Phytobiomes Journal

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.

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.

AbstractSoil microorganisms are crucial for plant survival and productivity, but factors governing rhizosphere recruitment across diverse regions remain unclear. This study investigated the rhizosphere microbiome of barley, using elite cultivars across seven location-year trials to evaluate the effects of environmental factors and crop genotype on bacterial and fungal community composition. Three locations were in the US northern Great Plains, and Hawaii was used as a contrasting environment. A greenhouse reciprocal transplant study determined the relative contributions of soil physicochemical factors and soil inoculum to rhizosphere community structure. Using 16S and ITS2 amplicon sequencing, the study characterized bacterial and fungal microbiomes and assessed the contribution of environment, soil chemistry, and barley genetics to microbial community assembly. In locations within the adapted range of barley, Actinobacteriota was the dominant phylum, while Proteobacteria was dominant in Hawaii. Variance partitioning showed that 73% of bacterial and 80% of fungal genera were associated with location-year effects while 53% of bacterial and 36% of fungal genera were responsive to soil factors. Enrichment analysis found 21.6% of bacterial and 51.4% of fungal ASVs were unique to specific barley genetic subpopulations. Results from the reciprocal transplant study validated field observations by demonstrating that 20.7% of the variation in community structure was explained by soil while 18.2% was explained by inoculum source. These findings demonstrate that environmental variation is the dominant constraint on rhizosphere community composition but within these constraints, barley genotype drives recruitment of distinct bacterial and fungal taxa. ImportanceThese findings underscore the complex interplay between plant genotype, environment, and microbial community assembly, providing insights into how barley recruits distinct microbial communities in the rhizosphere across different environments. These insights have the potential to be leveraged for management and plant breeding strategies to optimize plant-microbe interactions for enhancing agricultural sustainability.

Plant-associated microbiomes contribute to plant health and resilience, yet the extent to which host traits shape microbiome assembly remains poorly understood despite increasing interest in leveraging them for crop performance. Here, we investigated whether drought-response phenotypes are associated with reproducible patterns of microbiome assembly across peanut genotypes under field conditions. The cultivars represented three drought-response categories: water-savers with tighter stomatal regulation, water-spenders with deeper root systems, and drought-sensitive genotypes. Bacterial and fungal communities were characterized from bulk soil, rhizosphere, and root endosphere compartments of six non-stressed peanut cultivars. Both host genotype and drought-response phenotype were associated with microbiome composition, with phenotype-associated patterns remaining detectable across multiple genetic backgrounds. Unexpectedly, the strongest phenotype-associated differences occurred in bulk soil communities, suggesting plant-mediated effects extending beyond the immediate root zone. Community differences were driven primarily by shifts in the relative abundance of existing taxa rather than turnover of distinct microbial lineages. Fungal communities responded more strongly to host phenotype than bacterial communities, with water-spender genotypes supporting greater fungal diversity and uniquely enriched taxa in the rhizosphere and endosphere. Neutral community modeling indicated stronger deterministic filtering of fungi than bacteria. Together, these findings demonstrate that drought-response phenotypes shape reproducible microbiome variation before stress exposure. HighlightThis study investigates the potential for host phenotype-associated drivers of microbiome assembly in drought-tolerant peanut cultivars that represented different physiological mechanisms for drought tolerance.

Amplicon sequencing remains the benchmark technique for characterizing microbial communities, but the limitations of PCR bias and single-locus targets conspire to limit conclusions. Metagenomics, the primary alternative, shares with amplicon sequencing challenges of economic scaling at adequate sequencing depth. Targeted enrichment strategies can improve the data resolution and economics of sequencing efforts while reducing methodological bias. Here, we develop a target capture strategy for metagenomic characterization of eukaryotic ribosomal DNA and root nodule symbiosis genes and test it in the metagenomes of plant root nodules. We utilize biotinylated RNA probes to selectively capture genomic regions of interest from complex environmental DNA samples, avoiding forms of PCR bias that can undermine community characterization and overcoming the need for conserved priming sites often lacking in functional genes. We designed custom probe sets targeting conserved flanking regions of eukaryotic ITS and known root nodule symbiosis (RNS)-related genes and tested them on diverse root nodule metagenomes and a mock community. We observed high recovery of target loci from samples, very high on-target read proportions, and enhanced detection of low-abundance taxa compared to amplicon sequencing, including stronger alignment with known mock community compositions. This approach will enable deeper insights into the phylogenetic diversity of eukaryotic symbionts, their genomic adaptations, and the functional potential of symbiotic interactions in a cost-effective manner suitable for large-scale projects. This strategy advances our understanding of microbial community dynamics and symbiotic relationships in natural and anthropogenic ecosystems.

Rhizosphere microbial communities contribute to the growth and health of their host but may be altered by the incidence of soil-borne pathogens. In avocado, the oomycete Phytophthora cinnamomi, causal agent of Phytophthora root rot (PRR), has been shown to alter rhizosphere bacterial communities, although its effect on fungal communities has seldom been explored. Our objective was thus to determine whether P. cinnamomi induced shifts in diversity, composition and co-occurrence networks of fungal communities in the rhizosphere of avocado trees, and to identify potential antagonists of P. cinnamomi that could be further considered for disease management. Fungal communities associated with the rhizosphere of asymptomatic and PRR-symptomatic avocado trees were studied through ITS metabarcoding. Although -diversity metrics were not significantly different between asymptomatic and PRR-symptomatic trees, differences in {beta}-diversity of rhizosphere fungal communities were detected. Moreover, PRR led to the enrichment of saprotrophic taxa and opportunistic pathogens such as Fusarium, Cladosporium or Plectosphaerella in the avocado rhizosphere, which were possibly attracted by the release of resources from necrosed roots. Co-occurrence network analysis revealed that fungal networks in the rhizosphere of PRR-symptomatic trees were more complex and connected than those from asymptomatic trees, suggesting a response of fungal communities to the disturbance caused by the pathogen. Some connector taxa from the PRR-symptomatic networks (Gibellulopsis, Cladorrhinum or Mycenella) were also identified as members of the P. cinnamomi pathobiome. Their negative correlations with the pathogen indicate they may act as potential antagonists, which calls for further isolation efforts to confirm their biocontrol activity of PRR.

Nitrogen-fixing symbioses, particularly those occurring in root nodules, are among the most consequential mutualisms in natural and agricultural systems and represent a globally important source of bioavailable nitrogen. Despite their importance, patterns of diversity and composition among diazotrophic symbionts--and the processes structuring those patterns in natural systems--remain poorly resolved, with competing hypotheses emphasizing ecological, or phylogenetic constraints on host-symbiont associations. Here, using a broad survey of nodulating plants from the southeastern United States, we examine how diazotrophic symbiont communities vary across host plant phylogeny, habitat context, and geographic origin. We find that host phylogeny is the primary determinant of symbiont composition, outweighing effects of fine-scale taxonomic identity. Symbiont associations are therefore structured mainly at deeper phylogenetic levels, consistent with phylogenetically constrained, or "fuzzy," host specificity. Likewise, nodule community diversity--potentially reflecting variation in host control over infection--differs primarily among higher-level clades rather than among closely related taxa. Habitat context also shapes nodule communities, but its influence is secondary and most evident in undisturbed environments. As well, nonnative legumes harbor distinct symbiont assemblages despite occupying similar habitats, whereas distantly related legume clades share symbionts across habitats, highlighting interactions among phylogeny, ecology, and geographic history. Overall, our results show that host phylogeny exerts the strongest influence on nodule microbial communities, likely reflecting evolutionary divergence in symbiotic function across major host lineages.

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 growth-promoting bacteria are gaining significant attention as promising biofertilizers. However, the inconsistency between in vitro plant growth-promoting traits and actual field performance remains a challenge, driven partly by a limited understanding of in situ colonization. This study characterized the colonization patterns of Citrobacter sedlakii CESi7, a novel plant growth-promoting bacterium, isolated from oil palm waste compost, during Brassica rapa cultivation. The in situ behavior of CESi7 was observed in both sterilized medium and non-sterilized soil using fluorescence in situ hybridization with a strain-targeting probe. The results revealed that CESi7 can establish both epiphytic and endophytic populations that transiently colonize roots. In a sterilized medium, CESi7 was widely distributed throughout the root tissues. Conversely, in non-sterilized soil, the bacterium formed dense aggregates specifically at the root tips. This study provides direct microscopic evidence of the colonization strategy of CESi7, offering crucial insights for its development as an effective biofertilizer.

Plants form holobionts by associating with diverse microbiota. Self-organization gives rise to emergent properties of the holobiont, such as increased resistance to pathogens. However, the local factors contributing to the self-organization are not well understood. We hypothesized that nematode communities and their associated microbiota govern the rhizobiome of the model plant holobiont tomato in terms of its suppression of root invasion by the parasite Meloidogyne hapla, and that the soil legacy influences the suppressive potential mediated by these biota. In pot experiments, a resistant tomato holobiont was favored by assembly in the presence of a nematode community conditioned by tomato plants, compared to oilseed rape or fallow soil. Nematode communities conditioned by tagetes could enhance resistance even better than tomato. Microbiota from crushed tomato-conditioned nematode communities increased resistance of the tomato holobiont, compared to microbiota of nematode communities conditioned by maize, or heat-inactivated microbiota. The 0.2 micrometre filtered microbiota from crushed nematodes had the same effect, suggesting a role of nematode-associated bacteriophages in holobiont assembly. The results indicate that soil nematodes and their associated microbiota play a role in the local organization and stabilization of plant holobionts. They can influence the resistance of plants that subsequently grow in the same soil. From an applied perspective, crop rotation schemes that alter nematode-microbiota communities could be harnessed to engineer crop holobionts.

Fungal plant pathogens can be affected by the bacteria they interact with in their environment, yet the characterization of these interactions beyond direct antagonism is lacking, especially in the case of endohyphal bacteria (EHB). Though limited in characterized examples, EHB can alter disease severity of their fungal host, providing either a potential tool or target for control. We screened isolates of Fusarium oxysporum f. sp. niveum (FON), an important soil-borne watermelon pathogen, using 16S PCR and fluorescence in situ hybridization microscopy to identify novel EHB. A symbiont of FON AS124 was identified to be a Paenibacillus sp. through genome sequencing and average nucleotide identity. To begin characterizing this relationship, we conducted watermelon infection assays using FON cured of its symbiont, the native association, and a coinoculation of fungi and bacteria. Disease severity was reduced in watermelon seedlings inoculated with the native association, though not in the coinoculation, and Paenibacillus sp. CB74 did not alone promote plant growth or inhibit fungal growth. This study shows an important functional outcome, reduced disease, for a novel symbiosis between FON and Paenibacillus sp. CB74, setting up further investigation into the mechanisms behind this outcome and the application of this interaction. ImportanceFungi pose a challenge in both the field and hospital as antifungal resistance rises and chemical control is increasingly scrutinized. In plant pathogenic fungi, endohyphal bacteria may present alternative targets or mechanisms of fungal control. These relationships are observed across diverse groups of fungi and bacteria, though few have been studied to the point of understanding impact. To contribute to the small but growing catalog of known endofungal bacterial relationships, we identified a novel symbiosis and began characterizing its functional outcomes with plant infection assays. The identified bacterial symbiont does alter disease severity of the fungal host offering a new system for both application and study of fungal pathogenesis.

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.

O_LIIn natural and agricultural systems, legumes recruit rhizobia from diverse soil populations to fix nitrogen in root nodules. A few legumes, including the model legume Medicago truncatula, can select and enrich beneficial rhizobia. Here, we investigated whether its perennial relative, Medicago sativa (alfalfa), a globally important forage crop, also possesses this ability. C_LIO_LIWe developed a genetically variable collection of 117 Sinorhizobium meliloti strains sampled from three field-grown alfalfa varieties, performed multi-strain and single-strain inoculations in a nitrogen-free greenhouse experiment across the same hosts, and evaluated plant benefits and relative strain fitness in nodules. C_LIO_LIAlfalfa varieties differed in which strains best promoted plant growth and which strains had high fitness in nodules. Regressing strain fitness and host benefit revealed that two of three alfalfa varieties selected and enriched more beneficial strains during symbiosis, though the strength of selection was weak. In alignment with these results, no variety produced as much biomass in mixed inoculation as it did with the best-performing single strain. C_LIO_LILegumes ability to enrich beneficial rhizobial populations from field-representative strain diversity warrants further study to develop optimized varieties. Ultimately, identifying crop varieties that naturally select for beneficial bacteria could reduce the need for repeated inoculant applications. C_LI

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.

We describe a biodegradable-cup bioassay for detecting viable Plasmodiophora brassicae in soil samples. Soil samples either artificially inoculated with P. brassicae resting spores or collected from canola fields were aliquoted into biodegradable cups containing 20 g of soil per cup. Two cups representing the same soil sample or inoculum concentration were placed in each pot filled with Sunshine Mix. Six seeds of the canola cultivar Westar were sown into each cup and thinned to four seedlings per cup ten days after planting. After four weeks, roots were examined for the presence of clubroot galls. Across three independent inoculated-soil experiments, galls were observed in samples containing as few as 1 resting spore g-1 soil. In contrast, under a qPCR assay evaluated in parallel, consistent amplification across three technical replicates was obtained only at 100 resting spores g-1 soil or greater. In field samples, the bioassay produced galls from 11 qPCR-positive samples and seven of ten qPCR-negative samples. Although the bioassay is not intended for rapid diagnosis or direct quantification, it provides a practical tool for annual clubroot surveys and for studies requiring recovery, propagation, or characterization of viable P. brassicae from soil samples collected across diverse geographic regions.

Cover crops (CCs) are widely used to improve soil health, but their species-specific effects on microbial functional diversity and redundancy remain poorly understood. We evaluated how monocultures of field pea (Pisum sativum), forage radish (Raphanus sativus), and cereal rye (Secale cereale), as well as a three-species mixture (3spp) and a five-species mixture (5spp), influence rhizosphere bacterial and fungal communities in a field experiment. Amplicon sequencing and predictive functional profiling were used to assess microbial diversity, composition, functional potential, and functional redundancy (FR). Microbial alpha diversity showed little change across treatments, but community composition and predicted functional profiles were strongly influenced by CC identity. The 3spp rhizobiome showed the highest bacterial FR, broad metabolic capacity, and greater network connectivity, suggesting increased functional stability of all treatments. In contrast, the 5spp rhizobiome showed reduced bacterial FR and more widespread functional depletion, likely due to imbalances in plant functional types. Monocultures showed species-specific patterns, with rye supporting functionally efficient communities and radish promoting more competitive interactions and reduced functional diversity. Fungal communities responded differently from bacterial communities. While fungal taxonomic shifts were limited, they showed stronger compositional differences among treatments and more stable functional profiles. Notably, fungal FR was highest in the 5spp rhizobiome. Overall, these findings highlight that balanced plant functional composition, rather than greater species richness alone, is important for shaping rhizosphere microbial function and redundancy. Balanced mixtures promoted higher bacterial redundancy, while more diverse but functionally imbalanced mixtures did not consistently enhance microbial function. ImportanceThe benefits of using CCs in agricultural settings are mostly influenced by microbial communities, including nutrient cycling, organic matter turnover, and resilience to disturbance. However, CC mixtures are often promoted on the assumption that more species provide greater soil benefits. This work challenges that assumption by showing that the functional identity and balance of CC species may matter more than the number of species alone. This distinction is important for farmers, land managers, and researchers because CC seed mixes can be costly, and poorly balanced mixtures may fail to deliver intended microbial benefits. Understanding which plant combinations support functionally stable microbial communities can improve CC recommendations and help design agricultural systems that maintain soil processes under changing environmental conditions.

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.

Native ash trees are destined for functional extinction in North America due to the spread of the non-native emerald ash borer. Yet, the consequences of ash loss for soil fungi are unclear. To address this, we employed a factorial study of forest soil fungi in two hydropedological soil types beneath four canopy tree species -- including white ash (Fraxinus americana). Sporocarp surveys and community DNA metabarcoding from soil samples revealed patterns in fungal communities related to canopy tree species but not soil type. Ash trees supported a particularly rich soil fungal community that was distinguishable from communities beneath beech, birch, and maple. We identified over 100 fungal taxa (OTUs) that are at risk of decline or loss from the studied forest, due to their association with ash. Our results indicate that canopy tree species influence soil fungi much more broadly than just the species with which they have mycorrhizal associations.

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.

Seed transmission is a critical pathway for the dispersal of phytopathogenic bacteria. This transmission can occur through three main routes: floral, internal, and external. Yet the relative contribution of individual transmission routes remains poorly characterized. Using a pathosystem based on Xanthomonas citri pv. fuscans (Xcf) and common bean (Phaseolus vulgaris cv. Flavert), we quantified the efficiency of each route. Under our experimental conditions, the vascular route was the most efficient with 25% of contaminated seeds and population sizes averaging 107 CFU per contaminated seed. Deploying this experimental framework to ten seed-borne bacterial strains isolated from bean revealed that almost none transmitted to seeds through any route, or at best at low efficiency. However, most of the strains were capable of surviving and disseminating within the vascular system. A major bottleneck for seed transmission was identified for pod vascular organs colonization and the similar behavior of an Xcf mutant, deficient in the T3SS, suggested that plant immunity could be involved at this step. Co-inoculation of a consortium composed of the seed-borne strains with Xcf reduced the number of seeds contaminated by Xcf at the highest inoculum concentration, although other consortia members were never recovered from seeds. This suggests that the strains are recognized by the plant and trigger defense responses. These findings also raise questions about the mechanisms used by seed-associated bacteria to colonize seeds in situ.