The ISME Journal

1.Structured Abstract1.1 AbstractSoybean red crown rot, caused by the soil-borne fungus Calonectria ilicicola, causes substantial yield losses, but the response of the root-associated bacterial microbiome remains poorly understood. Here, we combined 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to characterize bacterial communities in soybean root-associated soils. 16S rRNA gene sequencing showed that diseased plants had rhizosphere and, more strikingly, rhizoplane microbiomes distinct from those of healthy plants, often with increased Enterobacterales. Shotgun metagenomics further revealed enrichment of genes associated with antibiotic resistance, particularly cationic antimicrobial peptide resistance, in diseased rhizoplane samples. Single-cell genomics recovered seven nonredundant Enterobacterales genomes and showed that plant pathogenicity-related genes were broadly distributed across these lineages. In contrast, dlt genes, which are associated with cationic antimicrobial peptide resistance, were detected only in the Enterobacterales lineages enriched in diseased rhizoplane soils. These results support a model in which soybean red crown rot is accompanied by microbiome restructuring and opportunistic enrichment of specific Enterobacterales lineages carrying putative cationic antimicrobial peptide resistance genes. More broadly, this study highlights the value of strain-resolved single-cell genomics for linking disease-associated community shifts to specific bacterial traits. 1.2 ImportanceUnderstanding crop disease requires resolving not only the primary pathogen but also the root-associated bacteria that respond to infection. Here, we used 16S rRNA gene sequencing, shotgun metagenomics, and single-cell genomics to examine the soybean rhizoplane microbiome under red crown rot. Diseased plants showed reproducible shifts in bacterial composition, including frequent enrichment of Enterobacterales and antimicrobial resistance-related functions. Strain-resolved genomes further revealed that the Enterobacterales lineages enriched in diseased rhizoplane soils specifically carried putative dlt-mediated resistance to cationic antimicrobial peptides, whereas general pathogenicity-related genes were broadly shared. These findings suggest that host defense-associated selection, rather than pathogenicity genes alone, may help shape disease-associated root microbiomes. This study demonstrates how single-cell genomics can uncover strain-level traits hidden within bulk community data and thereby clarify plant-pathogen-microbiome interactions.

Eukaryotes originated from the symbiosis of an Asgard archaeon, the alphaproteobacterial ancestor of mitochondria, and possibly additional bacterial contributions. This transition occurred in redox-transition environments such as microbial mats or shallow sediments [~]2 billion years ago, when atmospheric oxygen was far lower than today. We investigated Asgard-enriched microbial mats from the low-oxygen, sulfidic Catherine volcano lake (Afar region, Ethiopia), mimicking early Proterozoic conditions. 16S rRNA gene metabarcoding, metagenomics, and metagenome-assembled genome analyses across redox-stratified layers of in situ and mesocosm-maintained mats revealed that Asgardarchaeota thrived in the sulfate-reduction zone, mainly co-occurring with Desulfurobacterota-Myxococcota, among others. Lokiarchaeia and Thorarchaeia preferred anoxic layers. Within Heimdallarchaeia, Heimdallarchaeales were enriched in upper layers, correlating with oxygen-tolerant hydrogenase and sulfate-reduction genes, and Hodarchaeales, in anoxic layers, correlating with methanogenesis. Although reactive-oxygen-species defense mechanisms were widespread, Asgardarchaeota lacked aerobic respiration. These results support the idea that Asgard archaea engaged primarily in syntrophic interactions with sulfate-reducers under early-Earth-like conditions.

Members of the UBA868 group within the order Arenicellales are globally distributed marine Gammaproteobacteria predicted to participate in sulfur and carbon cycling, yet their physiology and ecological roles remain unknown due to the absence of cultured representatives. Here, we report the isolation and characterization of the first heterotrophic representative of the previously uncultured UBA868 group. Using dilution-to-extinction cultivation, we obtained four isolates from the Yellow Sea whose high-quality genomes represent a single UBA868 species. One strain, IMCC57338, maintained in axenic culture, exhibited small coccoid morphology and slow growth (doubling time [~]2.9 days), consistent with an oligotrophic lifestyle. Genome analysis revealed a predominantly aerobic chemoorganoheterotrophic lifestyle with a streamlined central carbon metabolism, including a complete glyoxylate shunt and limited carbohydrate utilization capacity, suggesting adaptation to low-nutrient conditions. The genome also encodes pathways for methylated amine oxidation coupled to formaldehyde assimilation via the serine cycle, indicating a capacity for methylotrophy. Genes encoding sulfur oxidation (Sox) and reverse dissimilatory sulfite reductase (rDsr) pathways further suggest a capacity for sulfur-based chemolithoheterotrophy. Global metagenomic and metatranscriptomic read recruitment showed that the species represented by IMCC57338 is widely distributed across ocean basins and pelagic depth layers, with higher abundance and transcriptional activity in mesopelagic waters. Our findings provide the first physiological and genomic insights into the UBA868 group and suggest that members of this lineage contribute to the cycling of organic carbon, C1 compounds, and sulfur in marine ecosystems.

Succession is an ecosystem building process in which a habitat and its community interact predictably by increasing diversity, habitat engineering, and ultimately reaching a climax community, where other ecological processes influence its dynamic. Key to succession is the establishment of primary producing habitat forming species, which drives niche differentiation leading to increasing diversity. Here, we use the primary colonizing and habitat forming seagrass, Halophila ovalis, to demonstrate that it drives bacterial succession in a meadow ecosystem, and its microbiome, both rhizoplane and phylloplane, are under host selection. Many of the characteristics attributed to plants for habitat modification are microbial processes such as nitrogen fixation and sulfide detoxification and succession is often extrapolated to such processes. To determine if succession (increasing diversity) or selection (reducing diversity) drives changes in diversity (16S rRNA gene) or habitat modifying processes (nifH, soxB, aprA, dsrA), molecular analysis was performed along chronosequences (as a proxy for succession) of seagrass patches. Bacterial communities were sampled within the meadow ecosystem and the microbiomes of H. ovalis (both rhizoplane and phylloplane). Genes involved in biogeochemical cycling are differentially impacted within the microbiome and meadow sediments, with only nifH under succession. All genes from all niches sampled for community analysis are under directional community trajectories, despite being subjected to distinct ecological processes, signifying that many ecological processes, including succession and host association, drive community assemblage.

Bacterivorous protists are central to aquatic food webs, mediating the transfer of carbon and nutrients to higher trophic levels through the microbial loop. In natural communities, a major challenge remains in linking protist grazing activity to environmental sequences and identifying which taxa are actively feeding at the community level. Here, we present the first application of quantitative stable isotope probing (qSIP) in a grazing experiment. By combining qSIP with 18S rRNA gene amplicon sequencing, we linked prey assimilation to the identity of active protist predators at the operational taxonomic unit (OTU) level. In a replicated 36-h bottle-experiment, live 13C, 15N-labeled Limnohabitans planktonicus cells were added to natural samples from a lake pelagic site and its main inlet stream. Although hydrologically connected, protist richness was higher in the inlet than in the lake, yet a similar number of taxa incorporated prey biomass, comprising 108 OTUs in the inlet and 107 OTUs in the lake, including both rare and abundant taxa. Of these, 26 OTUs were labeled at both sites. The most strongly labeled protist in the inlet was a putative phago-mixotrophic prasinophyte, whereas in the lake it was an uncultured chrysophyte. Across sites, prey incorporation occurred in a broad range of taxa, including heterotrophs (e.g., choanoflagellates, cercozoans, ciliates, centrohelids), putative mixotrophs (e.g., cryptophytes, chrysophytes, dictyochophytes), parasitic protists and fungi. These results demonstrate the potential of qSIP to resolve trophic interactions at fine taxonomic resolution in natural communities and highlight new opportunities to study complex microbial food webs across environmental systems.

Nitrogen fixing legume nodules are typically viewed as the product of a bilateral mutualism between host plants and nitrogen-fixing rhizobia, yet nodules also harbor diverse non-rhizobial endophytes whose functional significance remains poorly understood, especially in wild legumes and uncultivated soil. Here, using the wild Mediterranean shrub Calicotome villosa, we performed a soil inoculation experiment to test whether plant performance is linked to the functional composition of the nodule microbiome. Soil inocula from different natural sites produced strong differences in nodulation success, plant biomass, leaf nitrogen concentration, nitrogen fixation rate, and nodule allocation under otherwise uniform conditions. Although Bradyrhizobium dominated all nodules, species composition varied among inoculation sources, and non-rhizobial endophytes reached substantial abundance in some treatments. Functional profiles of the nodule microbiome were significantly associated with plant phenotype, with the strongest coupling observed for traits related to nodule investment. Targeted and genome-wide analyses identified trait-associated genes in both symbionts and endophytes, including genes involved in nitrogen cycling, ammonium transport, denitrification, pyrimidine degradation, sulfur assimilation, and type VI secretion systems. Several of these functions were not part of the canonical symbiosis machinery, yet were strongly associated with plant nitrogen status, biomass accumulation, or nodule mass fraction. Together, our results show that legume performance is better predicted by the collective functional composition of the nodule microbiome than by the primary symbiont alone. These findings support a broader view of nodules as multipartite microbial communities.

Plant leaves harbor diverse microbial communities influenced by environmental inputs and host traits, yet it remains unclear whether leaves act as passive substrates or active ecological filters that reorganize microbial functional capacity. Phylloplane pH regulation is one hostplant trait that has been traditionally underexplored. We used metatranscriptomics to examine microbial gene expression on the phylloplane and within whole leaves of five plant species spanning the extremes of baseline phylloplane pH, including hyperalkalinizing Gossypium species, weakly buffering Beta vulgaris, and hyperacidifying Nepenthes species. Young leaves were inoculated with a common soil-derived microbial community to quantify host-associated restructuring of taxonomic and functional profiles, and short-term pH perturbations were applied to test the effect of transient abiotic stress. Across both phylloplane and whole-leaf datasets, host species identity was the primary axis structuring microbial taxonomic composition and expressed functional repertoires. Leaf-associated communities diverged from the source inoculum, but retained a substantial shared functional backbone enriched for central biosynthetic and core metabolic pathways. Host-associated differentiation reflected selective retention and redistribution of reactions within this shared environmental pool rather than acquisition of novel metabolic capacity. Enriched pathway subsets were metabolically coherent and taxonomically distributed across multiple bacterial orders, consistent with functional redundancy and trait-based assembly. Among hosts, Gossypium exhibited the strongest restructuring relative to inoculum, suggesting comparatively stronger host-associated filtering. In contrast, short-term pH manipulation did not induce consistent community-wide functional reorganization. Microbial physiological responses to the phylloplane environment and external pH were observed at the organismal level. Together, these results position leaves as active ecological filters that reorganize microbial functional landscapes through host-specific trait regimes. This work begins to implicate some role of phylloplane pH regulation in microbial assembly and function.

Cross-feeding interactions are pervasive in microbial communities and profoundly shape community structure, stability, and function. While previous studies have explored how cross-feeding affects evolvability, this work has predominantly focused on bidirectional mutualistic interactions in engineered auxotrophic systems where both partners reciprocally exchange essential metabolites. However, most metabolic interactions in natural microbial communities are unidirectional, with organisms feeding on the metabolic waste products of other species. Our study addresses this gap by examining how a unidirectional cross-feeding interaction affects the evolutionary dynamics of both the producer (Acinetobacter johnsonii) and consumer (Pseudomonas putida) over 800 generations of experimental evolution. We found that co-culture constrained adaptive evolution in both species. Co-cultures exhibited lower {pi}N/{pi}S ratios (0.75 for P. putida; 1.04 for A. johnsonii) than monocultures (1.44 and 2.02, respectively) indicating stronger purifying selection against nonsynonymous mutations in the community context. Lineage tracking through whole genome sequencing of populations and clones revealed greater lineage diversity and complexity in monocultures, with more mutations showing significant parallelism across replicate populations. Additionally, P. putida evolved increased dependence on its partner; co-culture-evolved P. putida grew significantly worse than its ancestor when A. johnsonii was removed. These findings demonstrate that ecological interactions fundamentally reshape fitness landscapes and constrain adaptive evolution even when fitness benefits are unidirectional, with implications for understanding microbial community stability and predicting evolutionary dynamics in complex communities.

Desulfovibrio spp. are associated with inflammatory diseases and human health, yet limited representative genomes and isolates hinder our understanding of their role in disease. Here, we assembled a comprehensive database of 2,658 Desulfovibrio genomes across 90 diseases and 32 countries, including 24 human isolates. Genomic analyses showed extensive species diversity and revealed disease-associated functional traits, including flagellin and virulence genes (i.e. ureases). Flagellin-mediated Toll-like receptor 5 activation was species-specific and D. desulfuricans flagellin downregulated TGF-beta signalling in murine small intestinal organoids, suggesting impaired immune tolerance. Additionally, we investigated genomic capacity for hydrogen sulfide (H2S) production, a main Desulfovibrio metabolite. While health- and disease-associated Desulfovibrio spp. mainly encoded dissimilatory sulfate reduction, tetrathionate metabolism-encoding bacteria were exclusively detected in inflammatory bowel diseases, including Proteus mirabilis and Morganella morganii. Overall, our study provides a comprehensive genomic Desulfovibrio resource and identifies new links associating strain variation, functional traits and H2S-production with inflammatory diseases.

Plastic biodegradation in natural environments is increasingly recognized as a multi-organism process, yet the mechanisms enabling coordinated depolymerization and metabolism of polyethylene terephthalate (PET) remain poorly understood. Previously, we demonstrated that a full consortium containing three Pseudomonas and two Bacillus strains isolated from hydrocarbon-rich coastal soils of Galveston Bay, Texas, can synergistically depolymerize PET plastic and utilize it as a sole carbon source, a capacity not observed in individual isolates. In this report, using integrated comparative genomics, proteomics, and chemical analyses, we show that PET degradation in this system reflects exaptation of hydrocarbon metabolism reinforced by metabolic division of labor. Within this naturally occurring consortium, Bacillus strains persist under environmental stress, establish biofilms, and perform essential secondary hydrolysis, while Pseudomonas strains catabolize aromatic monomers and buffer oxidative stress. Genes supporting these functions are enriched within the accessory genomes of the consortium strains, indicating consortium-enriched horizontal gene transfer (HGT). In addition to the canonical two-step hydrolytic pathway well documented in PET biodegradation, we identify a secondary methylation-and redox-associated process, mechanisms where the full consortium acts on the oligomer mono(2-hydroxyethyl) terephthalate (MHET), yielding nearly complete conversion to terephthalic acid (TPA) and methylated MHET (MMHET). Together, these findings demonstrate how cooperation and competition within consortia facilitate targeted gene exchange, enabling emergent plastic biodegradation in natural microbial communities. IMPORTANCEEnvironmental plastic degradation is rarely accomplished by a single organism, yet the microbial mechanisms enabling community-level PET plastic breakdown remain poorly understood. This study shows that a bacterial consortium isolated from crude petroleum-contaminated beaches biodegrades PET through exaptation of ancestral hydrocarbon pathways, metabolic division of labor, and targeted gene exchange rather than specialized PET-specific metabolic pathways. Pseudomonas strains initiate PET cleavage, while stress-tolerant Bacillus strains persist long enough to clear inhibitory intermediates and enable downstream aromatic and diol metabolism. PET degradation is observed to be an emergent property of ecological interactions and distant evolutionary history. These findings provide a community-level model for understanding how natural microbial communities may adapt to novel anthropogenic substrates such as synthetic polymers, sustaining prolonged biodegradation.

The stability of gut bacterial communities is determined by complex inter-cellular interactions such as competition, cooperation and host dynamics. A mechanism proposed to mediate these interactions is bacterial secretion systems: specialized protein complexes that secrete effector molecules into neighbouring cells or the surrounding environment to influence community stability. However, the forces driving secretion system distribution and evolution in host-associated microbiomes remain unclear. Here, we show that secretion systems in the bee gut microbiome are predominantly vertically inherited and evolve primarily through recurrent gene loss rather than horizontal acquisition. Using comparative genomic analysis, we found that bee gut symbionts mostly encode type I, V, and VI secretion systems. In contrast, the pathogen-associated type II and III systems are missing, but they retain evolutionarily related pili and flagella. We found weak association between the presence of specific secretion systems and the bee hosts, suggesting that these systems are maintained for interbacterial interactions rather than host-specific adaptation. Co-phylogenetic analyses show congruence between bacterial strain phylogenies and most secretion system phylogenies, indicating a vertical mode of transmission. Only a subtype of the type VI system in the Orbaceae and the type IV system in Snodgrassella spp. show evidence of horizontal transmission. The lack of horizontal transfers means that losses of secretion systems is a permanent evolutionary event in almost all lineages of the bee gut microbiota. Our study provides a uniquely comprehensive analysis of secretion systems across an entire gut bacterial community, giving insight into how microbiomes evolve and maintain functional interactions within host-associated environments.

Plant growth-promoting rhizobacteria (PGPR) are key members of soil microbial communities, supporting nutrient cycling, plant health, and productivity. In agricultural soils, these beneficial bacteria are often exposed to multiple stressors simultaneously, including fungicides, antibiotics, and rising temperatures. Despite their ecological importance, little is known about how PGPR respond evolutionarily to such combined stressors. Here, we investigated how the model PGPR Pseudomonas fluorescens SBW25 evolves resistance to the fungicide formulation Fubol Gold (metalaxyl-M + mancozeb), under ambient or warming soil conditions. Using a 16-week experimental evolution in soil microcosms with a fully factorial design (fungicide {+/-} warming), we assessed the evolution of fungicide resistance via phenotypic assays and whole-genome sequencing. Fungicide exposure rapidly selected for increased fungicide resistance, detectable as early as week 4, and co-selected for multidrug resistance, likely through mutations in a mexS ortholog that cause efflux pump overexpression. Warming did not alter the evolution of fungicide resistance; however, populations subjected to both fungicide and warming stress went extinct more rapidly, indicating that population evolutionary rescue was less effective under dual stress. Our findings show that fungicides alone can drive multidrug resistance in beneficial soil bacteria, suggesting that strategies to tackle AMR in agriculture should also consider non-antibiotic drivers of resistance.

Endosymbiotic bacteria can affect many ecological attributes of their insect hosts, including (in herbivorous insects) how insects interact with plants where they feed. This raises the issue of whether deliberate endosymbiont introductions could be used to decrease crop damage caused by insect pests. Here we investigate how transinfecting Rickettsiella viridis and Regiella insecticola endosymbionts into a novel pest aphid host, the Russian wheat aphid (Diuraphis noxia), influences population growth, alate production, dispersal ability and crop damage. Both the Rickettsiella (originating from pea aphids) and Regiella (from green peach aphids) were stably maintained in their new host where they had contrasting effects. Rickettsiella increased the severity of aphid damage on wheat and barley, resulting in greater leaf loss, chlorotic streaking, and higher aphid populations, whereas Regiella reduced aphid population growth and the severity of feeding damage by aphids. Their effects on dispersal morphology also differed: Regiella had no detectable impact on alate incidence, while Rickettsiella consistently suppressed wing formation in small cages, and in larger mesocosms with multiple wheat plants this endosymbiont suppressed dispersal. Endosymbiont-mediated changes in feeding damage did not involve the main plant immune response pathways: transinfected and wild type aphids induced similar levels of jasmonic acid, jasmonic acid-isoleucine, and salicylic acid in plant tissues, even though these plant defenses were strongly activated during aphid feeding. Novel endosymbionts can therefore modulate the severity of plant feeding damage by aphids as well as influencing aphid dispersal. Potential applications in controlling pest D. noxia populations are discussed. Significance statementEndosymbiotic bacteria that live within insect cells can have wide-ranging effects on the reproduction and fitness of their insect hosts in different environments. In herbivorous insects this includes effects on host plant use. Here we test if novel endosymbionts in a pest aphid, the Russian wheat aphid, might be used to decrease crop damage and dispersal. We show that the damage caused to wheat and barley plants from aphid feeding is modulated by novel but stably transmitted introduced endosymbionts. One endosymbiont (Rickettsiella) increased the severity of damage but decreased aphid dispersal, while another (Regiella) decreased damage severity without impacting dispersal. These contrasting effects may be associated with changes in aphid population growth and wing formation but were not linked to key plant immune response pathways. We discuss implications of these findings for using endosymbionts in agricultural pest management. Classification: Applied Biological Sciences, microbiology

Bacteriophages of environmental bacteria remain underrepresented, lending paucity to phage-biofilm research beyond clinical and model species domains. Here, we present the Alpine Lotic Phage (ALP) collection, curated through an isolation campaign from biofilm-forming bacteria of alpine streams. We obtained 57 phage isolates, which were dereplicated to 28 unique genomes following sequencing. The collection consists of tailed phages infecting 14 bacterial host species with genomes spanning 37 to 363 kb while exhibiting diverse plaque morphologies, depolymerase activity, and distinct impacts on host biofilm architecture. Comparative analyses against public viral genomes and a curated planetary-scale contig database revealed limited sequence similarity, underscoring the novelty of ALP phages. Functional annotation resolved 9 - 54% of predicted genes which encoded viral structural components, nucleotide metabolism functions, anti-defence mechanisms, and auxiliary genes that facilitate viral infection and replication. Together, the ALP collection represents a foundational resource for investigating phage evolution and ecology in natural bacterial communities.

Plant invasion can modify soil microbial communities and ecosystem processes through plant-soil feedbacks, yet it remains unclear whether these effects are expressed mainly through taxonomic turnover or through shifts in microbial function and interaction structure. We tested how soil legacy generated by the invasive Conyza bonariensis, the native Helminthotheca echioides, or unplanted control soil influenced short-term microbial responses to standardized amendments and plant-derived inputs. In Experiment 1, conditioned soils were amended with water, cellulose, or ammonium and analyzed for extracellular enzyme activity, qPCR-based gene abundance, bacterial community composition, and family-level co-occurrence networks. In Experiment 2, the same soil legacies were exposed to water, glucose, or sterile root exudates from native or invasive plants. Native- and invasive-conditioned soils differed significantly in composition, but they were not consistently distinguished by strong indicator taxa, indicating that legacy effects were expressed mainly through redistribution of shared taxa rather than community turnover. In contrast, functional responses were clearer: enzyme activity and nirS abundance showed strong soil-legacy dependence, and network analysis revealed that invasive-conditioned soil supported a denser, more positive, and more compact family-level association structure than native-conditioned soil. In Experiment 2, invasive root exudates produced stronger short-term functional-based differentiation among soil legacies than native exudates, especially for extracellular enzymes. Together, the two experiments show that plant invasion can leave a persistent belowground legacy that is expressed primarily through functional filtering and network rewiring of a broadly shared microbiome, rather than through major taxonomic turnover alone.

Microbial communities of geothermal habitats are central to understanding the evolution of life on Earth. Metagenomics has provided insight into the role of viruses in shaping microbial diversity of complex environments. However, identification of novel viruses is constrained by lack of marker genes and low nucleotide similarities between related viral taxa. While microbial and viral diversity have been explored in terrestrial hot springs and hydrothermal vent systems, other volcanic features remain underexplored. Fumaroles (steam vents) are geothermal features that heat groundwater with magma, releasing steam and volcanic gases such as CO2 and H2S. Comparatively physicochemically dynamic to hot springs, fumarole temperatures and gas emissions rapidly fluctuate with volcanic activity. Here, we describe viruses identified metagenomically from microbial mats hosted near basaltic fumaroles on the Big Island of Hawai`i. To our knowledge, this is the first systematic survey of fumarole viruses. Our utilization of a sensitive profile-based approach for identification reveals high viral diversity in fumaroles, resulting in estimation of two undescribed order-level clades of Caudoviricetes (tailed phages). Viral metabolic genes provide evidence of viral-mediated adaptation of microbes to fumarole conditions. We describe patterns of viral diversity that diverge from the Bank model of viral ecology, hinting at viral dispersal between biofilms and high viral richness and evenness. Lastly, we provide a description of the first terrestrial geothermal environment dominated by Microviridae, previously only described in viral communities of deep ocean hydrothermal vents. This study offers important findings for exploration of viral ecology in extreme environments.

The stinkbug Plautia stali harbors essential gut symbiotic bacteria of the genus Pantoea, whose natural strains differ in cultivability and host benefits. Using this system, we evaluated how laboratory-evolved and genetically-engineered symbiotic Escherichia coli strains compete against native Pantoea symbionts and how they influence host fitness. In single infection assays, the native uncultivable symbiont Sym A conferred the highest host performance, whereas the evolved (CmL05G13) and artificial ({Delta}cyaA) symbiotic E. coli strains supported host survival at levels comparable to cultivable Pantoea symbionts (Sym C-F). In competitive co-infection assays, the symbiotic E. coli strains generally showed unexpectedly strong colonization ability. CmL05G13 outcompeted all the cultivable symbionts Sym C-F and even displaced the native uncultivable symbiont Sym A, whereas {Delta}cyaA and the nonsymbiotic control E. coli {Delta}intS were dominated by Sym A at the adult stage. Despite their superior infection competitiveness, the symbiotic E. coli strains provided limited reproductive benefits, behaving as "cheater-like" associates. They were able to invade and dominate the symbiotic organ but failed to match the fitness contributions of native symbionts. These results demonstrate that the experimentally evolved E. coli can rapidly acquire strong colonization ability surpassing that of the natural symbionts that have coevolved with P. stali in nature. At the same time, the mismatch between infection success and host fitness benefits highlights potential evolutionary conflicts and provides an experimental model for studying the dynamics of cheating, mutualism, and symbiont replacement in vertically transmitted symbioses. IMPORTANCEUnderstanding how novel symbionts invade and displace long-term mutualists is central to the evolution of symbiosis. This study demonstrates that Escherichia coli, originally a nonsymbiotic bacterium, can rapidly evolve potent colonization ability and even outcompete native Pantoea symbionts of the stinkbug Plautia stali. Meanwhile, these competitive E. coli strains confer markedly lower reproductive benefits compared with the native symbionts that have developed intimate mutualistic association with host P. stali over evolutionary time, revealing a striking decoupling between infection success and host fitness. This finding highlights the potential for cheater-like microbes to invade vertically transmitted symbioses and destabilize coevolved partnerships. By combining experimental evolution, controlled co-infections, and quantitative analyses, the P. stali-E. coli experimental symbiotic system provides a powerful model for studying the mechanisms and evolutionary dynamics of mutualism, cheating, and symbiont replacement.

The soil microbiome is key to plant health and nutrient acquisition, and viruses likely play important but largely unknown roles in these processes. To interrogate bulk and rhizosphere soil viral biogeography, we collected samples over a tomato growing season in California from an experiment testing arbuscular mycorrhizal fungi (AMF) treatment. We generated 78 viromes, 16S rRNA gene, and ITS1 amplicon datasets, and 33 rhizosphere metatranscriptomes. Of 67,038 DNA viral species genomes (vOTUs), 25% were previously identified, predominantely in agricultural systems, suggesting habitat filtering and greater viral homogeneity across agricultural compared to natural soils globally. Rhizospheres had significantly higher DNA viral richness than bulk soils, whereas no significant richness differences were observed for other biota. 60% of vOTUs were shared between compartments, compared to only 21-23% of bacterial and fungal taxa. Although bulk soil viral biogeography resembled that of prokaryotes, with significant structuring by moisture content, greater virome similarity between high-moisture bulk soils and rhizospheres suggests that conditions with high host activity selected for similar viral communities. In rhizospheres, while bacterial and fungal communities differed most over time, DNA and RNA viral communities differed most by sampling location, matching prokaryotic transcriptional patterns and further implicating host activity in viral biogeography. Similarly, AMF treatment induced changes in the prokaryotic transcriptome but, across biota, only significantly affected DNA viral communities. Overall, results indicate strong viral responses to spatiotemporally localized conditions, with viral biogeography reflecting both dispersal opportunities (high between neighboring bulk and rhizosphere soils, low across fields) and selection via local host activity.

Plant-associated microbiota are composed of hundreds of microbial species. For many of them, little is known about their individual functions and even less is known about their emergent community-level traits. While culture-independent methods provide valuable insights into the composition, diversity, and functional potential of plant-associated microbiota, culture-dependent methods are essential for reductionist lines of inquiry into the roles of individual species and their interactions within a community. Here, we present ZeaMiC, a publicly available culture collection of root-associated bacteria from Zea mays (maize). This resource comprises 88 isolates obtained from diverse soils and several maize genotypes, with live cultures available through DSMZ (German Collection of Microorganisms and Cell Cultures) both as single stocks and as cost-effective bundles (https://www.dsmz.de/collection/catalogue/microorganisms/microbiota/zeamic). To maximize relevance, isolates were selected to be representative of maize root-associated microbiomes in the Corn Belt of the United States, based on abundance-occupancy patterns from previously published root microbiome data, phylogenetic diversity, and literature-based evidence of functional importance. Whole-genome sequencing and annotation revealed genes associated with root colonization, plant growth promotion, and nutrient cycling, including functions such as chemotaxis, biofilm formation, secretion systems, hormone modulation, and phosphate solubilization. This collection serves as a community resource for future mechanistic studies of plant-microbe and microbe-microbe interactions, filling the gap in our understanding of the ecological interactions in plant microbiomes.

Invasive plants can reshape ecosystems by altering soil biogeochemistry and microbial functioning under global change. Competitive interactions between the invasive Conyza bonariensis and the native Helminthotheca echioides were evaluated under warming, nitrogen enrichment, and elevated CO2, together with rhizosphere microbial function in solitary versus competitive growth. Plants were grown alone or in interspecific competition under elevated temperature (27 vs 29 {degrees}C), ammonium-nitrate fertilization versus no fertilization, and ambient versus elevated CO2 (400 vs 720 ppm). Plant traits and relative growth rate (RGR) were measured alongside potential extracellular enzyme activities (EEA) of -D-glucosidase (C acquisition) and N-acetyl-{beta}-D-glucosaminidase (NAGase; N acquisition) and functional gene abundances (nirS and bacterial amoA). To relate enzyme signals to plant demand and microbial biomass, we calculated a growth-normalized rhizosphere investment metric (Specific Rhizosphere Index; SRI) and a biomass-normalized investment metric (Specific Enzyme Activity; SEA). Competition effects were summarized as {Delta}SRI and {Delta}Tax (change from alone to competition) to quantify how competition altered growth- and biomass-normalized investment. Plant responses were driver- and context-dependent. Elevated CO2 produced the largest changes in growth traits, especially for the invasive species. Warming effects were modest in solitary plants but became apparent under competition, where elevated temperature reduced competitive suppression via increased invasive leaf production and reduced constraints on native leaf expansion. Fertilization caused comparatively small shifts in plant endpoints. Microbial responses depended strongly on soil conditioning history. Potential EEA showed limited shifts with warming and fertilization, whereas elevated CO2 enhanced NAGase mainly in invasive-conditioned soils and increased nirS across soils. Despite overlap in ecoenzymatic stoichiometry, SRI and {Delta}Tax revealed treatment- and legacy-dependent patterns in how competition re-scaled microbial C and N acquisition relative to plant growth and microbial biomass. Together, these results indicate that global change can decouple plant growth from enzymatic investment and reconfigure invasive-native interactions through shifts in above-belowground coupling.