Environmental Microbiome

Plant roots host defined microbial communities that differ from those found in the surrounding soil and these communities shift dynamically in response to plant development and environmental changes. Whilst it is widely accepted that root exudates play a key role in the assembly and dynamics of root-associated microbial communities, the underlying mechanisms are not well understood. This is partly due to a lack of controlled experimental systems that monitor both exudate- and microbiome-dynamics simultaneously. Here, we compared two microcosm systems commonly used in either root microbiome (clay particle-based) or root exudate studies (glass bead-based) for their suitability to simultaneously monitor both aspects. We evaluated these systems based on plant performance, bacterial growth, and time-resolved community and exudate profiling. In both systems, we reveal an exudate effect, characterised by higher bacterial diversity and Pseudomonas abundances in proximity to plant roots. While clay particles impeded exudate recovery, even when plants were removed from microcosms for exudate collection, the glass bead set-up allowed us to uncover dynamic exudate shifts during bacterial community establishment. This highlighted a transient increase of glucosinolates upon root colonisation by initially dominant Pseudomonas species. Overall, the comparison proved only the glass bead-based semi-hydroponic system to be suitable for the paralleled study of exudate and root microbiome dynamics.

Endophytic bacteria are increasingly recognised for their roles in plant health through symbiosis. However, methodological challenges, such as inconsistent root sterilisation, inefficient microbial DNA extraction, and co-amplification of plant organellar DNA, limit accurate characterisation of these communities, especially in wild grassland plants and non model plant in general. To address this, we developed and tested a streamlined protocol for bacterial endophyte detection from wild grassland plant roots, encompassing surface sterilisation of roots, DNA extraction, clamping of plant internal mitochondrial and chloroplast DNA, and 16S rRNA amplicon sequencing. Our approach minimises plant DNA contamination and yields high-quality microbial profiles. The protocol is adaptable and specific to grassland plant species, offering a standardised foundation for endophyte studies in wild and non-model plants. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/706108v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@157df36org.highwire.dtl.DTLVardef@1ff645aorg.highwire.dtl.DTLVardef@1580ecorg.highwire.dtl.DTLVardef@1c31b89_HPS_FORMAT_FIGEXP M_FIG C_FIG (Haskins and Ajaz, 2026) https://BioRender.com/47gd2xr

IntroductionCaves represent unique, nutrient-limited windows into the deep biosphere, yet the microbiology of the deep terrestrial subsurface remains remarkably under-explored. In this work, we took advantage of a rare expedition into Gourgouthakas Cave (Crete, Greece), one of the worlds deepest vertical systems, which had remained untouched by humans for 19 years. MethodsWe performed a high-resolution vertical profiling of the caves microbiome by sampling rock surfaces across nine different depths down to 1,100 meters. Through extensive cultivation using various media and temperatures, we established a biobank of 820 bacterial isolates. ResultsTaxonomic identification of a 362-isolate subset revealed a diverse community spanning 25 genera and 4 phyla, dominated by Pseudomonas, Bacillus, and Stenotrophomonas. Beyond characterizing diversity, we explored the biotechnological potential of these subterranean microbes against major agricultural threats. Screening 70 representative isolates against six key pathogens, including Ralstonia solanacearum, Verticillium dahliae, and Phytophthora nicotianae, uncovered a significant group of strains with potent antagonistic activity, particularly within the Pseudomonas and Brevibacillus groups. Genomic sequencing of cave-derived Actinobacteria (Streptomyces and Nocardiopsis isolates) further highlighted this potential, revealing 142 biosynthetic gene clusters (BGCs); notably, over half of these showed little to no similarity to known clusters, suggesting a hidden reservoir of novel secondary metabolites. Finally, ex vivo trials showed that the Pseudomonas sp. SRL917 isolate, significantly reduced Botrytis cinerea infections on tomato leaves, even surpassing the performance of a commercial biocontrol agent. DiscussionCollectively, our results demonstrate that deep karstic systems are not just geological wonders but vital hotspots for microbial innovation with tangible applications for sustainable agriculture.

Favoured by global changes, freshwater cyanobacterial harmful blooms generate major ecological, economical and public health challenges. Microcystis, one of the most widespread cyanobacterial genera, grows within a phycosphere where specialised interactions with its microbiome occur, and are suspected to influence bloom appearance and its potential toxicity. Using a combination of metagenomic, metabolomic and metabolic modelling, we characterised the phycospheres of twelve Microcystis strains isolated from a French pond. The distribution of metabolic reactions within Microcystis was consistent with their genospecies, whereas the metabolic landscape at the community level diverged from cyanobacterial phylogeny indicating functional decoupling between cyanobacteria and their associated microbiomes. Phycosphere-associated bacteria substantially expand the metabolic repertoire of the system, while maintaining functional redundancy within and across communities. On the other hand, metabolomic profiles were largely driven by cyanobacterial metabolic outputs. Metabolic modelling, together with the identification of toxic specialised metabolites produced by specific biosynthetic gene clusters, further highlighted differences in metabolic potential among phycospheres. Together, these findings deepen the understanding of Microcystis phycosphere functioning, demonstrate the value of multi-omics systems biology approaches, and underscore the ecological relevance of interspecies and inter-phycosphere metabolic interactions as a structuring process in bloom-associated microbiomes.

BackgroundThe root microbiome plays a critical role in nutrient acquisition, stress tolerance and overall plant health. Rice, a staple food for more than half of the worlds population, is commonly cultivated under flooded conditions. Despite its agronomical importance, our current understanding of root-associated microbiomes in rice grown under flooded conditions is limited. On the other hand, nitrogen (N) and phosphorus (P) fertilizers are routinely applied to maximize rice yield. It is also well known that root colonization by arbuscular mycorrhizal (AM) fungi enhances mineral nutrition in plants, but whether mycorrhizal associations influence the composition of the rice root microbiome remains poorly understood. In this study, shotgun metagenomic sequencing was used to characterize the root endosphere and rhizosphere microbiomes in two temperate japonica rice varieties (cv. Bomba and JSendra) grown under flooded conditions. The impact of colonization by the AM fungus Rhizophagus irregularis on the root microbiome was investigated. ResultsRoot-associated compartments harbour distinct microbial communities in rice with bacterial taxa comprising approximately 95% of the total microbia in rice roots. At the Phylum level, the root bacteriome was primarily composed of Pseudomonadota (Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria) followed by Actinomycetota. The fungal microbiome was dominated by Ascomycota (Sordariomycetes, Eurotiomycetes and Dothideomycetes) and Basidiomycota. Not only the root compartment, but also the host genotype can shape the root microbiome. Recruitment of specific microorganism mainly occurs at the species level. Genotype-specific and compartment-specific associations of microbial species in mycorrhizal rice roots were also observed supporting that root colonization by an AM fungus contributes to variations in the root microbiome. Further, key microbial species primarily associated to methane production and nutrient cycling (e.g. Phosphate Solubilizing Bacteria and Nitrogen cycling bacteria) colonizing root compartments in each rice genotype and mycorrhizal condition are described. ConclusionsThe rice genotype, root compartment and mycorrhizal condition markedly influence the microbiome in roots of rice plants growing in flooded rice fields. These findings illustrate the potential of the plant to shape its associated root microbiome, thus, offering valuable insights for the development of microbiome-based strategies to improve growth and performance in rice plants under flooded conditions.

Despite the successful cultivation of many microbes from rich bacterial communities inhabiting alkaline soda lakes, members of the bacterial phylum Verrucomicrobiota have so far been detected only through metagenomics. Here, we used alginate as a selective substrate to enrich and isolate two strains of haloalkaliphilic Verrucomicrobiota. The isolates share identical 16S rRNA gene sequences representing a new genus lineage, and, together with other metagenome assembled genomes, a new family within Opitutales. Cells of strains AB-alg1T (from soda lakes) and AB-alg4 (from soda solonchak soils) are small and motile cocci forming submerged colonies in soft alginate agar. They are saccharolytic heterotrophs growing aerobically on polysaccharides (alginate, starch and inulin) and sugars (glucose, fructose, mannose, sucrose, melezitose, maltose and cellobiose). They also grow anaerobically by fermentation of alginate and D-mannose and by coupling incomplete denitrification to oxidation of alginate. Both isolates are obligately alkaliphilic and moderately salt-tolerant. The dominant membrane phospholipids include phosphatidylcholines and diphosphatidylglycerols (cardiolipins). The genome of AB-alg1T features polysaccharide lyases of the PL6, 7, 15, 17, 38, and 39 families for depolymerization of alginate. Based on distinct phenotype and phylogeny, we propose classification of strains AB-alg1T (JCM 35393T=UQM 41574T) and AB-alg4 as Verruconatronum alginivorum gen. nov., sp. nov. within a new family Verruconatronumaceae. ImportanceThe presented isolates are the first isolated representatives of an environmental family of Opitutales, part of the core microbiome of alkaline soda lakes. These bacteria feed on polysaccharides. We present the key enzymatic machinery for the polysaccharide breakdown. These enzymes are high-pH tolerant and have potential for industry applications, for example in washing powders and biomass waste recycling.

The Imperial Palace in Tokyo serves as a significant reservoir of biodiversity within the urban landscape; however, its soil microbial communities remain uncharacterized despite decades of macro-biological surveys. This study presents the first dataset profiling the soil microbiome of the Imperial Palace Outer Gardens, utilizing both 16S rRNA amplicon and shotgun metagenomic sequencing to fill this knowledge gap. We collected bulk soil samples from four distinct sites, including pond sediments and soils beneath ginkgo and pine trees, to capture a range of environmental conditions within this conserved greenspace. Both 16S rRNA amplicon sequencing and shotgun metagenomic sequencing revealed that Pseudomonadota and Actinomycetota were the predominant phyla across all samples. Notably, sites with monoculture vegetation, such as those beneath pine trees, exhibited lower microbial diversity than other locations. Functional annotation identified core metabolic pathways and detected specific antimicrobial resistance and virulence factor genes in selected samples. These datasets provide a critical baseline for future research into urban ecosystem dynamics, soil health, and the intersection of environmental conservation and public health.

O_LIGlacier forefields in the high-desert region of Ladakh (northwestern Himalaya) are colonized by a variety of interdependent organisms, including lichens, prokaryotes, fungi, mosses, and vascular plants, along a successional gradient. Together with bulk soil, these hosts and their associated microorganisms form a broader microbial metacommunity (holobiome) whose structure, interactions and functions remain poorly underexplored in one of the Earths most extreme and climate-sensitive environments. C_LIO_LIUsing a multidisciplinary approach combining glacial chronosequence transects, GIS-derived topographic variables, soil properties, and plot cover measurements, we assessed the abiotic and biotic factors influencing bacterial and fungal communities sequenced from different hosts and bulk soil (hereafter sources). Microbial composition was primarily shaped by source identity, though certain sources, such as biological soil crusts (BSCs), mosses, and plant rhizospheres, also showed relationships with moraine age in either bacterial or fungal communities. C_LIO_LIBacterial and fungal community congruence was tested using Procrustes analyses, revealing that mosses maintained tightly coupled inter-kingdom relationships throughout the glacier forefields. However, the degree of congruence in plant rhizospheres and bulk soils was influenced by topographic variation and moraine age, respectively. C_LIO_LICo-occurrence network analyses revealed that early successional microbial communities were assembled more stochastically, with bacteria being more interconnected than fungi. In contrast, late successional stages were more compartmentalized, being more structurally stabile, likely driven by increased plant cover and functional redundancy among microbial taxa. C_LIO_LIKeystone bacterial and fungal taxa were identified in plant rhizospheres and bulk soil using a dual-criteria approach related to inter-kingdom congruence and network node eigenvalues. Furthermore, some of these taxa were associated with environmental factors, suggesting topographic heterogeneity and successional age can promote or deter the influence of keystone taxa. C_LIO_LISynthesis: This study reveals the impact of both macroorganism colonization (i.e. plants, mosses, and lichens) and microcommunity establishment (BSCs and bulk soil), as abiotic and biotic sources, on microbial metacommunity assembly in glacier forefields. By adopting a broader approach across different spatial scales, we demonstrate that while plant colonization plays a central role in shaping microbial metacommunities, its effects are modulated by topographic variation along the chronosequence. C_LI

O_LIVolatile organic compounds (VOCs) emitted by soil bacteria influence interactions with other soil microbes and with plant roots. While their potential as plant-growth promoters is well recognized, their role in promoting plant resilience to abiotic stress and the underlying molecular mechanisms remains poorly understood. Here, we investigate the role of Pseudomonas VOCs in enhancing plant resilience to drought stress. C_LIO_LIArabidopsis thaliana plants were exposed to VOCs emitted by Pseudomonas strains under both control and osmotic-stress conditions. VOC exposure generally enhanced plant growth, and this effect was even more pronounced under both drought and salt stress. Transcriptomic analysis revealed that VOC exposure modulates key stress-responsive pathways, including those related to abscisic acid biosynthesis and signalling, sugar transport, iron uptake, aliphatic glucosinolate biosynthesis, and plant defences. Using Arabidopsis mutants, we identified abscisic acid and aliphatic glucosinolates as important components in mediating the plant response to VOCs. SWEET11/12 sugar transporters and ABA signaling genes were downregulated by VOCs exposure, in order to allow for a positive regulation of lateral root numbers (in case of SWEET genes) and plant growth in general under drought stress. In summary, using metabolomics, transcriptomics and functional analysis, we showed a negative cross-talk between the effects of VOCs on plant growth and glucosinolate production, whereas a positive interaction was observed between the biosynthesis of coumarins and VOCs. C_LIO_LINotably, VOCs also improved drought tolerance in soil-grown Brassica oleracea plants. We showed that VOC treatment altered the root-associated microbiome under drought, leading to a community composition more similar to that of well-watered plants. C_LIO_LIOur results show that Pseudomonas emitted VOCs can promote plant growth under drought conditions, linked to root transcriptional reprogramming and direct or indirect microbiome modulation. C_LI

Stentor is a genus of large ciliates that can be found in ponds, lakes, rivers, and fresh waters all over the world. Since their initial discovery in 1744, Stentor strains have been isolated from all populated continents. To date, over 50 individual strains have been identified, yet not a single isolate from a marine environment has been verified. Over 200 years since the initial description of the Stentor genus, our study entails the first concrete discovery of a fully marine Stentor species, as evidenced by its morphological, ecological, and phylogenetic positioning amongst Stentor. This new marine organism, which we have named Stentor hondawara, was verified to be a new species of the Stentor genus that appears to have fully adapted to a uniquely marine lifestyle in a high-salinity environment. Using comparative genomics analysis between the whole-genome sequences of Stentor hondawara and two freshwater species of Stentor, we further detected several intriguing differences in the enrichment of gene orthologs between the marine Stentor hondawara and the freshwater species, Stentor coeruleus and Stentor pyriformis. The gene groups specifically enriched in Stentor hondawara encode a variety of proteins, including ion channels, pH-responsive proteins, osmoprotectants, amino acid biosynthesis enzymes, and signaling receptors. Additionally, using metagenomics, we detected and isolated, from within our initial genome assembly, the genome of a novel marine bacteria, which we propose is an endosymbiont of Stentor hondawara. This bacterial species is an uncharacterized member of the order Rhodospirillales and appears to be a nutritional factory for the host Stentor hondawara. Taken together, our study provides insight into how Stentor hondawara adapted to a marine environment distinct from the habitats of all the other currently known Stentor species living in freshwater.

BackgroundBuilt environment microbiome studies have identified numerous factors that shape indoor microbiomes, yet the reproducibility of these findings across buildings, timepoints, and research groups remains unclear. Differences in sequencing protocols, sampling design, and environments pose major challenges for cross-study comparisons, particularly in low-biomass environments where technical variation can obscure biological signal. To address this gap, we constructed a simple ontology which groups samples into one of three categories: hand, hand-associated surfaces, and floor then applied it to four publicly available 16S rRNA gene datasets: a hospital, university dormitory, Air Force dormitory, and private residential houses. ResultsWe identified strong and reproducible separation between floors and surfaces with frequent human contact. We found that floors consistently harbored soil-associated taxa, including KD4-96, 67-14, Skermanella, and Sphingobacterium, whereas hands and hand-associated surfaces were enriched with skin-associated genera, such as Lawsonella and Cutibacterium. Within studies, these results were generally consistent across timepoints. Across studies, mixed-model PERMANOVA analysis revealed significant clustering by sample type, with modest effects of study, suggesting that biological signal outweighed differences in laboratory or sequencing methods. Leave-one-study-out random forest models achieved high AUCs for hand vs. floor comparisons (0.865 to 0.921), moderate AUCs for hand-associated vs. floor comparisons, and weaker performance for hand vs. hand-associated comparisons. Application of the batch-correction method DEBIAS-M did not improve effect sizes or classification performance, indicating that reproducible structure was already discernible without batch adjustment. ConclusionsDespite substantial temporal and environmental heterogeneity among studies, we found that the built environment microbiome has a reproducible bacterial signal. There was consistent enrichment of soil-derived taxa on floors and human-associated taxa on hands and hand-associated surfaces suggesting a stable microbiome despite differences in building type, occupancy, and methodology. These findings establish an important foundation for future studies, suggesting cross-study comparability, the accuracy of ecological inference, and the ability to support the development of predictive applications in indoor microbiome research.

BackgroundMicrobial communities in the rhizosphere are key drivers of plant immunity, mediating plant responses to stress. Under specific stresses plants are capable of recruiting beneficial microorganisms into their rhizosphere with the potential to alleviate these stresses. Among these stresses, herbivorous pests remain a major agricultural challenge. Despite this, the impact of leaf herbivory on root-associated microbiomes, and how this impact can shape plant defense phenotypes are still understudied. In this study, our main objective was to determine the extent to which leaf herbivory affects the rhizosphere microbiome, and whether and how these herbivory-induced changes modulate plant defense phenotypes through plant-soil feedback. To that end, we designed a two-phase assay in which we challenged sunflower (Helianthus annuus L.) with Spodoptera exigua and later tested the effect of the microbial legacy after infestation on sunflower defense phenotype, considering resistance and tolerance as major drivers. ResultsWe found that herbivory triggered significant changes in the bacteriome structure and dynamics, and microbiome functional profile, while effects on mycobiome were comparatively less pronounced. Under herbivory, several bacterial taxa and functional groups were enriched, the bacterial co-occurrence network was more complex and assembly processes were slightly more stochastic. Furthermore, after evaluating the plant-soil feedbacks of herbivory-induced microbiomes we observed no effect on plant resistance proxies such as herbivore growth and survival, and leaf phenolic and flavonoid content. We did observe differences on tolerance proxies, while plants grown on herbivore-challenged microbiome were overall smaller, the biomass loss to herbivory was significantly lower while the elemental nutrient content and photosynthetic pigments content was enhanced. ConclusionsOur study demonstrates that insect herbivory by S.exigua reshapes sunflower rhizosphere microbiome and generates a soil legacy that promotes herbivory tolerance on subsequent plant generations. This highlights the broader potential of microbiome-mediated plant-soil feedbacks in shaping plant adaptation to herbivory.

Mycorrhizal fungi form essential mutualisms with the majority of land plants, yet their role on plant community composition and diversification remains poorly understood. Orchids provide a model system for studying these interactions because the orchid life cycle obligatorily depends on mycorrhizae. Historically, studies have emphasized the role of niche partitioning and competition avoidance resulting in distinct mycobiome compositions among coexisting orchids. Since closely related orchid species have been found to associate with similar groups of fungi, it has been speculated that different fungal species are needed for coexistence but not for speciation. However, fungi have often been examined at lower resolution levels (i.e., class, order, family, or genus). Using third-generation long-read sequencing (PacBio) of the full ITS region, we characterized fungal communities at fine scale with high accuracy. We evaluated alpha- and beta-diversity of fungal communities in four closely related, narrowly endemic epiphytic orchid species from the rapidly diversifying genus Lepanthes in one of the worlds richest biodiversity hotspots. Our analyses reveal that orchid species have distinct mycobiont assemblages, with differences in composition unevenly distributed across species. These results suggest that shifts in fungal partners may contribute to speciation and rapid diversification in Lepanthes. This study highlights the potential evolutionary role of mycorrhizal fungi in orchid diversification and demonstrates the value of high-resolution sequencing in uncovering cryptic fungal diversity.

Microbial diversity remains largely unexplored across environments and scales, notably because at local scales many microbial taxa exist under a dormant state. Microbial biogeography is shaped by edaphic and ecological drivers, and shifts in microbial community composition are frequently associated with host community structure and health. Nontuberculous mycobacteria represent a striking example of environmental microorganisms with opportunistic pathogenic potential. Unfortunately, data on their diversity, distribution, and ecological interactions in aquatic environments remain limited. However, understanding competition for niche space and the role of abiotic and biotic factors shaping their biogeography is crucial for predicting disease emergence and transmission. Here we aimed at i) identifying microhabitat abiotic and biotic drivers influencing their distribution, ii) assessing the predictability of their diversity and distribution across continents, and iii) examining potential exclusion or associations between pathogenic and nonpathogenic mycobacterial species. By deploying an eDNA-based metabarcoding approach from freshwater samples collected in urban and rural sites in French Guiana and Cote dIvoire, we have boosted our understanding of environmental mycobacteria ecology by highlighting the influence of habitat type, abiotic factors, and microbial interactions on mycobacterial distribution. In addition, the detection of pathogenic species further highlighted the importance of environmental reservoirs in mycobacterial disease transmission.

Ophiostomatoids are an ecological group of microfungi that commonly associate with bark and ambrosia beetles. As well as being insect symbionts, they play significant ecological roles as plant pathogens, and include species responsible for major forest tree diseases. Despite their ecological similarities, ophiostomatoids are distributed across two quite distantly related orders, the Microascales and Ophiostomatales. Historically, these fungi were considered a single natural group; however, molecular studies have revealed their independent origins and convergent ecological strategies. Previous phylogenetic studies of these fungi have typically focused on resolving taxonomic issues or understanding individual lifestyles, such as beetle-cultivated ambrosia lineages or vascular wilt pathogens. As a result, we lack a comprehensive phylogenetic framework that integrates dense species-level sampling with ecological data across both orders. Such frameworks are essential for understanding the broader phylogenetic and ecological context in which key fungal lifestyles have evolved. Here, we assembled and analysed all available sequence data for the Microascales and Ophiostomatales from seven widely used fungal marker loci to reconstruct a densely sampled phylogeny for each order. We evaluated locus performance and showed that whilst individual loci fail to resolve many taxa, concatenated datasets produce robust, well-supported topologies consistent with published genomic studies. By mapping ecological traits onto these trees, we show that lifestyle diversity and beetle associations are much more variable in the Microascales than in the Ophiostomatales, despite comparable species richness. Presenting both orders together provides a unique comparative perspective on the ecology and evolution of ophiostomatoids. As metabarcoding datasets of ophiostomatoids become increasingly common, this integrative framework can offer a valuable resource for environmental sequence identification and investigating fungal lifestyle switches, which in turn can support future biodiversity and ecology studies.

Mucoromycotina fine root endophyte (M-FRE), although commonly present in cultivated crops, represent a largely overlooked symbiosis, and their diversity and ecological functions under natural field conditions remain poorly understood. The co-occurrence of M-FRE and Glomeromycotina arbuscular mycorrhizal fungi (G-AMF) was assessed in field-grown durum wheat (Triticum turgidum subsp. durum (Desf.) Husn.), testing the effects of combined water and nitrogen limitation on root colonization and fungal community diversity in roots, rhizosphere, and extra-radical hyphae. The M-FRE colonization was reduced under combined water and nitrogen stress but was unaffected by wheat genotype. In contrast, G-AMF colonization varied among genotypes and was insensitive to this combined stress. While G-AMF colonization correlated with root traits, M-FRE abundance was rather determined by soil properties and the applied stress. Remarkably, under stress, M-FRE but not G-AMF colonization correlated with nitrogen and phosphorus uptake in plant shoots. Partial 18S metabarcoding detected 74 G-AMF taxa and 12 M-FRE taxa, some shared across compartments, revealing active growth of M-FRE extra-radical hyphae. Stress had contrasting effects on diversity: G-AMF alpha diversity remained stable, whereas M-FRE diversity declined, with stress driving distinct community structures for both groups. These results indicate that M-FRE and G-AMF are shaped by divergent drivers, highlighting functional differences between these morphologically similar symbioses.

Endophytic microbiomes of crop wild relatives (CWRs) adapted to extreme environments, such as halophytes, are promising sources of plant-beneficial bacteria and secondary metabolites for sustainable food production. Here, we analyzed 25 Bacilli isolates obtained from CWRs, halophytes, and other plant species in Crete, Greece. Using a hybrid Illumina-PacBio sequencing approach, we generated high-quality genomes and performed comparative genomics, phylogenetic, and pangenome analyses, complemented by in vitro assays. We identified 312 biosynthetic gene clusters (BGCs), nearly 60% of which showed no similarity to known clusters, revealing extensive unexplored biosynthetic potential. These unique BGCs may constitute an adaptive feature enabling endophytic Bacilli to colonize and interact with host plants. The isolates spanned diverse genera (Bacillus, Paenibacillus, Peribacillus, Neobacillus, Cytobacillus, Rossellomorea), including three novel species. Phenotypic assays of our isolates demonstrated high salinity tolerance (up to 17.5% w/v NaCl) and strong antagonism against major bacterial and fungal phytopathogens. Genome mining further revealed a broad array of putatively plant-beneficial traits related to growth promotion, stress adaptation, host interaction and inhibition of pathogens. Together, these findings show that Bacilli endophytes from wild and halophytic plants possess exceptional phylogenetic novelty, functional diversity, and biosynthetic capacity, providing new genomic and ecological insights into Bacilli associated with plants inhabiting extreme environments.

Urbanisation is rapidly increasing worldwide, with increasing attention focused on its consequences for human populations and the environment. Despite the importance of outdoor urban environments for biodiversity and human wellbeing, their microbial ecology remains poorly characterised, particularly in relation to emerging microbial threats including antimicrobial resistance (AMR). Here, we present a citywide metagenomic study of outdoor public surfaces across Liverpool, United Kingdom, examining microbial community composition, diversity, and antimicrobial resistance gene (ARG) distribution across five distinct surface types. We show that patterns of human activity and surface use strongly influence both microbial community structure and AMR signatures in outdoor urban environments. Touchpoints were enriched for human-associated taxa and exhibited the highest overall resistome burdens, whereas Pathway and Waterside niches showed no strong taxonomic enrichment and exhibited low ARG prevalence. Refuse surfaces showed mixed patterns, characterised by sporadic but occasionally high-abundance ARG detections. Soil harboured the most distinct microbial communities but showed minimal ARG prevalence, which may partly reflect the limited representation of environmental taxa in current ARG databases. This study provides a baseline for understanding how urban infrastructure and behaviour shape microbial and resistance landscapes, and highlights the value of outdoor metagenomic surveillance for future environmental and public health research.

Soil microflora is fundamental to ecosystem functioning, yet their contribution in Miyawaki afforestation, a globally implemented ecological engineering approach, remains poorly characterized. In the present study, we examined the bacterial taxonomic diversity and their functional potential in a peri-urban Miyawaki mini-forest and compared with a nearby grassland the pre-existing ecosystem across dry and wet seasons. The Miyawaki plantation comprised of highly diverse native trees, sub-trees and shrubs spanning evergreen and deciduous varieties, potentiating nitrogen-fixation, diverse litter generation and rooting strategies resulting in pronounced functional heterogeneity. Notably relative to grassland, the Miyawaki forest was intensively managed and supplemented with organic amendments, and supportive irrigation, buffering the seasonal moisture stress. Using 16S rRNA amplicon sequencing of soil eDNA, we characterized seasonal variation in soil bacterial communities in both the systems. The observed soil bacterial community organization in forest as compared to the unmanaged grassland indicates combined influence of vegetation structure, dense canopy cover, continuous litter generation and root exudates. Microbial assemblages in the forest specialised in heterotrophic complex carbon degradation, biofilm formation, exopolysaccharide production and sporulation pathways which suggests adaptive abilities to anoxic microsites and other stressful conditions. In contrast, grassland soils harboured less diversified bacterial communities dominated by phototrophic and oxidative stress adaptation pathways consistent with sun lit, non-irrigated and moisture-variable conditions. Nonetheless, functional divergence in dry season reflects temporal reorganization of microbial communities marking a gradual trend towards soil ecosystem development. Together, these findings establish microbial baseline for Miyawaki forests revealing how tree-dense mini-forests restructure soil bacterial communities relative to grasslands highlighting the value of identifying soil microbial indicators for critically evaluating urban afforestation outcomes over extended time scales to inform sustainable design and policy. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/704982v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@15e1bb1org.highwire.dtl.DTLVardef@16c1dddorg.highwire.dtl.DTLVardef@11c99ecorg.highwire.dtl.DTLVardef@bd6d1d_HPS_FORMAT_FIGEXP M_FIG C_FIG Schematic representation of the study depicting the Miyawaki forest and nearby grassland.

O_LIPhyllosphere microbiomes are subject to microbial import from various sources and undergo substantial changes during phenological changes of plants. However, these processes are still poorly understood for forest canopies. We propose that phenology-driven changes in host properties, and rainwater-mediated, within-canopy transport shape the phyllosphere microbiome in temperate forests. Leaves and throughfall samples were collected from oak, ash and linden trees at top, mid, and bottom canopy positions at the Leipzig canopy crane facility (Germany) at time points representing early, mid and late phenological stages. Bacterial community composition was assessed by 16S rRNA gene amplicon sequencing. C_LIO_LIPhenological stages explained 19% of phyllosphere bacterial community variation, followed by tree species identity (12%) and canopy position (2%). Later phenological stages exhibited more homogeneous and functionally redundant phyllosphere communities along with a strong decline of plant pathogens and increasing potential for microbially mediated biocontrol mechanisms. Throughfall transported up to 1011 bacterial cells per litre with maximum bacterial fluxes at the canopy top. C_LIO_LIOur findings demonstrate that in temperate forests, phenology-driven effects on the phyllosphere microbiome are far more important than tree species specific effects. Extent and selectivity of throughfall-mediated mobilization may play a crucial role for the spatial heterogeneity of microbial communities in tree crowns. C_LI