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Background Understanding the genetic and environmental factors that structure plant microbiomes is necessary for leveraging these interactions to address critical needs in agriculture, conservation, and sustainability. Legumes, which form root nodule symbioses with nitrogen-fixing rhizobia, have served as model plants for understanding the genetics and evolution of beneficial plant-microbe interactions for decades, and thus have added value as models of plant-microbiome interactions. Here we use a common garden experiment with 16S rRNA gene amplicon and shotgun metagenomic sequencing to study the drivers of microbiome diversity and composition in three genotypes of the model legume Medicago truncatula grown in two native soil communities. Results Bacterial diversity decreased between external (rhizosphere) and internal plant compartments (root endosphere, nodule endosphere, and leaf endosphere). Community composition was shaped by strong compartment × soil origin and compartment × plant genotype interactions, driven by significant soil origin effects in the rhizosphere and significant plant genotype effects in the root endosphere. Nevertheless, all compartments were dominated by Ensifer , the genus of rhizobia that forms root nodule symbiosis with M. truncatula , and additional shotgun metagenomic sequencing suggests that the nodulating Ensifer were not genetically distinguishable from those elsewhere in the plant. We also identify a handful of OTUs that are common in nodule tissues, which are likely colonized from the root endosphere. Conclusions Our results demonstrate strong host filtering effects, with rhizospheres driven by soil origin and internal plant compartments driven by host genetics, and identify several key nodule-inhabiting taxa that coexist with rhizobia in the native range. Our results set the stage for future functional genetic experiments aimed at expanding our pairwise understanding of legume-rhizobium symbiosis toward a more mechanistic understanding of plant microbiomes. 4yG9Be_5fuxNDH9Lv9Ven5 Video Abstract

Background Rice-fallow agroecosystems face challenges such as poor soil fertility and low crop productivity. This study evaluates a multi-species bacterial consortium—comprising atypical rhizobia ( Pararhizobium giardinii , Ochrobactrum sp.) and non-rhizobial endophytes ( Serratia plymuthica , Serratia sp.)—for improving lentil cultivation in these soils. Multilocation field trials assessed its effects on plant growth, soil nutrients, microbial diversity, and nifH gene expression in nodules. The goal is to offer a sustainable, biologically based alternative to chemical fertilizers like diammonium phosphate (DAP), enhancing soil health and enabling lentil intensification on fallow land. Results In greenhouse pot experiments, the bacterial consortium increased lentil growth by 166% in sterile and 116% in non-sterile soils compared to the uninoculated control. Scanning electron microscopy (SEM) confirmed enhanced root hair development in treated plants. Further, the consortium treatment modulated the root exudate profiles, which demonstrated higher concentrations of fatty acids, triterpenes, and methyl esters. This markedly affected the composition of the rhizosphere microbial population. The consortium facilitated a proliferation of beneficial taxa, including Mesorhizobium , Rhizobium , and Bradyrhizobium , enhancing synergistic microbial interactions associated with enhanced plant development. The bacterial treatment increased nifH gene expression inside root nodules and augmented leghemoglobin content by 275%. Field experiments at five rice-fallow sites exhibited enhancements in soil pH, organic carbon (16%), nitrogen (6.5%), phosphorus (8%), and enzymatic activity. Meta-transcriptomic analysis demonstrated a 200% augmentation in nifH expression inside the root nodules, in the plots treated with the consortium. 16 S rRNA amplicon sequencing revealed that the microbiota in the rhizosphere and nodules was predominantly composed of Pseudomonadota, with elevated numbers of rhizobia in both environments. Microbiome alterations correlated with a significant yield increase, with lentil grain output reaching 1168 kg/ha, surpassing both the uninoculated control and DAP treatment. Conclusions The study showed that a multi-species bacterial consortium of atypical rhizobia and non-rhizobial endophytes (NREs) improved plant growth and soil health in rice-fallow soils. It enhanced nifH gene expression in root nodules and altered root exudation, fostering a beneficial, plant growth-promoting microbial community. Importantly, these effects were achieved without chemical fertilizers, highlighting the consortium’s potential for sustainable lentil intensification on fallow land.

Purpose Studying the legume nodule microbiome is important for understanding the development and nutrition of the plants inhabited by the various microbes within and upon them. We analyzed the microbiomes of these underground organs from both an important crop plant ( Medicago sativa ) and a related legume ( M. polymorpha ) using metagenomic and culture-based techniques to identify the main cultivatable contributors to plant growth enhancement. Methods Using high-throughput sequencing, culturing, and in planta techniques, we identified and analyzed a broad population of the bacterial taxa within Medicago nodules and the surrounding soil. Results Fifty-one distinct bacterial strains were isolated and characterized from nodules of both Medicago species and their growth-promoting activities were studied. Sequencing of 16S rRNA gene amplicons showed that in addition to Ensifer , the dominant genus, a large number of Gram-positive bacteria belonging to the Firmicutes and Actinobacteria were also present. After performing ecological and plant growth-promoting trait analyses, selecting the most promising strains, and then performing in planta assays, we found that strains of Bacillus and Micromonospora among others could play important roles in supporting the growth, health, and productivity of the host plant. Conclusion To our knowledge, the comparison of the biodiversity of the microbiota of undomesticated vs. cultivated Medicago roots and nodules is novel and shows the range of potential Plant Growth-Promoting Bacteria that could be used for plants of agricultural interest. These and other nodule-isolated microbes could also serve as inoculants with rhizobia with the goal of replacing synthetic fertilizers and pesticides for sustainable agriculture.

Pulses provide distinct health benefits due to their low fat content and high protein and fiber contents. Their grain production reaches approximately 93,210 × 103 tons per year. Pulses benefit from the symbiosis with atmospheric N2-fixing bacteria, which increases productivity and reduces the need for N fertilizers, thus contributing to mitigation of environmental impact mitigation. Additionally, the root region harbors a rich microbial community with multiple traits related to plant growth promotion, such as nutrient increase and tolerance enhancement to abiotic or biotic stresses. We reviewed the eight most common pulses accounting for almost 90% of world production: common beans, chickpeas, peas, cowpeas, mung beans, lentils, broad beans, and pigeon peas. We focused on updated information considering both single-rhizobial inoculation and co-inoculation with plant growth-promoting rhizobacteria. We found approximately 80 microbial taxa with PGPR traits, mainly Bacillus sp., B. subtilis, Pseudomonas sp., P. fluorescens, and arbuscular mycorrhizal fungi, and that contributed to improve plant growth and yield under different conditions. In addition, new data on root, nodule, rhizosphere, and seed microbiomes point to strategies that can be used to design new generations of biofertilizers, highlighting the importance of microorganisms for productive pulse systems.

Symbiotic associations between leguminous plants and their nodule microbiome play a key role in sustainable agriculture by facilitating the fixation of atmospheric nitrogen and enhancing plant stress resistance. This study aimed to decipher the root nodule microbiome of two halophytic legumes, Sesbania cannabina and Glycine soja, which grow in saline soils of the Yellow River Delta, China, using PacBio’s circular consensus sequencing for full-length bacterial 16S rRNA gene to obtain finer taxonomic information. The cultivated legume Glycine max was used for comparison. We identified 18 bacterial genera and 55 species in nodule samples, which mainly classified to Proteobacteria, and rhizobial genus Ensifer was the predominant group. The three legumes showed similarity in operational taxonomic unit (OTU) diversity but distinction in OTU richness, indicating that they harbor similar bacterial species with different relative contents. The results of principal coordinates analysis and ANOSIM tests indicated that G. soja and G. max have similar nodule bacterial communities, and these communities differ from that of S. cannabina. Wild legumes S. cannabina and G. soja both harbored a higher number of rhizobia, while G. max possessed more non-rhizobial bacteria. These differences could be associated with their adaptability to saline–alkali stress and revealed clues on the nodule endophytes with relative importance of culturable rhizobial symbionts.

Herbarium specimens are increasingly being used as sources of information to understand the ecology and evolution of plants and their associated microbes. Most studies have used specimens as a source of genetic material using culture‐independent approaches. We demonstrate that herbarium specimens can also be used to culture nodule‐associated bacteria, opening the possibility of using specimens to understand plant–microbe interactions at new spatiotemporal scales. We used historic and contemporary nodules of a common legume, Medicago lupulina, to create a culture collection. We were able to recover historic bacteria in 15 genera from three specimens (collected in 1950, 2004, and 2015). This work is the first of its kind to isolate historic bacteria from herbarium specimens. Future work should include inoculating plants with historic strains to see if they produce nodules and if they affect plant phenotype and fitness. Although we were unable to recover any Ensifer, the main symbiont of Medicago lupulina, we recovered some other potential nodulating species, as well as many putative growth‐promoting bacteria. Biological collections have been used to study the genetics and genomics of numerous organisms, including microorganisms. Additionally, herbaria have been used to resurrect extinct and extant plant species. Here we describe methods to resurrect historic microbes from herbarium specimens, allowing for many novel questions to be asked across temporal and spatial scales.

Background Pigeonpea ( Cajanus cajan ) is an important legume crop in semi-arid regions with multiple uses. The microbial diversity within its root nodules in Indian soils remains poorly explored. We investigated the bacterial diversity of pigeonpea root nodules across different genotypes and soil types to identify the factors driving their assembly. Using a metagenomic approach and high-throughput sequencing of the 16S rRNA gene, we analyzed the nodule microbiomes of three pigeonpea genotypes (Asha, Durga, and Mannem Konda Kandi) grown in three different soil types (Alfisol, Vertisol, and Inceptisol) and wild pigeonpea ( C. scarabaeoides ) in its native soil. Results Our results indicated that pigeonpea nodules harbor diverse rhizobial and non-rhizobial endophytes and that host genotype, nodule position, soil type, and other edaphic factors influence significant variation in the microbial community structure. The core nodule microbiome was dominated by Proteobacteria and Bacteroidetes. Bradyrhizobium and Ensifer were predominant among the rhizobial taxa, and non-rhizobial genera such as Pseudomonas , Chitinophaga , and Limnobacter were also abundant. Edaphic factors, particularly soil type, pH, and nutrient availability, had a stronger influence on the nodule bacterial community composition than the host genotype. Although bulk soil exhibited higher bacterial diversity, nodule microbiomes were less diverse but more specialized, indicating host-mediated selection. A comparison of the nodule microbiomes of wild and cultivated pigeonpea revealed distinct differences, with the core nodule microbiome of wild pigeonpea dominated by Bradyrhizobium , while that of cultivated pigeonpea exhibited a diverse bacterial community. Conclusions These findings demonstrate that soil properties play a more critical role than host genetics in shaping the pigeonpea nodule microbiome, emphasizing the importance of environmental conditions in symbiotic interactions. The differences between wild and cultivated genotypes suggest that domestication has altered microbial recruitment strategies. This study provides foundational insights into the factors driving microbial assembly in pigeonpea nodules, with implications for improving crop productivity through targeted microbial management. Future research should explore the functional roles of these microbial communities to optimize their use in sustainable agriculture.

PurposeThyroid cancer and thyroid nodules are the most prevalent form of thyroid endocrine disorder. The balance of gut microbiome is highly crucial for a healthy human body, especially for the immune and endocrine system. However, the relationship between gut microbiome and the thyroid endocrine disorders such as thyroid cancer and thyroid nodules has not been reported yet.MethodsA cohort of 74 patients was recruited for this study. Among them, 20 patients had thyroid cancer, 18 patients had thyroid nodules, and 36 were matched healthy controls. Gut microbiome composition was analyzed by 16S rRNA (16S ribosomal RNA) gene-based sequencing protocol.ResultsWe compared the gut microbiome results of 74 subjects and established the correlation between gut microbiome and thyroid endocrine function for both thyroid cancer and thyroid nodules. The results inferred that alpha and beta diversity were different for patients with thyroid tumor than the healthy controls (p < 0.01). In comparison to healthy controls, the relative abundance of Neisseria (p < 0.001) and Streptococcus (p < 0.001) was significantly higher for thyroid cancer and thyroid nodules. Butyricimonas (p < 0.001) and Lactobacillus (p < 0.001) displayed notably lower relative abundance for thyroid cancer and thyroid nodules, respectively. It was also found that the clinical indexes were correlated with gut microbiome.ConclusionOur results indicate that both thyroid cancer and thyroid nodules are associated with the composition of gut microbiome. These results may support further clinical diagnosis to a great extent and help in developing potential probiotics to facilitate the treatment of thyroid cancer and thyroid nodules.

• Nodule microbiota are dominated by symbiotic nitrogen-fixing rhizobia, however, other non-rhizobial bacteria also colonise this niche. Although many of these bacteria harbour plant-growth-promoting functions, it is not clear whether these less abundant nodule colonisers impact root–nodule symbiosis. • We assessed the relationship between the nodule microbiome and nodulation as influenced by the soil microbiome, by using a metabarcoding approach to characterise the communities inside nodules of healthy and starved Lotus species. A machine learning algorithm and network analyses were used to identify nodule bacteria of interest, which were re-inoculated onto plants in controlled conditions to observe their potential functionality. • The nodule microbiome of all tested species differed according to inoculum, but only that of Lotus burttii varied with plant health. Amplicon sequence variants representative of Pseudomonas species were the most indicative non-rhizobial signatures inside healthy L. burttii nodules and negatively correlated with Rhizobium sequences. A representative Pseudomonas isolate co-colonised nodules infected with a beneficial Mesorhizobium, but not with an ineffective Rhizobium isolate and another even reduced the number of ineffective nodules induced on Lotus japonicus. • Our results show that nodule endophytes influence the overall outcome of the root–nodule symbiosis, albeit in a plant host-specific manner.

Background and aims Forage legumes form mutualistic interactions with specialized soil rhizobia bacteria that inhabit root nodules and fix atmospheric nitrogen. However, legumes are sensitive to drought stress, which can interrupt nodulation and symbiotic nitrogen fixation (SNF). We hypothesize that drought-impaired SNF may influence soil nitrogen availability and soil microbial diversity. Methods Here, we evaluated the effects of drought on nodulation, plant growth, physiological parameters, SNF, soil nitrogen availability, soil extracellular enzyme activity, and soil microbiome of alfalfa ( Medicago sativa ) and red clover ( Trifolium pratense ). The drought treatments were imposed at the flowering stage by maintaining soil moisture contents at 20% field capacity (FC) (severe drought), 40% FC (moderate drought), and 80% FC (well-watered) for three weeks. Results Drought significantly reduced nodulation, root and shoot growth, and SNF in alfalfa and red clover. Soil available nitrogen was significantly increased following severe drought conditions. The enzyme assays showed reduced activity of N-acetyl-glucosaminidase and β-D cellobiosidase enzymes under drought stress in alfalfa and red clover, respectively. Microbiome data showed shifts in the relative abundance of some key bacterial taxa under drought stress. Conclusion Overall results indicate that drought has deleterious effects on SNF and plant growth, affecting carbon and nitrogen cycling enzymes, soil nitrogen availability, and soil microbial diversity.