, while the surface community had a higher potential for degrading organic matter. These findings suggest that the buried organic soil in the deep may not simply be an accumulation of past organisms, but rather an active carbon sink driven by a chemolithotrophic microbiome that differs from the organotrophic microbiome of the surface soil.

Dryland ecosystems experience seasonal cycles of severe drought and moderate precipitation. Desert plants may develop symbiotic relationships with root endophytic microbes to survive under the repeated wet and extremely dry conditions. Although community coalescence has been found in many systems, the colonization by functional microbes and its relationship to seasonal transitions in arid regions are not well understood. Here we examined root endophytic microbial taxa, and their traits in relation to their root colonization, during the dry and wet seasons in a hot desert of the southwestern United States. We used high-throughput DNA sequencing of 16S rRNA and internal transcribed spacer gene profiling of five desert shrubs, and analyzed the seasonal change in endophytic microbial lineages. Goodness of fit to the neutral community model in relationship to microbial traits was evaluated. In summer, Actinobacteria and Bacteroidia increased, although this was not genus-specific. For fungi, Glomeraceae selectively increased in summer. In winter, Gram-negative bacterial genera, including those capable of nitrogen fixation and plant growth promotion, increased. Neutral model analysis revealed a strong stochastic influence on endophytic bacteria but a weak effect for fungi, especially in summer. The taxa with higher frequency than that predicted by neutral model shared environmental adaptability and symbiotic traits, whereas the frequency of pathogenic fungi was at or under the predicted value. These results suggest that community assembly of bacteria and fungi is regulated differently. The bacterial community was affected by stochastic and deterministic processes via bacterial response to drought (response trait), beneficial effect on plants (effect trait), and likely stable mutualistic interactions with plants suggested by the frequency of nodule bacteria. For fungi, mycorrhizal fungi were selected by plants in summer. The regulation of beneficial microbes by plants in both dry and wet seasons suggests the presence of plant-soil positive feedback in this natural desert ecosystem.

Mega-fires of unprecedented size, intensity, and socio-economic impacts have surged globally due to climate change, fire suppression, and development. Soil microbiomes are critical for post-fire plant regeneration and nutrient cycling, yet how mega-fires impact the soil microbiome remains unclear. We had a serendipitous opportunity to obtain pre- and post-fire soils from the same sampling locations because the 2016 Soberanes Fire, a mega-fire burning >500 Km2, burned with high severity throughout several of our established redwood-tanoak plots. This makes our study the first to examine microbial fire response in redwood-tanoak forests. We re-sampled soils immediately post-fire from two burned plots and one unburned plot to elucidate the effect of mega-fire on soil microbiomes. We used Illumina MiSeq sequencing of 16S and ITS1 to determine that both bacterial and fungal richness were reduced by 38-70% in burned plots, with richness unchanged in the unburned plot. Fire altered composition by 27% for bacteria and 24% for fungi, whereas the unburned plots experienced no change in fungal and negligible change in bacterial composition. We observed several pyrophilous taxa previously observed in Pinaceae forests, indicating that these microbes are likely general fire-responders across forest types. Further, the pyrophilous taxa that positively responded to fire were phylogenetically conserved, suggesting shared evolutionary traits. For bacteria, fire selected for increased Firmicutes and Actinobacteria. For fungi, fire selected for the Ascomycota classes Pezizomycetes and Eurotiomycetes and for a Basidiomycota class of heat-resistant Geminibasidiomycete yeasts. We hypothesize that microbes share analogous fire response to plants and propose a trait-based conceptual model of microbial response to fire that builds from Grime’s Competitor-Stress tolerator-Ruderal framework (C-S-R) and its recent applications to microbes. Using this framework and established literature on several microbial species, we hypothesize some generalizable principals to predict which microbial taxa will respond to fire.

A better understanding of how nitrogen (N) cycling genes are involved in ecological processes is one of the crucial areas of microbial ecology. Currently, most molecular biological techniques investigating N cycling genes in the environment heavily rely on the accuracy of the polymerase chain reaction (PCR) primers; however, their specificity and coverage have not been comprehensively evaluated. Here, we collected a sequence database, NcycFunGen, aimed for primer evaluation and redesign. NcycFunGen was based on hidden Markov model profiles for 22 marker genes involved in N cycling, which included 607,359 paired nucleotide and protein sequences with their taxonomic information. Then, a total of 608 published primers were fully evaluated through NcycFunGen, as well as against full‐length sequences collected from KEGG. The new primers were designed by DegePrime. In the experiment, the updated ureC gene primer pair ureC607F/ureC898R and nifH gene primer pair nifH107F64/nifH379R64 was applied to a urea amendment site using droplet digital PCR and high‐throughput amplicon sequencing. The results showed that the majority of primer pairs cover less than 30% sequences of target genes and that 22.55% were inappropriate for quantitative PCR and amplicon sequencing (<100 bp or >550 bp). In general, this in‐silico evaluation demonstrated that although many primers have been adopted in published studies, some of them should be validated and updated as needed according to the updated gene database. Therefore, new degenerate primer pairs for ureC targeting urease, bacterial and archaeal amoA targeting ammonium monooxygenase, and nifH targeting nitrogenase were designed through NcycFunGen. These new primer pairs showed higher coverage and amplification efficiency, as well as amplicon lengths that were applicable for high‐throughput amplicon sequencing. Furthermore, the experimental results displayed better characteristics than commonly used published ureC and nifH gene primer pairs. In conclusion, primer evaluation and redesign are highly recommended to improve the accuracy of primers targeting N cycling genes, which could facilitate amplicon‐based N cycling studies in various environments. The bioinformatics framework developed in this study can also be applied to build functional gene databases for other biogeochemical pathways.

The soil microbial community actively drives biogeochemical cycling even in the plant-dormant season of winter in temperate forests. The northern ecosystems are experiencing considerable winter climate change, which causes the snowpack to become thinner and the soil freeze-thaw cycles to occur more frequently in winter. These climatic and edaphic changes may affect the microbial community function. This study aimed to characterize the soil microbial community's response to winter climate change and its consequences in nitrogen (N) cycling. We conducted a large-scale snow removal experiment in a cool-temperate forest in northern Japan to simulate a winter climate change and assessed the abundance of total bacteria and fungi and ammonia oxidizers and the bacterial community composition throughout a year. This experiment indicated that snowpack decline prolonged the soil freeze-thaw period, which increased the carbon (C) availability to soil microbes in winter. The soil microbial community then sensitively responded by increasing in abundance and shifting the composition based on each taxon's absolute and relative abundances in the way that was phylogenetically patterned, which further activated microbial N cycling. However, the soil microbial community's high resilience driven by the C availability prevented the functional and compositional responses to winter climate change from persisting into the plant-growing season, which left no apparent cascading effect on the soil microbial community and N content during the plant-growing season. This study highlights the sensitive and phylogenetically patterned response of the soil microbial community to the change in soil nutrient availability and its high resilience under winter climate change in a forest.

Nitrous oxide (N2O), an ozone-depleting greenhouse gas, is generally produced by soil microbes, particularly NH3 oxidizers and denitrifiers, and emitted in large quantities after N fertilizer application in croplands. N2O can be produced via multiple processes, and reduced, with the involvement of more diverse microbes with different physiological constraints than previously thought; therefore, there is a lack of consensus on the production processes and microbes involved under different agricultural practices. In this study, multiple approaches were applied, including N2O isotopocule analyses, microbial gene transcript measurements, and selective inhibition assays, to revisit the involvement of NH3 oxidizers and denitrifiers, including the previously-overlooked taxa, in N2O emission from a cropland, and address the biological and environmental factors controlling the N2O production processes. Then, we synthesized the results from those approaches and revealed that the overlooked denitrifying bacteria and fungi were more involved in N2O production than the long-studied ones. We also demonstrated that the N2O production processes and soil microbes involved were different based on fertilization practices (plowing or surface application) and fertilization types (manure or urea). In particular, we identified the following intensified activities: (1) N2O production by overlooked denitrifying fungi after manure fertilization onto soil surface; (2) N2O production by overlooked denitrifying bacteria and N2O reduction by long-studied N2O-reducing bacteria after manure fertilization into the plowed layer; and (3) N2O production by NH3-oxidizing bacteria and overlooked denitrifying bacteria and fungi when urea fertilization was applied into the plowed layer. We finally propose the conceptual scheme of N flow after fertilization based on distinct physiological constraints among the diverse NH3 oxidizers and denitrifiers, which will help us understand the environmental context-dependent N2O emission processes.

Nitrous oxide (N 2 O) is an important greenhouse gas and an ozone-depleting substance. Due to the long persistence of N 2 O in the atmosphere, the mitigation of anthropogenic N 2 O emissions, which are mainly derived from microbial N 2 O-producing processes, including nitrification and denitrification by bacteria, archaea, and fungi, in agricultural soils, is urgently necessary. Members of mesofauna affect microbial processes by consuming microbial biomass in soil. However, how microbial consumption affects N 2 O emissions is largely unknown. Here, we report the significant role of fungivorous mites, the major mesofaunal group in agricultural soils, in regulating N 2 O production by fungi, and the results can be applied to the mitigation of N 2 O emissions. We found that the application of coconut husks, which is the low-value part of coconut and is commonly employed as a soil conditioner in agriculture, to soil can supply a favorable habitat for fungivorous mites due to its porous structure and thereby increase the mite abundance in agricultural fields. Because mites rapidly consume fungal N 2 O producers in soil, the increase in mite abundance substantially decreases the N 2 O emissions from soil. Our findings might provide new insight into the mechanisms of soil N 2 O emissions and broaden the options for the mitigation of N 2 O emissions.

Purpose As a potential tool for the biodegradation of nitrogen contaminants, including nitrate, nitrite, and ammonium, in pickled foods with high salinity, the halophilic and denitrifying archaeal strain Halomicrobium sp. ZPS1 was isolated from edible salt particles. Methods Under anaerobic and static culture conditions, Halomicrobium sp. ZPS1 could simultaneously degrade nitrate, nitrite, and ammonium in liquid medium with 18% salinity and generate N2O. To gain insight into these physiological characteristics, the complete genome of Halomicrobium sp. ZPS1 was sequenced to reveal the mechanism of nitrogen metabolism associated with salt-tolerance. Result The complete genome sequencing revealed a genome size of 3,094,203 bp with a circular chromosome and a GC content of 65.64%. Based on gene annotation, 3191 CDSs, 6 rRNA genes, and 76 tRNA genes were identified. Moreover, 28 genes were annotated as related to salt tolerance, ammonium assimilation, and a truncated denitrification pathway. Conclusion The annotated functional genes indicate that Halomicrobium sp. ZPS1 could be a candidate strain for the simultaneous removal of nitrate, nitrite, and ammonia in extremely high salt environments.

Nitrification–denitrification processes in the nitrogen cycle have been extensively examined in rice paddy soils. Nitrate is generally depleted in the reduced soil layer below the thin oxidized layer at the surface, and this may be attributed to high denitrification activity. In the present study, we investigated dissimilatory nitrate reduction to ammonium (DNRA), which competes with denitrification for nitrate, in order to challenge the conventional view of nitrogen cycling in paddy soils. We performed paddy soil microcosm experiments using 15N tracer analyses to assess DNRA and denitrification rates and conducted clone library analyses of transcripts of nitrite reductase genes (nrfA, nirS, and nirK) in order to identify the microbial populations carrying out these processes. The results obtained showed that DNRA occurred to a similar extent to denitrification and appeared to be enhanced by a nitrate limitation relative to organic carbon. We also demonstrated that different microbial taxa were responsible for these distinct processes. Based on these results and previous field observations, nitrate produced by nitrification within the surface oxidized layer may be reduced not only to gaseous N2 via denitrification, but also to NH4+ via DNRA, within the reduced layer. The present results also indicate that DNRA reduces N loss through denitrification and nitrate leaching and provides ammonium to rice roots in rice paddy fields.

Novoherbaspirillum sp. strain UKPF54, a plant growth-promoting rhizobacterium with the ability to mitigate nitrous oxide emission from agriculture soils, has been successfully isolated from paddy soil in Kumamoto, Japan. Here, we report the whole-genome sequence of this strain.

Ammonium (NH4+) and nitrate (NO3–) concentrations and production rates in forest soil vary by hillslope position due to variation in ammonia-oxidizing microorganism concentrations, soil chemistry, and surface soil moisture. These spatial distributions have a significant effect on nutrient cycles and streamwater chemistry. Soil moisture conditions significantly restrict microbial activity, influencing the spatial distribution of NO3– concentrations on forest hillslopes. However, studies linking forest hydrological processes to nitrogen cycling are limited. Therefore, we investigated the determinants of spatial variation in soil moisture and evaluated the effects of soil moisture fluctuations on spatial variation in NO3– concentration and production rate. The study sites were the Fukuroyamasawa Experimental Watershed (FEW) and Oyasan Experimental Watershed (OEW) in Japan. The two have similar topographies, climates, and tree species. In each watershed, a 100 m transect was set up from the ridge to the base of the slope, and soil moisture sensors were installed at soil depths of 10 cm and 30 cm at both the top and bottom of the slope. We collected surface soil samples at a depth of 10 cm at the top, middle, and bottom of the slopes using 100 cm3 cores, and measured soil physical properties, particle size distribution, volcanic ash content, chemical properties (pH, NO3–, NH4+, nitrification rate, and mineralization rate), and microbial content (archaeal content). Spatial and temporal changes in soil moisture on the hillslope were calculated using HYDRUS-2D to examine contributing factors of soil moisture. At FEW, high NO3– concentrations and nitrification rates were observed only at the slope bottom and middle, and no NO3– concentrations were detected at up slope. By contrast, at OEW, high NO3– concentrations and nitrification rates were observed at all points. NH4+ concentrations were similar at all points in both watersheds. At FEW, 10 cm surface soil moisture fluctuated within 25–40% at the slope top but was within 40–50% at the slope bottom. At OEW, surface soil moisture was 30–40% at both the slope top and bottom, with no significant differences according to slope position. It was confirmed that soil moisture was significantly involved in NO3– concentration and nitrification rates. Model simulations showed that the difference in soil moisture fluctuations between FEW and OEW was mainly explained by the spatial variation in soil physical properties. In particular, volcanic ash influenced soil moisture along the entire slope at OEW, resulting in high water retention, but only influenced soil moisture at the slope bottom at FEW. These findings indicate that spatial variability in soil physical properties has a significant effect on soil moisture fluctuation and leads to a spatial distribution of NO3– production.

Recent studies demonstrated that phylogenetically more diverse and abundant bacteria and fungi than previously considered are responsible for denitrification in terrestrial environments. We herein examined the effects of land-use types on the community composition of those denitrifying microbes based on their nitrite reductase gene (nirK and nirS) sequences. These genes can be phylogenetically grouped into several clusters. We used cluster-specific PCR primers to amplify nirK and nirS belonging to each cluster because the most widely used primers only amplify genes belonging to a single cluster. We found that the dominant taxa as well as overall community composition of denitrifying bacteria and fungi, regardless of the cluster they belonged to, differed according to the land-use type. We also identified distinguishing taxa based on individual land-use types, the distribution of which has not previously been characterized, such as denitrifying bacteria or fungi dominant in forest soils, Rhodanobacter having nirK, Penicillium having nirK, and Bradyrhizobium having nirS. These results suggest that land-use management affects the ecological constraints and consequences of denitrification in terrestrial environments through the assembly of distinct communities of denitrifiers.