In root nodule symbiosis, symbiosome compartments accommodate nitrogen-fixing rhizobia inside the plant cell. Differentiated into bacteroids, the rhizobia are surrounded by a peribacteroid space and a plant-derived peribacteroid membrane, which separates them from the plant cytoplasm but allows signal and nutrient exchange between host and microbe. The morphological features of symbiosomes are primarily determined by ultrastructural single focal plane imaging, with limited information about spatial details. This study combines 2D and 3D imaging, using transmission electron microscopy and focused ion beam scanning electron microscopy as complementary techniques to analyse the symbiosome ultrastructure and organisation in Lotus japonicus wild-type plants. The 3D model of a mature colonised root nodule cell region demonstrates a dense, puzzle-like arrangement of symbiosomes relative to one another and adjacent plant organelles. The symbiosome shape and size depends on the orientation and number of bacteroids within the compartment and features connective tubular structures. Furthermore, vesicular structures, some likely of bacterial origin, were present at the interface. The study presents a multi-angled analysis of symbiosome-related structures, highlighting their volumes, spatial distribution, and pronounced compactness. Interface associated vesicles, protrusions and connective structures hint towards a dynamic and flexible system that contributes to the plant-microbe crosstalk.

Soil organic carbon is well known to shape microbial communities. However, the mechanisms by which carbon source quality filters diazotroph assemblages in agroecosystems remain poorly understood. We incubated two contrasting agricultural soils with ten distinct carbon substrates to investigate whether carbon quality functions as a dominant, trait-based filter for diazotroph assembly. Carbon type accounted for a larger proportion of the variation in diazotroph community structure than either soil type or incubation time. Across both soils, carbon amendments induced convergent shifts in key soil properties, including dissolved organic carbon, pH, inorganic nitrogen and available phosphorus, as well as diazotroph community trajectories. High-quality carbon inputs (glucose, sucrose and citric acid) reduced diazotroph diversity and shifted communities toward r-strategist dominance. In contrast, low-quality substrates (oxalic acid, cellulose, lignin and crop residues) maintained higher diversity and favored K-strategists. Amino acids exerted a unique dual effect, maintaining diversity by simultaneously alleviating both carbon and nitrogen limitations. Azotobacter consistently emerged as the predominant r-strategist taxon, showing strong competitive dominance under labile carbon enrichment. These findings demonstrate that carbon quality functions as a primary trait-based filter, regulating the trade-off between diazotroph growth rate and community diversity. This framework helps predict how diverse organic amendments, from simple sugars to complex residues, restructure diazotroph communities in agricultural soils.

Arbuscular mycorrhizal fungi (AMF) structure soil microbiomes and support plant growth. However, the stability of established AMF-microbiome interactions to biotic perturbation by introduced microbial inocula is unclear. We investigated how AMF shape soil microbial communities and transcriptional activity when challenged by the introduction of foreign microbial inocula. Using established maize-AMF mesocosms, we introduced microbial communities from forest and agricultural soils and assessed taxonomic composition and metatranscriptomic profiles. Despite clear differences between introduced inocula, neither microbiome treatment altered resident microbial community composition or transcriptional profiles. Instead, AMF exerted a strong and consistent influence on microbial gene expression and bacterial taxa abundances, with effects scaling quantitatively with the degree of mycorrhizal colonisation. Co-expression analyses revealed coordinated transcription among plant, AMF, and microbial genes, suggesting multi-kingdom interactions. Overall, we find AMF drive the structure of soil microbial communities, with biotic perturbation exerting limited influence under the conditions tested.

The Fabaceae encompasses key agricultural species such as soybean, barrel medic (Medicago), and common bean, which are valued for their contributions to global food security and their ability to perform symbiotic nitrogen fixation. Although substantial progress has been made in sequencing economically important legumes, the taxonomic coverage remains uneven, with a disproportionate emphasis on model crops. Furthermore, most genome assemblies lack telomere-to-telomere (T2T) resolution, limiting insights into complex genomic regions and regulatory mechanisms. Emerging T2T technologies offer a transformative opportunity to overcome these limitations. By generating complete T2T assemblies for a representative range of Fabaceae species, including underrepresented lineages, researchers should obtain novel comparative and functional insights into the genetic and epigenetic bases of symbiotic nitrogen fixation. These high-resolution assemblies potentially facilitate the identification of conserved and divergent regulatory networks, shed light on the evolution of nodulation processes, and deepen our understanding of agronomically important traits.

Legume nodulation is initiated when soil bacteria rhizobia infect root hairs and is tightly regulated by host-derived mechanisms that restrict nodule numbers to balance the benefits of symbiotic nitrogen fixation with the plant’s growth and metabolic demands. However, how plants actively promote the initiation of nodulation to counterbalance these restrictive mechanisms and maintain an optimal level of nodulation remains largely unknown. Here, we report a systemic regulatory mechanism through which soybean (Glycine max) promotes rhizobial infection. We show that inoculation of soybean roots with rhizobia suppresses the biogenesis of microRNA miR4416-5p in shoots, a mobile microRNA that is transported from shoots to roots. The resulting reduction of miR4416-5p levels in roots enhances the expression of a vegetative lectin gene Lectin 3 (GmLe3), which promotes rhizobial infection, thereby enhancing nodule formation and improving plant productivity under low nitrogen conditions. We further demonstrate that suppression of miR4416-5p biogenesis in shoots is triggered by the root-derived C-TERMINALLY ENCODED PEPTIDE 7 (GmCEP7), establishing a long-distance GmCEP7-miR4416-5p-GmLe3 regulatory loop that is critical for desirable symbiotic synergy and plant productivity. Comparative genomic analysis reveals that this miR4416-5p-mediated regulatory module is absent in the model legumes Medicago truncatula and Lotus japonicus but appears to be conserved in economically important legume crops common bean (Phaseolus vulgaris) and pigeonpea (Cajanus cajan), suggesting an evolutionary innovation in nodulation control. These findings uncover a systemic mechanism that promotes rhizobial infection and highlight an evolutionary innovation in regulation of nodulation with potential implications for improving legume crop productivity under nitrogen-limited conditions.

Epidermal patterning factor-like (EPFL) peptides are regulators of stomatal development across land plants, yet their below-ground functions remain poorly understood. Here, we show that two EPFL family members from Medicago truncatula, MtEPFL9 and MtEPFL14, act as positive regulators of nodule organogenesis during nitrogen-fixing symbiosis. Rhizobia and purified Nod factors transcriptionally induced MtEPFL9 and MtEPFL14 genes in a common symbiosis-signaling-pathway-dependent manner. Exogenous application of synthetic MtEPFL9 and MtEPFL14 peptides promoted root development and root nodule formation by promoting cell division and growth during nodule primordium formation. Synthetic MtEPFL peptides partially restored nf-ya1 mutant nodulation defects and suppressed the nodule-to-root conversion phenotype in the noot1/noot2 mutant. Transcriptomic analyses revealed that MtEPFL9 and MtEPFL14 peptides act on rhizobia-induced programs associated with gibberellin, auxin, and cytokinin signaling. Together, our results identify MtEPFL9 and MtEPFL14 as regulators of meristematic activity and organ growth in nodules and lateral roots of Medicago truncatula and uncover previously unrecognized roles for EPFL peptides as host-derived signals promoting symbiotic organogenesis.

Aims
Associative nitrogen fixation represents a promising alternative to chemical fertilizers. However, diazotrophs that combine high nitrogen-fixation efficiency with strong rhizosphere colonization ability in cereal crops remain extremely scarce. This study aimed to isolate and characterize novel associative diazotrophs from the rhizosphere of white clover (Trifolium repens L.) and to evaluate their potential to promote plant growth.

Methods
The associative diazotrophic consortium was enriched from the white clover rhizosphere and screened for nitrogenase activity. The most efficient isolate, identified as a potentially novel species within the genus Paenibacillus (designated as strain SY6), was characterized through acetylene reduction assays, plant growth-related trait analysis, and whole-genome sequencing. Maize was used as a model crop to evaluate rhizosphere colonization and growth promotion under nitrogen-limited hydroponic conditions.

Results
The strain SY6 exhibited high nitrogenase activity (123.39 nmol C₂H₄ h⁻1 mL⁻1), significantly exceeding that of the reference strain Klebsiella variicola W12 (P < 0.05) and reported associative diazotrophs. Genome analysis revealed complete molybdenum-dependent (nif) and vanadium-dependent (vnf) nitrogenase systems, an indole-3-pyruvic acid (IPyA) pathway for IAA biosynthesis, and multiple amino acid synthesis genes. Inoculation of maize with SY6 resulted in over 20% increase in biomass and a 16—30% elevation nitrogen content, demonstrating effective rhizosphere colonization and enhanced nitrogen assimilation.

Conclusions
Paenibacillus sp. SY6 represents a potentially novel, highly efficient associative diazotroph with dual nitrogenase systems and phytohormone-producing capacity. Its robust cross-host colonization and significant plant growth promotion suggest strong potential as a microbial biofertilizer for sustainable agriculture.

Soybean (Glycine max (L.) Merr.) is one of the important legume crops, rich in protein, vegetable oil, maintain soil fertility and used as human food and livestock feed. Despite its importance, adoption and productivity of soybean in South Ethiopia is limited by poor agricultural practices and poor access to inputs like biofertilizers. This work aimed at trapping and screening elite rhizobia strains to use as inoculants in farmers’ fields for improved soil health and crop productivity. Forty-four rhizobia strains isolated from different locations in South Ethiopia region were screened along with a commercial inoculant Bradyrhizobium japonicum strain (obtained from Menagesha Biotech Industry, Addis Ababa) in greenhouse using modified Leonard jars (MLJ). Three top performing rhizobia strains SB19, SB22, and SB24 based on the MLJ experiment and the commercial inoculant were further evaluated in farmers’ fields at different locations with varying eco-physiological conditions because environmental factors differ from place to place, affecting how living things grow, survive and function. MLJ experiment revealed that the new isolates SB19, SB22, and SB24 had significantly higher relative symbiotic effectiveness (SE%) (p < 0.05) than un-inoculated and N-fertilized control plants. Except SB19, the top performing strains did not differ from the commercial strains regarding SE%. Strain SB19 produced 34 to 61 number of nodules, while the commercial strain induced 22 to 49 nodules. In the first field experiment (2023) inoculation of soybean with SB19 resulted in average grain yields of 3.1 and 2.92 tons ha⁻1 at Arba Minch University (AMU) demo farm and Abaya campus experimental sites to be consistent with, respectively. In experiment 2 (2024), SB19 strain resulted in the average grain yields of 2.39 and 2.45 tons ha-1 at Abaya and AMU demo sites, respectively. Across all locations, the commercial strain produced an average yield of 2.25 to 2.40 tons ha⁻1, which was significantly lower (p < 0.05) than that of the native strains, but higher than the control plants, which yielded 1.87 to 2.02 tons ha⁻1. Among the evaluated strains, SB19 consistently exhibited the most promising performance across all fields and locations compared to the commercial one and others. This finding highlights the presence of highly effective, locally adapted rhizobial strains capable of nodulating soybean in South Ethiopian soils, and demonstrate their promise for selection and improvement into superior inoculant strains to enhance soybean productivity.

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.

The successful conquest of land by ancient streptophyte algae is closely related to the structural features and functional roles of their extracellular matrix and associated evolutionary innovations. Terrestrialization 500+ million years ago required adaptation to multiple and novel abiotic and biotic stressors. The establishment of a sessile habit which encompasses adhesion likely required special cell surface components like arabinogalactan protein-like macromolecules. The secretion of polysaccharide-rich extracellular polymeric substance (EPS) provided multiple services including establishment of a platform for interactions with surrounding microorganisms, i.e., biofilms. Protection against harmful UV radiation likely included production of phenolic compounds, cuticular components and sporopollenin. Innovations to the various components of the cell wall contributed to the evolution of multicellularity and large thallus sizes, water retention, defense, wound response and interactions with surrounding microorganisms. Yet we are only in an infancy stage in understanding how specific cell wall components contributed to the invasion of land by streptophyte algae. Future studies are needed that encompass much larger taxonomic screening, detailed glycomics and proteomics, mining of biosynthetic pathways and comprehensive analyses of cell wall integrity especially in response to abiotic and biotic stressors of terrestrial habitats.

Arbuscular mycorrhizal fungi form symbioses with ~70% of plant species, building hyphal networks that exchange nutrients for host-derived carbon. These tubular networks move ~1 billion metric tons of carbon per year into Earth’s soils. However, we have no quantitative understanding of the hyphal infrastructure required to carry out this resource transfer. We assembled data from 322 studies representing more than 16,000 soil cores across nine biomes and developed machine-learning models to predict hyphal densities globally. With robotic imaging of more than 300,000 hyphae, we calibrated a biomass model from our spatial predictions. We estimate that global topsoils contain 1.10 × 1017 ± 0.13 × 1017 SD kilometers of living hyphae, weighing ~300 ± 60 SD megatons, ~4- to 6-fold the biomass of humans. Our uncertainty analyses identified undersampled ecosystems that require additional empirical attention.

Arbuscular mycorrhizal (AM) symbiosis is conserved across land plants and is the default nutrient uptake strategy in nature. Within roots, AM colonisation is tightly patterned and dynamically tuned by nutritional cues. Multiple genetic modules contribute to this regulation, including the phosphate starvation response, DWARF14-LIKE (D14L) karrikin signalling, and the common symbiosis signalling pathway (CSSP). Transcriptional overlap among these has led to the hypothesis that phosphate starvation and D14L signalling act upstream of the CSSP. Here, we examined the epistatic relationship between D14L and CSSP in rice. Overexpression of an autoactive gain-of-function CCaMK (gofCCaMKox) restored AM colonisation and symbiosis marker gene expression in d14l mutants to wild-type levels or above, whereas overexpression of wild-type CCaMK did not, confirming that CSSP operates downstream of D14L signalling. However, gofCCaMKox did not rescue the d14l mesocotyl elongation phenotype, supporting a bifurcation of D14L into developmental and symbiotic outputs. Unexpectedly, gofCCaMKox also expanded fungal access to normally restrictive tissue domains (the meristematic zone and endodermis) assigning a role for CCaMK activation in defining root zone and cell-type competence for AM colonisation. Despite restored colonisation, introduction of gofCCaMKox into d14l produced arbuscules, which however were less developed and had increased hyphal septation, revealing a CCaMK-independent role for D14L in intraradical colonisation and arbuscule development. Transcriptome profiling resolved AM-relevant genes into modules controlled by CCaMK activation alone, in combination with D14L, or requiring additional colonisation-associated cues, and further suggested CCaMK primarily acts through AP2 transcription factors. Together, these findings reinforce CCaMK as a master regulator of AM symbiosis at the genetic, transcriptomic and anatomical levels while uncovering CCaMK-independent functions of D14L in arbuscule development.