Legume Root Research Articles

Bacterial diversity and community structure in native legume root nodules in intact and mining-disturbed Arctic tundra

Mining restoration in Arctic regions is challenging due to harsh environmental conditions with slow natural ecosystem recovery taking decades or even centuries. Identifying native species that can both colonize disturbed areas and contribute to soil development are critical to hasten restoration timelines. Nodules were collected from four native legume species (<i>Astragalus alpinus</i>, <i>Hedysarum americanum</i>, <i>Oxytropis arctica</i>, and <i>Oxytropis maydelliana</i>) observed to be naturally colonizing gravel quarries within a mine footprint near Rankin Inlet, Nunavut, Canada. Samples came from both gravel quarries and adjacent intact tundra. Next-generation sequencing of the 16S and <i>nifH</i> regions was used to characterize the nodule bacterial community composition and diversity. Despite the large differences in soil conditions between gravel substrates and intact tundra, no significant effects of soil environment were found on bacterial community composition within plant nodules. Further, few differences were observed in nodule communities between the plant species. Overall, the study suggests that the microbial propagules necessary for successful nodulation are present in gravel quarries. While restoration efforts involving native legumes may succeed without commercial inoculants, further research is needed to determine whether the rhizobia in these environments can provide sufficient nitrogen to support robust host plant growth.

First Report of Clonostachys rhizophaga Causing Root Rot on Lentil in France and Cross-Pathogenicity on Pea.

Root rot affects legumes such as lentil (Lens culinaris subsp. culinaris Medik.) and pea (Pisum sativum L.) (Chatterton et al. 2019). In France, legume root rot occurs in 65% of cultivated areas and cause up to 60% yield loss (Augagneur et al. 2021; Moussart el al. 2011). Soil was sampled from plots where root rot disease was previously observed. Samplings were conducted in April 2019 from two plots in central France (46.59 N, 2.05 E; 46.57 N, 2.3 E). Pre-germinated lentil (cv. Anicia) seeds were transplanted in both soils and symptomatic plants were collected after two months. They showed yellowing and wilting symptoms associated with necrotic spots on the root system. The light brown color on the roots turned to dark brown as the necrotic spots progress. Diseased root fragments were surface-disinfected by soaking for 30 s in 70% ethanol, rinsed with sterile water and placed on malt extract agar (MEA). Fungal colonies growing from the root fragments were purified by single-spore sub-culturing and preserved in the Microorganisms of Interest for Agriculture and Environment (MIAE) collection (INRAE Dijon, France). They were transferred to potato dextrose agar and carnation leaf agar for macroscopic and microscopic observations. The colonies developed a dense and cottony aerial mycelium, pearly in color with pink reflections (Fig. S1). From below, the colonies were orange-pink. Under the microscope, Verticillium-like conidiophores arising on the same whorl released asymmetrical ovoid single-celled conidia, from 4 to 7 µm long and 2.5 to 3 µm wide, similar to the description of Clonostachys rhizophaga by Schroers (2001). Isolates MIAE08192 and MIAE08209 were identified as Clonostachys sp. based on their internal transcribed spacer (ITS) sequences (GenBank accessions OR902030 and OR902031). They were further identified based on their sequence of the translation elongation factor 1-α gene (TEF-1α) (Moreira et al. 2016) (GenBank accessions OR947648 and OR947649). A BLAST search identified the two isolates as C. rhizophaga based on 100% identity of their sequences to the published sequence KX184992 (382 bp out of 382 bp and 377 bp out of 377 bp, respectively). A phylogenetic analysis combining ITS and TEF-1α sequences confirmed that the two isolates clustered within the C. rhizophaga species (Fig. S2). Two strains MIAE07881 and MIAE07884 of C. rhizophaga have been reported to cause root rot on pea (Gibert et al. 2022). Due to equivalent symptoms on lentil and pea, and the fact that the strains belonged to the same taxon led to test cross-pathogenicity on the two plant species with the two strains collected from lentil and the two strains collected from pea. Surface-disinfected lentil (cv. Anicia) and pea (cv. Firenza) seeds were placed on MEA, and after 72 h, the germinated seeds were transferred into sterile glass tubes containing 30 mL of Hoagland's No. 2 basal salt mixture at 1.6 g.L-1 added with agar 8 g.L-1. Three days later, they were inoculated with 1 mL of a conidial suspension at 105 conidia.mL-1 or sterile deionized water for the control plantlets. Twelve tubes per strain and twelve negative control tubes were prepared. The tubes were incubated in a growth chamber at 22°C day/18°C night and 12 h light for four weeks. The bioassay was performed twice. The control plants were asymptomatic. Whatever their origin, the four isolates inoculated on lentil or pea caused necrotic areas on 100% of the plants leading to strongly degraded root system. Koch's postulates were verified by reisolating the inoculated fungi from one symptomatic plant among the twelve replicates. Our results confirm the absence of specificity of this interaction and suggests the possible pathogenicity of C. rhizophaga on other legumes (Abang et al. 2009). To our knowledge, this is the first report of C. rhizophaga as a causal agent of root rot on lentil and of its cross-pathogenicity on lentil and pea.

Annexin- and calcium-regulated priming of legume root cells for endosymbiotic infection

Legumes establish endosymbioses with arbuscular mycorrhizal (AM) fungi or rhizobia bacteria to improve mineral nutrition. Symbionts are hosted in privileged habitats, root cortex (for AM fungi) or nodules (for rhizobia) for efficient nutrient exchange. To reach these habitats, plants form cytoplasmic cell bridges, key to predicting and guiding fungal hyphae or rhizobia-filled infection thread (IT) root entry. However, the underlying mechanisms are poorly studied. Here we show that unique ultrastructural changes and calcium (Ca2+) spiking signatures, closely associated with Medicago truncatula Annexin 1 (MtAnn1) accumulation, accompany rhizobia-related bridge formation. Loss of MtAnn1 function in M. truncatula affects Ca2+ spike amplitude, cytoplasmic configuration and rhizobia infection efficiency, consistent with a role of MtAnn1 in regulating infection priming. MtAnn1, which evolved in species establishing intracellular symbioses, is also AM-symbiosis-induced and required for proper arbuscule formation. Together, we propose that MtAnn1 is part of an ancient Ca2+-regulatory module for transcellular endosymbiotic infection.

Isolation and characterization of non-rhizobial bacteria and arbuscular mycorrhizal fungi from legumes

This study investigates non-rhizobial endophytic bacteria in the root nodules of chickpea (Cicer arietinum L), faba bean (Vicia faba), and cowpea (Vigna unguiculata L. Walp), as well as arbuscular mycorrhizal fungi in the rhizospheric soil of chickpea and faba bean. Out of the 34 endophytic bacterial populations examined, 31 strains were identified as non-rhizobial based on nodulation tests. All strains were assessed for their plant growth-promoting (PGP) activities in vitro. The results revealed that most isolates exhibited multiple PGP activities, such as nitrogen fixation, indole-3-acetic acid (IAA) and ammonia (NH3) production, phosphate solubilization, and exopolysaccharide production. The most effective PGP bacteria were selected for 16S rRNA analysis. Additionally, a total of 36 species of native arbuscular mycorrhizal fungi (AMF) were identified. Acaulospora (100%) and Scutellospora (91.66%) were the most prevalent genera in Cicer arietinum L. and Vicia faba L. plants, respectively. Acaulospora also exhibited the highest spore density and relative abundance in both plants. Moreover, the root colonization of Cicer arietinum L. and Vicia faba L. plants by hyphae, vesicles, and arbuscules (HVA) was significant. The findings of this study provide valuable insights into non-rhizobial endophytic bacteria associated with legume root nodules and the diversity of AMF. These organisms have great potential for PGP and can be manipulated by co-inoculation with rhizobia to enhance their biofertilizer effectiveness. This manipulation is crucial for promoting sustainable agriculture, improving crop growth, and advancing biofertilizer technology.

Antibiotics Resistance and PGPR Traits of Endophytic Bacteria Isolated in Arid Region of Morocco

This study aimed to characterize endophytic bacteria isolated from legume nodules and roots in the rhizosphere soils of Acacia trees in Morocco’s arid regions. The focus was on identifying bacterial strains with plant growth-promoting rhizobacteria (PGPR) traits and antibiotic resistance, which could enhance legume productivity under various abiotic stresses. Autochthonous legumes were used to harbor the endophytic bacteria, including chickpea (Cicer arietinum), faba bean (Vicia faba), lentil (Lens culinaris), and common bean (Phaseolus vulgaris). In a previous study, seventy-two isolates were obtained, and molecular characterization grouped them into twenty-two bacterial isolates. These twenty-two bacterial isolates were then further analyzed for their antibiotic resistance and key PGPR traits, such as phosphate solubilization, indole-3-acetic acid (IAA) production, and siderophore production. The results revealed that 86.36% of the isolates were resistant to erythromycin, 45.45% to ciprofloxacin, 22.73% to ampicillin-sulbactam, and 9.09% to tetracycline, with ciprofloxacin and tetracycline being the most effective. All isolates produced IAA, with HN51 and PN105 exhibiting the highest production at 6 µg of IAA per mg of protein. The other isolates showed varying levels of IAA production, ranging from moderate to low. Siderophore production, assessed using CAS medium, indicated that the strains PN121, LR142, LNR146, and HR26 exhibited high production, while the rest demonstrated moderate to low capacities. Additionally, 18.2% of the isolates demonstrated phosphate solubilization on YED-P medium, with PR135 and LNR135 being the most efficient, achieving solubilization indices of 2.14 and 2.13 cm, respectively. LR142 and LNR146 showed a moderate solubilization efficiency. Overall, these findings indicate that these isolated endophytic bacteria possess significant potential as biofertilizers, owing to their antibiotic resistance, IAA production, siderophore production, and phosphate solubilization abilities. These characteristics position them as promising candidates for enhancing legume growth under abiotic stress and contributing to sustainable agriculture in arid regions.

Functions of the Sinorhizobium meliloti LsrB Substrate-Binding Domain in Oxidized Glutathione Resistance, Alfalfa Nodulation Symbiosis, and Growth.

To successfully colonize legume root nodules, rhizobia must effectively evade host-generated reactive oxygen species (ROS). LsrB, a redox regulator from Sinorhizobium meliloti, is essential for symbiosis with alfalfa (Medicago sativa). The three cysteine residues in LsrB's substrate domain play distinct roles in activating downstream redox genes. The study found that LsrB's substrate-binding domain, dependent on the cysteine residue Cys146, is involved in oxidized glutathione (GSSG) resistance and alfalfa nodulation symbiosis. LsrB homologues from other rhizobia, with Cys172/Cys238 or Cys146, enhance GSSG resistance and complement lsrB mutant's symbiotic nodulation. Substituting amino acids in Azorhizobium caulinodans LsrB with Cys restores lsrB mutant phenotypes. The lsrB deletion mutant shows increased sensitivity to NCR247, suggesting an interaction with host plant-derived NCRs in alfalfa nodules. Our findings reveal that the key cysteine residue in the LsrB's substrate domain is vital for rhizobium-legume symbiosis.

Potential Nitrogen Fixing Rhizobia Isolated from Some Wild Legumes of Nagaland Based on RAPD with Nif-directed Primer and Their Biochemical Activities

Wild legumes are widely dispersed and can survive in challenging environments as bacteria dwell in their nodules and help each other. Although Nagaland is home to many wild legume varieties, research on the microbial diversity that goes along with them is still in its infancy. This work aimed to characterize several wild legume root nodules and distinguish possible rhizobial isolates using RAPD and nif-directed RPO1 primer. Nodule bacteria were isolated in Yeast extract culture media. Based on their colony morphology, 150 isolates were selected for performing RAPD with nif-directed RPO1 primer. Eighty-four isolates were bonded with RPO1 primer, and a few biochemical tests were conducted on RPO1-positive isolates. Activities that promoted plant development were also investigated for these isolates. Of all the isolates, 18 exhibited phosphate solubilization capacity, while 38 isolates were found to produce IAA. This study entails a large variety of rhizobia in the nodules, which were able to promote growth. Hence, these isolates promise to be bio-fertilizers that could improve agricultural operations.

Rhizosphere flavonoids alleviates the inhibition of soybean nodulation caused by shading under maize-soybean strip intercropping

The flavonoids produced by legume roots are signal molecules that induce nod genes for symbiotic rhizobium. Nevertheless, the promoting effects of flavonoids in root exudates in intercropping system on soybean nodulation are still unknown. A two years of field experiments was carried with maize soybean strip intercropping, i.e., the interspecific row spacing of 30 cm (MS30), 45 cm (MS45), 60 cm (MS60), and sole soybean/maize:SS/MM, and root interaction, i.e., root no barrier (NB) and root polythene-plastic barrier (PB), to evaluate relationships between flavonoids in root exudates and nodulation. We found that root-root interaction between soybean and maize enhances the nodules number and fresh weight in intercropped soybean. This enhancement increase gradually with expansion of interspecific distance. Proportion of nodules with diameter greater than 0.4cm was higher in intercropped soybean with NB than with PB. The expressions of nodules-related genes (GmENOD40, GmNIN2b and GmEXPB2) were up-regulated. Furthermore, compared with monocropping, isoflavones secretion of soybean roots reduced, flavonoids and flavonols secretion of maize and soybean roots increased under intercropping. The secretion of differential metabolites of flavonoids in the rhizosphere of maize and soybean declined with root barrier. The expressions of GmCHS8 and GmIFS1 in soybean roots were up-regulated and GmICHG was down-regulated under root interaction. The most of the flavonoids and flavonol compounds were positively correlated with nodule diameter. The nodules number, the nodules fresh weight and the proportion of nodules with a diameter greater than 0.2 cm increased in different genotypes of soybean treated with maize root exudate, which promoted the improvement of nitrogen fixation capacity. Therefore, maize-soybean strip intercropping combined with reasonable spacing to enhance the positive effect of underground root interaction, and improve the nodulation and nitrogen fixation capacity of intercropping soybean.

Adaptation of Rhizobium leguminosarum sv. trifolii strains to low temperature stress in both free-living stage and during symbiosis with clover

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. This process occurs within special new structures, called nodules, formed mainly on legume roots. Soil bacteria, commonly known as rhizobia, fix atmospheric dinitrogen, converting it into a form that can be assimilated by plants. Various environmental factors, including a low temperature, have an impact on the symbiotic efficiency. Nevertheless, the effect of temperature on the phenotypic and symbiotic traits of rhizobia has not been determined in detail to date. Therefore, in this study, the influence of temperature on different cell surface and symbiotic properties of rhizobia was estimated. In total, 31 Rhizobium leguminosarum sv. trifolii strains isolated from root nodules of red clover plants growing in the subpolar and temperate climate regions, which essentially differ in year and day temperature profiles, were chosen for this analysis. Our results showed that temperature has a significant effect on several surface properties of rhizobial cells, such as hydrophobicity, aggregation, and motility. Low temperature also stimulated EPS synthesis and biofilm formation in R. leguminosarum sv. trifolii. This extracellular polysaccharide is known to play an important protective role against different environmental stresses. The strains produced large amounts of EPS under tested temperature conditions that facilitated adherence of rhizobial cells to different surfaces. The high adaptability of these strains to cold stress was also confirmed during symbiosis. Irrespective of their climatic origin, the strains proved to be highly effective in attachment to legume roots and were efficient microsymbionts of clover plants. However, some diversity in the response to low temperature stress was found among the strains. Among them, M16 and R137 proved to be highly competitive and efficient in nodule occupancy and biomass production; thus, they can be potential yield-enhancing inoculants of legumes.

Periodic cytokinin responses in Lotus japonicus rhizobium infection and nodule development.

Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeatedly at the region behind the tip of the growing roots. Here, live-imaging and transcriptome analyses revealed oscillating host gene expression with approximately 6-hour intervals upon bacterial inoculation. Cytokinin response also exhibited a similar oscillation pattern. Cytokinin signaling is crucial to maintaining the periodicity, as observed in cytokinin receptor mutants displaying altered infection foci distribution. This periodic regulation influences the size of the root region responsive to bacteria, as well as the nodulation process progression.

Nitrogen and Nod factor signaling determine Lotus japonicus root exudate composition and bacterial assembly

Symbiosis with soil-dwelling bacteria that fix atmospheric nitrogen allows legume plants to grow in nitrogen-depleted soil. Symbiosis impacts the assembly of root microbiota, but it is unknown how the interaction between the legume host and rhizobia impacts the remaining microbiota and whether it depends on nitrogen nutrition. Here, we use plant and bacterial mutants to address the role of Nod factor signaling on Lotus japonicus root microbiota assembly. We find that Nod factors are produced by symbionts to activate Nod factor signaling in the host and that this modulates the root exudate profile and the assembly of a symbiotic root microbiota. Lotus plants with different symbiotic abilities, grown in unfertilized or nitrate-supplemented soils, display three nitrogen-dependent nutritional states: starved, symbiotic, or inorganic. We find that root and rhizosphere microbiomes associated with these states differ in composition and connectivity, demonstrating that symbiosis and inorganic nitrogen impact the legume root microbiota differently. Finally, we demonstrate that selected bacterial genera characterizing state-dependent microbiomes have a high level of accurate prediction.

Shining a Light on Symbiosis: N-Fixing Bacteria Boost Legume Growth under Varied Light Conditions

Legumes are a diverse and important group of plants that play a vital role in agriculture, food security, and environmental sustainability. Rhizobia are symbiotic bacteria that form nitrogen-fixing nodules on legume roots, providing the plant with a valuable source of nitrogen. Phenolic acids are a group of secondary metabolites produced by plants that have a wide range of biological functions, including defense against pests and diseases, tolerance to abiotic stresses, and nutrient uptake. In the context of climate change and the imperative for sustainable agriculture, this study delves into the dynamic responses of legume species to varying light intensities and their intricate interactions with soil microorganisms. We investigated the impact of light intensity and rhizobial inoculation on the biomass, nitrate reductase, acid phosphatase, and production of phenolic acids in the roots of four legume species, Trifolium repens, Vicia sativa, Ornithopus compressus, and Coronilla juncea. Plants were grown under three light intensity regimes (low, medium, and high) and inoculated with either rhizobia or a non-inoculated control. The results highlight that shaded-light-adapted species, T. repens and V. sativa, increased root exudate production when exposed to high light intensity. This response aligns with their mining strategy, effectively allocating resources to optimize nutrient acquisition under varying conditions. In contrast, species hailing from well-illuminated environments, O. compressus and C. juncea, displayed distinct strategies by significantly increasing biomass under high irradiance, capitalizing on the available light and nutrients. The mining strategy of legumes emerged as a central theme, influencing biomass production, nitrogen dynamics, and enzymatic activities. The strong correlations between biomass and total nitrogen accumulation underscore the role of the mining strategy in efficient nutrient acquisition. Inoculated plants, which rely more on biological nitrogen fixation (BNF), exhibited lower δ15N values, indicative of a successful mining strategy to acquire and utilize atmospheric nitrogen. Enzymatic activities and phenolic acids exhibited significant interspecies variations, reflecting the adaptability of legumes to different light conditions. The findings of this study could be used to develop new strategies for improving legume stress tolerance, nutrient uptake capacity, and rhizosphere health.

Species Diversity, Nitrogen Fixation, and Nutrient Solubilization Activities of Endophytic Bacteria in Pea Embryos

Endophytic bacteria, especially those that participate in nitrogen fixation, play critical roles in supplying essential nutrients for legume plant growth. Despite that there have been numerous investigations targeting bacterial microbiomes in legume roots and nodules, little is known about embryonic bacteria that facilitate plant nutrient utilization after seed germination. Here, we collected and investigated endophytic bacterial microbiome in edible pea (Pisum sativum) embryos using five representative cultivars and a pea sprout (shoot of pea [SHP]) control. Twenty-six nitrogen-fixing bacteria (NFB) were isolated from pea embryos, with three strains found in fresh grain pea (FGP) and snow pea (SP) exhibiting the strongest nitrogenase activity of above 85 nmol C2H4/mL/h. Some NFB isolates are also potassium-solubilizing bacteria (KSB) or phosphorus-solubilizing bacteria (PSB) utilizing inorganic and/or organic phosphorus. All 26 NFB showed variable levels (0.41 to 7.10 μg/mL) of indole-3-acetic acid (IAA) secretion. The nutrient-solubilizing NFB identified in our research are potential targets for biofertilizer development. They could be useful in converting nitrogen, potassium, and/or phosphorus into usable forms for the plants. At the microbiome level, high-throughput 16S ribosomal RNA (rRNA) sequencing of 40 bacterial collections from pea embryos generated 4234 operational taxonomic units (OTUs) using 97% identity as the threshold for clustering high-quality effective reads (valid tags). Analysis of OTU annotation results revealed similar species community structures, abundance, and diversity in most samples. Our embryo-derived endophytic bacterial pool provides a microbiome platform for seed dormancy and germination research of edible peas, as well as for digging new biofertilizer resources in general.

Novel Insights into the Promoted Accumulation of Nitro-Polycyclic Aromatic Hydrocarbons in the Roots of Legume Plants.

Substituted polycyclic aromatic hydrocarbons (sub-PAHs) are receiving increased attention due to their high toxicity and ubiquitous presence. However, the accumulation behaviors of sub-PAHs in crop roots remain unclear. In this study, the accumulation mechanism of sub-PAHs in crop roots was systematically disclosed by hydroponic experiments from the perspectives of utilization, uptake, and elimination. The obtained results showed an interesting phenomenon that despite not having the strongest hydrophobicity among the five sub-PAHs, nitro-PAHs (including 9-nitroanthracene and 1-nitropyrene) displayed the strongest accumulation potential in the roots of legume plants, including mung bean and soybean. The nitrogen-deficient experiments, inhibitor experiments, and transcriptomics analysis reveal that nitro-PAHs could be utilized by legumes as a nitrogen source, thus being significantly absorbed by active transport, which relies on amino acid transporters driven by H+-ATPase. Molecular docking simulation further demonstrates that the nitro group is a significant determinant of interaction with an amino acid transporter. Moreover, the depuration experiments indicate that the nitro-PAHs may enter the root cells, further slowing their elimination rates and enhancing the accumulation potential in legume roots. Our results shed light on a previously unappreciated mechanism for root accumulation of sub-PAHs, which may affect their biogeochemical processes in soils.

Arbuscular mycorrhizal hyphae facilitate rhizobia dispersal and nodulation in legumes.

In soil ecosystems, rhizobia occupy the rhizosphere of legume roots to form nodules, a process triggered by microbial recognition of specific root-derived signals (i.e. flavonoids). However, soil conditions can limit bacterial motility, restricting signal perception to the area directly influenced by roots. Legumes, like most plants of agricultural interest, associate with arbuscular mycorrhizal fungi, whose hyphae develop extensively in the soil, potentially providing an effective dispersal network for rhizobia. We hypothesized that mycelial networks of arbuscular mycorrhizal fungi play a role in signal transmission and act as a highway, enabling rhizobia to migrate from distant soil to the roots of leguminous plants. Using in vitro and greenhouse microcosm systems, we demonstrated that Rhizophagus irregularis helps Shinorhizobium meliloti to migrate towards the legume Medicago truncatula, triggering nodulation, a mechanism absent without the arbuscular mycorrhizal fungus. Metabolomics analysis revealed eight flavonoids unique to the compartment containing extraradical hyphae of the arbuscular mycorrhizal fungus linked to M. truncatula roots, associated with Sinorhizobium meliloti growth and nod gene expression. Rhizobia plated on the extraradical hyphae connecting two plants (the legume M. truncatula and non-legume Solanum tuberosum) by a common mycelium network, showed preference for the legume, suggesting the chemoattraction by specific signals transported by the fungus connected to the legume. Simultaneously, S. meliloti stimulated the cytoplasmic/protoplasmic flow in the hyphae, likely increasing the release of nutrients and signals. Our results highlight the importance of extraradical hyphae (i.e. the mycorrhizal pathway) of arbuscular mycorrhizal fungi for the migration of rhizobia over long distances to the roots, leading to nodulation.

An isoflavone catabolism gene cluster underlying interkingdom interactions in the soybean rhizosphere.

Plant roots secrete various metabolites, including plant specialized metabolites, into the rhizosphere, and shape the rhizosphere microbiome, which is crucial for the plant health and growth. Isoflavones are major plant specialized metabolites found in legume plants, and are involved in interactions with soil microorganisms as initiation signals in rhizobial symbiosis and as modulators of the legume root microbiota. However, it remains largely unknown the molecular basis underlying the isoflavone-mediated interkingdom interactions in the legume rhizosphere. Here, we isolated Variovorax sp. strain V35, a member of the Comamonadaceae that harbors isoflavone-degrading activity, from soybean roots and discovered a gene cluster responsible for isoflavone degradation named ifc. The characterization of ifc mutants and heterologously expressed Ifc enzymes revealed that isoflavones undergo oxidative catabolism, which is different from the reductive metabolic pathways observed in gut microbiota. We further demonstrated that the ifc genes are frequently found in bacterial strains isolated from legume plants, including mutualistic rhizobia, and contribute to the detoxification of the antibacterial activity of isoflavones. Taken together, our findings reveal an isoflavone catabolism gene cluster in the soybean root microbiota, providing molecular insights into isoflavone-mediated legume-microbiota interactions.

Methods for Studying Swimming and Surface Motilities in Rhizobia.

Rhizobia are soil proteobacteria able to establish a nitrogen-fixing interaction with legumes. In this interaction, rhizobia must colonize legume roots, infect them, and become hosted inside new organs formed by the plants and called nodules. Rhizobial motility, not being essential for symbiosis, might affect the degree of success of the interaction with legumes. Because of this, the study of rhizobial motility (either swimming or surface motility) might be of interest for research teams working on rhizobial symbiotic performance. In this chapter, we describe the protocols we use in our laboratories for studying the different types of motilities exhibited by Sinorhizobium fredii and Sinorhizobium meliloti, as well as for analyzing the presence of flagella in these bacteria. All these protocols might be used (or adapted) for studying bacterial motility in rhizobia.

ENHANCED HEAVY METAL EXTRACTION FROM POST-MINING CONTAMINATED SOIL THROUGH IRON OXIDE NANOPARTICLE ADSORPTION AND SUBSEQUENT PHYTOREMEDIATION

ABSTRACT. In the current paper, the iron oxide nanoparticles (IONPs) have been demonstrated to facilitate the extraction of heavy metals from post-mining contaminated soil due to their water solubility and adsorption capabilities for the target contaminants. Furthermore, IONPs were subsequently removed from the treated soil through phytoremediation and bioaccumulated in the roots and stems of legume plants. For that purpose, 20 nm IONPs coated with an organic shell of humic acid were engineered and their adsorption capacity was assessed to heavy metals as cadmium (Cd2+), lead (Pb2+), zinc (Zn2+), manganese (Mn2+), copper (Cu2+), and nickel (Ni2+), which are common pollutants of acid mine drainage. In the framework of phytoremediation employing legume plants, the IONPs were directly introduced into the experimental soil samples alongside compost. Subsequently, it was noted that the legume roots absorbed the nanoparticles, acting as carriers for the captured heavy metals, thus aiding in their translocation to the aboveground portions of the plants. When introduced into soil environments, humic acid-coated iron oxide nanoparticles in combination with compost may exert positive effects on rhizosphere microbial populations. This can occur through the reduction of toxic metal ion levels and the potential side reaction of degrading or transforming organic pollutants into less harmful substances. Ultimately, the heavy metals become immobilized within the plant biomass, which can subsequently be harvested and removed from the contaminated site. This process leads to soil purification and remediation. The report presented here outlines a promising avenue for practical implementation, combining organic-coated IONPs with select plants to enhance bioaccumulation of heavy metals. This approach aims to develop future cost-effective passive treatment systems for the phytoremediation of soils contaminated postmining sites.

Convergent gene pair dSH3 and irr regulate Pi and Fe homeostasis in Bradyrhizobium diazoefficiens USDA110 and symbiotic nitrogen fixation efficiency

The nitrogen-fixing bacteroids inhabit inside legume root nodules must manage finely the utilization of P and Fe, the two most critical elements, due to their antagonistic interactions. While the balance mechanism for them remains unclear. A double SH3 domain-containing protein (dSH3) in the Bradyrhizobium diazoefficiens USDA110 was found to inhibit the alkaline phosphatase activity, thereby reducing P supply from organophosphates. The dSH3 gene is adjacent to the irr gene, which encodes the iron response repressor and regulates Fe homeostasis under Fe-limited conditions. Their transcription directions converge to a common intergenic sequence (IGS) region, forming a convergent transcription. Extending the IGS region through Tn5 transposon or pVO155 plasmid insertion significantly down-regulated expression of this gene pair, leading to a remarkable accumulation of P and an inability to grow under Fe-limited conditions. Inoculation of soybean with either of the insertion mutants resulted in N2-fixing failure. However, the IGS-deleted mutant showed no visible changes in N2-fixing efficiency on soybean compared to that inoculated with wild type. These findings reveal a novel regulative strategy in the IGS region and its flanking convergent gene pair for antagonistic utilization of P and Fe in rhizobia and coordination of N2-fixing efficiency.

Nodule-specific Cu+ -chaperone NCC1 is required for symbiotic nitrogen fixation in Medicago truncatula root nodules.

Cu+ -chaperones are a diverse group of proteins that allocate Cu+ ions to specific copper proteins, creating different copper pools targeted to specific physiological processes. Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required. MtNCC1 is a nodule-specific Cu+ -chaperone encoded in the Medicago truncatula genome, with a N-terminus Atx1-like domain that can bind Cu+ with picomolar affinities. MtNCC1 is able to interact with nodule-specific Cu+ -importer MtCOPT1. MtNCC1 is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper-dependent cytochrome c oxidase activity. A subset of the copper proteome is also affected in the ncc1 mutant nodules. Many of these proteins can be pulled down when using a Cu+ -loaded N-terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper-dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.