Phytobiomes Journal

O_LIThe above-ground plant microbiome (the phyllosphere) is increasingly recognized as an important component of plant health. We hypothesized that phyllosphere interactions may be disrupted in a greenhouse setting, where microbial dispersal is limited, and that adding a microbial amendment might yield important benefits to the host plant. C_LIO_LIUsing a newly developed synthetic phyllosphere microbiome for Tomato, we tested this hypothesis across multiple trials by manipulating microbial colonization of leaves and measuring subsequent plant growth and reproductive success, comparing results from plants grown in both greenhouse and field settings. C_LIO_LIWe confirmed that greenhouse-grown plants have a depauperate phyllosphere microbiome and that the addition of the synthetic microbial community was responsible for a clear and repeatable increase in fruit production in this setting. We further show that this effect is synergistic with the addition of micronutrient-based soil amendments, with important implications for agriculture. C_LIO_LIThese results suggest that greenhouse environments have poor phyllosphere microbiome establishment, with negative impacts on the plant. The results also implicate the phyllosphere microbiome as a key component of plant fitness, emphasizing that these communities have a clear role to play in the ecology and evolution of plant communities. C_LI

Tomatoes are an important crop worldwide and phytopathogens can cause devastating losses. Our study describes a treatment, consisting of Azospirillum brasilense Sp7 and either tomato seedling exudate or the exudate compound cytidine. The combination of A. brasilense Sp7 with cytidine showed a remarkable reduction of 83.4% in disease severity of tomatoes challenged with Pseudomonas syringae pv. tomato DC3000. Replacing cytidine with exudate was less effective at 71%, but the reduction in disease severity was still larger than by A. brasilense Sp7 alone at 55%. This reduction in disease severity was not paralleled by a decrease in P. syringae in leaf homogenates. Cytidine caused a 6.7 fold increase in A. brasilense Sp7 16S rDNA in root homogenates. In phyllosphere and rhizosphere, treatments modulated the microbial composition. In the phyllosphere, A. brasilense together with cytidine or tomato seedling exudate reduced the presence of eight human and/or plant pathogens in P. syringae challenged test groups. In the rhizosphere, principal component analysis revealed that variation along PC1 was dominated by the presence or absence of A. brasilense. Intriguingly, the inoculant caused an increase in the abundance of other Azospirillales species. Les tomates sont une culture dimportance mondiale, et les phytopathogenes peuvent entrainer des pertes devastatrices. Notre etude decrit un traitement compose dAzospirillum brasilense Sp7 et soit dun exsudat de plantules de tomate, soit de la cytidine, un compose particulier provenant des exsudats et suppose avoir un effet chimiotactique. Lassociation de A. brasilense Sp7 avec la cytidine a entraine une reduction remarquable de 83,4 % de la severite de la maladie parmi les plantes de tomate infectees par Pseudomonas syringae pv. tomato DC3000. Le remplacement de la cytidine par des exsudats etait moins efficace (71 %), bien que la reduction de la severite de la maladie ait demeure superieure a celle obtenue avec A. brasilense Sp7 seul (55 %). Cette diminution de la severite de la maladie na toutefois pas ete accompagnee dune reduction de la concentration de P. syringae dans les homogenats foliaires. La cytidine a provoque une augmentation de 6,7 fois de lADNr 16S de A. brasilense Sp7 dans les homogenats racinaires. Dans la phyllosphere et la rhizosphere, les traitements ont module la composition microbienne. Dans la phyllosphere, A. brasilense associe a la cytidine ou aux exsudats de plantules de tomate a reduit la presence de huit agents pathogenes humains et/ou vegetaux. Dans la rhizosphere, lobservation la plus remarquable est que la composante (PC1) de lanalyse en composantes principales (ACP) etait fortement influencee par la presence ou labsence de linoculum. De maniere interessante, A. brasilense Sp7 a entraine une augmentation de labondance dautres especes dAzospirillales.

The phyllosphere is a restrictive environment for microbes, resulting in microbial communities typically dominated by select taxa with specific adaptations for success in this niche. However, biotic stress, especially from plant disease, could disrupt this environment in ways that alter the resulting phyllosphere community, with potential consequences for plant and human health. Additionally, plant disease resistance, through both broad (pattern-triggered immunity) and specific (effector-triggered immunity) resistance, could affect non-pathogenic communities directly or indirectly via effects on disease progression. Here, we tested how transgenic ETI and PTI resistance genes affected the phyllosphere communities of tomato plants in the face of infection by Xanthomonas perforans and the resulting bacterial spot disease. We found that the expression of the Bs2 transgene (ETI) had major effects on phyllosphere communities, while the EFR (PTI) transgene did not. The effect of the Bs2 resistance gene could be largely attributed to the change in disease symptoms. Diseased leaves harbored reduced bacterial diversity and reductions in major phyllosphere inhabitants (e.g. Sphinogmonas and Methylobacterium), while a limited number of bacterial genera showed increased relative abundance on diseased leaves. These results suggest that phyllosphere communities are sensitive to the direct and indirect effects of plant disease and resistance, and the consequences of these shifts for plant and human health deserve further investigation.

The European hazelnut, Corylus avellana, is one of the most economically important tree nut crops globally. The biotrophic ascomycete pathogen Anisogramma anomala, found naturally associated with wild C. americana, continues to pose a significant threat to European hazelnut production across North America. Here, metagenomics was used to examine the taxonomic and functional features of the rhizosphere microbial communities of hazelnut trees differing in their levels of resistance to A. anomala: highly tolerant Corylus americana, and resistant and susceptible Corylus avellana. No statistically significant differences in microbial alpha diversity or beta diversity were noted between the three rhizosphere groups. Compared to bulk soil, all three rhizosphere groups were enriched for the fungal phylum Basidiomycota and bacterial phylum "Candidatus Rokubacteriota". At the genus level, the bacterial genera Actinospica, Occallatibacter, and "Candidatus Sulfotelmatobacter" were under-represented, while the genus Rhizobacter was over-represented, in the resistant and susceptible C. avellana rhizosphere samples compared to the bulk soil. A total of 45 dereplicated, high-quality metagenome-assembled genomes (MAGs) were generated, corresponding to 41 bacteria and 4 archaea. Many of the MAGs carried multiple biosynthetic gene clusters, including MAGs corresponding to the genera Lysobacter and Actinospica. Overall, the low differentiation of the rhizosphere microbiomes suggest that differences in A. anomala disease expression are likely not associated with differences in the rhizosphere microbiome. Nevertheless, the results shed new light on the rhizosphere communities of two species of hazelnut, and woody perennials more broadly, and identify potential avenues for future research into the development of microbial inoculants for Corylus spp..

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.

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

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.

Phyllosphere exudates create specialized microhabitats that shape microbial community diversity. Here, we explore the microbiome associated with two sorghum phyllosphere exudates, the epicuticular wax and aerial root mucilage. We hypothesized that these exudates selectively enrich for microbiome members that support host resilience to stress. Thus, we assessed the microbiome associated with the epicuticular wax from sorghum plants under non-limiting and limiting water conditions, and the aerial root mucilage from nitrogen-fertilized and non-fertilized plants. In parallel, we isolated and characterized hundreds of bacteria from wax and mucilage, and integrated data from cultivation-independent and -dependent approaches to gain deeper insights into phyllosphere functions and phenotypes. We found that Sphingomonadaceae and Rhizobiaceae families were the major taxa in the wax regardless of water availability to plants and that plant development only modestly affected wax bacterial community structure. The mucilage-associated bacterial microbiome contained several described diazotrophic species, and its structure was strongly influenced by sorghum development but only modestly influenced by fertilization. In contrast, the fungal community structure of mucilage was strongly affected by the year of sampling but not by fertilization or plant developmental stage, suggesting a decoupling of fungal-bacterial dynamics in the mucilage. Our bacterial isolate collection from wax and mucilage increased phylogenetic diversity of non-rhizosphere, plant-associated bacteria by ~20% from previous work, and several isolates matched 100% to detected amplicon sequence variants. This work expands our understanding of the microbiome of phyllosphere exudates and advances our long-term goal of translating microbiome research to support sorghum cultivation for biofuel production.

Similar to the human gut microbiome, diverse microbes colonize the plant rhizosphere, and an imbalance of this microbial community, known as dysbiosis, may negatively impact plant health. This study aimed to investigate the influence of rhizosphere dysbiosis on above-ground plant health using tomato plants (Solanum lycopersicum L.) and the foliar bacterial spot pathogen Xanthomonas perforans as model organisms. Four-week-old tomato plants rhizospheres were treated with streptomycin (0.6 g x L-1), or water (negative control) and spray-inoculated with X. perforans (105 cells x mL-1) after 24 h. Half of the plants treated with streptomycin and X. perforans received soil microbiome transplants (SMT) from uninfected plant donors 48 h after streptomycin application. Streptomycin-treated plants showed a 26% increase in disease severity compared to plants that received no antibiotic, while plants that received the SMT had an intermediate level of disease severity. Antibiotic-treated plants showed a reduced abundance of rhizobacterial taxa like Cyanobacteria from the genus Cylindrospermum as well as down-regulation of genes related to plant primary and secondary metabolism and up-regulation of plant defense genes associated with induced systemic resistance (ISR). This study highlights the crucial role of beneficial rhizosphere microbes in disease resistance, even to foliar pathogens.

Microbes in floral nectar can impact both their host plants and floral visitors, yet little is known about the nectar microbiome of most pollinator-dependent crops. In this study, we examined the abundance and composition of the fungi and bacteria inhabiting Vaccinium spp. nectar, as well as nectar volume and sugar concentrations, hypothesizing that nectar traits and microbial communities would vary between plants. We compared wild V. myrsinites with two field-grown V. corymbosum cultivars collected from two organic and two conventional farms. Differences in nectar traits and microbiomes were identified between V. corymbosum cultivars but not Vaccinium species. The microbiome of cultivated plants also varied greatly between farms, whereas management regime had only subtle effects, with higher fungal populations detected under organic management. Nectars were hexose-dominant, and sugars were depleted in nectar with higher cell densities. Bacteria were more common than fungi in blueberry nectar, although both were frequently detected and co-occurred more often than would be predicted by chance. Cosmopolitan blueberry nectar microbes that were isolated in all plants, including Rosenbergiella sp. and Symmetrospora symmetrica, were identified. This study provides the first systematic report of the blueberry nectar microbiome, which may have important implications for pollinator and crop health. One-sentence summaryParallel analysis of blueberry crops and a wild relative offers insight into the impacts of management and domestication on the nectar microbiome O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/556904v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@15f36caorg.highwire.dtl.DTLVardef@63496org.highwire.dtl.DTLVardef@1667a6eorg.highwire.dtl.DTLVardef@eff26e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Agricultural management practices act as ecological disturbances that can restructure soil and plant-associated microbial communities, but the functional consequences of these microbial shifts on crop performance remain poorly understood. Here, we examined how common orchard inputs, including wood mulch, glyphosate, and humic acid, affect citrus root and rhizosphere microbiomes and tree performance over a three-year field experiment. Mulch emerged as the dominant driver of microbiome structure, significantly altering bacterial and fungal community composition and increasing rhizosphere alpha diversity. Root microbiomes remained comparatively stable, suggesting stronger host selective forces within root tissues. Mulched rhizospheres were enriched with saprotrophic fungi and metabolically diverse bacteria, while non-mulched soils contained taxa typically associated with nutrient cycling, like Rhizobium, Sphingomonas, and Nitrososphaera. Interactions between mulch and glyphosate further reshaped bacterial communities and corresponded with reduced tree physiological performance, including photosynthesis rates. To verify whether these microbial shifts were contributing to these plant phenotype changes, we conducted a greenhouse experiment using field-derived soil microbiota. Active microbiota from mulch-treated soils reduced citrus seedling establishment and root growth relative to microbiota from non-mulched soils, whereas heat-killed controls eliminated these negative effects, demonstrating a causal relationship between management-induced microbiota changes and decreases in plant performance. In contrast, humic acid influenced plant growth primarily through direct abiotic effects rather than microbial community-level traits. Together, our results show that orchard management practices can restructure citrus microbiomes and generate community-level traits that influence plant performance, highlighting the importance of incorporating microbial ecology and microbiome information when designing and testing crop management strategies.

The seed microbiome plays a key role in the assembly of the plant microbiome, which has major impacts on plant functioning. Nonetheless, little is known about the origin of the seed microbiome. We investigated the relative contributions of two hypothesized transmission routes: sexual inheritance (via reproductive organs) and asexual inheritance (via the plant vascular system). To do that, we sampled shoot endophytes both before bloom and at seed maturity stages, apple flower ovaries and pollen sacs, and mature seeds from field-grown apple trees (Malus domestica BO_SCPLOWORKHC_SCPLOW. cv Gala Galaxy Selecta). We showed that bacterial richness, diversity and composition differ among tissue types, with shoots before bloom harboring a higher diversity than ovary and pollen. Source tracking revealed that both sexual (30.3%) and asexual (23.8%) pathways contributed to seed microbiome assembly, with shoots at seed maturity being the dominant source. Notably, a large proportion (49.5%) of the seed microbiome originated from unknown sources. Lastly, the transmission pathways significantly differed among bacterial genera, with Pseudomonas primarily linked to shoots, Rhizobacter to pollen and Burkholderia to the ovary. Insights into seed microbiome origin offers new opportunities to enhance seed health and crop productivity through microbiome-assisted breeding.

Plant root microbiomes play a central role in plant health, yet their responses to viral infection remain poorly understood. Here, we investigated how Turnip mosaic virus (TuMV) alters root-associated bacterial and fungal communities in two Arabidopsis thaliana genotypes (Col-0 and Mar-12) grown in natural soils. Using 16S and ITS amplicon sequencing, we assessed changes in diversity, taxonomic composition, enriched microbial taxa, and co-occurrence network structure to distinguish between plant-mediated recruitment ("cry-for-help") and pathogen-induced dysbiosis. TuMV infection caused a pronounced reduction in bacterial diversity and a restructuring of bacterial community composition, whereas fungal communities remained largely stable. Viral infection also led to genotype-specific shifts in enriched bacterial genera, with opportunistic and stress-tolerant taxa proliferating differently in each genotype. Despite the initial perturbation, bacterial networks recovered connectivity and, in some cases, reached higher complexity than those of healthy plants, indicating strong microbial resilience. Together, these results reveal that TuMV infection acts as a selective filter on bacterial, but not fungal, root communities and that the surviving taxa can reorganize into functional networks. Our study provides one of the most comprehensive assessments of virus-induced microbiome restructuring and highlights the importance of host genotype in shaping microbial responses to biotic stress.

O_LISeed banks are a vital resource for preserving plant species diversity globally. However, seedling establishment and survival rates from banked seeds can be poor. Despite a growing appreciation for the role of seed microbiota in supporting seed quality and plant health, our understanding of the effects of conventional seed banking processes on seed microbiomes remains limited. C_LIO_LIWe investigated the composition and functional potential of the epiphytic seed microbiome of a native plant species using both 16S rRNA gene sequencing and culture-based approaches. C_LIO_LIComparing the bacterial community composition of freshly collected seeds and those sourced from seed banking organisations, we found stored seeds hosted significantly less diverse bacterial populations, with substantial reductions in both low-abundance taxa and some core community members identified in unstored seeds. Bacteria with key plant growth promoting traits including IAA production, ACC deaminase activity, phosphate solubilisation, siderophore activity, and nitrogen fixation were identified in seed epiphytic communities, but these beneficial traits were less prevalent in stored seed compared to fresh seeds. C_LIO_LIOverall, these results suggest that epiphytic seed microbiomes may undergo significant changes during the storage process, selecting for bacteria tolerant to storage conditions, and potentially reducing the population of plant-growth promoting bacteria on seeds. C_LI

Metabolic engineering of crops can redirect host carbon flux, but its consequences for microbiomes remain unclear. Here, we show that engineering oilcane for triacylglycerol (TAG) accumulation reshapes rhizosphere microbial guilds across greenhouse and field environments while preserving functional capacity. Using 36 rhizosphere metagenomes from wild-type sugarcane and engineered oilcane accessions, we reconstructed metagenome-assembled genomes and linked community turnover with shifts in functional potential. Oilcane rhizospheres exhibited taxonomic restructuring relative to wild-type plants, driven primarily by turnover rather than nestedness and marked by genotype-dependent replacement of microbial guilds. These patterns were strongest in accession 1566 and amplified under field conditions. Despite these compositional shifts, broad patterns of functional potential remained similarly distributed, whereas pathway-level differences were evident in energy production and conversion, lipid transport and metabolism, secondary metabolite biosynthesis, transport and catabolism, and signal transduction. These findings extend evaluation of engineered crops beyond host traits alone to include microbiome-scale responses.

Understanding the resident microbial communities and their above and below ground interactions with plants will provide necessary information for crop disease protection and stress management. In this study, we show how diversity of core microbiome varies with disease susceptibility of a crop. To test this hypothesis, we have focused on identifying the core microbial species of Cotton Leaf Curl Disease (CLCuD) susceptible Gossypium hirsutum and CLCuD resistant Gossypium arboreum under viral infestation. Derivation of core membership is challenging as it depends on an occupancy threshold of microbial species in a sampling pool, whilst accounting for different plant compartments. We have used an abundance-occupancy distribution approach where we dynamically assess the threshold for core membership, whilst marginalizing for occupancy in four compartments of the cotton plant, namely, leaf epiphyte, leaf endophyte, rhizosphere, and root endophyte. Additionally, we also fit a neutral model to the returned core species to split them into three groups, those that are neutral, those that are selected by the plant environment, and finally those that are dispersal limited. We have found strong inverse relationship between diversity of core microbiome and disease susceptibility. A deeper understanding of this association will aid in the development of biocontrol agents for improving plant immunity against biotrophic pathogens.

Bacterial culture collections are essential resources for exploring the diversity of microorganisms and their interactions with each other and their hosts. Here, we report on the sequencing of the first 129 bacterial isolates, representing 34 genera, from a culture collection of more than 600 bacterial strains originally isolated from leaves of a naturalised Arabidopsis thaliana population from [O]tautahi (Christchurch), Aotearoa New Zealand. Epiphytic (leaf surface), and endophytic (apoplastic) bacteria were isolated separately from the same leaves, providing complementary insights into both compartments. The recovered isolates encompass the dominant taxa typically associated with the Arabidopsis phyllosphere, including Pseudomonas, Sphingomonas, Methylobacterium, and Flavobacterium. Their full genome assemblies (BUSCO average completeness > 99%, checkM average completeness > 97% and average contamination < 1%) were analysed and compared to assess genomic features across epiphytic and endophytic lineages. While the epiphytic and endophytic strain collections did not show large genomic differences, certain functional categories differ, such as terpene biosynthesis and biofilm formation being enriched in epiphytic strains, while arginine biosynthesis and carbohydrate degradation were associated with endophytic strains. These data provide a genomic foundation for future experimental work on leaf-associated microbial ecology and plant-microbe interactions. To our knowledge, this is the first Arabidopsis leaf culture collection established from a Southern Hemisphere source.

The emerging field of plasma agriculture investigates the potential benefit of non-thermal plasma (NTP) for agricultural practices. NTP-treated water, referred to as plasma activated water (PAW), has been proposed as a sustainable alternative to conventional nitrogen (N) fertilizers. Growing demand for N fertilizer is concomitant with increased global food demands. PAW contains nitrate (NO3-) and reactive oxygen species, such as hydrogen peroxide (H2O2), which are fixed from atmospheric molecules via NTP. While early studies report positive effects of PAW on plant growth, its influence on plant-associated microbial communities remains poorly understood. Here, we compared the impacts of PAW or NO3- solutions on the rhizosphere microbial community of Arabidopsis thaliana and Solanum lycopersicum. PAW was generated by a radio frequency (RF) glow discharge plasma source and contained no measurable ROS, while the control solution contained an equivalent concentration of NO3-. No significant differences in alpha diversity were detected in either plant species microbiome after 5 weeks of treatment when grown in non-commercial potting substrate. Significant dissimilarity was found in terms of beta diversity, but the relative abundance of the sequenced genera suggested no functional differences in rhizosphere communities. Overall, PAW treatment did not adversely impact the rhizosphere microbiome in either Arabidopsis or tomato. These results support the use of PAW as an alternative N-fertilizer, though outcomes may differ for PAW solutions containing ROS.

It has become increasingly clear that the microbiome plays a critical role in shaping the host organisms response to disease. There also exists mounting evidence that an organisms ploidy level is important in their response to pathogens and parasites. However, no study has determined if or how these two factors influence one another. We investigate the effect of whole-genome duplication in Arabidopsis thaliana on their above-ground (phyllosphere) microbiome, and determine the interacting impacts of ploidy and the microbiome on disease outcome. Using seven independently derived synthetic auto-tetraploid Arabidopsis accessions, a synthetic leaf-associated bacterial community, and the model pathogen Pseudomonas syringae pv. Tomato DC3000, we confirm that polyploids are generally more resistant to pathogens, but illustrate that this resistance may be in part due to a decrease in the reliance on beneficial bacteria. Polyploids fare better against the pathogen than diploids regardless of microbial inoculation, while we observed that diploids harboring an intact microbiome have lower pathogen densities than those without. We then use RNA sequencing to show that diploids have many more differentially expressed defense-related genes in the presence of their phyllosphere microbiota, while polyploids exhibit constitutively activated defenses regardless of exposure to the synthetic community. These results imply that whole-genome duplication can disrupt historical host-microbiome associations, and suggest that a potential cause or consequence of disruption is a heightened capacity for pathogen defense that is less impacted by the microbiome.

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.