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.

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.

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.

The perennial grass Miscanthus x giganteus (miscanthus) offers a sustainable alternative to traditional biomass feedstocks while improving key soil health parameters, including aggregation. Aggregate stability results from dynamic soil-plant-microbe interactions, yet the relative importance of each factor remains an active research question. Building on previous observations that miscanthus alters soil structure to improve water-holding capacity and aggregate stability, we characterized the communities of soil bacteria and arbuscular mycorrhizal fungi (AMF) across three sites in Iowa, USA, comparing miscanthus to annual maize (Zea mays L.) and non-cropped perennial turfgrass (Poa spp.). We examined whether microbiomes co-varied with soil aggregation and, if so, whether plant cover identity or life history categorization better explained the observed patterns. Bacterial and AMF communities varied across sites and plant types, with signals that life history and plant cover identity both mattered. Aggregate stability aligned with a perennial-annual divergence in microbial beta diversity, while finer-scale differences in community composition and network structure were plant-specific. Soils under perennial plants were enriched in microbial groups positively correlated with aggregate stability; we identified 61 bacterial and 8 AMF "architect" taxa for future study. Within- and cross-kingdom co-occurrence network analysis revealed greater complexity under perennial plants: 1.9-fold more network links in miscanthus bacteria-bacteria networks than in maize, and 1.7-fold more in turfgrass AMF-AMF networks. Miscanthus fundamentally shapes microbial interactions, particularly among bacteria, relating to improved soil physical structure. Understanding these soil-plant-microbe feedbacks advances the development of biomass feedstocks with a portfolio of soil health benefits for next-generation biofuels and bioproducts. IMPORTANCEPerennial bioenergy crops can provide the raw material for biofuels and bioproducts while simultaneously improving soil health. Miscanthus x giganteus (miscanthus) efficiently stabilizes soil aggregates, potentially leading to higher water retention and erosion resistance. Understanding the microbial contributions to these outcomes is key to building resilient, sustainable bioenergy systems. This study highlights the connections between communities of soil microbes--bacteria and arbuscular mycorrhizal fungi--across three sites and three plant covers, including miscanthus, maize, and turfgrass. We identify a guild of potential "microbial architects" linked to soil aggregation and show more interconnected microbial networks under the perennial plant covers compared to annual maize. These insights shed light on the interactions between soil biological communities and soil physical and chemical properties. More broadly, the results may inform efforts to harness plant-associated microbiomes for sustainable biomass production.

Seeds link parental environments to offspring establishment, but whether seed-associated bacteria retain signatures of farming and climate legacies across plant generations remains unclear. Here, we characterized epiphytic and endophytic bacterial communities of winter wheat seeds collected from the Global Change Experimental Facility (GCEF) in Germany across three harvest years representing contrasting climates. We then tested how farming (organic versus conventional) and climate (ambient versus future) legacies experienced by maternal plants were associated with offspring rhizosphere bacterial communities and plant performance under drought in the greenhouse. Harvest year was the dominant driver of grain dry weight and seed-associated bacterial communities. Climate legacy additionally affected seed epiphytic communities, whereas farming legacy was expressed in the endophytic diversity. Germination was higher overall for seeds from the conventional than the organic farming legacy and from the ambient than the future climate legacy. A small subset of unique seed-associated ASVs was detected in offspring rhizospheres. Although these ASVs occurred at low relative abundance in seeds (<1%), they accounted for up to approximately 40% of rhizosphere relative abundance under drought. Together, these findings show that seeds retain bacterial signatures of parental farming and climate legacies and that a subset of seed-associated ASVs remains detectable and can become abundant in offspring rhizospheres under drought.

Forest-associated Saccharomycotina occur at low relative abundance, limiting inference about their diversity and dynamics. We sampled leaf litter weekly for 47 weeks across a subtropical forest in northern Taiwan. Enrichment, isolation and ITS sequencing recovered 687 isolates, including 613 Saccharomycotina representing 56 described species and 77 putatively novel operational taxonomic units. Rarefaction indicated unsampled culturable diversity. Among litter traps, community dissimilarity was high and dominated by taxon replacement, but neither topography nor geographic distance was associated with composition, and turnover matched randomised expectations. Richness peaked during warm, wet periods and declined in winter, and minimum temperature showed the strongest statistical association. Composition was associated with maximum temperature, minimum relative humidity, precipitation and solar radiation. Selected isolates' thermal optima covaried with collection-week temperatures, and two October Magnusiomyces magnusii isolates had higher optima than four winter isolates. Together, these findings reveal substantial culturable diversity and seasonal community restructuring consistent with temperature-related filtering.

Full-length 16S rRNA gene sequencing using Oxford Nanopore Technologies has emerged as a promising approach to improve species-level resolution in microbiota studies. However, the accuracy of taxonomic assignment remains highly dependent on the bioinformatics s used to process Nanopore long-read data. Therefore, the only way to ensure a good level of certainty in obtained results is to use positive controls in the form of mock communities in the experimental designs. In this study, we compared the performance of Epi2Me 16S (using Minimap2 or Kraken2) workflows provided by Oxford Nanopore Technologies and an EMU workflow for full-length 16S rRNA gene analysis. Using a commercial mock community sequenced across multiple Nanopore runs, taxonomic assignment accuracy and reproducibility was evaluated. Epi2Me-Kraken2 exhibited 18 % of incorrect genus-level assignments and failed to identify 3 species present in the mock community. While Epi2Me-Minimap2 achieved an excellent genus-level classification, reporting 9 % of sequences assigned to a genus not in the mock community, species-level assignments were inconsistent for several community members such as Listeria. In contrast, EMU provided accurate and consistent species-level taxonomic profiles, with all species correctly identified while keeping the number of genus absent from the mock community at 1.2%. ImportanceThese results highlight that Epi2Me integrated workflows are not the best option for specie-level taxonomic assignation. More importantly, this paper underscores the importance of routine inclusion of positive controls for microbiota studies, in the form of mock communities, as a critical safeguard for accurate data interpretation. Without the use of a mock community, a paper published would be at risk of reporting wrong observations and inaccurate conclusions.

Corals associate with many bacteria whose evolutionary histories and holobiont roles are unknown due to a lack of genomic resources. An example is the BD1-7 clade, which is found in some microbial metabarcoding libraries of corals and has been speculated to be phototrophic. To evaluate its phylogenetic position and assess its metabolic capabilities, we assembled and annotated the genome of an octocoral associate classified as BD1-7. Its full genome revealed that it instead represents a distinct and divergent clade of widespread coral associates. We propose the name Ca. Steroidedax gorgoniicola for this associate of Swiftia exserta. Unlike the true BD1-7 clade, its genome encoded no pathways to generate ATP from light and instead reveals that it is likely a heterotroph that can degrade steroids, chitin, and collagen as well as produce toxins or antimicrobial compounds and detoxify several reactive oxygen and nitrogen species. In addition, we identified several genes that were likely horizontally transmitted from Endozoicomonadaceae, including transposases and genes involved in virulence and cell adhesion. This work sheds light on the potential role of horizontal genetransfer in the evolution of symbiosis and highlights the importance of obtaining genomes to resolve coral-associated lineages and their metabolic capabilities.

Protists play important roles in food chains and symbioses in soil and aquatic environments, displaying an enormous morphological and functional diversity. While most commonly found protist species are well known to science, our global-scale environmental DNA survey across soil, water, and sediments reveals dozens of novel, phylum-level phylogenetic lineages that remain to be characterized for basic morphology and function. A vast majority of these undescribed taxa occur in marine water and sediments, but some are common in soil. Most of these novel taxa have distinct substrate and habitat preferences and biogeographic patterns. To accord these lineages scientific agency and enable unambiguous scientific communication, we propose formal names for 150 species to phylum-level taxa from 25 deep lineages based on eDNA and rRNA gene long-read sequence information.

The Southern Ocean harbors diverse marine microbial communities shaped by both local oceanographic conditions and dispersal limitations. However, this knowledge is mainly based on coastal Antarctic sites, whereas circumpolar Antarctic open sea and subantarctic ecosystems remain poorly explored. Here, we characterize marine microbial communities (using 16S rDNA high-throughput sequencing) and marine oceanographic data across two regions: the Subantarctic, involving two localities (the Magellan Strait and the Beagle Channel), and Antarctic open sea, involving two localities (Eastern Indian and Central South Pacific). We found extensive differences across regions and localities, characterized by distinct taxonomic patterns, alpha diversity, microbial composition, and enriched taxa profiles. Despite these differences, Clade Ia, Amylibacter, NS5 marine group, and NS2b marine group exhibited high prevalence across regions. Oceanographic parameters had variable relationships with microbial alpha diversity across regions: Sea surface temperature and salinity had a negative and positive correlation, respectively, in the Magellan Strait during 2024. In the Antarctic region, dissolved oxygen displayed a negative correlation in the Indian Ocean during 2024, whereas salinity displayed a more variable relationship in the Indian Ocean: positively correlated during 2024, while negatively correlated during 2025. Collectively, our results highlight a strong microbiological biogeographic structure in the Southern Ocean, both across broad scales (between Subantarctic and Antarctic regions) and within regions. Furthermore, our results show dynamic relationships between oceanographic variables and marine microbial diversity across Antarctic and Subantarctic regions.

Plant growth-promoting bacterial endophytes represent a sustainable strategy for enhancing agricultural productivity while reducing reliance on synthetic fertilizers and pesticides. This study focused on the genomic and functional characterization of two endophytic bacterial strains, R11F and R19M, isolated from bean and maize roots, respectively. Comparative analyses based on 16S rRNA gene sequences, average nucleotide identity (ANI), and genome-to-genome distance calculations (GGDC) classified both isolates as Pseudomonas palleroniana. Comparative genomic analyses revealed highly conserved genomes containing genes associated with plant colonization, phosphate solubilization, stress adaptation, heavy metal resistance, and hydrocarbon degradation. Genome mining further identified 17 and 18 biosynthetic gene clusters (BGCs) in R11F and R19M, respectively, including non-ribosomal peptide synthetases (NRPS), pyoverdine, NRP-metallophores, RiPP-like compounds, arylpolyenes, {beta}-lactones, terpenes, NAGGN, and hydrogen cyanide. Strain-specific BGCs associated with syringomycin and viscosin biosynthesis were identified in R11F, whereas R19M harbored clusters related to asplenin and kolossin biosynthesis. In vitro assays confirmed indole production, phosphate solubilization, and siderophore production, as well as the ability of both strains to grow in nitrogen-free medium. Both strains significantly inhibited the growth of Fusarium oxysporum, Phytophthora cinnamomi, and Colletotrichum gloeosporioides. Furthermore, plant inoculation assays demonstrated host-dependent growth promotion, with R11F showing the most consistent improvements in plant growth parameters in tomato, wheat, and lentil. Overall, the integration of comparative genomics and experimental validation demonstrates that P. palleroniana R11F and R19M possess complementary traits associated with plant growth promotion, pathogen suppression, saline stress adaptation, and bioremediation.

Climate driven aridification and intensified human activity are placing increasing pressure on Mediterranean freshwater ecosystems. These impacts propagate from land to water, altering nutrient regimes and reshaping aquatic microbial communities. We analysed Arcellinida diversity across 363 lentic inland saline and freshwater sediment samples spanning broad gradients of land use, water chemistry, soil properties, and climate in southern Spain. Random forest models identified terrestrial land use intensity followed by water chemistry as main predictors of community diversity. Diversity declined sharply in sites with population densities above [~]33 inhabitants/km{superscript 2} and under eutrophic conditions, but peaked in oligotrophic systems with stable, carbon rich soils. These threshold responses demonstrate that aquatic protist assemblages integrate both long term terrestrial pressures and current water conditions. Overall, our findings show that landscape transformation and its cascading effects on water quality dominate community assembly, and that the combination of community level diversity metrics with selected taxon-level indicators capture ecosystem degradation more consistently than relying on a single metric.

Marine sponges are known sources of bioactive natural products (NPs), many of which are produced by associated bacterial symbionts via encoded biosynthetic gene clusters (BGCs). A particularly interesting subclass of sponge-derived NPs is comprised of small, brominated alkaloids, which are recovered from diverse habitats and host sponge taxonomies. Despite having been described decades ago, most of these NPs do not have an elucidated biosynthetic origin. We queried metagenomes of several sponge species by making use of a minimal set of core enzymes that we postulate to be necessary to produce these small peptidic NPs: an FADH2-dependent halogenase and an AMP-binding adenylation enzyme. This revealed a variety of novel BGC architectures, many of which showed conservation among sponge host phylogenies and were encoded in the genomes of diverse sponge-associated bacteria. Furthermore, we identified a BGC in the sponge G. barretti that is potentially linked to the production of the iconic barettins, given its enzymatic machinery and specific acidobacterial origin. The present work contributes to the challenging quest to link orphan brominated NPs to their parent BGCs in the sponge holobiont and beyond.

Grassland microbial communities are central to mediating ecosystem function and stability, yet how long-term grazing reshapes root-associated microbiomes across contiguous soil-root habitats remains poorly understood. This limits our ability to identify robust microbial bioindicators for grassland health monitoring. In this study, we investigated the community assembly and functional variation of root-associated microbiomes of Stipa breviflora, a dominant perennial clonal grass in desert steppes, across a 17-year continuous grazing experiment with four grazing intensity treatments (no grazing, light, moderate, and heavy grazing). We show that grazing intensity induces niche-specific restructuring of microbial communities, with the most profound compositional and functional shifts occurring in the rhizosphere, followed by root endophytic compartments and bulk soil. Light and moderate grazing significantly enriches the phylum Bacillota in rhizosphere and endophytic compartments, whereas the genus Pseudomonas dominates ungrazed grassland soils and is markedly depleted under grazing conditions. Microbial community responses to grazing follow a unimodal intermediate disturbance pattern, with moderate grazing triggering the strongest microbial community differentiation, enhanced microbial network connectivity and modularity, and the highest abundance of grazing-responsive microbial biomarkers. Notably, grazing-induced microbial community variation is decoupled from intraspecific phenotypic changes in S. breviflora. Our findings demonstrate that long-term grazing acts as a strong selective filter partitioning core beneficial microbial taxon, establishing Bacillota and Pseudomonas as complementary bioindicators for evaluating desert steppe ecosystem health. This study advances the understanding of plant-microbe interactions under anthropogenic disturbance and provides microbiome-based insights for sustainable grassland management.

Bacterial exopolysaccharides (EPS) are key components in biofilm formation, stress protection, and symbiosis in Rhizobiaceae. While EPS structural diversity is extensive, experimental characterization remains limited. In this study, we experimentally determined and compared four distinct EPS structures produced by ten Rhizobium alamii strains. Using genomic data, we bioinformatically identified supra-operonic clusters (SOCs) responsible for these EPS biosynthesis. We introduced a computational framework to predict, score, and compare EPS SOCs across 84 Rhizobium and Sinorhizobium species, linking gene content to structural and functional EPS diversity. A total of 743 EPS SOCs was selected for network analyses, allowing the identification of 36 major groups of orthologous EPS SOCs, successfully recovering all known EPS biosynthetic loci and two novels SOCs potentially encoding uncharacterized EPS (xEPS-I, xEPS-II). Profiles of EPS SOCs correlated with taxonomical groups, with a single EPS SOC conserved through all 84 genomes and distinct additional EPS SOCs depending on the group, but do not strictly explain symbiotic capacity. Genetic comparisons of transporters (Wzx, Wzy) and glycosyltransferase sequences indicated these proteins as key markers of EPS structure. Overall, this computational framework accurately identified and classified EPS SOCs, providing a scalable, genome-based method for predicting EPS biosynthetic potential in Rhizobiaceae and usable in other microbial genera.

Nutritional homeostasis in many leafhoppers (Cicadellidae) is largely attributed to ancient obligate symbionts, yet the facultative bacteria these insects carry and if whether they contribute to digestion, remains poorly understood. This question is especially relevant in mesophyll cell-rupture feeders of the subfamily Typhlocybinae, which are reported to lack classical obligate associations. The potato leafhopper, Empoasca fabae, is a polyphagous, migratory Typhlocybine that feeds on more than 200 plant species. Metagenomic analysis of field-collected E. fabae recovered four complete metagenome-assembled genomes corresponding to the opportunistic plant-pathogenic Enterobacterales Enterobacter mori, Kosakonia cowanii, Pantoea agglomerans, and Pantoea ananatis, each highly similar to its type strain. Species-specific PCR across a five-year window showed that E. mori and K. cowanii were detected in every field sample and persistent in an inbred colony, demonstrating likely recurrent and maintained associations, whereas the two Pantoea species were detected intermittently. All four genomes encoded broad carbohydrate-processing repertoires, including sucrose phosphotransferase systems, glycolysis, and aromatic amino acid biosynthesis, suggesting a capacity to synthesize aromatic amino acids-essential for the host. Among 614 glycoside hydrolases, two putatively secreted GH5-25 cellulases were further examined, with recombinant K. cowanii KcGH5-1 hydrolyzing carboxymethyl cellulose at acidic pH, signifying a functional bacterial endoglucanase. These results identify recurrent plant-pathogenic Enterobacterales as carriers of complementary digestive functions, and as candidate contributors to the exceptional dietary breadth of a major migratory agricultural pest.

Delivery of therapeutic biomolecules into plant vascular tissues remains a challenge in management of vector-borne plant pathogens. The symbiont concept uses reprogrammed Agrobacterium tumefaciens galls (called symbionts) to produce biomolecules while remaining connected to host vasculature. We evaluated whether symbionts expressing antimicrobial peptides (AMPs) suppress Candidatus Liberibacter solanacearum (CLso), the causal agent of potato zebra chip disease. Symbionts were engineered to express a Streptococcus mutans bacteriocin associated with bacterial membrane disruption (Blp-Sm), or an AMP isolated from finger lime and associated with resistance to citrus greening disease (MaSAMP). Effects of AMP-producing symbionts on CLso titers, infection incidence, pathogen movement, and disease symptoms were evaluated in tomato and potato. In tomato, neither AMP significantly reduced CLso titers or infection incidence. However, in potato, AMP-producing symbionts reduced CLso accumulation and movement from CLso-inoculated source shoots into non-inoculated sink shoots connected through underground tubers. Blp-Sm produced the strongest reduction in CLso accumulation and infection incidence in sink tissues. In separate assays where symbionts were established directly on potato seed tubers, MaSAMP significantly reduced CLso titers in stems and tubers and reduced zebra chip symptoms in tubers, despite no reduction of CLso titers in terminal leaves. These findings demonstrate that AMP-producing symbionts suppress vascular pathogen accumulation and movement within plants and highlight the symbiont concept as a potential platform for managing diseases caused by vascular-restricted pathogens. Further, they show the potato-CLso system is a promising infection model to both refine and improve symbiont technology, and to test additional AMPs for potency against related pathogens.

Recognizing the central role of microorganisms in greenhouse gas (GHG) cycling in aquaculture systems, we provide a genome- and gene-centric perspective on the metabolic potential for CO2 and CH cycling in prawn aquaculture ponds across seasons and contrasting culture practices. Using TaxVAMB, we recovered 78 high- and medium-quality metagenome-assembled genomes (MAGs), including previously underappreciated taxa such as Bathyarchaeia and Terriglobia. Metabolic profiling revealed that CO2 and CH cycling constitute a minor fraction of the ponds metabolic potential, dominated instead by heterotrophic processes such as fermentation, oxygen metabolism, and iron reduction. The relative metabolic weight of these carbon-cycling pathways was lower than that reported for permafrost, wetland, peatland, deep-sea, and human gut microbiomes. An integrated metabolic network suggested that genetic potential for CO2 production is primarily driven by pyruvate and acetyl-CoA oxidation, while methanogenesis and methane oxidation genes together encode the potential for internal carbon-recycling loops via canonical archaeal and bacterial pathways. Seasonal dynamics, rather than management treatment, strongly influenced functional gene abundances, with CO2 fixation and CH4 oxidation genes increasing toward the late season. Bathyarchaeia emerged as the most versatile taxon for CO2 cycling and methanogenesis, with stable relative abundance across seasons and treatments. This study underscores the role of seasonally evolving microbial networks in regulating carbon turnover and the potential for CO2 and CH4 emissions in prawn aquaculture ponds.