Soil rotifers, a phylum of microscopic, multicellular animals, play a critical role in various important functions across global ecosystems. However, our understanding of the processes shaping the large-scale distribution of soil rotifers, particularly in relation to plants and environmental factors, remains limited. Through the analysis of 570 soil samples collected across China, we found that alpha-diversity of rotifers was significantly higher in rhizosphere soils than in bulk soils, and their beta-diversity also exhibited significant differences, highlighting the critical role of the rhizosphere effect in shaping rotifer community structures. The beta-diversity of rotifers in rhizosphere soils was primarily driven by geographical location, whereas rotifer communities in bulk soils showed weaker responses to environmental variables. Stochastic processes predominantly shaped the community assembly of rotifer communities in both rhizosphere and bulk soils. Our study provides new insights into the rotifer community assembly in agricultural ecosystems and suggests that plant hosts can have significant impacts on soil rotifer communities.

Soil tardigrades (water bears) are widespread microfauna and important members of soil food webs, yet their role in linking community attributes to ecosystem multifunctionality (EMF) remains poorly understood. Environmental heterogeneity across soil layers and resource shifts during stand development may jointly shape tardigrade communities and soil functioning. Here, we investigated tardigrade diversity, extraction- and counting-based absolute abundance, sequencing-based relative abundance, and their contribution to EMF across soil layers and stand ages in soils from subtropical plantations in China. Tardigrade communities were quantified using microscopic counting and high-throughput 18S rRNA amplicon sequencing. Tardigrade richness decreased significantly from the litter layer to deeper mineral soil layers, with the highest values observed in the litter layer, whereas stand age had no significant effect. Soil pH and nutrient availability were significant predictors of tardigrade richness and relative abundance, while soil water content was negatively associated with richness and abundance. Both absolute and relative abundances of tardigrades were positively correlated with soil available phosphorus (AP), acid phosphatase (ACP) and β-1,4-glucosidase (BG), indicating close associations with phosphorus availability and ecosystem functions related to carbon decomposition and phosphorus mineralization. The EMF declined with soil depth and was positively associated with tardigrade abundance. Structural equation models (SEMs) further identified tardigrade relative abundance as a key contributor to EMF. Together, these results suggest that soil layer and soil physicochemical properties shape tardigrade community attributes in subtropical forest soils, and highlight the potential contribution of soil tardigrades to EMF, particularly in carbon and phosphorus cycling.

Manual isolation of tardigrades for molecular analysis is time-consuming and limits large-scale biodiversity studies. To enhance the efficiency of soil tardigrade molecular research, we developed a membrane filtration method to enrich soil tardigrades and other microfauna (nematodes and rotifers) for DNA extraction and the subsequent sequencing analyses. Filtration accelerated the workflow and detected more tardigrade species than manual isolation, although all membrane extraction methods underestimate their relative abundance. Filtration with a 15 mu m membrane yielded the highest richness, while community differences were mainly driven by soil type rather than extraction method. Our results highlight the potential of filtration approaches in saving sample processing time and improving the detection of tardigrade species richness.

Nitrous oxide (N2O) is a potent greenhouse gas predominantly emitted from grazed pasture through denitrification, driven by soil oxygen (O-2) availability and urine-derived nitrogen (N). Pasture soils are vulnerable to compaction from animal treading, restricting gas diffusion and enhancing N2O emissions. Although subsoiling alleviates compaction, its impact on soil O-2 status and N2O emissions, particularly under high urine N load, remain poorly understood and rarely investigated. This in-situ field study (March-August 2023) evaluated the effect of subsoiling on soil moisture, O-2 content, relative gas diffusivity (D-p/D-o), functional gene abundance, N2O emissions, and pasture production. Treatments included non-subsoiling or subsoiling, each with or without synthetic ruminant urine (713 kg N ha(-1)). Subsoiling improved macroporosity, enhanced O-2 availability, increased D-p/D-o at 5, 10 and 20 cm depth (P < 0.001), and reduced moisture at 10 cm depth (P < 0.001). Subsoiling significantly reduced N2O emissions by 52% and 81% of non-subsoiled plots for non-urine and urine treatments, respectively (P < 0.05). D-p/D-o was strongly correlated with N2O fluxes during the first 15 days following urine application (R-2 = 0.59-0.87), suggesting its utility as a predictive indicator under high substrate availability. Molecular analysis showed reduced nirK gene abundance under subsoiling, with limited response for other denitrification genes. Subsoiling had no significant effect on pasture yield or N uptake. Overall, subsoiling mitigates N2O emissions by improving soil aeration and D-p/D-o while maintaining productivity, offering a promising strategy for sustainable N management in grazed pasture soils.

Soil net nitrogen (N) mineralization rate (Nmin) is closely linked to N availability in forests, while the combined impact of N and phosphorus (P) deposition on Nmin remains unclear in subtropical forests. In this study, the impact of N and P input on the dynamics of Nmin via laboratory incubation approach as well as the controlling factors along the soil depths (i.e., 0–20, 20–40, 40–60, 60–80 cm), were investigated based on a long-term N and P simulated field experiment in a subtropical forest. The addition of N significantly increased soil Nmin by 44

Soil microorganisms mediate critical ecosystem processes, including nutrient cycling and climate regulation. However, the extent to which their functional resilience and microbial food web dynamics respond uniformly to organic amendment across two land-use types remain poorly understood. In this study, we conducted a 30-day microcosm experiment to investigate how exogenous resources addition restructures the assembly of primary microbial functional groups (bacteria, fungi, and protists) in urine-amended soils under contrasting land-use regimes: intensively managed maize fields and natural woodlands. Results showed that straw addition consistently reduced both OTU richness and Shannon diversity of bacteria (by 8

Forest soils serve as vital terrestrial carbon sinks, in which microbial carbon use efficiency (CUE) plays a central role in regulating soil carbon storage. While anthropogenic deposition exerts profound influences on forest carbon cycling, the depth-dependent responses of microbial CUE to nitrogen (N) and phosphorus (P) deposition remain poorly understood. Here, we examined how soil microbial CUE responded to N and P inputs along a soil profile (0-80 cm) in a subtropical forest, using a long-term simulated N and P deposition experiment. Our results demonstrated a consistent increase in microbial CUE with depth across all treatments. Specifically, N addition significantly enhanced CUE by 27 % and 10 % at the depth of 40-60 cm and 60-80 cm, respectively, relative to the non-N group (which controlled for P variation). Furthermore, when compared directly to control, N addition increased CUE by 12.7 % and 13.5 % at these depths. In contrast, P addition suppressed CUE by 14 % and 16 % at the depth of 0-20 cm and 20-40 cm, respectively, compared to the non-P group (which controlled for N variation). Similarly, direct comparison with the control showed that P addition decreased CUE by 8.1 % and 10.9 % at these topsoil layers. Regression analyses revealed significant associations between microbial CUE and soil pH, dissolved organic C, dissolved organic N, and available P. Variance partitioning analysis further elucidated that microbial CUE was predominantly governed by the interplay of soil substrates (primarily carbon and nutrient availability) and microbial enzymatic activities in surface layers, and N input was a major environmental driver for CUE variation in deep soils. These findings uncover contrasting nutrient-depth interactions in regulating microbial CUE and underscore the pivotal role of subsoil C dynamics under escalating N deposition.

Heterotrophic nitrification is considered a potentially important N cycling process, contributing to nitrate production in terrestrial ecosystems. The specific impacts of land-use change on this process, and its underlying microbial communities, are not well characterised. Here we report on a study examining the contribution of heterotrophic nitrification to total nitrification in four land use soils (dairy pasture, sheep pasture, long-term cropland and pine forestry), using 15N isotope labelling ((15NH4)2SO4 and 15N-glycine) incubation, a nitrification inhibitor, and molecular techniques. The results showed that the pine forest soil had the highest contribution (>78%) of heterotrophic nitrification to total nitrification, despite having the lowest overall nitrification. The addition of glycine enhanced the contribution of heterotrophic nitrification in cropland compared to dairy and sheep pastures, with this value in cropland being approximately 1.9-fold higher. The contribution of heterotrophic nitrification to total nitrification was positively correlated with soil fungal abundance and the fungi to bacteria ratio, indicating that fungi might be the main contributor of heterotrophic nitrification in the studied soils. Known heterotrophic nitrifiers, Penicillium was a potential candidate to play a significant role in pine forest soil with a relative gene abundance of 9.7%, whereas Mortierella was potentially prevalent in the dairy pasture (12.7%), sheep pasture (19.4%) and cropland (14.0%) soils, suggesting that the composition of potential heterotrophic nitrifiers varied between pine forest and agricultural (pasture and cropland) soils. The filamentous Ascomycota fungus Sagenomella, not previously known to perform heterotrophic nitrification may be a potential heterotrophic nitrifier in the studied forest soil, which warrants further investigation. This research improves our comprehensive understanding of heterotrophic nitrification in different land use soils, important for understanding the nitrogen cycle in different terrestrial ecosystems.

Pasture soils are major sources of nitrous oxide (N2O), a potent greenhouse gas mainly produced through microbial denitrification. However, interactions between soil aeration status and microbial responses involved in regulating N2O emissions remain poorly understood. We investigated denitrifier gene expression together with N2O and dinitrogen (N2) emissions using repacked cores of three pasture soils with contrasting bulk densities incubated across ten matric potentials (−1 to −10 kPa). Peak N2O fluxes occurred at matric potentials of −1 to −2 kPa and were strongly related to relative gas diffusivity (Dp/Do) and water-filled pore space (WFPS). In contrast, the expression of nirS, norB, and nosZ was more closely associated with matric potential and volumetric water content than with Dp/Do and WFPS. However, denitrifier gene transcription alone did not explain the observed gaseous N emissions, indicating that microbial transcriptional responses and measured N fluxes were decoupled under changing soil aeration conditions. These findings suggest bulk soil aeration indices (e.g. Dp/Do or WFPS) do not fully represent the local conditions regulating microbial response, whereas measured gaseous N emissions are additionally constrained by soil physical processes governing gas transport. Overall, these results highlight the importance of integrating microbial responses with soil physical processes to improve our mechanistic understanding of N2O emissions from grazed pasture soils.

Land-use conversion exerts substantial effects on ecosystem functioning in coastal wetlands; however, its impacts on nitrogen (N)-related functional communities and associated biogeochemical processes remain unclear. In this study, we investigated the abundance and diversity of N-cycling functional communities and their environmental drivers following the conversion of coastal wetlands (CW) to tidal flats (TF), agricultural land (AL), and fallow land (FL) using a metagenomic approach. The results showed that the abundance of key functional genes involved in assimilatory nitrate reduction, denitrification, and nitrification declined significantly following the conversion of CW to AL or FL. Shannon diversity index of N-cycling related taxonomic groups also declined markedly in response to land-use conversion. Across all the land-use types, denitrification process was the predominant N transformation pathway, accounting for 50.9% to 52.7% of total N-cycling potential. Structural equation modeling further revealed that soil electrical conductivity was the primary environmental driver influencing the abundance and diversity of N-related functional genes, explaining 79.1% and 66.6% of the variation in functional genes and taxonomic groups, respectively. Collectively, these findings demonstrate that land-use conversion substantially alters the composition and diversity of N-cycling functional communities in wetland ecosystems, with important implications for wetland management and ecological restoration for sustaining soil multifunctionality.

The phyllosphere, the aboveground interface between plant leaves and their microbial residents, plays a vital yet underappreciated role in crop productivity. While root- and soil-associated microbiomes are well-studied, the ecological assembly and yield-related effects of host-mediated phyllosphere microbial communities remain largely understudied, particularly under field conditions. This study investigates the phyllosphere microbiomes of sorghum cultivars resistant and susceptible to nutrient deficiency, focusing on how host genotype mediates microbial community assembly, keystone enrichment, and yield outcomes. The β-diversity of phyllosphere microbiomes differs significantly between resistant and susceptible cultivars, with resistant lines also showing more modular co-occurrence networks enriched in keystone taxa. These cultivars supported a higher abundance of keystone beneficial specialists (KBS), predominantly affiliated with Bacteroidia and Bacilli, and their abundance was positively correlated with yield. In contrast, susceptible cultivars exhibited lower and more taxonomically dispersed KBS, with a negative correlation between KBS and yield. Structural equation modeling suggested that while soil properties consistently promoted yield across cultivars, the impact of KBS on yield was genotype-dependent. These findings reveal a host-driven microbial mechanism linking phyllosphere composition to yield performance and highlight KBS as potential targets for microbiome-informed breeding or foliar microbial applications to improve crop productivity in sustainable systems.

Protists are ubiquitous in soils and play vital roles in biogeochemical cycling in forest ecosystems. While their sensitivity to nitrogen (N) addition is well-documented, the responses to phosphorus (P) enrichment remain less understood. In this study, we examined the seasonal (i.e., spring and autumn) variations in six-year simulated N and P addition, including control, N, P and NP treatments, on the diversity and community composition of soil bacteria, fungi and protists in a subtropical forest. Results showed a significant increase in the alpha-diversity of soil protists with N or P addition across both seasons, while bacterial and fungal alpha-diversity remained unchanged compared to the control. Principal coordinate analysis revealed significant differences in microbial community structure across treatments and seasons. Consumer protists dominated the community (90% of total abundance), with their relative abundance showing seasonally consistently positive responses to both N and P additions. Phylum Cercozoa dominated the protistan community in both seasons, with its relative abundance showing a seasonally stable increase in the N treatment compared to the control. The relative abundances of the classes Sarcomonadea and Imbricatea were significantly higher in the samples with N addition. Random forest and structural equation modelling analyses identified soil nutrients (i.e., NH4+-N, available P, and total P) as significant drivers of protist community compositions in both seasons. These findings revealed seasonal sensitivity of soil protists to nutrient addition, indicating their divergent resource-use strategies and adaptive plasticity in response to environmental disturbance.

Microbial communities play a significant role in maintaining the health of Great Barrier Reef (GBR) ecosystems, however, the influence of sediment composition and other environmental factors such as temperature and wave regime on microbial communities are largely unknown. Here we show how sediment composition and exposure influences bacterial communities across the inner section of the GBR (Cleveland Bay, Halifax Bay and Dunk Island) between 2016 and 2018. Sediment traps were installed and routinely deployed ( every 3 months) at eight sites in the inshore GBR and analysed for water chemistry, sediment geochemistry and organic characteristics and associated bacterial communities. Results showed a significant variation in water turbidity, sediment collection rate and geochemistry across the trap sites. Bacterial communities also significantly varied along the inner GBR, with the shift in relative abundance of Actinobacteria, Acidobacteria, Planctomycete, Verrucomicrobia and Chloroflexi being the main cause of the bacterial community dynamics. The variation in spatial patterns of bacterial communities was highly correlated with water turbidity and the geochemical characteristics of associated sediments (e.g., K, Fe, Mn, Co, Al, Cr, Ca) collected across the marine trap sites. Our findings indicate that sediment composition and collection rate (and linked water turbidity) can change the spatial patterns of bacterial communities by creating environmental gradients along the inner section of the GBR.

Phosphorus (P) is an essential nutrient for plant and microbial growth. Phosphatases play a crucial role in catalyzing the hydrolysis of phosphate esters in organic compounds, thereby regulating the bioavailability of soil P pools. The aim was to identify the optimal concentration of phosphatase activator to increase soil P availability in four soil types. Microcosm experiments were conducted on black soil from Northeast China with the addition of different concentrations (0, 0.05, 0.1, 0.3, 0.4, and 0.6

Wetlands can be a significant source of N2O under current global climate change regime with the soil-water interface representing a biogeochemical hotspot for microbial activity. However, the role of soil-water interface in controlling N2O emissions remains poorly understood. We hypothesized that the millimeter-scale redox gradient across the soil-water interface generates corresponding distinct niche for N-cycling microorganisms that collectively regulate the production and consumption of N2O over the same spatial scale. The abundance, transcriptional activity and spatial organization of different N-cycling guilds across the soil-water interface were characterized in mesocosms from three different paddy soils with different N2O emissions. Results demonstrated millimeter-scale stratification of N-cycling microbial activity across the soil-water interface, and in particular within the first 10 mm of flooded soils. Ammonia-oxidizing microorganisms were only transcriptionally active in the top 4 mm, suggesting a previously underestimated contribution to N2O emissions from wetlands. Variation in N2O accumulation was observed across the soil-water interface, with the highest concentrations measured at either the soil-water interface or in the deeper anoxic layer of paddy soils. Despite this difference, N2O-reducing microorganisms exhibited high transcriptional activity at the soil-water interface in all soils, suggesting that there is a microbial-mediated sink for N2O across the soil-water interface that can reduce N2O produced from both oxic and anoxic layers. This work demonstrate an underappreciated and essential role of the microbial hot zones at soil-water interface in regulating N2O emissions from wetlands.

Plant types and soil depths are key drivers shaping soil microbial networks. Protists, the most diverse group of eukaryotes, act as keystone organisms in soil ecosystems, contributing to nutrient cycling, plant productivity, and soil health. However, how plant types and soil depths affect soil protist networks complexity in wetland ecosystems remains largely unexplored. Here, we collected soil samples from five dominant plant types and a bare tidal flat at three depths in a subtropical estuarine wetland in China. We applied high-throughput sequencing combined with ecological network and statistical analyses to investigate protist communities. Our results revealed that protist network complexity varied in both plant types and soil depths. Specifically, plant types had a stronger influence on network complexity than soil depths. To further explore the underlying mechanisms, we examined the correlation between environmental factors and protist community. Random forest analysis identified total nitrogen (TN) was the key predictor of protist community structure. Moreover, structural equation model (SEM) demonstrated that plant types affected protist network complexity through their influence on soil properties, protist diversity and community structure. Together, these findings enhanced our understanding of the protists ecological roles and highlighted the importance of plant types in shaping microbial interactions in wetland ecosystem.

Nitrification plays a crucial role in glacial-degraded forelands as it influences nitrogen cycling and nutrient availability. This study investigated the community composition and microbial interaction patterns of comammox bacteria, as well as canonical ammonia-oxidizing bacteria (AOB) and archaea (AOA), across glacialdegraded forelands. The results revealed that the majority of comammox bacterial sequences fell within Nitrospira clade B (84.44 %) through phylogenetic analysis. Moreover, a significant positive coexistence relationship was observed among the three ammonia-oxidizing microbial groups, with comammox bacteria occupying a central position within the ammonia-oxidizing microbial network of glacial forelands. These findings highlight the central role of comammox bacteria in glacial ammonia-oxidizing microbial networks, and advancing our understanding of microbial succession dynamics related to nitrogen cycling in alpine environments.

The contrasting response of AOA, AOB, and comammox Nitrospira amoA transcript abundance to temperature, moisture, and nitrogen was investigated using soil microcosms. The moisture, temperature, and nitrogen treatments were selected to represent conditions typically found in a New Zealand (NZ) dairy farm. AOB dominated all synthetic urine treated soils. Peak AOB amoA transcript abundance was positively correlated with estimated soil ammonia availability. While AOB gDNA abundance and nitrification rate trends were similar. AOA were strongly influenced by soil temperature. At 20°C, AOA amoA peak transcript abundance averaged over 1 order of magnitude higher than at 8°C. Within the AOA community a member of the Nitrosocosmicus clade was positively correlated with ammonium and estimated ammonia concentrations. The presence and relative increase of an AOA community member in a high nitrogen environment poses an interesting contrast to current scientific opinion in NZ. Comammox Nitrospira abundance showed no correlation with soil moisture. This suggests that previously found associations are more complex than originally thought. Further research is required to determine the drivers of comammox Nitrospira abundance in a high moisture environment. Overall, these results indicate that AOB are the main drivers of nitrification in New Zealand dairy farm soils.

Fertilization practices could exert significant influence on the diversity, interactions, and functions of soil microorganisms. However, little is known about how specific microbial groups and their interactions adapt or evolve in response to agricultural practices, especially long-term mineral fertilization. Here we explored the community assembly process shaping the microbial community and co-occurrence networks of abundant and rare groups based on a high-throughput sequencing approach in a field experiment with 40 years of mineral nitrogen (N) and phosphorus (P) fertilization. The results indicated that fertilization (25-51 %) had a strong impact on microbial community structure, while little difference were found between rhizosphere and bulk soils irrespective of abundant and rare microbial groups. Deterministic processes primarily govern the assembly of both abundant and rare bacterial and fungal taxa. Random forest analysis revealed that soil pH and N-related nutrients (i.e. nitrate nitrogen (NO3--N), dissolved organic nitrogen (DON) and ammonium nitrogen (NH4+-N)) were the key factors influencing microbial community structure. Structural equation modeling and mantel test further indicated that deterministic factors, particularly soil pH, influence co-occurrence network complexity by modulating the microbiome. Overall, these findings provide insights into factors shaping the microbial community assembly and co-occurrence network dynamics in agroecosystems subjected to long-term fertilization.

Subsoiling is a common practice for improving soil structure and has recently been recognised for its potential to reduce nitrous oxide (N2O) emissions. However, its impact on nitrate (NO3--N) leaching must also be considered if it is to be used as a mitigation strategy. This study investigated N2O emissions, NO3--N leaching and grass yield in an Italian ryegrass pasture soil with a compacted subsoil layer using a lysimeter study. Additionally, a separate field study examined mineral nitrogen (N) concentration and the abundance of nitrifying and denitrifying genes. Both studies have four treatments, with non-subsoiling (NS), subsoiling (SS), non-subsoiling and urine (NSU), subsoiling and urine (SSU), and the urine treatments were applied at a rate of 700 kg N ha(-1). The results showed that 6 months after subsoiling, soil macroporosity in all treatments remained elevated at a depth of 0-20 cm (p < 0.01) compared with all non-subsoiling treatments. This created more aerobic conditions, which suppressed N2O emissions from denitrification by 20.3% (p < 0.05) during 190 days after urine application (NSU vs. SSU). Additionally, subsoiling had no significant effect on NO3--N leaching in the presence of winter-active Italian ryegrass. Molecular analysis of N-cycling microbial communities revealed that subsoiling had no effect on the abundance of amoA gene-carrying microorganisms involved in nitrification but reduced the abundance of nirS denitrifier genes, indicating that subsoiling primarily affected the denitrification process. These findings suggest that subsoiling can effectively reduce N2O emissions without increasing NO3--N leaching when combined with winter-active ryegrass.