Soil Biology and Biochemistry

Climate change is reshaping soil microbial communities, yet the impact of warming in bacterial-fungal interactions (BFIs) remains underexplored. We investigated whether heatwave temperature influence BFIs and the mechanism supporting the interaction. Using co-culture experiments with two bacterial and two fungal strains isolated from heathland soil, we compared mono- and co-cultures final abundances under ambient (18{degrees}C) and heatwave (25{degrees}C) soil temperatures. Our results revealed strongly asymmetric interactions, where fungi benefited by around 5% from bacterial presence, while bacterial abundance was inhibited by around 68%, regardless of temperature. Analyses of pH confirmed that acidification by fungi was probably the main cause of this inhibition. Moreover, warming did not affect the strength or direction of these interactions, though it slightly increased fungal abundance. These findings provide direct experimental evidence that fungi can impact bacteria via acidification, and that the interaction is unaffected by temperature. Understanding these mechanisms is crucial for improving predictions of microbial community dynamics and ecosystem functioning in warming environments.

Soil harbours a wide diversity of microbes responsible for essential functions, such as depolymerizing the C and N in organic matter through the production of exoenzymes. Some of these exoenzymes are universal, whereas others are specific to certain microbes. We hypothesized that higher microbial alpha diversity is associated with greater depolymerization capacity, specifically for protein and cellulose depolymerization, which will result in more N being mineralized. We therefore diluted two soil microbial communities, one from a forest soil and one from an agricultural soil, to create a diversity gradient. After nine weeks, we transferred these communities to a synthetic soil in which microbial necromass was the only nitrogen source. Before the transfer and two weeks after, we quantified protease, deaminase and {beta}-glucosidase potential activity, characterized the bacterial and fungal communities, and measured the quantity of nitrogen mineralized. The dilution had very little effect on the processes measured, with no clear trend. For identical alpha diversity values, some communities had high process rates, while other not. It appeared that these communities varied widely, a side effect of the dilution approach, and that this variation was significantly linked to process rates. This shows that community composition (beta diversity) is more strongly related to enzymatic potential and mineralization than species richness (alpha diversity) following necromass addition. In conclusion, the relationship between diversity and depolymerization of microbial necromass is not simply a matter of a linear decrease along with diversity but is rather linked to how reduced diversity results in more stochastic microbial communities. Highlights- Community composition (beta diversity) influence more microbial necromass depolymerization than species richness - Abundance of specific microbes explained ammonification and nitrification processes - Mineralization rates is different between crop and forest soil

Nitrous oxide (N2O) is a potent greenhouse gas and is largely produced by incomplete denitrification. Although we know many of the microbial species that denitrify, we are still unable to reliably predict N2O production from soils. Recent work in microbial ecology has shown that when key microbes are considered as members of functional ensembles rather than isolated, the predictive power linking their activity to emergent properties increases dramatically. We hypothesized that the active microbial community during high N2O production would be taxonomically distinct from the inactive portion and increases in N2O production rates would correlate more strongly with increased abundance across multiple active taxa than with dominance by a single active species. We conducted a microcosm experiment where agricultural soil was incubated in anaerobic vials for up to 15 hours while tracking N2O production. Using bioorthogonal non-canonical amino acid tagging paired with fluorescence-activated cell sorting and 16S rRNA amplicon sequencing (BONCAT-FACS-Seq), we probed the active subset of the microbial community throughout the incubation period. Analysis of 16S rRNA gene amplicons revealed that the active and inactive fractions contained distinct taxa, and the taxonomic composition of the active fraction shifted over time. We found that less than 1% of the microbial community was responsible for N2O flux rates as high as 3.84 {micro}g N2O-N g dry soil-1 hr-1. The level of activity (median fluorescent intensity of active cells) correlated well with N2O production rates. The Ensemble Quotient Optimization for Microbiomes (mEQO) tool was used to identify an ensemble of eight organisms whose combined abundance best correlated with N2O fluxes. Overall, our results reveal that N2O fluxes are driven not by changes in a single taxon but by shifting ensembles of active microorganisms whose combined functional potential supports consistent emissions. This study applied a novel conceptual and methodological framework with a distinct focus on the active microbial community, rather than the entire community; if our observation that N2O flux rates are correlated with an ensemble of organisms is broadly confirmed, then framing denitrification as a community trait may increase predictability of this key process.

Viruses are abundant and ecologically important in soils, yet the persistence and production dynamics of extracellular virions remain poorly understood. We applied a genome-resolved stable isotope probing viromics (SIP-viromics) approach, combining H 18O labeling with viral metagenomics, to track virion turnover in seasonally dry grassland soils following rewetting. We identified 354 viral populations (vOTUs) using individual-sample and combined metagenome assemblies. Only 22% of vOTUs exhibited significant 18O enrichment, indicating active replication and new virion production during the 1-week incubation; the majority (78%) persisted without detectable replication, consistent with a viral seed bank. Active vOTUs accounted for 4.76-5.15% of total virions per gram of soil, with viral loads ranging from 3.15 x 1010 to 6.59 x 1010 virions per gram. Probabilistic and deterministic sensitivity analyses spanning viral DNA fraction and genome length reinforced that persistent virions represented the majority of the extracellular viral pool post-wet-up, regardless of parameter assumptions. Host predictions linked both active and persistent vOTUs primarily to Actinomycetota and Pseudomonadota--bacterial groups known to rapidly resuscitate following rewetting--suggesting that some viruses exhibit rapid turnover while others persist over longer timescales, forming a stable viral pool capable of reinitiating infections during favorable conditions. These results demonstrate that SIP-viromics can distinguish newly produced from persistent virions and reveal host-associated patterns of lytic infection and virion production. Our findings advance understanding of soil virus-host interactions and highlight the ecological role of persistent virions as a genetic reservoir contributing to microbial turnover and biogeochemical cycling following environmental disturbance. ImportanceUnderstanding the persistence and production dynamics of soil viruses is critical for elucidating their roles in microbial community dynamics and nutrient cycling, yet these processes have remained largely uncharacterized due to methodological limitations. By integrating stable isotope probing with viromics, this study provides a robust framework for directly distinguishing newly produced from persistent virions in situ. Unlike conventional viromics, which only catalogs viral diversity, SIP-viromics enables quantification of active viral replication and persistence under natural soil conditions. Our results demonstrate that most virions in a seasonally dry soil persisted through a rewetting event, with active replication limited to a minority of viral populations. Persistent virions were primarily linked to dominant bacterial groups, indicating that host ecophysiology and environmental stability strongly influence lytic infection. Collectively, these findings highlight viruses as long-term reservoirs of genetic material, capable of shaping microbial dynamics and ecosystem processes over time. This work establishes SIP-viromics as a powerful approach for studying virus-host interactions and their ecological significance in terrestrial environments.

Soil microbial diversity is shaped by the spatial scale at which communities are sampled, yet standard sampling practices often homogenize samples, obscuring fine-scale spatial structure and diversity patterns. To better understand how sampling effort, spatial extent, and physical homogenization influence plot-level microbial richness estimates, we sampled 57 forest and grassland sites across three regions in Germany using a 14-core cross-transect design and performed 16S rRNA gene metabarcoding. We simulated sampling efforts and a range of spatial extents and compared diversity estimates to those from physically homogenized composite samples. Plot-level richness increased continuously with sampling effort and spatial extent, with no evidence of saturation. However, when sequencing depth was held constant, sampling completeness declined with increasing sampling effort, meaning that more diversity is not captured. Composite samples substantially underestimated plot-level richness and altered apparent diversity relationships between ecosystems; individual cores identified forests as richer than grasslands, whereas homogenized samples suggested the opposite relationship. These results demonstrate that sampling effort, spatial extent, and homogenization fundamentally shape soil microbial diversity estimates. Homogenized composite samples cannot substitute for individual cores when the goal is to reliably quantify plot-level richness or compare diversity across ecosystems.

Global change drivers are reshaping agroecosystems and their sustained functions worldwide. While soil microorganisms underpin the resilience of these systems, the individual and interactive effects of multiple anthropogenic stressors on microbial community structure and function using large-scale field experiments remain poorly understood. Here, we utilize a full-factorial field experiment in a subtropical agroecosystem to investigate how land-use intensity, cattle grazing intensity, and altered precipitation regimes interact to shape soil microbiomes. Combining microbiome sequencing with network analyses and functional bioinformatics, we evaluated effects of these drivers on prokaryotic and fungal diversity, composition, predicted functional profiles, and community structure. Land-use intensity emerged as the primary driver of microbial responses, explaining 25% and 13% of the total variation in community composition for prokaryotes and fungi, respectively. Compared to intensively managed pastures, semi-natural pastures had significantly different community composition for prokaryotes and fungi and exhibited 22% higher fungal diversity. Semi-natural pastures were enriched with decomposer-associated taxa and metabolic pathways related to energy and lipid metabolism indicating enhanced microbial activity. Surprisingly, intensively managed pastures showed higher network modularity but lower network richness, suggesting a trade-off between community compartmentalization and complexity under intensive land management. Grazing and precipitation manipulations induced core microbiome changes within land-use intensities but had no impact on overall community structure and no significant interactions with land-use. Together, these findings suggest that long-term land-use legacies exert a persistent influence on soil microbial community structure, function, and organization, shaping the context within which other global change drivers operate in subtropical agroecosystems.

Microbial communities regulate carbon and nitrogen (N) cycling, yet their long-term responses to chronic global changes remain unclear. Using 12 years of grassland litter samples from the Loma Ridge Global Change Experiment in Irvine, California, we tested whether interactions between experimental drought and N deposition, and previously observed temporal variability are driven by background climatic conditions, including precipitation and temperature. Consistent with short-term studies, drought and N addition had relatively small effects on bacterial community composition compared to pronounced seasonal and interannual variability, with drought-by-year interactions explaining more variation than drought alone. Seasonal shifts were largely driven by short-term fluctuations in rainfall and temperature, whereas the substantial interannual variability in community composition was not captured by site-level climate metrics. Contrary to expectations, drought effects were influenced more by background temperature than precipitation, with the strongest effects observed in cooler years. Lastly, a bacterial taxons sensitivity to climate variability under ambient conditions did not predict its response to chronic drought. Together, our findings show that bacterial responses to drought are temporally dynamic and influenced by background temperature, underscoring the need for long-term longitudinal studies of soil microbial communities to better predict microbial responses under future global change. ImportanceMicrobial responses to global change, particularly drought and nitrogen addition, are often inferred from short-term studies (< 2 years), yet natural temporal variability may overshadow experimental effects. Using a 12-year dataset of grassland leaf litter communities, we show that temporal variability, both seasonal and interannual, exert a stronger influence on bacterial community composition than chronic drought or nitrogen deposition. These findings challenge assumptions about the magnitude of drought effects, particularly in naturally drought-affected ecosystem such as California grasslands and highlight the importance of long-term datasets for predicting microbial responses to climate change. By demonstrating that bacterial communities are strongly shaped by background climatic variability (baseline precipitation and temperature independent of imposed chronic treatments) and may be buffered to sustained drought, this work improves forecasts of ecosystem responses and informs the design of global change experiments and restoration strategies in future research studies.

Soil microbial communities have a variety of mechanisms to deal with emerging drought stress. One well-documented mechanism is increased microbial production of extracellular polymeric substances (EPS), which can potentially change the soil density and water holding capacity. Yet little is known about how microbial diversity influences the functional capacity of EPS formation and the resulting outcomes in water dynamics. To understand more about communal microbiome EPS production, we set up sterile mesocosms where we examined the effects of microbial diversity (high or low treatments) and nutrient input (supplement or deficient treatments) on these processes. To capture the microhydrology of the mesocosms, we measured water holding (WH), infiltration, evaporation, and soil properties we believe microbes are altering (EPS, soil aggregation). Our hypothesis stated that if diversity was artificially manipulated, then soil-water properties will be altered via production of EPS. We predicted that low diversity systems would have lower functional diversity, leading to less EPS production, moisture storage, and minimal changes from inert soil media. As predicted, we found that the high-diversity systems had a higher water retention and lower rates of water loss over time than low-diversity systems. This trend was magnified in the nutrient-supplemented treatment, suggesting that EPS production and subsequent water-holding traits are emergent features of the microbiome. Unexpectedly, we observed a correlation between the amount of water retained and the quantity of lipid EPS produced. This suggests that EPS composition, rather than quantity, is determinative of a biofilms function. In conclusion, it appears that microbial diversity influences soil properties that are important to moisture retention within these systems. To date, the role that microbes and their diversity play in soil hydrology has been severely understudied, so this work aims to build ecological understandings of these systems. These findings are valuable, for if we learn how microbes manipulate soil moisture, we can apply these functions to advance sustainable agricultural practices and enhance ecosystem resilience to water scarcity in arid regions. Open Research StatementUpon publication data, and code will be made available through Zenodo. Sequencing data will be uploaded to NCBI SRA.

Interactions between amoebae and bacteria are increasingly viewed as key drivers of zoonotic pathogen emergence in rodent-dwelling burrows, yet the environmental factors shaping these interactions remain poorly understood. Here, we analyzed soil characteristics and used absolute quantitative high-throughput sequencing to assess microbial communities in active burrow, inactive burrow, and off-burrow soils across four rodent species (marmot, squirrel, gerbil, and vole) in the Hulunbuir grassland of Inner Mongolia, China. This study demonstrates that rodent activity creates chemically distinct soil microhabitats, with nitrate (NO --N) enrichment in active burrow soils consistently observed across rodent species. Elevated soil NO3--N was associated with reduced microbial phylogenetic diversity and reorganization of amoeba-co-occurring bacterial assemblages. Both absolute abundance-based correlations and functional prediction of co-occurring bacteria indicated that amoebae were primarily associated with nitrogen-cycling bacteria in off-burrow soils. In burrow soils, amoebae increasingly interacted with bacterial taxa associated with pathogenicity while retaining ties to nitrogen-cycling taxa. Structural equation modeling and mediation analysis revealed that NO3--N enrichment indirectly linked to increased infectious disease-related functional potential by amoeba-associated bacterial restructuring and coordinated shifts in nitrogen cycling, independent of changes in bacterial abundance. Together, our findings highlight the importance of rodent-driven soil heterogeneity in shaping amoeba-bacteria interactions and suggest that rodent-mediated NO --N enrichment may promote the emergence and persistence of potentially pathogenic bacteria, with broader implications for soil ecosystem functioning and disease-related processes in terrestrial ecosystems.

Permafrost thaw exposes ancient organic matter to microbial degradation, which is predicted to release globally significant quantities of greenhouse gases into the atmosphere. Though microorganisms drive these processes, the relative importance of biotic (taxonomic and functional community composition) versus environmental (e.g., soil physicochemistry) drivers and their interactions are unknown. Using a novel in situ thaw experiment conducted at the Cold Regions Research and Engineering Laboratorys Permafrost Tunnel near Fairbanks, Alaska, we experimentally separated the effects of soil physicochemistry and microbial communities under "real-world" thaw conditions. To simulate thaw, active layer soil, Holocene permafrost (2 kya), and Pleistocene permafrost (40 kya) were sterilized, inoculated with microbial communities from the different soils, enclosed in 0.22 {micro}m membrane bags to prevent immigration, and buried in the active layer. We retrieved the bags after two weeks and two months of thaw and characterized microbial community structure (16S rRNA and ITS2 amplicon sequencing), functional potential (metagenome sequencing), and soil organic matter (OM) composition at the molecular level (FT-ICR MS). Soil had a stronger effect on bacterial community and gene assemblages than inoculum, and the effects of inoculum were stronger and longer-lasting on community structure than functional potential. Pleistocene permafrost initially contained approximately eleven times more dissolved organic carbon than the other soils, and was enriched in OM derived from microbial necromass and low molecular weight organic acids. This carbon was rapidly depleted during thaw and OM compositional characteristics became increasingly similar to active layer and Holocene permafrost, paralleling shifts in Pleistocene permafrost functional gene profiles and bacterial community structure towards those of other soils. Overall, this work provides new insights into the susceptibility of OM to microbial degradation in compositionally distinct permafrost soils, and ways in which Pleistocene Yedoma permafrost carbon is likely to be particularly vulnerable to permafrost thaw.

Soil protists shape microbial food webs and nutrient cycling, yet methods for measuring their population growth in soil have lagged behind the taxonomic resolution available from 18S rRNA gene sequencing. Microscopy-based approaches can estimate abundance and growth, but with limited taxonomic resolution. Here, we tested whether quantitative stable isotope probing (qSIP) can provide reproducible, sequencing-resolved growth measurements for soil protists. We incubated soil with natural-abundance or {superscript 1} O-labeled water and measured taxon-specific 18O enrichment in DNA using three common 18S rRNA gene primer sets. 18O enrichment values were positively correlated across datasets, with relationships closest to 1:1 after poorly resolved taxonomic assignments were excluded, indicating that qSIP provides reproducible population-level growth signals across marker choices. We then compared 18S amplicon profiles from unfractionated DNA with qSIP-derived growth measurements across a soil moisture gradient. Amplicon profiles showed small shifts in relative abundances of major protist groups, whereas qSIP revealed a large moisture response in the growing community: 7 ASVs were growing at 20% field capacity compared with 143 at 80% field capacity, representing 1.6% and 63.3% of total protist relative abundance, respectively. Average growth was <1% day{square}1 in the two driest treatments, increasing to 2.1% day{square}1 at 60% and 5.6% day{square}1 at 80% field capacity; among growing taxa, rates averaged 8.7% and 8.3% day{square}1 in the two wetter treatments. By pairing taxonomic resolution with isotope-based growth estimates, qSIP with 18O-H2O moves soil protist ecology toward quantitative population dynamics: identifying which taxa grow, how fast, and how growth responds to the environment.

Soil microbial inoculants have the potential to improve crop yield, enhance agricultural sustainability, and support soil restoration, but they often display unpredictable in-field performance across varied soil conditions. Cyanobacteria-dominated soil surface consortia (SSCs) offer a tractable model for studying inoculant-soil interactions because their visible surface growth enables direct observation and sampling after application. Here, we introduced the SSC "DG1," dominated by the diazotrophic cyanobacterium Nostoc linckia, into soil microcosms differing in resident microbiome diversity (low vs. high diversity) and urea fertilization history, (+urea vs. -urea). We used 16S rRNA gene sequencing and genome-resolved metatranscriptomics to assess inoculant establishment and functioning. Resident microbiome diversity did not affect total N. linckia gene expression, but heterotrophic DG1 members showed reduced expression in high-diversity soils. Soil diversity and urea history drove broad shifts in DG1 transcription and significantly affected transcription of key N. linckia carbon and nitrogen metabolism genes. High-diversity soils with urea were associated with increased transcription of photosynthesis, CAZyme, and nitrogen cycling genes, whereas low-diversity soils without urea promoted increased nitrogenase transcription and reduced carbon and nitrogen metabolism transcription. These results show that inoculant outcomes depend not only on establishment, but also how soil conditions and biology shape post-establishment functioning.

Modern agriculture is characterized by substantial fertilizer nitrogen (N) losses from soils, resulting in low crop N-use efficiency. Biological nitrification inhibitors (BNIs) are studied as a strategy to improve N retention in soils by suppressing nitrification. However, the impacts of applying exogenous BNIs to crops with unknown intrinsic BNI capacity remain poorly understood. In this study, we evaluated the impacts of adding three BNIs (methyl 3-(4-hydroxyphenyl) acrylate [MHPA], 6-methoxy-2(3H)-benzoxazolone [MBOA], and limonene), their mixture, and the synthetic nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on barley (Hordeum vulgare L.) growth, plant and soil N dynamics, and soil microbial communities. Using a rhizobox system with planted and bare-soil compartments, combined with 15N isotope tracing and molecular microbial community analyses, we assessed the spatio-temporal dynamics of N transformations, losses, plant N uptake, and microbial community responses in an alkaline agricultural soil. Independent of inhibitor application, the applied fertilizer N was lost primarily through NO- leaching (3-9% of the applied N). In contrast, N2O emissions represented only 0.001-0.028% of the applied N and varied with inhibitor type. MHPA increased dissolved inorganic N soil pools without affecting plant biomass or 15N uptake or strongly shifting microbial community composition. MBOA reduced NO3- concentrations in soil pore water without influencing plant growth or N uptake but shifted soil microbial community composition. In contrast, limonene reduced plant growth and 15N uptake and most significantly altered microbial community composition, without significantly changing N availability. Applying a BNI mixture, as well as limonene alone, was detrimental to plant growth and 15N uptake. DMPP showed only minor effects on N pools, plant growth, plant N uptake and microbial community composition. Overall, our results reveal both the potential and limitations of exogenous BNI application for improving N retention in crop systems.

Soil microbial communities (SMCs) play an important role in various ecological processes, including plant growth, carbon cycling, and greenhouse gas production and consumption. There have been many prior studies of soil microbiome function and structure. However, soil is a complex environment in which to conduct biological studies. Therefore, simplified SMC models, often adapted to liquid culture, have been employed in the laboratory to study specific microbial interactions and individual microbial functions. Specific advantages of these laboratory liquid SMC models include the ability to modulate community membership, control environmental conditions, and employ high-throughput assay techniques. The disadvantages of current laboratory liquid SMC models include long cycles for growing bacteria in vitro, the obligatory use of strains that are culturable in isolation, intricate media requirements, and complex community assembly protocols. To address some limitations of current liquid SMC models, we sought to create a streamlined process for extracting and maintaining a liquid culture of an existing SMC. Soil-Extracted Solubilized Organic Matter (SESOM) was made from four different soil types, including rich organic potting soils and environmental samples, and filtered to maintain the SMC. These SESOM liquid SMC models were cultured for 28 days, and SMC composition was measured by 16S rDNA sequencing. The SESOM SMCs maintain high alpha and beta diversity over time, including strains that are not culturable in isolation, with the greatest stability correlated with higher soil organic carbon. Further, the SESOM SMCs maintain unique signatures of their starting solid soils, suggesting that drift in SMC composition over extended time in liquid culture does not eliminate the defining microbial relationships of a given soil type. Network analysis of SESOM SMCs relative to solid soils suggests the functional roles of bacterial taxa were maintained in the liquid models over time. We further demonstrate that the platform can be applied to monitor the survival and persistence of a model engineered microbe - the common synthetic biology chassis Pseudomonas putida - within a native SMC. We conclude that the SESOM model is a valuable tool for facilitating the study of SMCs in the laboratory.

High-severity wildfires of increasing size and frequency result in release of carbon dioxide and loss of timber resources, reduction in biodiversity, loss of soil, diminished water quality, and reduced recreational opportunities. Forest recovery strongly depends on the reestablishment of soil microbial communities, motivating research on how restoration of soil microbiomes in burned forests can be accelerated. Here, we used a high intensity burn pile experiment to test the effectiveness of post-fire native soil amendment. This design enabled us to sequentially and simultaneously sample unburned, burned, and inoculated burned soils while holding post-fire abiotic factors constant. All conditions were sampled at six time points across an annual hydrological cycle and analyzed using 16S and ITS rRNA amplicon sequencing, genome resolved metagenomics, metatranscriptomics, and soil chemistry. Fire sharply reduced bacterial and fungal diversity and eliminated ectomycorrhizal and ericoid symbiotic fungi. Inoculating the burned soil with native microbes accelerated recovery of microbial diversity and of functions associated with nutrient cycling, especially nitrogen transformations. Despite introducing the full diversity of soil microbes from adjacent unburned forest, only a small subset of adapted organisms were engrafted. Native soil inoculation stimulated reestablishment of mycorrhizal fungi, including genera that form essential symbioses with conifers, although this response was not persistent over the full year. Nonetheless, reestablishment of mycorrhizal fungi for even a window of time may facilitate early forest regrowth. We conclude that, by microbial inoculation, recovery that would otherwise rely on dispersal from distant sites is accelerated, potentially enhancing reforestation efforts.

O_LIA productivity-driven higher nutrient demand of trees in diverse mixtures is frequently reported. Yet, it remains unclear how tree diversity influences microorganisms-plants interactions, in which microbes facilitate tree nutrient acquisition in exchange for carbon (C) to meet the resource demand of both. C_LIO_LIUsing a long-term tree diversity experiment in the subtropics, we assessed microbial investment in C-, nitrogen (N)-, and phosphorus (P)-acquiring enzymes in litter and mineral soil, testing the effects of tree species richness and mycorrhizal type (arbuscular (AM)- vs. ectomycorrhizal (EcM)-associated tree species). C_LIO_LIWith increasing tree species richness, microbial investment in C acquisition decreased, while investment in N and/or P acquisition increased in litter and in mineral soil. In mineral soil of AM-associated tree mixtures, ecoenzymatic stoichiometry revealed a shift from microbial investment in C toward P acquisition as tree species richness increased. C_LIO_LIOur findings suggest that tree diversity strengthens microbe-tree interactions in terms of C-for-nutrient exchange. This highlights the key role of soil microorganisms, particularly in AM symbiosis, shaping tree diversity-biogeochemical feedbacks. C_LI

Soils are recognized as reservoirs of antibiotic resistance genes (ARGs) with the potential to transfer to clinical pathogens, creating antimicrobial resistance (AMR) that poses a threat to human health. While large-scale AMR surveys have profiled how diverse biomes shape soil resistomes, less is known about the influence of specific soil properties. Here, we combined metagenomics and 16S rRNA amplicon sequencing with isolate-based approaches to investigate drivers of soil AMR across a tropical watershed from beach to mountaintop in Waimea Valley, Oahu, Hawai{square}i. We leveraged functional- and taxonomic-classification of resistances to unravel how soil properties interact with bacterial taxa to structure resistomes. Metagenomic- and isolate-resistomes showed remarkable consistency, including a general gradient of increasing AMR from ridge to beach. Resistome functional composition was significantly correlated with total bacterial community structure. The relationship between resistances and soil properties was primarily dictated by taxonomic composition of each resistance. Rifampin- and Vancomycin-ARGs associated with Actinomycetes negatively correlated with soil physical properties, while resistant genes and isolates from Gammaproteobacteria positively correlated with enzymatic activity metrics. These findings indicate that soil properties structure the resistome indirectly through taxonomic filtering of microbial hosts and challenge the notion that AMR is decoupled from phylogenetic relatedness.

Streptomyces are abundant soil inhabitants with extensive secondary metabolism and antibiotic resistance traits. Yet, their ecological role in shaping soil antibiotic resistome dynamics remains understudied. Here, we investigated how two different bio-based fertilizers harbouring Streptomyces shaped soil resistome and mobilome by combining genome analysis of eight Streptomyces isolates to metagenomic profiling of soils before fertilization, within 48 hours after fertilizer application, and six weeks after. Streptomyces genomes showed linkages among antibiotic resistance genes, carbohydrate-active enzymes, and antibiotic-production-associated biosynthetic gene clusters, connecting resistance and biosynthesis to broader metabolic strategies. Relationships between carbon degradation and biosynthesis associated with specific enzyme families, indicating that carbon availability shapes secondary metabolism. We confirmed experimentally that antibacterial potential varied with carbon source, suggesting that microbial activity during manufacturing of the bio-based fertilizers may create localized selection pressures before fertilizers enter the soil. Fertilization with the studied materials induced modest but consistent shifts in resistome and mobilome without major changes in dominant taxa or overall bacterial abundances, indicating functional reorganization within soil communities. Diversity of antibiotic resistance genes and mobile genetic elements increased, whereas abundance changes were small. Mobile genetic element composition showed stronger responses that were associated with fertilizer inputs, Streptomyces abundance, and taxa linked to faecal and resistance sources. Together, our results show that bio-based fertilizers shape soil resistome primarily through ecological restructuring of resident soil communities, while carbon-dependent microbial activity within fertilizers may enrich resistance. These factors should be considered in manufacturing of bio-based fertilizer as well as in designing agricultural practices.

Northern peatlands store large carbon stocks but are sensitive to disturbance. Hydrology, vegetation, herbivory and snow conditions may affect the soil microorganisms driving methane (CH) and nitrous oxide (N2O) cycling. We investigated how reindeer exclusion and snow depth (increased and reduced relative to ambient) manipulations (ongoing for three seasons) influenced archaeal and bacterial communities in a boreal rich fen. Metagenomic (MG) and metatranscriptomic (MT) sequencing were combined with pore-water chemistry and CH flux measurements to link the microbiome to ecosystem processes. Microbial communities differed between outside and inside the exclosure. However, these patterns primarily reflected underlying hydrological variation. Slightly wetter inside plots showed higher expression of denitrification genes (norB, nosZ) and lower (nirS+nirK)/nosZ ratios, indicating greater potential for complete denitrification to N2 instead of N2O. Methane dynamics were mainly associated with vegetation: plots associated with Carex rostrata exhibited lower pmoA/mcrA ratios and elevated CH fluxes. Snow manipulations had subtle effects: reduced snow depth decreased the expression of taxa dependent on microbial interactions, while the effect to the investigated metabolic marker genes was small. Overall hydrology, leading to variations in redox conditions and nutrient availability, together with vegetation appeared as the primary drivers on microbial greenhouse gas processes in this peatland.

Secondary forest succession following agricultural abandonment is a dominant land-use transition across the tropics, yet whether soil microbial communities recover toward old-growth forest reference states remains poorly understood, particularly in West Africa. Here, we investigated the successional dynamics of bacterial and arbuscular mycorrhizal (AM) fungal communities along post-agricultural chronosequences spanning 1 to 43 years across six classified forests in Cote dIvoire, using Bayesian hierarchical models applied to amplicon sequencing data. Both guilds attained moderate to high alpha diversity within the first decade of succession; AM fungal diversity showed moderate evidence of age-related increase thereafter while bacterial diversity showed no directional trend. Pairwise turnover analyses revealed progressive internal convergence in AM fungal communities with plots farther apart in successional time becoming more compositionally similar, while bacterial communities showed only a weak and uncertain tendency in the same direction. Beta-dispersion modelling further indicated progressive within-forest homogenisation of AM fungal communities across abundance-weighted metrics, while bacterial assemblages showed no such stabilisation. Despite this internal convergence, compositional distances to old-growth reference plots remained persistently high for both guilds throughout the chronosequence, with no statistical evidence of recovery toward old-growth states across any dissimilarity metric or guild within the 40-year window. Indicator species analysis identified no robust stage-specific taxa after correction for multiple testing. These results indicate that microbial succession in post-agricultural West African forests is characterised by rapid early reorganisation followed by stabilisation into site-specific assemblages that remain persistently distinct from old-growth reference communities. This outcome challenges the direct application of classical vegetation successional theory to soil microbiomes and suggests that passive regeneration alone is unlikely to restore old-growth microbial communities within restoration-relevant timescales.