Phytobiomes Journal

Phyllosphere microbiomes are increasingly recognized as key regulators of plant health and stress responses, although they are also known to change considerably over both space and time. In the phyllosphere, members of the genus Methylobacterium are often abundant and ecologically important as plant growth promoting bacteria. However, knowledge about the temporal abundances and community dynamics of Methylobacterium in agricultural systems remains limited. To address this gap, we characterized seasonal shifts in Methylobacterium-specific and total phyllosphere bacterial loads and community structure on two common summer crops and one overwintering cover crop. Leaf samples of Zea mays (corn), Glycine max (soybean), and Thlaspi arvense L. (pennycress) plants were collected over one year in Minnesota, USA and analyzed with host-associated microbial PCR (hamPCR). Microbial loads and community composition varied strongly among hosts and across growing seasons. Corn supported the highest Methylobacterium and total bacterial loads, increasing towards senescence, while pennycress exhibited the lowest loads and the most distinct communities. While there were strong host-specific patterns, a group of most abundant genera were shared across all crops (Methylobacterium, Sphingomonas, Pseudomonas, and Massilia) and the most abundant Methylobacterium amplicon sequence variants were present on all three hosts. Our findings highlight how microbial loads and community composition change during phyllosphere assembly across diverse summer and overwintering crops, with a small core of versatile taxa dominating multiple agricultural hosts. Understanding these host and season-linked patterns provides a foundation of harnessing Methylobacterium strains to enhance crop productivity and resilience.

Seed microbiota play a crucial role in plant health and development, yet remain understudied compared to other plant-associated microbial communities. This study aimed to characterize seed microbiota diversity across four major crops (common bean, rapeseed, tomato, and wheat) and establish a comprehensive strain collection of seed-borne microorganisms (bacteria and fungi). We employed a combination of culture-dependent and culture-independent approaches to analyze 68 seed samples representing diverse genotypes and production modes. Our results revealed highly variable seed microbiota, with bacterial colonization ranging from 10 to 100 million bacterial CFUs per gram of seeds, and microbial richness varying from 4 to 351 bacterial and 16 to 138 fungal amplicon sequence variants (ASVs) per sample. Both plant genotype and production mode significantly influenced microbiota composition, with each seed sample produced harboring a distinct microbial assemblage. Interestingly, seeds produced in confined environments exhibited lower bacterial colonization but higher microbial richness compared to field-produced seeds. We observed divergent ecological drivers shaping bacterial and fungal communities. Bacterial assemblages were more host-specific and variable, while fungal communities showed greater stability and a substantial core microbiome shared across plant species. Our culturomics approach yielded a collection of 2,510 bacterial and 837 fungal isolates, representing 10-21% of the seed microbiota diversity detected by metabarcoding and the majority of the prevalent and abundant taxa. Notably, 44-60% of cultured bacterial isolates were not detected by metabarcoding, highlighting the complementary nature of these approaches to detect rare or under amplified taxa in PCR. This study provides insights into the complexity and variability of seed microbiota across different crops and production conditions. Our findings emphasize the importance of combining culturomics and sequencing methods for comprehensive characterization of seed microbiota to uncover the potential of seed-borne microorganisms as bioinoculants for sustainable agriculture.

Drought threatens food security globally. Adaptive root phenotypes and microbiomes can improve maize (Zea mays L.) water uptake and tolerance to drought. However, synergisms between root phenotypes and microbiomes remain underexplored. We aimed to investigate the association between varying root phenotypes and rhizosphere microbiomes under field-scale drought. We grew 22 maize inbred lines in the field under optimal water availability and drought imposed by excluding rain with rainout shelters. We quantified grain yield and measured root architectural and anatomical phenotypes on root crown and cross-section images obtained by laser ablation tomography, respectively. We characterized rhizosphere prokaryotic and fungal communities with DNA metabarcoding of ribosomal markers. Rhizosphere microbial diversity predominantly associated with root anatomy rather than root architecture. Cortical parenchyma wall width explained 13.1% of the variance of the prokaryotic {beta}-diversity and correlated with grain yield under control conditions. Under the same conditions, number of cortical cell files and metaxylem vessels explained 1.4-2.1% of the variance of prokaryotic and fungal {beta}-diversities. No effect of the root phenotypes was observed under drought. We found 248 significant correlations between microbial taxa abundances and root anatomical phenotypes, especially cortex-related phenotypes such as number of cell files and living cortical area. Overall, a greater number of correlations was found under control conditions. We identified root phenotypes explaining a small but significant percentage of the variance of the microbial {beta}-diversity, mostly under optimal water availability. We showed that especially root anatomy is associated with rhizosphere microbial diversity in field-grown maize.

Antibiotic use in agricultural systems can unintentionally disrupt beneficial rhizosphere microorganisms, yet the consequences of this dysbiosis for plant fitness remain insufficiently understood. Building on previous findings that application of streptomycin to the roots decreases cyanobacteria and increases tomato plant susceptibility to foliar Xanthomonas infection, this study aimed to determine whether this relationship reflects causation or correlation. We evaluated whether targeted inoculation with the filamentous nitrogen-fixing cyanobacterium Cylindrospermum sp. (CI) or a complex rhizosphere microbiome transplant (RMT) could mitigate antibiotic-induced dysbiosis. As expected, streptomycin treatment significantly increased bacterial spot disease severity and reduced microbial richness in the rhizosphere, marked by a pronounced decline in cyanobacterial and Cylindrospermum operational taxonomic units. Co-occurrence network analysis revealed that this dysbiotic state was defined by reduced community connectivity and increased negative associations, indicating a breakdown in cooperative microbial relationships. Notably, both CI and RMT reduced plant disease severity, though they caused distinct rhizosphere community reassembly outcomes. While RMT relied on microbial functional redundancy, the targeted CI approach achieved more robust colonization and effectively "patched" the functional gap left by dysbiosis. Microbiome restoration directly influenced host physiology, significantly reducing the overactivation of ethylene-mediated defense genes, such as ERF1, and partially reinstating auxin-responsive signaling pathways (IAA21) that were disrupted under dysbiosis. These findings suggest that targeted microbial inoculation could reverse dysbiosis and enhance plant resilience under pathogen pressure as effectively as complex microbial transplants. This work highlights a shift in microbiome management: from the complex rebuilding of communities to the strategic repair of specific functional gaps.

Common bean (Phaseolus vulgaris) is an important crop in Canada and globally. Like other legumes, common bean (Phaseolus vulgaris) establishes symbiotic interactions with nitrogen fixing bacteria called rhizobia. However, nitrogen fixation by rhizobia in association with common bean is often suboptimal, constraining its productivity and necessitating the application of nitrogen fertilizer. To support the development of high-performing, locally adapted rhizobial inoculants for Ontario common bean growers, we isolated 216 common bean-nodulating rhizobia from southern Ontario soils using a nodule trapping approach with four common bean cultivars. Whole genome sequencing followed by phylogenomic analyses of the 216 rhizobial isolates revealed substantial diversity, assigning them to 11 Rhizobium species, including two novel species. Nearly all isolates belong to the symbiovar phaseoli, spanning the nodC {gamma}-a, {gamma}-b, and alleles, with four isolates belonging to the symbiovar gallica. Soil origin had a significant impact on the species-level community composition recovered during the nodule trapping experiments, indicative of biogeographical structuring of common bean-nodulating rhizobia across southern Ontario. In contrast, host trapping cultivar had only a minor influence of the recovered Rhizobium population diversity. Greenhouse assays demonstrated that one of the novel Rhizobium species exhibited the highest average symbiotic effectiveness, although high-quality isolates were found across multiple species. Together, these results revealed a diverse and genomically variable Rhizobium community capable of forming effective symbioses with common bean in southern Ontario soils. Importantly, our genome-sequenced Rhizobium collection will serve as a valuable resource for identifying competitive and high-quality strains for the development of inoculants tailored to Ontario common bean production. IMPORTANCECommon bean is a globally important food crop, yet its productivity is often limited by suboptimal nitrogen fixation, forcing growers to rely on synthetic fertilizers. Consequently, identifying high-performing, locally adapted inoculant strains is essential for reducing dependence on synthetic nitrogen fertilizers and improving the sustainability of temperate agroecosystems. Our study provides a genome-sequenced collection of common bean-nodulating Rhizobium from southern Ontario, revealing substantial species and genomic diversity across sampling locations. Greenhouse studies allowed us to identify multiple isolates, including isolates from a novel Rhizobium species, that consistently fix nitrogen with, and enhance the growth of, common bean plants. Our findings highlight strong biogeographical structuring of rhizobial communities and demonstrate that Ontario soils already harbour strains with high symbiotic potential. In addition, our Rhizobium collection represents a foundational resource to support future inoculant development and enables future work on the ecology, evolution, and applied optimization of legume-rhizobium symbioses.

Plant-associated bacteria can improve plant performance under abiotic stress, but beneficial functions in plant microbiomes may depend on defined combinations of microorganisms rather than individual isolates alone. Here, we developed a cube-based screening strategy to identify functional synthetic microbial communities (SynComs) from 135 quinoa-associated bacterial isolates while preserving combinatorial diversity and traceability of isolate-level contributions. The isolates were divided into five 27-isolate sets, each arranged as a 3 x 3 x 3 cube in which each 3 x 3 layer was defined as a 9-isolate SynCom, generating 45 SynComs in total. Screening under 100 mM NaCl identified SynCom DY1 (SCDY1) as a candidate salt stress-mitigating consortium. SCDY1 consisted of nine taxonomically diverse isolates and exhibited a multifunctional profile, including siderophore production, phosphate solubilization, carboxymethyl cellulose degradation, indole compound production, and growth under saline conditions. In Arabidopsis thaliana, SCDY1 promoted primary root elongation and biomass accumulation in a salinity-dependent manner, with the clearest effect under 120 mM NaCl, and at least a subset of constituent bacteria was recoverable from inoculated seedlings. RNA sequencing and targeted RT-qPCR indicated that SCDY1 modulated host gene expression under moderate salinity stress, with responsive genes associated with oxidative stress, water- and oxygen-related processes, phenylpropanoid biosynthesis, glutathione metabolism, and root epidermis-related processes. Root hair phenotyping further showed that SCDY1 enhanced root hair-related traits and shifted visible root hair formation closer to the root apex. These findings identify a quinoa-derived SynCom that improves plant performance under salinity stress and provide a practical, traceable framework for discovering beneficial microbial consortia from plant-associated bacterial collections. Scope statementThis manuscript fits the Research Topic "Harnessing Plant Microbiomes for Climate Resilience: From Ecological Insight to Synthetic Community Design" in Frontiers in Plant Science because it presents a traceable strategy for discovering functional synthetic microbial communities from a stress-adapted plant-associated bacterial collection. We developed a cube-based screening strategy using 135 quinoa-associated bacterial isolates and identified a nine-isolate synthetic microbial community, SCDY1, that promotes Arabidopsis growth under moderate salinity stress. The study integrates microbiological screening, characterization of plant growth-promoting traits, bacterial re-isolation, plant growth phenotyping, RNA-seq, RT-qPCR, and root hair phenotyping. These analyses link SCDY1 treatment to salinity-dependent growth promotion, recoverable bacterial members, stress- and redox-associated transcriptional changes, phenylpropanoid-related responses, and modulation of root epidermal phenotypes. By connecting a defined SynCom with host transcriptional and root epidermal responses, this work advances understanding of beneficial plant-microbe interactions under salt stress. The cube-based design also provides a practical and traceable framework for discovering functional SynComs from large plant-associated bacterial collections, which should be of interest to researchers studying plant symbiosis, microbiome engineering, abiotic stress tolerance, and sustainable crop improvement.

Endophytic bacteria from arid medicinal plants represent a promising source of stress-adapted, plant growth-promoting (PGP) microorganisms. Here, we investigated the cultivable endophytic microbiota of Peganum harmala using both nutrient-rich and diluted media to maximize taxonomic recovery. Isolates were dominated by Bacillota and Gammaproteobacteria, with higher diversity in roots than in shoots. Venn analysis revealed a shared core fraction between compartments, forming the basis for consortium assembly. Nine representative strains belonging to Phyllobacterium, Bacillus, Brevibacillus, Burkholderia, Ralstonia, and Amycolatopsis were selected for functional screening. Pairwise antagonism assays showed high compatibility among Bacillus-related strains, whereas certain taxa exhibited inhibitory interactions, guiding rational consortium design. Functional characterization demonstrated complementary PGP traits, including nitrogen-related activity, phosphate, potassium, and silicate solubilization, siderophore and indole-3-acetic acid production, and ammonia production. No single isolate performed optimally across all traits, supporting a consortium-based strategy. A synthetic bacterial consortium (C2), reconstructed from the core endophytic microbiota using compatibility-guided selection, was evaluated in two crop systems. In vitro flax germination assays showed accelerated radicle emergence and improved vigor index, particularly with C2. Under greenhouse conditions, C2 significantly enhanced flax shoot and root biomass, root architecture, leaf area expansion, and photosystem II performance in sterile soil. In faba bean under natural soil, C2 increased leaf number (p = 0.02) relative to the control. These results indicate that consortia derived from core endophytes of arid medicinal plants can promote plant growth across diverse crops and soil contexts, although effects remain context-dependent and require rigorous field validation. IMPORTANCEEndophytic bacteria can serve as sustainable bioinoculants to enhance crop performance under stress conditions. This study demonstrates that the core microbiota of the arid medicinal plant Peganum harmala can be rationally assembled into a functionally complementary consortium that improves germination, nutrient acquisition, and whole-plant physiological performance in flax and faba bean. By combining compatibility-guided assembly with functional screening, we show that consortium-based strategies may outperform single-strain inoculants. These findings provide insight into the development of scalable, plant growth-promoting microbial consortia and highlight the importance of testing microbial inoculants under multiple environmental contexts to ensure consistent benefits.

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

The rhizosphere microbiome, one of the most diverse and metabolically active microbial ecosystems known, plays fundamental roles in plant health and productivity. However, the ecological dynamics occurring during the transition between germination and the establishment of the first true leaves, a developmental window associated with the onset of active photosynthesis and rapid root expansion, remain poorly understood. Here, we investigated rhizosphere microbiome assembly during the first four weeks of tomato development by sampling communities arising from seven distinct natural soil inocula twice weekly to obtain fine-scale temporal resolution. Bacterial load, richness, evenness and phylogenetic diversity all increased significantly during plant development, indicating progressive increases in rhizosphere ecosystem complexity. In addition, diverse initial microbial communities differentially influenced both host plant development and the bacterial carrying capacity of the resulting rhizosphere ecosystem. Although temporal effects on rhizosphere microbiome composition were significant, assembly trajectories remained strongly constrained by the initial inoculum. Temporal analysis nevertheless revealed significant taxonomic turnover despite limited global compositional restructuring. In particular, Proteobacteria and Pseudomonadaceae decreased over time, whereas Actinobacteria, Acidobacteria and Streptomycetaceae increased. However, communities did not become progressively more similar or divergent over time. Altogether, our results indicate that early rhizosphere microbiome assembly involves rapid ecological succession within inoculum-constrained compositional trajectories, with early copiotrophic Proteobacteria progressively giving rise to more diverse and phylogenetically structured communities. These findings suggest that the first weeks of plant development may represent a critical ecological window for microbiome-based manipulation strategies in agriculture.

The honey bee (Apis mellifera) gut microbiome plays a central role in host health, yet its variation across agricultural landscapes remains poorly resolved. This study investigates how major environmental stressors, particularly pesticide exposure and RNA virus loadings, shape the honey bee gut microbiome in a large-scale field study conducted across Canada, spanning diverse agroecosystems from British Columbia to Quebec. We identify consistent associations between specific bacterial taxa and major RNA viruses, including enrichment of Serratia marcescens with SBV and depletion of Bombella intestini with BQCV. Pesticide exposure is likewise linked to reproducible shifts in key microbial taxa. Together, these findings reveal that interacting stressors jointly shape the bee gut microbiome and enable prediction of microbiome responses in agroecosystems. HighlightsDistinct associations identified between gut bacteria and major bee RNA viruses (BQCV, SBV, LSV, IAPV) Pesticide exposure is linked to reproducible shifts in key microbial taxa Combined virus-pesticide effects form coordinated clusters that predict microbiome variation and specific bacterial responses Integrated modeling demonstrates that environmental stressors can jointly explain microbiome structure beyond crop effects Graphical abstractSchematic overview of potential links between pesticide exposure and RNA virus infection and their effects on the bee gut bacterial community. Solid arrows indicate associations supported by the present study, whereas dashed arrows indicate hypothesized or unresolved interactions. Associations between the presence of specific bee RNA viruses (left) or pesticide residues (right) and changes in the relative abundance of particular gut taxa (pink {uparrow}, increased; blue {downarrow}, decreased). The pesticide subtype is indicated by the icon in the cell (leaf - herbicide, hyphae - fungicide and insect - insecticide). Several bacterial taxa showed reproducible associations with specific viral or pesticide variables, including Bombella intestini, Serratia marcescens, Melissococcus plutonius, Paenibacillus alvei, Apibacter sp. wkB309, and Gilliamella sp. A7. Abbreviations: BQCV Black queen cell virus; LSV, Lake Sinai virus; SBV, Sacbrood virus; IAPV, Israeli acute paralysis virus. (p/n/b) indicate the sample matrix in which the pesticide was detected, namely pollen, nectar, and bee tissue, respectively. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/731697v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@cb92a4org.highwire.dtl.DTLVardef@1087045org.highwire.dtl.DTLVardef@102cabforg.highwire.dtl.DTLVardef@4ce2f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Aridification and climate stress threaten global plant productivity, but the survival strategies of desert plants remain only partly understood. In this study, we examined how the microbiome of Citrullus colocynthis, a hardy desert cucurbit valued for its ecological and medicinal benefits, may influence the plants ability to withstand harsh conditions. Using 16S rRNA amplicon sequencing, shotgun metagenomics, and culture-based methods, we analyzed microbiome changes across two regions of the UAE during the rainy and dry seasons. Leaf and root bacterial communities showed clear seasonal shifts, with greater richness in winter and higher evenness in summer, while soil microbiomes remained stable. Dominant bacterial groups, Actinomycetota and Pseudomonadota, varied seasonally, indicating trade-offs between stress tolerance and metabolic flexibility. Fungal communities (mainly Ascomycota and Basidiomycota) were stable at the phylum level but reorganized by order between seasons; archaeal populations showed little change. Among 24 cultured bacterial isolates, including three potential new species, we identified multiple stress tolerance and plant growth-promoting traits. Genomic data revealed biosynthetic clusters for antimicrobial and stress-protective functions, as well as adaptation genes in Pseudomonas orientalis. These results demonstrate that the dynamic, functionally diverse microbiome of C. colocynthis enhances its resilience to desert stress, offering potential for arid-land agriculture.

Viruses are abundant and ecologically important in soils, yet the persistence and production dynamics of extracellular virions remain poorly understood. We applied a genome-resolved stable isotope probing viromics (SIP-viromics) approach, combining H 18O labeling with viral metagenomics, to track virion turnover in seasonally dry grassland soils following rewetting. We identified 354 viral populations (vOTUs) using individual-sample and combined metagenome assemblies. Only 22% of vOTUs exhibited significant 18O enrichment, indicating active replication and new virion production during the 1-week incubation; the majority (78%) persisted without detectable replication, consistent with a viral seed bank. Active vOTUs accounted for 4.76-5.15% of total virions per gram of soil, with viral loads ranging from 3.15 x 1010 to 6.59 x 1010 virions per gram. Probabilistic and deterministic sensitivity analyses spanning viral DNA fraction and genome length reinforced that persistent virions represented the majority of the extracellular viral pool post-wet-up, regardless of parameter assumptions. Host predictions linked both active and persistent vOTUs primarily to Actinomycetota and Pseudomonadota--bacterial groups known to rapidly resuscitate following rewetting--suggesting that some viruses exhibit rapid turnover while others persist over longer timescales, forming a stable viral pool capable of reinitiating infections during favorable conditions. These results demonstrate that SIP-viromics can distinguish newly produced from persistent virions and reveal host-associated patterns of lytic infection and virion production. Our findings advance understanding of soil virus-host interactions and highlight the ecological role of persistent virions as a genetic reservoir contributing to microbial turnover and biogeochemical cycling following environmental disturbance. ImportanceUnderstanding the persistence and production dynamics of soil viruses is critical for elucidating their roles in microbial community dynamics and nutrient cycling, yet these processes have remained largely uncharacterized due to methodological limitations. By integrating stable isotope probing with viromics, this study provides a robust framework for directly distinguishing newly produced from persistent virions in situ. Unlike conventional viromics, which only catalogs viral diversity, SIP-viromics enables quantification of active viral replication and persistence under natural soil conditions. Our results demonstrate that most virions in a seasonally dry soil persisted through a rewetting event, with active replication limited to a minority of viral populations. Persistent virions were primarily linked to dominant bacterial groups, indicating that host ecophysiology and environmental stability strongly influence lytic infection. Collectively, these findings highlight viruses as long-term reservoirs of genetic material, capable of shaping microbial dynamics and ecosystem processes over time. This work establishes SIP-viromics as a powerful approach for studying virus-host interactions and their ecological significance in terrestrial environments.

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

Bacterial canker of tomato, caused by Clavibacter michiganensis (Cm), remains difficult to control due to lack of effective management options. In this study, a collection of over 500 bacterial isolates was screened in vitro for antagonistic activity against Cm and plant growth-promoting (PGP) traits. Based on these results, 32 candidates were evaluated in planta, leading to the identification of three highly effective strains: Pantoea agglomerans SO16PY and two Pseudomonas marginalis sensu lato strains, IRDA16 and SO16PC, which consistently enhanced tomato vegetative growth. Notably, P. agglomerans SO16PY delayed disease onset in Cm-inoculated plants by up to 7 days and significantly reduced wilting severity, lowering the disease severity score from 85% to 45%. Strains IRDA16 and SO16PC also restricted disease development, reducing severity scores to 67.5% and 57.5%, respectively. Whole-genome sequencing and comparative genomics revealed that strains IRDA16 and SO16PC form a distinct, specialized rhizosphere lineage within the Pseudomonas marginalis group, exhibiting average nucleotide identity (ANI {approx} 96%) and digital DNA-DNA hybridization (dDDH {approx} 69.5%) values near species delineation thresholds. Genome mining identified diverse biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), the lipopeptide viscosin, and terpenes, which likely drive the biostimulant and antagonistic traits of this novel Pseudomonas lineage. Together, these findings characterize promising bacterial candidates with dual biostimulant and biocontrol capacities while uncovering a genomically distinct Pseudomonas lineage optimized for beneficial plant-microbe interactions in sustainable agriculture. IMPORTANCEClavibacter michiganensis (Cm) is a major bacterial pathogen of tomato and poses a significant economic threat to global production. It is classified as an A2 quarantine pathogen by the European and Mediterranean Plant Protection Organization (EPPO). Current management strategies rely largely on chemical control, including copper-based compounds (e.g., Bordeaux mixture, copper oxychloride), mancozeb, and antibiotics like streptomycin. However, these approaches raise increasing concerns related to environmental contamination, phytotoxicity, and the development of resistant pathogen populations. As a sustainable alternative, plant growth-promoting bacteria (PGPR) have emerged as promising biocontrol agents. In this study, we identified bacterial strains exhibiting antagonistic activity against Cm both in vitro and in planta. Notably, these strains also enhanced tomato growth parameters, demonstrating their dual functionality. Given the environmental drawbacks associated with chemical inputs, the use of such beneficial microorganisms represents a promising strategy for advancing sustainable and ecofriendly tomato production systems.

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

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

BackgroundGlobal warming increasingly challenges plant performance and ecosystem function, and plant responses to elevated temperature are shaped not only by intrinsic plasticity, but also by interactions with rhizosphere microbial communities. However, it remains unclear how warming-induced microbiome reorganization relates to plant performance, whether microbiome community composition predicts plant phenotypic responses to elevated temperature better than simple host-microbiome origin matching, and which bacterial or fungal community features contribute most strongly to this prediction. ResultsWe implemented a full factorial design manipulating plant genotype (P), microbial inoculum (M), and temperature (T) using natural Arabidopsis thaliana ecotypes and their corresponding rhizosphere microbiomes from contrasting climatic regions. By integrating high-throughput plant phenotyping, microbial community profiling, microbiome-phenotype coupling analyses, and predictive modeling, we found that elevated temperature induced a coherent thermomorphogenic shift in plant architecture, while plant phenotypic variation remained determined primarily by genotype over the 14-day vegetative growth period covered in this study. Overall, cold-origin genotypes showed larger temperature-induced trait shifts than warm-origin genotypes. Rhizosphere microbial communities were structured predominantly by inoculum origin, but were also affected by warming. Evidence for a increased thermomorphogenic growth when genotypes were combined with inoculum from their home site was limited. Instead, microbiome-phenotype associations were better captured by community composition than by matching status, with fungal community variation showing stronger and more consistent associations with plant phenotype than bacterial variation and providing more robust predictions of plant phenotypes across models. ConclusionsWarming reorganized plant-rhizosphere systems within persistent host- and inoculum-associated baselines. Plant phenotypic variation during vegetative development under elevated temperature was linked more closely to microbiome composition, especially in fungi, than to simple host-microbiome matching of origins. These findings provide a framework for identifying microbiome features associated with plant performance under climate warming.

Plants inhabitng in arid and semi-arid ecosystems, such as Citrullus colocynthis (L.) Schrad., are adapted to drought, heat, salinity, and nutrient limitation. Their associated microbial communities may further support plant persistence under these harsh conditions. Here, we characterized the bacterial communities associated with leaf endosphere, rhizosphere and roots of C. colocynthis growing in a semi-arid region of Moroccan using 16S rRNA gene amplicon sequencing, culture-dependent isolation, and genome-informed functional profiling of selected isolates. The results revealed a structured microbiome, with rhizosphere harboring the highest bacterial diversity, roots representing an intermediate selective habitat, and the leaf endosphere containing a more restricted assemblage. Communities were dominated by members of the phyla Pseudomonadota, Actinomycetota, Bacillota, and Bacteroidota. Several families associated to plant colonization, nutrient mobilization, and stress tolerance, including Pseudomonadaceae, Microbacteriaceae, Rhizobiaceae, Devosiaceae, and Xanthomonadaceae, showed compartment-specific enrichment. Although soil physicochemical properties influenced bacterial community structure, they explained only part of the variation observed, suggesting that bacteriome assembly is shaped by both environmental conditions and host filtering processes. Culture-bdependant analyses recovered diverse endophytic genera, mainly Achromobacter, Pseudomonas, and Glutamicibacter, most of which were also detected in the amplicon sequencing dataset. Genome-based profiling identified traits related to stress response, osmoprotection, nutrient-related metabolism, colonization, and plant-microbe interactions. Together, these findings highlight C. colocynthis as a reservoir for functionally relevant bacterial diversity with ecological and biotechnological potential in semi-arid environments. ImportanceUnderstanding how plants survive in arid and semi-arid ecosystems is increasingly important in the context of climate change and land degradation. This study demonstrates that Citrullus colocynthis hosts a structured and functionally diverse bacteriome across the leaf endosphere, rhizosphere, and root compartments. By combining amplicon sequencing, cultivation, and genome-informed functional analyses, we identified bacterial taxa and traits associated with stress tolerance, nutrient acquisition, and plant colonization. The recovery of cultivable endophytes with adaptive genomic features highlights the potential of desert plant-associated microbiota as a source of beneficial microorganisms for sustainable agriculture and biotechnological applications in water-limited environments.

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

IntroductionMany plant species secrete nectar to attract beneficial animals. The chemical composition of floral nectar influences pollinator nutrition and behavior, as well as microbial growth in flowers. Yet factors that predict nectar composition across plant species, as well as chemical compounds determining microbial growth in nectar, remain poorly understood. MethodsWe used both targeted and untargeted metabolomics to compare the nectar chemical profiles across 31 phylogenetically diverse plant species that span a range of floral morphologies. We examined the common classes of compounds detected in nectar and patterns of co-occurrence among them. We combined newly collected chemical data with previously published data on microbial growth in nectar of the same plant species to examine how nectar chemistry is associated with microbial growth. ResultsPlant species and clades varied in amino acid, minor sugar, and secondary metabolite composition and concentration. Sampled rosids and lilioids generally contained higher amino acids while asterids contained greater concentrations of oligosaccharides and sugar alcohols. Across plant species, proteinogenic amino acids frequently co-occurred in nectar but many were negatively associated with sucrose concentration. Plant species with greater concentrations of amino acids and other nitrogen-containing compounds hosted greater microbial density in nectar, while some other compound groups were negatively associated with microbial diversity. ConclusionsNegative correlations between nectar amino acid and sucrose concentration across species suggest ecological tradeoffs or physiological constraints in nectar composition. Given that the growth of common nectar microbes is limited by amino acid concentration, these findings suggest an ecological cost to amino acid production in nectar. Finally, we document variation among species in nectar vitamins, non proteinogenic amino acids and secondary metabolites with hypothesized yet currently untested ecological roles.