Environmental Microbiome

Alkaline and calcareous soils can induce iron (Fe) limitation in plants, yet the responses of root-associated microbial communities to beneficial rhizobacteria under these conditions remain poorly understood in cotton. Here, we investigated the effects of Variovorax paradoxus on plant performance, Fe nutrition, and root microbiome dynamics in cotton exposed to bicarbonate-induced Fe limitation. In this study, V. paradoxus inoculation under bicarbonate-induced Fe limitation significantly improved photosynthetic parameters, growth parameters, and tissue Fe status. Interestingly, V. paradoxus partially suppressed the Fe-deficiency-induced increase in root ferric-chelate reductase activity without further increasing rhizosphere siderophore activity. This response suggests that improved Fe availability reduced the demand for maximal activation of the intrinsic Strategy I response. Despite improved plant health, V. paradoxus reduced root C levels, suggesting altered belowground carbon utilization associated with bacterial inoculation and stress conditions. Split-root experiments further showed that inoculating both root compartments showed substantially greater recovery than unilateral inoculation, indicating that broader root exposure to V. paradoxus enhanced the beneficial response. Although bacterial alpha diversity remained unchanged, V. paradoxus significantly altered bacterial community composition and enriched Cellvibrio together with the fungal taxa Funneliformis and Dominikia under Fe limitation. Exploratory analysis identified the plant-beneficial fungal hubs Funneliformis and Serendipita in the V. paradoxus-treated community under indirect Fe deficiency, along with the core genera Pseudomonas, Hydrogenophaga, and Funneliformis and the indicator taxa Shinella and Aquabispora. Spearman correlation analysis further associated Streptomyces with root Fe accumulation and biomass, while Epicoccum and Sordariales were positively associated with siderophore production in cotton exposed to bicarbonate-induced Fe limitation and inoculated with V. paradoxus. These findings demonstrate the potential of V. paradoxus and identify candidate microbial partners for microbiome-informed biofertilizers to improve Fe nutrition in cotton grown in calcareous soils.

Background and AimsAbiotic stress is a major constraint for citrus production in Mediterranean environments, where water deficit and salinity frequently occur. This is particularly relevant for perennial crops, like citrus, where limited options for stress avoidance exist. Rootstocks are extensively employed to enhance stress resilience; however, their influence on the root microbiome under abiotic stress remains largely unexplored. Here, we investigated the effects of water stress and salinity on the diversity, composition, and interactions of bacterial and fungal communities in two citrus rootstocks with reported contrasting phenotypes, such as Bitters, which has been described as exhibiting a promising tolerance to both water and salt stress, and Carrizo, which is generally reported to be highly sensitive to these conditions. MethodsThe distinct rootstocks have been subjected to either water stress or salt stress and compared with the non-stressed rootstocks. At the end of stress period, they were profiled and then integrated with recorded plant morphological (i.e. root volume), physiological (water potential, abscisic acid, chlorophyll and chlorophyll content meter) and biochemical measurements (abscisic acid and catalase). In parallel, we used a high-throughput amplicon sequencing to profile bacterial and fungal communities inhabiting the rhizosphere and endorhizosphere microhabitats of the rootstocks in both stresses and in non-treated conditions. Finally, we used correlations and multivariate analysis to determine relationships between plant performance and microbiome putatively underpinning stress adaptation and tolerance. ResultsAcross all treatments, microbial community composition was primarily shaped by microhabitat, with clear differentiation between rhizosphere and endorhizosphere. Abiotic stress significantly restructured microbial communities, particularly in the rhizosphere, while the endorhizosphere exhibited stronger genotype-dependent patterns. Bacterial communities showed pronounced stress-driven enrichments of taxa belonging to the main phyla (such as Proteobacteria, Actinobacteriota and Bacteroidota), with selective recruitment of taxa putatively associated with stress adaptation, whereas the response of fungal taxa (more represented by Ascomycota, Basidiomycota and Glomeromycota phyla) was less consistent and mainly microhabitat-driven. Notably, the two rootstocks exhibited distinct physiological strategies, with Bitters by increased proline accumulation and root volume and Carrizo characterized by enhanced ABA and catalase. ConclusionsOur findings showed Bitters outperform Carrizo in terms of tolerance to both water and salinity stress. In both rootstocks, specific bacterial taxa such as high abundant core or rare members, were associated with distinct phenotypic parameters, highlighting the importance of integrating plant and microbiome perspectives for improving stress resilience in citrus.

Fungi are key members of microbial communities, yet microbiome surveys often lack trait-based information required for ecologically meaningful interpretation of mycobiome data. To demonstrate the value of fungal trait-based phenotyping in microbiome research, we re-analyzed N=3,221 samples across four case studies spanning human, agricultural, and environmental systems. In human cancer and vineyard datasets, trait-based analysis detected fungi producing macroscopic fruiting bodies, likely introduced via airborne spore dispersal, indicating widespread contributions of transient or contaminant fungi that can confound interpretation of sequencing data from tumor biopsies and grape berries. In sourdough fermentations, filamentous fungi were highly abundant alongside traditionally-recognized yeast and occupied distinct ecological niches. In forest soils, increasing habitat disturbance was associated with increased prevalence and abundance of plant pathogens, and a marked decline in ectomycorrhizal and lichenized fungi. These changes were accompanied by a shift toward large-spored taxa in urban soils, consistent with enhanced stress tolerance. To facilitate broader adoption of fungal phenotyping in microbiome studies, we introduce q2-fungal-traits, a QIIME2 plugin for automated integration of fungal taxonomy derived from marker-gene or shotgun metagenome sequencing surveys with ecological and functional trait data. The plugin assigns lifestyle-related traits and spore size estimates through hierarchical taxonomic matching and integrates directly into standard microbiome workflows. Our case studies demonstrate that integrating trait-based ecology with mycobiota datasets can generate novel findings and testable hypotheses, enabling inference of the functional (ir)relevance of community constituents. Our work contributes to bridging the gap between descriptive community profiling and functional ecology in microbiome research.

Root-associated microorganisms play a pivotal role in helping plants adapt to drought stress. However, the underlying mechanisms of the rhizospheric microbiome under limiting soil moisture remain largely unresolved. Integrating total and active microbiome analyses enables a more accurate interpretation of microbial responses to climate change-associated water stress. We assessed the effect of reduced rainfall on two wheat genotypes, drought-tolerant (DT) and drought-sensitive (DS), using rainout shelters that allowed 100%, 75%, 50%, and 25% of natural precipitation to reach the crop. At the peak of the growing season, rhizosphere samples were collected for metagenomic (MG) and metatranscriptome (MT) sequencing. In parallel, rhizosphere volatile organic compounds (VOCs) were collected and analysed. Differential expression analysis of metatranscriptomic data using metagenomic abundance as a cofactor was performed by comparing all treatments to the 100% precipitation control. Our results demonstrate that particularly oxidative stress-related transcripts intensify in DS as rainfall decreases. Transcriptomic shifts primarily involved upregulation of transcripts associated with antioxidant (catalase, superoxide dismutase), heat shock proteins (Hsp10, Hsp60, DnaK/DnaJ, GroEL, GroES), as well as microbial functions related to osmoregulation, proline and glycine betaine (PutA, PutP, OpuBB), and plant growth-promoting traits such as auxin production, phosphate solubilization. Moreover, volatile organic compound (VOC) emissions differed significantly between the control and drought treatments, with higher emissions, particularly acetates, in the DS genotype than in the DT genotype. Overall, pronounced drought-induced shifts in active microbial functions and VOC emissions indicate high sensitivity and functional plasticity of the active microbiome, whereas the total microbiome remains robust under medium drought.

Despite their agricultural potential, how bacterial-fungal consortia reshape root microbiomes and improve crop performance in sorghum remains poorly understood. Here, we investigated how individual and combined inoculation with Bacillus subtilis and Trichoderma afroharzianum influenced sorghum performance and root microbiome assembly. The in vitro co-culture assay demonstrated the compatibility of B. subtilis and T. afroharzianum as a microbial consortium. The B. subtilis-T. afroharzianum consortium demonstrated the highest CPPI (composite plant performance index) and shoot fresh weight in sorghum, while all inoculation treatments improved multiple growth and physiological traits. Split-root analysis demonstrated that bilateral root co-inoculation was necessary to maximize whole-plant growth benefits. Also, B. subtilis-T. afroharzianum co-inoculation increased carbon levels in both roots and leaves, accompanied by enhanced rhizosphere siderophore production consistent with improved nutrient status. In microbial community analysis, neither bacterial nor fungal alpha or beta diversity differed significantly among treatments; instead, inoculation selectively restructured root microbial communities. The B. subtilis-T. afroharzianum consortium selectively enriched plant growth-promoting Actinoplanes, siderophore-producing Enterobacter, and the plant-beneficial fungal genus Podospora. Co-occurrence network analysis identified Rhodoplanes, Serendipita, and Zopfiella among hub taxa associated with B. subtilis-T. afroharzianum co-inoculation, suggesting potential roles in microbial community connectivity and organization. Furthermore, the persistence of Streptomyces and Serendipita, particularly the latter, suggests the presence of a beneficial microbial core that may contribute to sustained rhizosphere functioning. In addition, Bacillus and Serendipita were among the indicator taxa associated with inoculated treatment combinations, suggesting that the inoculants selectively assembled a distinct plant-beneficial microbiome. Devosia was associated with chlorophyll content, siderophore production, and shoot height, whereas Serendipita correlated with shoot biomass under the B. subtilis-T. afroharzianum co-inoculation. Taken together, B. subtilis-T. afroharzianum consortium promotes sorghum growth by selectively reshaping the root microbiome, highlighting its potential as a next-generation microbial biofertilizer.

Amplicon-based techniques provide a rapid and cost-effective approach for profiling microbial communities. However, the observed microbial diversity is influenced by a wide range of factors, encompassing pre-analytical steps such as the choice of primers and target regions, as well as the bioinformatic pipeline, including the selection of tools, reference databases, and parameter settings. Several benchmarks are already available in the literature, but the updates to important tools and databases, namely LotuS3, the Ribosomal Database Project and GreenGenes2, prompted our investigation. In this study, we conducted a comprehensive benchmark of the main bioinformatic tools and databases. Using seven regions for three publicly available mock communities of increasing complexity, we tested 38 possible combinations of sequence resolution algorithms (DADA2 stand-alone, LotuS3 (DADA2/UPARSE)), taxonomic classifiers and search tools (Kraken2, DECIPHER, RDP, MMseqs2, Lambda, and Metaxa2), and databases (SILVA, GreenGenes2, RDP, RefSeq, and Metaxa2). The region V1-V3, coupled with DADA2+MMseqs2+SILVA, DADA2+Metaxa2, or LotuS3 (DADA2)+RDP yielded the highest-quality estimates of the true diversity according to the metrics. We also demonstrated that even certain dominant genera remain difficult to detect, and that the quantification of all genera can be substantially over- or under-estimated, even when using optimal combinations of tools and reference databases.

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.

Improving environmental management in the offshore Oil & Gas sector requires approaches that capture the ecosystem services (ES) provided by marine sediments, particularly their roles in carbon and nutrient cycling. Microbial communities are central to these processes, and molecular tools offer new opportunities to assess their functional diversity. To explore how different sequencing approaches inform environmental impact assessment, we compared taxonomic and functional prediction based on 16S metabarcoding, shotgun metagenomics and messenger RNA-based metatranscriptomics, targeting prokaryotic communities. Our aims were to evaluate the ability of each approach to detect impact and to determine how well they captured functions relevant to ES. All approaches revealed clear differences in community composition between impacted and non-impacted sediments at both taxonomic and functional levels, with impact significantly associated with hydrocarbon and barium content. Functional inventories showed substantial overlap across the three approaches, and 48-55 ES-related processes were detectable in all datasets. While metagenomics provided the strongest statistical discrimination between impact groups, metatranscriptomics resolved the actively expressed pathways underpinning ES, yielding the most biologically meaningful functional profiles despite its lower statistical power. All approaches indicated that Oil & Gas activity drives shift towards anaerobic, hydrocarbon-degrading, and sulfur-respiring microbial communities, with hydrocarbon degradation, sulfur cycling, and metal detoxification being the dominant ES processes in impacted sediments. Metabarcoding was confirmed as a cost-effective option for impact assessment when focused on taxonomic composition. However, functional prediction from metabarcoding data proved less reliable, as several ES showed contrasting associations to impact category between metabarcoding and shotgun sequencing approaches.

Predatory protists are important in shaping terrestrial microbial ecosystems, but their roles in the phyllosphere, or the communities on aerial plant surfaces, are poorly understood. Previous work found that the order Colpodida dominated heterotrophic protist communities in the phyllosphere. While most protists were sporadically present, a few Colpodida variants were prevalent and abundant, indicating that these variants may represent species adapted to the phyllosphere. To identify these organisms, we cultured colpodids from field-collected tomato leaves and performed phylogenetic analysis of the 18S rRNA gene. Five of nine independent isolates matched the most prevalent Colpodida variant previously identified as leaf-enriched through amplicon sequencing, and these isolates comprised a novel clade of Paracolpoda steinii. When compared to a maize root isolate of Colpoda inflata, an abundant rhizosphere ciliate, a P. steinii isolate was similar in size and growth yield on E. coli, but grew to higher yields and formed large cyst clusters when incubated with model phyllosphere bacteria prey Erwinia and Pseudomonas. We developed and validated quantitative PCR (qPCR) methods for detection and cell abundance estimation of the P. steinii phyllosphere clade, C. inflata, and the order Colpodida in environmental samples. In inoculated greenhouse plants, qPCR-estimated protist populations matched measured inoculum levels, and protist inoculum was still detectable after five days. In an uninoculated tomato field, P. steinii was detected on all plants, with greatest abundances observed in lower leaves and after a rain event. P. steinii comprised up to 18.7% of total leaf Colpodida populations, which were estimated at up to [~]1400 organisms per gram of fresh weight. The findings demonstrate that Colpodida communities are consistently present on tomato leaves, dynamically affected by the abiotic environment, and include significant populations of P. steinii. We propose that the P. steinii isolates and qPCR tools presented can be used as a model system to investigate colonization and distribution patterns, biotic interactions, genetic adaptations, and agricultural applications of leaf predation.

Plant-microbe interactions in the rhizosphere are central to nutrient cycling and ecosystem functioning. Sedimentary ancient DNA (sedaDNA) is a promising yet underexplored tool for reconstructing past microbial communities and investigating ecological interactions among plants, animals, and microorganisms. Here, we reanalyse the previously published Kap Kobenhavn Formation (Northern Greenland) sedaDNA dataset to move beyond taxonomic ecosystem reconstruction and test whether ancient sediments preserve structured, rhizosphere-compatible plant-microbe association signals. Our results show that this ancient boreal ecosystem hosted several rhizosphere-associated taxa, comparable to those in modern boreal soils. Several bacterial genera co-occurred repeatedly with specific plant families, forming a rhizosphere-like taxonomic core with predicted plant-growth-promoting traits related to nutrient acquisition, colonisation, and stress tolerance. Although sedaDNA co-occurrence cannot demonstrate direct symbiosis, the consistency of taxonomic, network, and functional signals suggests that ancient sediments preserve interconnected ecological structure. Our findings extend sedaDNA-based ecosystem reconstruction beyond taxonomy and provide a possibility for investigating plant-microbe association signals in deep time.

Increasing global droughts exert large but poorly understood effects on the microbial communities and ecology of soil. Microbial communities generally show resilience and return to pre-drought conditions when short-term droughted soils are rewet; soils exposed to long-term drought, however, often show a lag upon rewetting, after which microbial communities may or may not return to their pre-stressed conditions. Though short-term droughts have been widely studied, long-term drought manipulation experiments remain rare, especially those that compare microbial response to short-term and long-term drought in tandem. We conducted a 1000-day drought simulation in controlled laboratory conditions with soil cores collected from a tidal freshwater ecosystem in Washington state, USA, and subsequently exposed them to rewetting for two weeks. We also included short-term (30-day and 90-day) drought and rewet treatments to directly compare microbial community and organic matter responses across drought durations. We found distinct microbial taxa belonging to Firmicutes and Actinobacteria enriched after the 1000-day drought, but not after the short-term droughts. While we hypothesized that the microbial community would recover from a short-term drought after rewetting to resemble pre-drought conditions, our results revealed community dissimilarities between rewet and pre-drought conditions across all drought durations. These findings suggest unique microbial life history strategies within certain microbial phyla that make them successful colonizers during an extended drought period, and the influence of environmental and physiological context on microbial responses to rewetting. ImportanceDroughts are increasing in frequency and intensity globally with severe implications for ecosystem services and soil functions. It is important to understand how long-term drought impacts soil microbial communities and organic matter chemistry to better predict future ecosystem responses to sustained moisture deficit conditions. We subjected soils to short-term (30 and 90 days) and long-term (1000 days) drought treatments and subsequently rewetted them to understand microbiome recovery to pre-drought conditions. Our results showed that prolonged drought drastically changes the microbial community and soil organic matter profile compared to short-term drought. While we expected the soil microbiome to recover upon rewetting after short-term drought, our results showed an altered microbiome composition, compared to pre-drought conditions, for both short-and long-term drought, suggesting microbial responses to soil rewetting was independent of drought duration imposed. These results provide important insights into soil biological and chemical functions that remain sensitive to change under fluctuating soil moisture conditions and future drought scenarios.

Thermal stress can disturb microbial communities associated with host species. As microbes can support essential functions related to host metabolism, physiology, nutrition and immunity, changes in microbial communities can have severe fitness consequences for the host. Although the effects of thermal conditions on host-associated microbiomes have been demonstrated in controlled laboratory settings, how climate change might affect the structure and functionality of microbial communities in wild populations remain poorly understood. Here, we took advantage of the well-characterized long-term field survey of the Glanville fritillary butterfly (Melitaea cinxia) metapopulation on the [A]land islands, in the Baltic Sea, to fill this gap. We investigated whether bacterial communities associated with larvae show signs of gradual temporal change in response to slow environmental warming across a 28-year period, or whether these communities responded through abrupt change following a sudden drought event that triggered bottlenecks in their butterfly host population. Using a combination of 16S rRNA metabarcoding and metagenomic sequencing, we first showed that M. cinxia harbours a set of stable resident bacteria, including Pseudomonas, Telluria, and Enterobacteriaceae bacteria. But we also characterized a gradual shift in the M. cinxia associated bacterial community over three decades of increasing temperatures and decreasing precipitations. This shift was not unidirectional for all bacterial taxa, as the dominant Telluria and Pseudomonas showed opposing responses to environmental trends. Additionally, the 2018 extreme drought, which triggered acute host population bottlenecks, was associated with a severe disruption of M. cinxia microbiota, and the loss of key Enterobacteriaceae taxa. However, the M. cinxia bacterial community seemed to be able to recover towards pre-drought structure in subsequent years, suggesting a degree of resilience to acute climatic perturbations in this microbial system.

Defined microbial communities (also known as synthetic communities) are showing promising results for plant health but are lacking an efficient lab to field transition. This is the due to limited knowledge on how to efficiently modulate plant microbiota by considering complex environments and multi-kingdom interactions. In this study, we aimed to better understand the transmission and impact of multi-kingdom synthetic communities (SynComs) from the seed to seedling stage. We constructed 20 different SynComs using both a priori and random approaches, from pool of diverse strains including 24 bacteria, 11 yeasts and 10 filamentous fungi. SynComs were inoculated on Brassica napus seeds and we monitored both transmission and impact on the microbiota of 15-day-old seedlings grown in non-sterile soil. Optimization of the inoculation protocol showed that alginate coating improved bacterial, yeast and filamentous fungi concentrations by more than 2 log compared with other approaches. With this inoculation method, we observed contrasted seedling colonization profiles, with SynComs members representing between 1.1-45.7% of bacterial community and 3.2-36.6% of fungal community. Our multiple SynCom design revealed that strain selection is a more critical determinant of SynCom performance than assembly strategy. Even randomly assembled communities performed well, as long as they are drawn from a pool of ecologically relevant, well-adapted taxa. Based on these evidences, we identified key bacterial and fungal traits explaining efficient seedling colonization such as high abundance on inoculated seed and low in vitro lag-time. Despite low colonization levels, we observed that SynCom inoculation altered seeding bacterial community assembly in 14 SynComs. A total of 82 native bacterial ASVs were identified as responsive to SynCom inoculation, most likely originating from the soil. This shift indicates that SynComs influence community assembly by modulating the recruitment of environmental taxa, especially when SynCom strains were more integrated in multi-kingdom network structures. Finally, we identified four distinct SynComs profiles which either colonized strongly or not seedlings while shifting of not native microbiota. Altogether, these findings provide actionable directions for improving SynCom design, suggesting that leveraging ecological processes such as host adaptation, optimal inoculation density, and network integration could enhance both colonization efficiency and plant phenotypic outcomes.

The rhizosphere microbiome plays fundamental roles in plant health and productivity, yet the ecological rules governing microbiome assembly remain poorly understood. Here, we investigated early rhizosphere community assembly in tomato using a replicated combinatorial community coalescence framework, in which seven distinct natural bacterial communities were inoculated individually and in all possible pairwise and triplet combinations. Single inoculum communities clustered according to inoculum identity, indicating a strong effect of source community composition on assembly trajectories. However, when all communities were analyzed jointly, samples formed a continuous compositional landscape with no clear evidence of discrete community states. Despite major differences in source community composition, rhizosphere communities consistently converged toward the same uneven rank abundance structure, with two ASVs accounting for 50% and a median of nineteen ASVs for 90% of total abundance. While assembly was dominated by a very small number of Pseudomonas ASVs, limited evidence of alternative dominant states was observed. Increasing inoculum complexity did not increase stochasticity but instead promoted stronger convergence toward a global rhizosphere compositional centroid. Moreover, dominance hierarchies emerging from community coalescence closely mirrored the distance of source communities to this centroid. Communities derived from orchard soils consistently showed the highest dominance, suggesting that historical contingency and prior adaptation to horticultural crop rhizospheres may influence competitive success. Together, these results support the existence of a canonical rhizosphere attractor in both community composition and abundance architecture, with patterns consistent with assembly occurring under a limited number of dominant ecological niches imposed by the tomato rhizosphere.

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.

Sponges are among the earliest-diverging metazoans, and their evolutionary success has been strongly linked to their symbiosis with microorganisms. While marine sponge-microbiome associations have been extensively characterized, freshwater sponges remain comparatively understudied, particularly in tropical systems such as the Amazon, where sponges undergo seasonal flood pulses and urbanization-derived disturbance. Here we characterized the microbiomes of two freshwater sponge species, Drulia brownii and Tubella paulula, from two contrasting sites in the Tapajos River (Amazon basin), a non-urbanized and an urbanized site, at two time points, the rainy and dry seasons. The whole metagenome was sequenced using a long-read shotgun approach, and metabarcoding was employed to characterize the gemmule microbiome. The analyses revealed that microbiome composition was primarily influenced by season, with urbanization exerting a secondary but significant effect. In non-urbanized rainy-season sponges, the microbiome was dominated by Pseudomonadales and Bacillales, whereas urbanized sponges showed more diverse profiles enriched with Burkholderiales, a possible symbiont. During the dry season, communities converged across sites, with Burkholderiales becoming the dominant taxon, including in gemmules. The 23 high-quality MAGs recovered revealed symbiosis-related genes, broad biosynthetic repertoires, and a distinctive carbohydrate-active enzyme profile. Functional analyses suggest that Pseudomonadales and Burkholderiales may play complementary roles across seasons, and Bacillales may be associated with organic matter turnover. These results show that Amazonian freshwater sponges harbor stress-sensitive but resilient microbiomes, with seasonality driving major compositional shifts and urbanization accelerating convergence toward Burkholderiales-dominated consortia, and highlight the central role of bacterial symbionts in nutrient acquisition, photoprotection, and chemical defense.

Microbial succession can be used to estimate the postmortem interval (PMI); however, the impact of spatial variability within the cadaver decomposition island (CDI) is not well understood. This study examined spatial variation in necrobiome communities where soil samples were collected over time and across spatial locations from the CDIs of two human body donors. Microbial communities were characterized using 16S rRNA sequencing and statistical modelling of variation and PMI were conducted. Necrobiome community metrics showed no significant differences across anatomical sampling sites within the CDI at a single timepoint. Temporal modelling identified 11 taxa with significant relationships to PMI in one donor, with spatial sampling having a minimal impact on the PMI relationships. Non-linear approaches also identified taxa with likely PMI signals in the second donor. These findings demonstrate that opportunistic sampling can capture robust linear and non-linear PMI signals in later decomposition stages.

Many wet tropical soils alternate frequently between fully oxygenated and anaerobic conditions, constraining the terminal electron acceptors available for microbial metabolism, and the mineral-organic matter interactions that regulate many aspects of soil carbon (C) cycling. However, it is still unclear how fluctuating soil redox conditions influence the microbial community composition and if microbially mediated C flux is sensitive to extended oxic or anoxic periods like those observed during drought and flooding respectively. Using a 44-day redox manipulation of tropical soils that experience daily-to-weekly oxygen (O2) fluctuations in the field, we measured how different redox regimes shape soil biogeochemistry and microbial and metabolite composition. Replicate microcosms were exposed to four treatments (static oxic, static anoxic, high frequency fluctuation (4 day oxic/4 day anoxic), or low frequency fluctuation (8 day oxic/4 day anoxic)) regimes, and harvested for microbial, metabolite, carbon dioxide (CO2) flux, and biogeochemical assays at multiple timepoints. Oxic and fluctuating redox conditions caused the microbial community to shift in a manner correlated with soil iron content and directly orthogonal to communities from anoxic soils. The identity of both iron oxidizers and iron reducers was distinct in static anoxic soils but was resilient to redox fluctuation and prolonged O2 exposure. The total amount of CO2 respired was similar across all four redox regimes. Water-extractable organic matter composition was distinct across redox treatments, with anoxic soils accumulating higher levels of carbohydrate-, proteins-, amino sugar-, and lignin-like compounds consistent with reduced enzymatic decomposition and release of mineral-associated organic matter via iron reduction, while oxic soils showed elevated lipid- and unsaturated hydrocarbon-like compounds indicative of greater microbial biomass turnover. The microbial community adapted to dynamic redox conditions and the results substantiate cycling of distinct C compounds under varying redox conditions resulting from varying bioavailability (driven by mineral-OM dynamics) and/or shifted microbial metabolism.

Bacteria inhabiting leaf surfaces - the phyllosphere - are crucial to plant health and ecosystem functioning. Methylobacterium is a taxonomically diverse, growth-promoting genus, ubiquitous on leaves. Different plant species host distinct Methylobacterium communities, but the genomic and functional basis of Methylobacterium symbiotic associations with particular host species remains poorly understood. Here, we used a metapangenomic approach to quantify the influence of host species on Methylobacterium assemblages, to identify genes potentially involved in Methylobacterium adaptations to host species, and to evaluate the contribution of Methylobacteriums accessory pangenome to these adaptations. We sequenced the metagenomes of 25 phyllosphere communities spanning five host species in a temperate forest in Quebec, Canada, and mapped these metagenomes onto Methylobacteriums pangenome to obtain nucleotide-level coverage and composition for each population on each individual host. We revealed strong divergences in the species- and gene-level community structure of Methylobacterium, driven by host phylogeny and plant form. Conifer communities were notably enriched in genes involved in amino acid, lipid, and carbohydrate metabolism; broadleaves, in genes involved in cell membrane, signalling, defense, and chemotaxis; and trees, in genes related to photosynthesis, oxidative phosphorylation, and translation. The shrub Corylus cornuta was a reservoir of Methylobacterium taxonomic diversity, and harboured numerous accessory genes under positive selection. Methylobacteriums accessory pangenome, evolving under weaker purifying selection, contributed importantly to gene-host associations, supporting its adaptive role. By linking genes to phyllosphere niches, our study shed light on the genetic basis of host adaptation and highlighted the crucial role of forest biodiversity in shaping microbial ecology and evolution.

Plant-associated microbiomes contribute to plant health and resilience, yet the extent to which host traits shape microbiome assembly remains poorly understood despite increasing interest in leveraging them for crop performance. Here, we investigated whether drought-response phenotypes are associated with reproducible patterns of microbiome assembly across peanut genotypes under field conditions. The cultivars represented three drought-response categories: water-savers with tighter stomatal regulation, water-spenders with deeper root systems, and drought-sensitive genotypes. Bacterial and fungal communities were characterized from bulk soil, rhizosphere, and root endosphere compartments of six non-stressed peanut cultivars. Both host genotype and drought-response phenotype were associated with microbiome composition, with phenotype-associated patterns remaining detectable across multiple genetic backgrounds. Unexpectedly, the strongest phenotype-associated differences occurred in bulk soil communities, suggesting plant-mediated effects extending beyond the immediate root zone. Community differences were driven primarily by shifts in the relative abundance of existing taxa rather than turnover of distinct microbial lineages. Fungal communities responded more strongly to host phenotype than bacterial communities, with water-spender genotypes supporting greater fungal diversity and uniquely enriched taxa in the rhizosphere and endosphere. Neutral community modeling indicated stronger deterministic filtering of fungi than bacteria. Together, these findings demonstrate that drought-response phenotypes shape reproducible microbiome variation before stress exposure. HighlightThis study investigates the potential for host phenotype-associated drivers of microbiome assembly in drought-tolerant peanut cultivars that represented different physiological mechanisms for drought tolerance.