FEMS Microbiology Ecology

The contribution of predation between bacteria to microbial community dynamics in agricultural fields has hardly been investigated. Here. dynamics of general prokaryotes (GEP) and of the predators Bdellovibrionales (Bd) and Bacteriovoracales (Bac) (Bdellovibrio-and-Like Organisms, BALOs) were studied in two agricultural fields differing in organic and mineral input regimes, for one year. Season, but not fertilization, affected absolute sizes of GEP and of BALO communities. 16S rRNA gene community analysis identified numerous novel Bd and Bac lineages, with none of the dominant BALOs related to characterized isolates. A few dominant BALO amplicon sequence variants (ASVs) persisted year-round, whereas others showed seasonal- or treatment specific responses. GEP, Bd, and Bac ASV a-diversity was mostly influenced by season, with some changes due to fertilization in Bd, and Bac communities. Seasonal changes, site, and fertilization regimes influenced {beta}-diversity of GEP, Bd and Bac communities and determined the structure of BALO-gram-negative bacteria interaction networks, signaling that niche segregation acts at the microbiome-BALO interface. Accordingly, we suggest that shifts in GEP community structure triggered by environmental changes and agricultural practices cascade to BALO predators, in turn affecting BALO-microbiome interactions. These dynamics may be harnessed to manipulate the soil microbiome to benefit sustainable environmental and agricultural outcomes.

Profiling microbiomes is an important way to understand the function and composition of communities in the wild, but natural microbiomes are often highly complex and often unamendable to experimentation to reveal cause and effect relationships. By using a small group of cultivable strains to represent those found in the wild, synthetic communities are one solution to this problem. Here we describe the MAize Rhizosphere Synthetic Community (MARSc), a genome-enabled 31-member bacterial community representative of the diversity found on the roots of maize grown in Iowa soils. This community is built around Pseudomonas putida KT2440, a model maize rhizosphere colonist and synthetic biology chassis. We characterized microbe-microbe interactions and biofilm formation of MARSc members in a variety of environmental contexts, finding that both behaviors are broadly controlled by nutrient levels. Genomic analysis and microbiome profiling of these organisms revealed that annotated biofilm genes (such as surface attachment and exopolysaccharide production) correlated to rhizosphere colonization, but neither trait correlated to in vitro biofilm formation. In vitro interactions assay findings were surprisingly consistent with co-correlations of rhizosphere abundance amongst MARSc members on roots. Finally, we found that when applied to the roots, MARSc can increase maize growth under nitrogen-limiting conditions. Altogether, MARSc is a useful tool for identifying some of the factors influencing rhizosphere microbiome assembly and will be a strong foundation for further work in this area.

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

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.

Plant roots are broadly colonized by endophytic fungi with saprotrophic capabilities, but our understanding of whether they function in ways that are beneficial or detrimental to the host remains limited to model organisms. We hypothesized that endophytic fungi broadly affect plant access to soil nutrients, particularly organic forms that are typically not directly available to the plant. To address this, we paired 41 fungal endophytes with switchgrass (Panicum virgatum L.) and provided either inorganic or organic forms of nitrogen (N) and phosphorus (P). We evaluated how the fungi affected plant tissue N and P as well as plant growth. We also examined if these outcomes could be predicted from fungal phylogenetic relationships, in vitro traits of the fungi, or characteristics of the habitat from which fungi were isolated. There was substantial variation in both plant N (0.05-0.63%) and P (0.02-0.10%) acquisition that depended on the interaction of fungus and nutrient treatment. More fungi were beneficial for plant N than for P and shoot nutrients generally increased more than root nutrients from fungal associations. However, fungal effects on plant nutrients were not predicted by fungal traits, habitat traits, or fungal phylogenetic relationships. This unpredictability highlights a key challenge for incorporating endophytes into nutrient management strategies. Improving our ability to predict endophyte impacts on host nutrient acquisition will require identifying the mechanisms underlying observed beneficial effects and scaling up to realistic, diverse root microbial communities.

Conventional anaerobic digestion emits methane from organic waste. Here, we investigate a sulfate-based alternative that suppresses methane production and generates alkaline solutions that may sequester carbon by carbonate precipitation. Although methanogenesis is known to occur when reduced organic carbon is replete and sulfate is limiting, it remains unclear whether methane emissions during microbial conversion of waste gypsum are primarily driven by community composition or organic availability. By comparing fluxes of electrons from organic matter toward sulfate or methane in microbial communities grown on different organic loads, we show that community composition, microbial growth, and organic availability collectively determine sulfide and methane fluxes. Lower organic loads increase the importance of syntrophic interactions with fermenters and competition between sulfate reducing bacteria and methanogens due to scarcity of substrates. Microbes present in the original sewage sludge reduce less sulfate, produce more methane, and generate less alkalinity compared to the communities enriched by multiple cycles of growth in the presence of sulfate and sewage sludge. The inoculation of communities enriched at low organic loadings in the presence of sulfate decreases the production of methane by enabling the growth of sulfate reducing bacteria from the order Desulfobacterales that can oxidize acetate to CO2 and compete with methanogens for acetate. The use of such enrichments in sludge treatment systems can stimulate the removal of organic substrates and waste gypsum, while suppressing methane production, over timescales comparable to those in the current sludge treatment systems that do not contain sulfate.

Rice, a staple crop of nearly half of the world population, is grown predominantly in flooded paddies which are one of the largest contributors to methane emissions. An effective approach is to minimise the anaerobic flooded conditions that favour the growth of methanogenic archaea. Empirical measurements showed that controlled irrigation regime reduces methane emissions by 70% to 90%. The soil microbiomes of both flood and drip irrigated soil were characterised using whole-genome shotgun metagenomics. Controlled irrigation was shown to suppress methanogens and lower methane emissions. While emissions are correlated with mcrA gene abundance, empty flooded fields exhibited relatively high mcrA levels above baseline despite undetectable methane emissions. Rice cultivar genotype had no significant effect on the soil microbiomes. Co-occurrence network analysis indicates that soil microbial communities stratify according to their oxygen preferences along a gradient. Methanogens were increased in flooded paddies, and methane production attributed to the microorganisms involved in the anaerobic decay of organic matter. Controlled irrigation altered the microbiome by raising the soil redox potential by enhancing aeration and promoting ammonia oxidation and nitrification pathways. IMPORTANCEThe temporal dynamics of microbial communities in drip-irrigated rice fields remain poorly characterized to-date. Empirical measurements demonstrate that controlled drip irrigation effectively suppresses methanogens and lowers methane emissions by up to 90%. Statistical analysis further revealed a moderate correlation between methane emissions and the mcrA gene with R = 0.6 and p-value = 2.9e-05. The correlation plot showed that the outliers corresponded to samples from empty flooded fields, where high mcrA gene abundance was observed despite low methane emissions. Methane produced in the soil is likely released into the atmosphere via transport through the aerenchyma of rice plants. Controlled irrigation is shown to be climate friendly as it reduces methane emissions by improving soil aeration and increasing the soil redox potential.

Host-associated microbiomes play vital roles in organismal health, ecological interactions, and evolution, yet little is known about how microbial communities shift during the transition from aquatic to increasingly terrestrial habitats. Here, we characterize the skin microbiomes of three combtooth blenny species (Blenniella paula, Praealticus labrovittatus, and Alticus arnoldorum) that occupy distinct positions along the intertidal gradient in Guam--from fully subtidal (and exclusively aquatic) to intertidal (amphibious) and supratidal environments (exclusively terrestrial). Using 16S rRNA amplicon sequencing, we compared skin-associated bacterial communities with those in surrounding seawater and substrate biofilms to assess habitat influences on microbiome structure. Skin microbiomes were distinct from environmental microbial communities, indicating strong ecological filtering by the host. The divergence between skin and substrate microbiomes in the three species parallels their distribution along progressively higher zones of the intertidal gradient. The most divergent skin microbiome was that of the supratidal fish A. arnoldorum, characterized by higher Gammaproteobacteria abundance and enrichment of epiphytic and mucus-associated taxa. Across all species, we identified 32 microbial orders significantly enriched on the skin relative to environmental samples, including taxa commonly associated with fish mucosa (e.g., Vibrio, Alteromonas, Cetobacterium) and others rarely reported in aquatic marine fish (e.g., Rubritalea, Granulosicoccus). Several rare taxa with potential pathogenicity were also detected at low abundances. Together, these findings suggest that habitat-specific selective pressures strongly shape fish skin microbiomes along subtidal (aquatic) to supratidal (terrestrial) habitats and suggest that microbial symbionts may contribute to the ecological and physiological adaptations enabling amphibious lifestyles. This study provides the first comparative assessment of skin microbiome divergence across amphibious fish species along an intertidal gradient and offers a framework for predicting microbiome responses to environmental change.

Urban agriculture increasingly relies on compost-based substrates for sustainable production, yet we lack a clear characterization of how these systems respond to biological amendments aimed at introducing beneficial microbiota. Here we investigated how developmental stage and co-inoculation with arbuscular mycorrhizal fungi (AMF) and phosphate-solubilizing bacteria (PSB) reshape rhizosphere microbial function in Solanum lycopersicum grown in compost-based urban farm substrate. Using plant physiology assays, 16S rRNA amplicon sequencing, and metagenome-informed metaproteomics, we characterized tomato physiological responses and rhizosphere microbial activity during flowering and fruiting across control, single AMF, single PSB, and AMF and PSB co-inoculation treatments. Co-inoculation synergistically enriched beneficial taxa, improved fruit nutrient accumulation, elevated nutrient transporter and quorum sensing protein production, and drove stress-driven dormancy in competitively excluded taxa, with responses varying between developmental stages. Our findings establish metagenome-informed metaproteomics as essential for resolving stage-specific rhizosphere microbiome functional responses to tomato development and AMF and PSB co-inoculation.

Scalable soil microbiome monitoring requires sampling methods that are reproducible across operators, field sites, and logistical constraints. Here, we evaluated three key methodological choices that commonly limit comparability in agricultural rhizosphere studies: how the rhizosphere sampling unit is operationally defined, sample pooling strategies, and preservation methods. We introduce the RhizoCore, a standardized root-zone soil core defined by core diameter, depth, position relative to the plant, and subsample volume, as a practical proxy for traditional rhizosphere sampling. The RhizoCore method captured more than 92% of the sequencing depth found in traditional rhizosphere samples, with differences limited predominantly to low-abundance taxa. Preservation methods significantly affected bacterial communities, while sample pooling showed greater impact on fungal diversity and substantially reduced within-group variability across all treatments. Despite these effects, differential abundance analysis revealed minimal compositional changes, with only a small fraction of microbial taxa significantly affected by either pooling or preservation method. Our findings demonstrate that the RhizoCore method provides a reproducible, and scalable approach for rhizosphere sampling that balances scientific rigor with practical field implementation, offering a framework for large-scale soil microbiome monitoring programs and for improving comparability among agricultural microbiome studies across diverse environmental conditions.

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.

Bacteriophages are abundant and diverse in soils, playing a major role in regulating bacterial communities and consequently affecting biogeochemical cycles. Such host-phage interactions may be influenced by fluctuations in soil moisture, as observed in wetlands soils which constitute a key feature of the ongoing climate change. Here, we investigated the spatial and temporal dynamics of both bacteria and T4-type bacteriophage community structures and diversities in soil of a freshwater wetland. Soil was sampled in three sites across a proximal soil transect presenting an increase moisture content at seven dates over an 18 months period with contrasted flooding periods. DNA was extracted and we applied amplicon sequencing of the bacterial 16S rRNA gene and viral g23 gene. Bacterial community composition varied across the proximal soil transect, with Methylomirabilia and Gammaproteobacteria being significantly enriched in the wettest site and comprising ASVs affiliated to methanotroph and denitrifying bacteria, respectively. We identified a large diversity of T4-type phages, among which a fraction was novel, while others were similar to phages previously sequenced from various biomes. These findings suggest that T4-type phages are capable of successfully colonizing diverse niches in the biosphere, contributing to their ubiquity and diversity. Viral community was however dominated by few vASVs, which were highly represented in one or two of the three studied sites supporting the Bank model. All together our results indicate that T4-type phages have broad host ranges and more likely follow bacterial population dynamics. The present study provides new insights into the role of phages in soil, highlighting their interactions with bacterial hosts involved in carbon and nitrogen cycles, interactions that are likely regulated by fluctuations in soil moisture, as observed in wetlands. HighlightsO_LIBoth bacterial and T4-type phages were structured across proximal sites C_LIO_LIBacterial 16S rRNA gene copy number was inversely correlated to the soil moisture C_LIO_LI26 viral ASVs did not cluster with reference sequences C_LIO_LIviral ASVs seem to be primarily controlled by host availability C_LIO_LISoil bacteria and phage diversities were significantly lower in the wettest site C_LI

Bacterial bloomers,populations that experience rapid and significant increases in abundance in response to environmental triggers, briefly dominate marine microbial communities, potentially impacting the ecosystem by channeling large amounts of nutrients and affecting carbon fluxes. Due to their ephemeral nature, bacterial bloomers are challenging to capture, and it remains unknown whether they are restricted to specific taxonomic groups or whether they exhibit recurrent patterns. We analyzed a decade-long time series from the Blanes Bay Microbial Observatory (BBMO, NW Mediterranean Sea) to investigate bacterial bloomers in two size fractions (free-living (0.2-3 um) and particle-attached (3-20 um) communities. We identified 57 Amplicon Sequence Variants (ASVs), less than 1% of the total bacterial richness, exhibiting recurrent or chaotic blooming-like behavior. Bloomers spanned diverse phyla, though some taxonomic coherence appeared within families containing multiple blooming taxa. Monthly sampling detected bloom events on average 4.6 +- 1.9 times per year across both size fractions. Once seasonality was accounted for, blooms showed weak associations with biological and physicochemical variables, likely a consequence of monthly sampling resolution. Nonetheless, a marked shift in the blooming community within the particle-attached size fraction coincided with ecosystem disturbances from the nearby harbour restoration, suggesting that bloomers may act as disturbance sentinels. Metagenomic data showed that blooms led to marked shifts in the community functional potential. Overall, our findings underscore the importance of investigating bloom dynamics to understand microbial contributions to biogeochemical cycles and stress the need for higher-frequency sampling to accurately capture these transient but ecologically relevant events.

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.

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.

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.

Crop species may shape soil chemical properties and root-associated microbiota, but direct comparisons between contrasting crops remain limited. We compared soils and root-associated bacterial communities of Welsh onion (Allium fistulosum) and sweet potato (Ipomoea batatas) under the same field context. Sweet potato soil showed significantly lower electrical conductivity, inorganic nitrogen, and Mg saturation than control soil. Root-associated communities differed between crops, whereas alpha diversity did not. Proteobacteria-related taxa were more represented in Welsh onion roots, whereas Actinomycetia-related taxa were more represented in sweet potato roots, providing a basis for future studies on crop-specific soil microbial management.

Microbial diversity across ecosystems may be impacted by dispersal pathways and selection due to physiochemical conditions, and so it is hard to predict the extent of strain sharing across heterogeneous environmental compartments. Evaluation of patterns of strain-level overlap requires genome-level comparisons using extensive datasets collected over large spatial scales. Here, we sampled microbiomes beneath (hyporheic zone) and within the East River (Colorado, USA) and found little or no strain overlap at sites along the river corridor, despite connection by river flow, suggesting selection due to the local environment. Comparisons involving microbiomes from hillslopes and riparian zone soil yielded essentially no strain-level overlap with the microbiomes of river and hyporheic zones. We also sampled a nearby groundwater well and found near-perfect genotypic overlap with strains in one hyporheic zone location that exhibited active groundwater upwelling. Given an absence of direct connectivity between these two locations via discrete hydrologic flow paths, we conclude that groundwater strains are widely dispersed in the aquifer. As hyporheic zone microbiomes in zones with low river flow share no strains with groundwater microbiomes, we infer that strains introduced by groundwater mixing in the hyporheic zone are transitory. We conclude that despite evidence for mixing of river-and-hyporheic zone water, and river-and-groundwater on short time scales, establishment of transported strains in the hyporheic zone is minimal.

Anthropogenic soil degradation is a major challenge for sustainable food production, particularly in tropical agricultural systems where excessive fertilizer use contributes to soil deterioration and greenhouse gas emissions. Microbiome-based agricultural technologies offer a potential strategy to improve fertilizer-use efficiency while maintaining crop productivity. Here, we characterized the taxonomic composition and predicted functional potential of a commercial microbiome-based fertilizer additive (humus) deployed across more than 1.4 million hectares in Paraguay and Uruguay, and compared it with root-associated microbiomes. In parallel, we evaluated agricultural and forest soil microbiomes from eastern Paraguay. Microbial communities were analyzed using 16S rRNA gene sequencing and PICRUSt2-based functional prediction. The humus microbiome displayed enrichment of pathways associated with degradation of organic compounds, nutrient cycling, and plant-growth-promoting activities. Furthermore, humus and root-associated microbiomes shared over 350 predicted microbial pathways, indicating substantial functional overlap despite differences in specific bacterial taxa, and suggesting that the consortium may function as a rhizosphere-like microbial community capable of providing functions commonly associated with plant-associated microbiomes. In agricultural soils, significant taxonomic differences were observed between high- and low-productivity fields, whereas predicted functional profiles remained largely conserved, consistent with functional redundancy within soil microbial communities. Productive soils were enriched in the superpathway of demethylmenaquinol-6 biosynthesis II, a microbial vitamin K2-related pathway involved in respiratory metabolism. Together, these findings provide the first detailed taxonomic and predicted functional characterization of a large-scale commercial microbiome-based fertilizer additive and establish a baseline for understanding microbial diversity and functional potential across productive agricultural soils in Paraguay. ImportanceSoil degradation and inefficient fertilizer use are major constraints to sustainable agriculture, particularly in tropical systems where nutrient losses and greenhouse gas emissions are high. Microbiome-based agricultural inputs are increasingly proposed as tools to enhance soil functioning and improve nutrient cycling efficiency, yet their ecological characteristics and functional potential remain poorly understood. This study provides the first detailed taxonomic and predicted functional characterization of a large-scale commercial microbiome-based fertilizer additive deployed in South American agriculture, and places it in the context of native forest and agricultural soil microbiomes. By integrating 16S rRNA gene sequencing with predictive functional profiling, this work reveals substantial functional overlap between the microbial consortium and plant-associated microbiomes, suggesting ecological convergence toward rhizosphere-like functions. In addition, the identification of conserved functional profiles across soils with contrasting productivity highlights the potential role of functional redundancy in maintaining ecosystem processes under different management regimes. Together, these findings provide a foundational framework for understanding microbiome-based agricultural inputs and soil microbial functional stability in subtropical agroecosystems, specifically Paraguay, contributing to the development of more sustainable agricultural practices.

The soil priming effect plays an important role in the global carbon cycle. Although well-studied, the mechanisms behind it remain elusive. So far, studies measured the phenomenon at the bulk soil scale, neglecting that it arises from spatially explicit processes that take place at the microscale. Here, we present a novel approach using a microfluidic device to directly assess the response of soil microbes living on a patch of complex substrate to a pulse of easily available substrate. Using time-resolved fluorescence microscopy, we tracked motility, position, shape characteristics and attached biomass of green fluorescent protein expressing Bacillus subtilis cells living on a transparent carboxymethylcellulose substrate patch exposed to a pulse of growth medium with differing concentrations. Assessing CMC decomposition via Congo-Red staining after 42 days of incubations with constant observation, we observed increased decomposition upon addition of enough labile substrate, resembling a priming effect. The pulse triggered a transient increase in bacterial motility, indicating the formation of exploring and growing subpopulations respectively. We observed a concentration-dependent growth response, resulting in different behaviors. High concentrations led to high biomass and decomposition of CMC, however growth was quickly limited, possibly by depletion of necessary nutrients and waste accumulation. Intermediate concentration, however, resulted in a more sustained attached biomass, that showed evidence of spatial self-organization, leading to similar decomposition. We present a novel experimental model system to study the behavior of soil microbes decomposing complex substrate and provide a unique view into the response of such a population to a labile substrate pulse.