Phytobiomes Journal

Harnessing plant-microbe interactions offers a promising path to reduce chemical inputs and enhance crop resilience in agricultural systems. However, microbial inoculants often fail to persist or function consistently across soils, which limits their broad utility. Here, we explore whether legume cover crops can be used to create microbial legacies that improve nodulation and nitrogen fixation in downstream legume cash crops. In a greenhouse experiment, we inoculated four cash crops with rhizosphere and nodule microbiomes derived from different legume cover crops, then used 16S rRNA and nifH amplicon sequencing to profile bacterial and diazotroph communities, respectively. Host identity shaped cover crop rhizosphere and nodule microbial communities, and specific taxa within these communities predicted nodulation and growth in some cash crops. Host specificity varied widely across cash crops, with alfalfa narrowly dependent on a specific symbiont and common bean and fava bean forming more permissive, taxonomically diverse nodule communities. Increased nodulation did not consistently improve biomass, and outcomes in some cash crops depended on more than symbiont compatibility alone. In particular, common bean growth was predicted by both rhizobial and non-rhizobial taxa, while soybean nodulation was shaped by its compatible symbiont as well as a mismatched rhizobial taxon associated with other hosts. Together, these results suggest that cover crops can shape cash crop microbiomes and productivity in host-specific ways, requiring precise symbiont matching in selective hosts but offering more flexible, multi-taxon management opportunities in permissive hosts.

Pyrroloquinoline quinone (PQQ) is known to promote plant growth, yet how the rhizosphere microbiome varies with PQQ concentration remains poorly understood. We applied metagenomic sequencing to characterize the taxonomic composition and functional potential of the tobacco rhizosphere microbiome across PQQ treatments at 0.5, 1 and 2 mol/L plus an untreated control. The rhizosphere microbiome was dominated by Pseudomonadota and Actinomycetota and differed among treatments in a concentration-associated, non-monotonic manner. The most pronounced differences were observed at 1 mol/L, where alpha diversity showed dimension-specific variation and beta-diversity analysis resolved the most distinct community profile. Taxonomic and functional profiles varied concurrently, with Streptomyces, Sphingomonas, Rhodanobacter and Lysobacter, together with functions related to biosynthesis, energy metabolism, colonization and environmental sensing, showing higher relative abundance at this concentration. No broad-spectrum antimicrobial effect was detected at the community level, and antibiotic resistance gene abundance showed no significant differences among treatments after correction for multiple testing. These findings provide a basis for further evaluation of PQQ in modulating the rhizosphere microbiome in continuous cropping systems.

Microbial community assembly on leaves (the phyllosphere) is influenced by deterministic, stochastic, and historical processes. However, the importance of historical processes such as priority effects, where the timing and order of colonization influence community assembly, has not been extensively investigated in field-based phyllosphere studies. We designed a field experiment in a temperate forest in Quebec where we inoculated five strains of Methylobacterium onto the leaves of red maple and American beech trees, either early or late during the growing season. We quantified microbial community structure throughout the season using metabarcoding of a Methylobacterium-targeting rpoB primer as well as a 16S rRNA primer targeting the entire bacterial community. We show that the effects of inoculation on bacterial communities depend on inoculation timing: early-season inoculation led to strain-specific shifts in community composition, while late-season inoculation had no significant effect. Although the effect of early-season inoculation weakened over time, early- and late-season inoculation still led to different community compositions at the end of the growing season. Our study demonstrates that priority effects contribute to the assembly of phyllosphere bacterial communities under field conditions, with arrival timing and strain identity shaping community trajectories. Considering historical processes will thus be crucial for successful microbiome management.

Background and aimsExcess nitrate (NO3-), from fertilizer overuse and intensive agriculture, can pollute water and contribute to greenhouse gas production (nitrous oxide - N2O). Plant metabolites from pastural herbs such as Plantago lanceolata (plantain) can inhibit microbial nitrification of ammonium to NO3- (biological nitrification inhibition - BNI) and change soil nitrogen cycle dynamics (lower potential nitrification rate - PNR). The main aim was to investigate differential plant metabolite expression associated with BNI and lowered PNR in different soil types. MethodsSix plantain cultivars were tested for BNI potential and screened for metabolites that correlated with inhibition of the ammonia oxidising bacterium (AOB) Nitrosospira multiformis. PNR and microbiome change was then investigated in four different New Zealand soils under the plantain cultivar Agritonic and ryegrass cultivar One50. ResultsPNR under plantain was 11 to 41% lower than fallow soil while PNR under ryegrass was 0 to 39% lower. In addition to verbascoside and aucubin, plantain metabolites associated with lower PNR included plantamajoside, riboflavin 3- and 5-sulfate, plantagoguanidinic acid. Chlorogenic acid was associated with lowered PNR under ryegrass. PNR reductions, microbiome structure and the ratio of ammonia oxidising archaea (AOA) relative to AOB was modulated by soil type. ConclusionPlantain and ryegrass lowered the PNR in four different soils and was correlated with metabolites beyond just aucubin and verbascoside. Based on candidate BNI-associated metabolites identified, it was hypothesised that lowered PNR is likely indirect through mechanisms such as chelation and appears to be dependent on both plant physiology and soil physicochemistry.

Plant microbiomes are assembled from environmental pools that differ substantially in composition, yet whether their responses to stress follow general rules or depend on the resident community remains unclear. We tested the generality of root microbiome responses to stress by growing three tomato (Solanum lycopersicum) genotypes in 20 independently sourced microbial communities under controlled abiotic and biotic stress. Microbial communities were transferred into a common sterile substrate, allowing to vary microbial community identity independently of soil physicochemical properties, and plants were exposed to drought, the foliar pathogen Pseudomonas syringae pv. tomato, or no stress. We analyzed the root microbiota using 16S rRNA amplicon sequencing. Stressor identity explained more variation in bacterial community composition than inoculum identity or plant genotype. Hierarchical models showed that many bacterial taxa responded consistently across distinct starting communities, with taxon identity contributing far more variation in stress response than microbial community of origin. Both stressors shifted between-community dissimilarity from taxon turnover toward nestedness, indicating increasingly similar patterns of taxon loss, but the pathogen produced stronger effects and increased variability among replicate plants. Drought reduced phylogenetic redundancy, whereas pathogen increased dispersal limitation. Together, these results show that stress imposes reproducible ecological filters across diverse starting microbiomes, while the magnitude and resulting community state remain contingent on the resident microbial community. This provides a basis for identifying transferable microbial targets for microbiome-based crop stress management, while also managing the local microbiome to maximize their beneficial effects.

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.

Abstract Background: Soil microbiomes play critical roles in plant health and resilience, yet it is still poorly understood how disruptions in local microbial communities, such as during soil management, impact these ecological functions. In this study, we investigated how compost addition, a widely used soil management practice, and microbiome origin influence microbiome-mediated plant resistance to insects. We combined surveys of working organic farms across New York with controlled laboratory experiments, amplicon sequencing, plant-insect bioassays, and mass spectrometry. Results: Compost use was associated with shifts in the relative abundance of 20 fungal and 2 bacterial genera across ten working farms. However, no consistent changes in bacterial or fungal diversity or community structure in fields with compost were observed across the farms. Under controlled conditions using a common compost source, compost increased rhizosphere microbial diversity and significantly altered rhizosphere community structure, including the relative abundance of 6 fungal and 41 bacterial genera. Despite these substantial community-level changes, compost did not affect populations of green peach aphids (Myzus persicae) on kale (Brassica oleracea). Instead, aphid populations and plant salicylic acid concentrations were more strongly associated with microbiome origin, demonstrating that compost can substantially restructure soil and rhizosphere microbial communities without corresponding changes in microbiome-mediated plant resistance to insects. Conclusions: Our findings demonstrate that microbial diversity and compositional responses to management are not necessarily reliable indicators of all microbiome functions. More broadly, this work highlights the importance of local soil microbiome variation in shaping plant-insect interactions and emphasizes the need to link community-level responses to direct functional measurements when evaluating microbiome responses to agricultural management.

Carbon farming practices are increasingly adopted as part of regenerative agriculture to enhance soil carbon sequestration and improve agricultural sustainability. However, their effects on the phyllosphere microbiome, and how these interact with field biodiversity, remain poorly understood. We investigated how field biodiversity and carbon farming practices (cover crops, all-in mixes, adaptive grazing and ley mixture) influence leaf microbial communities across 19 Finnish farms using ITS and 16S amplicon sequencing. Foliar fungal - but not bacterial - diversity increased with plant species richness. Carbon farming altered both fungal and bacterial community composition, but only fungal community was positively associated with disease load. Network analysis identified three candidate keystone taxa: Alternaria sp. (FASV 34) and Sporobolomyces roseus (FASV 2070), which were mutually exclusive, and Methylobacterium sp. (BASV 75), which co-occurred with FASV 2070. Together, these findings demonstrate that different carbon farming practices shape distinct leaf microbiomes cover crop systems tending to favor potential fungal pathogens, whereas ley mixture and adaptive grazing strategies promote microbial taxa associated with healthier phyllosphere communities. Our study underscores the importance of understanding the complex microbial interactions within leaf ecosystems to inform sustainable agricultural practices.

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.

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.

Nitrogen fixation performed by rhizosphere bacteria has the potential to improve the sustainability of cereal crop cultivation. Deciphering the role of interspecies interactions on nitrogen fixation is crucial for devising strategies to enhance this process. To unravel the contributions of interspecies interactions, we constructed synthetic microbial communities from the bottom-up that contain diazotrophic bacteria that fix nitrogen and maize rhizosphere bacteria that do not have this capability. Interactions that impacted nitrogenase activity via growth-independent mechanisms were prevalent in the system. Nitrogenase activity increased and eventually saturated as a function of the number of inoculated diazotrophs. Using a tailored machine learning model for microbiome dynamics and explainable artificial intelligence, we deciphered species contributions on nitrogenase activity and diazotroph growth. We identified a community containing Klebsiella variicola, Herbaspirillum seropedicae, and Stutzerimonas stutzeri as a starting point for developing microbial inoculants for cereal crops. Taken together, these results provide insights into the role of interspecies interactions on nitrogenase activity.

Tomato worldwide production is increasingly challenged by complex disease outbreaks involving multiple interacting pathogens. In Israel, recent years have seen a marked rise in vascular collapse symptoms in greenhouse-grown tomatoes, coinciding with the widespread emergence of tomato brown rugose fruit virus (ToBRFV) and pepino mosaic virus (PepMV). Here, we investigated the bacterial and viral agents associated with these outbreaks and examined how viral infection influences the development and severity of bacterial diseases that cause vascular collapse. Surveys conducted between 2021 and 2026 revealed that tomato pith necrosis outbreaks were associated with a diverse bacterial community dominated by members of the Pseudomonadales and Enterobacterales, while bacterial canker outbreaks were exclusively linked to Clavibacter michiganensis. Multilocus sequence analysis showed that pith necrosis-associated Pseudomonas isolates clustered primarily within the P. syringae, and P. corrugata phylogroups. Pathogenicity assays demonstrated that only a subset of pith necrosis-associated bacteria, P. mediterranea, P. capsici, P. viridiflava, and Xanthomonas euvesicatoria pv. perforans, induced pith necrosis under controlled conditions, with high variability in symptom severity. Co-inoculation experiments showed that ToBRFV- and PepMV-infected plants exhibited a 40-100% increase in lesion size following inoculation with pith necrosis-associated bacteria, without a corresponding increase in bacterial colonization, whereas the same viral infections attenuated wilt symptoms caused by C. michiganensis. Together, our findings demonstrate that endemic viral infections differentially modulate bacterial disease outcomes, either exacerbating or attenuating symptoms depending on the pathogen. These results highlight the importance of multi-pathogen interactions in disease severity and have important implications for tomato disease management.

Plant-associated microbial communities exhibit pronounced specificity across biological and spatial scales. While the patterns and accompanied functions have been well documented across and within plant species, the functional importance of intra-individual variation remains underexplored. Particularly in trees that experience strong environmental gradients within single crowns, stratum-specific microbiota may significantly contribute to plant performance. We experimentally tested whether variation in microbiota within the crown of Quercus robur is related to host performance. In mesocosm experiments, we transferred microbial communities derived from sun and shade leaves to germ-reduced clonal individuals of the same species and applied UV radiation simulating conditions that matched or mismatched the origin of the microbial inoculum (environmental matching). Our results demonstrate that matching microbiota-environment combinations increased plant performance compared to mismatching combinations. We infer that pronounced intra-individual variation of leaf-associated microbial communities not only reflects environmental heterogeneity along canopy strata but is functionally relevant for the plant host.

Mixing plant functional groups can boost plant productivity, but the underlying rhizosphere microbial dynamics remain unclear. We tested when to expect divergence from additive expectations by measuring plant traits and active and total rhizosphere microbiomes (via BONCAT-FACS and 16S rRNA gene amplicon sequencing) in monocultures and mixtures of legumes, brassicas, and grasses under varying nitrogen. While shoot biomass was predictable from monocultures, legume-containing mixtures yielded greater-than-expected root biomass and N-fixation. Each plant species supported distinct active and total microbiomes, and nitrogen addition increased microbiome divergence among plant species and their mixtures. In most plant mixtures, microbiomes were additive combinations of those associated with the individual species; legume-grass combinations were a notable exception, showing non-additive shifts in microbial composition, microbial activity, and the abundance of Rhizobium sp. This divergence from additive expectations coincided with non-additive biomass gains in legume-grass mixtures, suggesting that emergent plant-microbe interactions may underlie synergistic belowground responses.

AO_SCPLOWBSTRACTC_SCPLOWEndogenous viral elements (EVEs) are widespread across animal genomes, yet the processes governing EVE evolution and diversification remain poorly understood. Here, we characterize the evolution of densoviral EVEs and exogenous densoviruses across the aphid tribe Macrosiphini, an agriculturally important group in which exogenous densoviruses and their endogenous derivatives have been linked to the plastic production of wings. Using new genome assemblies, transcriptomics, and phylogenetic analysis, we find that EVE content varies extensively across species. Moreover, we discovered a novel densovirus that is vertically transmitted, but phylogenetic incongruence between other densoviruses and their hosts suggests that horizontal transmission may also occur. Finally, we show that EVE-mediated regulation of wing plasticity extends across species that use different environmental signals to induce winged offspring. Our study shows that in this system, the evolution of EVEs is highly variable and lineage-specific, generating genomic patterns that cannot be predicted from host evolutionary relationships.

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.

Barley yellow dwarf virus (BYDV), transmitted by the bird cherry-oat aphid (Rhopalosiphum padi L.), is among the most damaging viral diseases of barley, but the mechanisms underlying resistance to both the virus and its vector remain poorly understood. Here, we investigated resistance associated with the Hordeum bulbosum-derived Ryd4 introgression in the barley hybrid SY Kestrel by integrating behavioural, electrophysiological, physiological and multi-omics analyses with functional validation of defence metabolites. SY Kestrel exhibited constitutive volatile-mediated antixenosis together with strong post-settlement antibiosis characterised by impaired phloem feeding, reduced aphid fitness and suppression of BYDV gene expression 10 days after transmission. Integrated transcriptomic, metabolomic and small RNA analyses revealed coordinated immune activation, chloroplast remodelling and defence metabolism associated with the resistance introgression. Candidate immune regulators were identified both within the refined Ryd4 interval and the surrounding introgressed region. Maintenance of photosystem II function was accompanied by reprogramming of -linolenic acid-derived oxylipin metabolism, while phenylpropanoid and branched-chain amino acid/lysine pathways generated metabolites that directly reduced aphid survival. We demonstrate that the Ryd4 introgression coordinates constitutive vector deterrence with host defence reprogramming to restrict aphid colonisation and suppress BYDV establishment. These results provide a mechanistic framework for improving durable resistance to aphid-transmitted viruses in cereals.

Xylella fastidiosa is one of the most important plant pathogens worldwide, infecting more than 700 plant species and causing major economic losses in affected agricultural regions. In the absence effective curative treatments under field conditions, experimental research is essential. However, research efforts are severely constrained by strict phytosanitary regulations and the pathogen's restricted geographic distribution, as well as its demanding culture requirements. To the best of our knowledge, no studies have compared different host infection models under controlled growth-chamber conditions. To address these limitations, we developed and compared a microinjection-based inoculation protocol across herbaceous and woody hosts maintained in biosafety growth chambers. Stems of Vitis vinifera, Nicotiana tabacum, N. benthamiana, and Medicago sativa were inoculated with X. fastidiosa by microinjection and maintained under controlled growth-chamber conditions. Pathogen establishment was assessed by qPCR analysis of petiole tissue, together with disease progression, plant growth, and leaf scorch development. Approximately one month after inoculation, bacterial detection rates reached 100% in V. vinifera and 80% in N. tabacum. Both species subsequently exhibited measurable disease development, whereas bacterial establishment and phenotypic responses were less consistent in N. benthamiana and M. sativa.

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.

Antibiotic-induced disruption of plant-associated microbiomes has the potential to alter host health beyond the directly exposed generation, yet whether the effects of dysbiosis are transmitted through the seed microbiome remains unknown. Here, we investigated the intergenerational impacts of streptomycin-induced dysbiosis in tomato (Solanum lycopersicum), demonstrated that seed microbiome transfer (SMT) restores progeny microbiome function and disease resistance, and characterized the underlying physiological and genetic mechanisms. Parental streptomycin exposure altered the composition of progeny rhizosphere bacterial communities, reduced expression of defense-associated genes, and increased susceptibility to Xanthomonas perforans. Suppression of immune gene expression was strongly associated with increased disease severity, indicating that parental dysbiosis impaired progeny plants ability to mount effective immune responses. Transfer of the seed microbiome from healthy plant donors partially restored rhizosphere community composition, reduced disease severity and recovered defense gene expression of three genes. Together, our findings demonstrated that antibiotic exposure microbiome disturbance generates intergenerational legacy effects that influence plant immunity and disease susceptibility and seed microbiome transfer can counteract this dysbiosis across generations.