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Leguminous tree root nodule nitrogen-fixing bacteria are critical for recuperation of soil C and N cycle processes after disturbance in tropical forests, while other nodule-associated bacteria (NAB) may enhance nodule development and activity, and plant growth. However, little is known of these root nodule microbiomes. Through DNA analysis, we evaluated the bacterial taxa associated with the root nodules of the 1-year-old, 2-year-old, 13-year-old, and old growth Inga punctata trees in a cloud forest. Bradyrhizobium diazoefficiens was the dominant taxon found in all nodules at 63.16% to 85.71% mean percent sequences (MPS) of the total nodule bacterial DNA and was found in the youngest nodules examined (1 year old), suggesting that it is the primary nodular bacteria. There were 26 other NAB genera with collective MPS levels between 7.4% to 12.2%, while 15 of these genera were found in the Bulk Forest soils at collective MPS levels of 4.6%. These bacterial community compositions were different between the NAB and Bulk Forest soils, suggesting the NAB became concentrated within the root nodules, resulting in communities with different compositions from the Bulk Forest soils. Twenty-three of the 26 NAB genera were previously identified with the potential to perform 9 plant growth promoting (PGP) activities, suggesting their importance in root nodule development and plant growth. These NAB communities appeared to successionally develop over time into more complex taxonomic communities, which is consistent with the outcome of advanced microbial communities following succession. The presence of both B. diazoefficiens and the NAB communities in the nodules across all ages of tree roots, and the potential for PGP activities linked with most of the NAB genera, suggest the importance of B. diazoefficiens and the NAB community for nodule development and enhanced development and growth of I. punctata throughout its lifespan, and most critically in the younger plants.

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Background: Desmodium species used as intercrops in push-pull cropping systems are known to repel insect-pests, suppress Striga species weeds, and shift soil microbiome. However, the mechanisms through which Desmodium species impact the soil microbiome, either through its root exudates, changes in soil nutrition, or shading microbes from its nodules into the rhizosphere, are less understood. Here, we investigated the diversity of root-nodule microbial communities of three Desmodium species- Desmodium uncinatum (SLD), Desmodium intortum (GLD), and Desmodium incanum (AID) which are currently used in smallholder maize push-pull technology (PPT). Methods: Desmodium species root-nodule samples were collected from selected smallholder farms in western Kenya, and genomic DNA was extracted from the root-nodules. The amplicons underwent paired-end Illumina sequencing to assess bacterial and fungal populations. Results: We found no significant differences in composition and relative abundance of bacterial and fungal species within the root-nodules of the three Desmodium species. While a more pronounced shift was observed for fungal community compositions compared to bacteria, no significant differences were observed in the general diversity (evenness and richness) of fungal and bacterial populations among the three Desmodium species. Similarly, beta diversity was not significantly different among the three Desmodium species. The root-nodule microbiome of the three Desmodium species was dominated by Bradyrhizobium and Fusarium species. Nevertheless, there were significant differences in the proportion of marker gene sequences responsible for energy and amino acid biosynthesis among the three Desmodium species, with higher sequence proportions observed in SLD. Conclusion: There is no significant difference in the microbial community of the three Desmodium species used in PPT. However, root-nodule microbiome of SLD had significantly higher marker gene sequences responsible for energy and amino acid biosynthesis. Therefore, it is likely that the root-nodules of the three Desmodium species host similar microbiomes and influence soil health, consequently impacting plant growth and agroecosystem functioning.

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Vicia villosa (VV) and Vicia sativa (VS) are legume forages highly valued for their excellent nitrogen fixation. However, no research has addressed the mechanisms underlying their differences in nitrogen fixation. This study employed physiological, cytological, and comparative transcriptomic approaches to elucidate the disparities in nitrogen fixation between them. Our results showed that the total amount of nitrogen fixed was 60.45% greater in VV than in VS, and the comprehensive nitrogen response performance was 94.19% greater, while the nitrogen fixation efficiency was the same. The infection zone and differentiated bacteroid proportion in mature VV root nodules were 33.76% and 19.35% greater, respectively, than those in VS. The size of the VV genome was 15.16% larger than that of the VS genome, consistent with its greater biomass. A significant enrichment of the flavonoid biosynthetic pathway was found only for VV-specific genes, among which chalcone-flavonone isomerase, caffeoyl-CoA-O-methyltransferase and stilbene synthase were extremely highly expressed. The VV-specific genes also exhibited significant enrichment in symbiotic nodulation; genes related to nodule-specific cysteine-rich peptides (NCRs) comprised 61.11% of the highly expressed genes. qRT‒PCR demonstrated that greater enrichment and expression of the dominant NCR (Unigene0004451) were associated with greater nodule bacteroid differentiation and greater nitrogen fixation in VV. Our findings suggest that the greater total nitrogen fixation of VV was attributed to its larger biomass, leading to a greater nitrogen demand and enhanced fixation physiology. This process is likely achieved by the synergistic effects of high bacteroid differentiation along with high expression of flavonoid and NCR genes.

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The symbioses between leguminous plants and nitrogen-fixing bacteria known as rhizobia are well known for promoting plant growth and sustainably increasing soil nitrogen. Recent evidence indicates that hopanoids, a family of steroid-like lipids, promote Bradyrhizobium symbioses with tropical legumes. To characterize hopanoids in Bradyrhizobium symbiosis with soybean, we validated a recently published cumate-inducible hopanoid mutant of Bradyrhizobium diazoefficiens USDA110, Pcu-shc::∆shc. GC-MS analysis showed that this strain does not produce hopanoids without cumate induction, and under this condition, is impaired in growth in rich medium and under osmotic, temperature, and pH stress. In planta, Pcu-shc::∆shc is an inefficient soybean symbiont with significantly lower rates of nitrogen fixation and low survival within the host tissue. RNA-seq revealed that hopanoid loss reduces the expression of flagellar motility and chemotaxis-related genes, further confirmed by swim plate assays, and enhances the expression of genes related to nitrogen metabolism and protein secretion. These results suggest that hopanoids provide a significant fitness advantage to B. diazoefficiens in legume hosts and provide a foundation for future mechanistic studies of hopanoid function in protein secretion and motility.A major problem for global sustainability is feeding our exponentially growing human population while available arable land decreases. Harnessing the power of plant-beneficial microbes is a potential solution, including increasing our reliance on the symbioses of leguminous plants and nitrogen-fixing rhizobia. This study examines the role of hopanoid lipids in the symbiosis between Bradyrhizobium diazoefficiens USDA110, an important commercial inoculant strain, and its economically significant host soybean. Our research extends our knowledge of the functions of bacterial lipids in symbiosis to an agricultural context, which may one day help improve the practical applications of plant-beneficial microbes in agriculture.

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Introduction: The rhizosphere microbiome is critical to plant health and resistance. PGPR are well known as plant-beneficial bacteria and generally regulate nutrient utilization as well as plant responses to environmental stimuli. In our previous work, one typical PGPR strain, Pseudomonas chlororaphis IRHB3, isolated from the soybean rhizosphere, had positive impacts on soil-borne disease suppression and growth promotion in the greenhouse, but its biocontrol mechanism and application in the field are not unclear. Methods: In the current study, IRHB3 was introduced into field soil, and its effects on the local rhizosphere microbiome, disease resistance, and soybean growth were comprehensively analyzed through high-throughput sequencing and physiological and molecular methods. Results and discussion: We found that IRHB3 significantly increased the richness of the bacterial community but not the structure of the soybean rhizosphere. Functional bacteria related to phosphorus solubilization and nitrogen fixation, such as Geobacter, Geomonas, Candidatus Solibacter, Occallatibacter, and Candidatus Koribacter, were recruited in rich abundance by IRHB3 to the soybean rhizosphere as compared to those without IRHB3. In addition, the IRHB3 supplement obviously maintained the homeostasis of the rhizosphere microbiome that was disturbed by F. oxysporum, resulting in a lower disease index of root rot when compared with F. oxysporum. Furthermore, JA-mediated induced resistance was rapidly activated by IRHB3 following PDF1.2 and LOX2 expression, and meanwhile, a set of nodulation genes, GmENOD40b, GmNIN-2b, and GmRIC1, were also considerably induced by IRHB3 to improve nitrogen fixation ability and promote soybean yield, even when plants were infected by F. oxysporum. Thus, IRHB3 tends to synergistically interact with local rhizosphere microbes to promote host growth and induce host resistance in the field.

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Sulfur deficiency severely limits soybean growth, inhibiting the rhizobia nitrogenase and soybean protein synthesis. This study assessed the impact of sulfur fertilization and rhizobia inoculation on soybean growth and nitrogen fixation through bacterial culture and hydroponic experiments. We selected three rhizobia strains for bacterial cultures and used six sulfur levels. The test demonstrated severe inhibition of Rhizobium USDA110 growth without sulfur. In hydroponic experiment, we employed five sulfur levels with USDA110 as the inoculum strain. Soybean growth, nitrogen fixation, yield, and root morphology-related parameters, and root nodule growth, were significantly inhibited without sulfur. Following Rhizobium inoculation, low sulfur concentrations (0.15-0.60 mM) stimulated early-stage (V9) root growth and increased shoot nitrogen accumulation, but inhibited root growth at R5 stage. Furthermore, Rhizobium inoculation notably enhanced soybean growth, nitrogen fixation, and yield, especially within the recommended low sulfur concentration range (0.15-0.30 mM). The maximum nodule nitrogenase activity at R5 stage and highest yield was recorded at a 0.3 mM sulfur concentration with Rhizobium inoculation, which was 9.51-1222.07% higher than other treatments. These findings highlight that low sulfur concentration and rhizobia inoculation enhance soybean growth, nitrogen fixation, and yield but reduce soybean root efficacy, increasing reliance on root nodules.

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The present review highlights both the fundamental questions of calcium localization, compartmentation, and its participation in symbiosome signaling cascades during nodule formation and functioning. Apparently, the main link of such signaling is the calmodulin…calcium- and calmodulin-dependent protein kinases…CYCLOPS…NIN…target genes cascade. The minimum threshold level of calcium as a signaling agent in the presence of intracellular reserves determines the possibility of oligotrophy and ultraoligotrophy in relation to this element. During the functioning of root nodules, the Ca2+-ATPases activity maintains homeostasis of low calcium concentrations in the cytosol of nodule parenchyma cells. Disturbation of this homeostasis can trigger the root nodule senescence. The same reasons determine the increase in the effectiveness of symbiosis with the help of seed priming with sources of calcium. Examples of calcium response polymorphism in components of nitrogen fixing simbiosis important in practical terms are shown.

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Long-term excessive application of chemical fertilizers can cause many problems, such as soil degradation and environmental pollution. Therefore, we reduced conventional nitrogen fertilization and added organic fertilizers in some cases to investigate the response of photosynthetic characteristics, root nodules and yield on reduced nitrogen fertilization. Compared to conventional nitrogen fertilization, the 25% and 35% nitrogen reduction treatments reduced the leaf area index, net photosynthetic rate, 100-fruit weight, 100-kernel weight and the yield of peanut, but had no significant effect on the kernel rate. With constant N fertilizer, adding organic fertilization alone increased leaf area index, chlorophyll, net photosynthetic rate and yield of peanut. In compounded treatments of nitrogen and organic fertilizer, the highest yields were achieved in the 25% N reduction with the 3000 kg/hm-2 organic fertilizer treatment (T3) and the 4500 kg/hm-2 organic fertilizer treatment (T4); furthermore, the net photosynthetic rate, leaf area index, yield and fertilizer contribution were significantly higher in these two treatments than in the conventional fertilizer treatments. Nitrogen fertilizer had significant effects on the quantity and fresh weight of root nodules. Concretely, nitrogen reduction increased the quantity and fresh weight of root nodules of peanut in the early stage of fertility but decreased them in the harvest stage. Nitrogen reduction with an additional organic fertilizer in the late stage of fertility increased the quantity and fresh weight of root nodules of peanut. Considering the property of root nodules was significantly positively correlated with net photosynthetic rate and yield, the arguments above may be the mechanism of the highest yields found in T3 and T4. This work can provide empirical and instructional support for a balanced fertilization strategy in peanut agriculture and high-yielding and efficient cultivation of peanut.

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Alnus cremastogyne Burkill (Betulaceae), an actinorhizal plant, can enter a mutualistic symbiosis with Frankia species that leads to the formation of nitrogen fixing root nodules. Some primary metabolites (carbohydrates, dicarboxylic acids, amino acids, citrulline and amides) involved in carbon and nitrogen metabolism in actinorhizal nodules have been identified, while specialized metabolites in A. cremastogyne root nodules are yet to be characterized. In this study, we isolated and identified three undescribed 3-pentanol glycosides, i.e., 3-pentyl α-l-arabinofuranosyl-(1''→6')-ß-d-glucopyranoside, 3-pentyl α-l-rhamnopyranosyl-(1''→6')-ß-d-glucopyranoside, and 3-pentyl 6'-(3-hydroxy3-methylglutaryl)-ß-d-glucopyranoside, as well as seventeen known compounds from A. cremastogyne root nodules. 3-Pentanol glycosides are abundantly distributed in root nodules, while they are distributed in stems, roots, leaves and fruits at low/zero levels. A. cremastogyne plants treated by root nodule suspension emit 3-pentanol. This study enriches the knowledge about specialized metabolites in the actinorhizal host, and provides preliminarily information on the signal exchange in the actinorhizal symbiosis between A. cremastogyne and Frankia.

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Antimicrobial silver nanoparticles (AgNPs) are popular in consumer and industrial products, leading to increasing concentrations in the environment. We tested whether exposure to AgNPs could be detrimental to a microbe, its host plant, and their symbiotic relationship. When subjected to 10 µg/mL AgNPs, growth of Bradyrhizobium japonicum USDA 110 was halted. Axenic nitrogen-fertilized Glycine max seedlings were unaffected by 2.5 µg/mL of 30 nm AgNPs, but growth was inhibited with the same dose of 16 nm AgNPs. With 2.5 µg/mL AgNPs, biomass of inoculated plants was 50% of the control. Bacteroids were not found in nodules on plants treated with 2.5 µg/mL AgNPs and plants given 0.5-2.5 µg/mL AgNPs had 40-65% decreased nitrogen fixation. In conclusion, AgNPs not only interfere with general plant and bacterial growth but also inhibit nodule development and bacterial nitrogen fixation. We should be mindful of not releasing AgNPs to the environment or to agricultural land.

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Nodule-associated nitrogen-fixing microorganisms (diazotrophs) residing in legume root nodules, and they have the potential to enhance legume survival. However, the succession characteristics and mechanisms of leguminous diazotrophic communities remain largely unexplored. We performed a high-throughput nifH amplicon sequencing with samples of root nodules and soil in the three developmental phases (young nodules, active nodules and senescent nodules) of the Sophora davidii (Franch.) Skeels root nodules, aiming to investigate the dynamics of nodule-endophytic diazotrophs during three developmental phases of root nodules. The results demonstrated the presence of diverse diazotrophic bacteria and successional community shifting dominated by Mesorhizobium and Bradyrhizobium inside the nodule according to the nodule development. The relative abundance decreased for Mesorhizobium, while decreased first and then increased for Bradyrhizobium in nodule development from young to active to senescent. Additionally, strains M. amorphae BT-30 and B. diazoefficiens B-26 were isolated and selected to test the interaction between them in co-cultured conditions. Under co-culture conditions: B. diazoefficiens B-26 significantly inhibited the growth of M. amorphae BT-30. Intriguingly, growth of B. diazoefficiens B-26 was significantly promoted by co'culture with M. amorphae BT-30 and could utilize some carbon and nitrogen sources that M. amorphae BT-30 could not. Additionally, the composition of microbial community varied in root nodules, in rhizosphere and in bulk soil. Collectively, our study highlights that developmental phases of nodules and the host microhabitat were the key driving factors for the succession of nodule-associated diazotrophic community.

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(1) Background: Alfalfa is an important legume forage throughout the world. Although alfalfa is considered moderately tolerant to salinity, its production and nitrogen-fixing activity are greatly limited by salt stress. (2) Methods: We examined the physiological changes and proteomic profiles of alfalfa with active nodules (NA) and without nodules (NN) under NaCl treatment. (3) Results: Our data suggested that NA roots showed upregulation of the pathways of abiotic and biotic stress responses (e.g., heat shock proteins and pathogenesis-related proteins), antioxidant enzyme synthesis, protein synthesis and degradation, cell wall degradation and modification, acid phosphatases, and porin transport when compared with NN plants under salt stress conditions. NA roots also upregulated the processes or proteins of lipid metabolism, heat shock proteins, protein degradation and folding, and cell cytoskeleton, downregulated the DNA and protein synthesis process, and vacuolar H+-ATPase proteins under salt stress. Besides, NA roots displayed a net H+ influx and low level of K+ efflux under salt stress, which may enhance the salt tolerance of NA plants. (4) Conclusions: The rhizobium symbiosis conferred the host plant salt tolerance by regulating a series of physiological processes to enhance stress response, improve antioxidant ability and energy use efficiency, and maintain ion homeostasis.

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Various legume plants form root nodules in which symbiotic bacteria (rhizobia) fix atmospheric nitrogen after differentiation into a symbiotic form named bacteroids. In some legume species, bacteroid differentiation is promoted by defensin-like nodule-specific cysteine-rich (NCR) peptides. NCR peptides have best been studied in the model legume Medicago truncatula Gaertn., while in many other legumes relevant information is still fragmentary. Here, we characterize the NCR gene family in pea (Pisum sativum L.) using genomic and transcriptomic data. We found 360 genes encoding NCR peptides that are expressed in nodules. The sequences of pea NCR genes and putative peptides are highly variable and differ significantly from NCR sequences of M. truncatula. Indeed, only one pair of orthologs (PsNCR47-MtNCR312) has been identified. The NCR genes in the pea genome are located in clusters, and the expression patterns of NCR genes from one cluster tend to be similar. These data support the idea of independent evolution of NCR genes by duplication and diversification in related legume species. We also described spatiotemporal expression profiles of NCRs and identified specific transcription factor (TF) binding sites in promoters of "early" and "late" NCR genes. Further, we studied the expression of NCR genes in nodules of Fix- mutants and predicted potential regulators of NCR gene expression, one among them being the TF ERN1 involved in the early steps of nodule organogenesis. In general, this study contributes to understanding the functions of NCRs in legume nodules and contributes to understanding the diversity and potential antibiotic properties of pea nodule-specific antimicrobial molecules.

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AIM: In this study, 16S rRNA amplicon sequencing analyses were performed to determine the diversity of the bacterial community present in the soil, rhizosphere region, root nodules and seeds of the horse gram plant. METHODS AND RESULTS: We observed the dominance of Proteobacteria, Actinobacteria, Firmicutes, Acidobacteria, Bacteroidetes, Planctomycetes and Gemmatimonadetes across all four domains of the horse gram plant. For community analyses, the significance of the alpha diversity was estimated using the Shannon index, Simpson index and Chao1 index, which revealed no significant difference among the samples. However, the estimation of the beta diversity indicated a significant difference among the samples, with p < 0.001 and R2  = 1. A strong positive correlation was found between the rhizosphere and root nodule samples. Comparative genomics of the 16S rRNA gene showed that ammonium-oxidizing metabolism (amoA), nitrite-reducing metabolism (nirK) and nitrogen-fixing metabolism (nifH) were prominent mechanisms in all samples. The genes involved in the biosynthesis of amino acids, purine metabolism and nitrogen metabolism were identified as the key genes associated with the functional traits of microbial domains in horse gram. CONCLUSION: The culturable microbes associated with horse gram can be used as a substitute for synthetic fertilizers to maintain soil fertility and ecological health in agricultural practices. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the survival strategies of bacterial communities that positively respond to multiple gate selection helps in understanding the structural diversity and functional traits primarily focused on the development of beneficial microbial consortium for promoting plant growth.

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In addition to the rhizobia, other non-rhizobial endophytes (NREs) have been simultaneously isolated from the root nodules. The existence of NREs in leguminous root nodules is a universal phenomenon, and they have the potential to enhance legume survival, especially under conditions of environmental stress. However, the diversity and biogeographic patterns of microbial communities inhabiting root nodules are not well studied or understood. Here, we explored and characterized the diversity of NRE bacteria by using 16S rRNA gene high-throughput amplicon sequencing. Additionally, we compared the biogeography and co-occurrence patterns in review of the bacterial microbiota inhabiting the rhizosphere, the bulk soil and the root nodule bacterial communities associated with Sophora davidii, a native N-fixing wild leguminous shrub in hilly and gully regions of the Loess Plateau of China. The results showed the presence of a large diversity of bacteria belonging to 81 phyla, 154 classes, 333 orders, 463 families, and 732 genera inside the nodules. Proteobacteria were dominant in the nodule and rhizosphere soil samples, and Actinomycetes were dominant in the bulk soil samples. Mesorhizobium was the dominant genus in the nodules, accounting for between 60.15 and 83.74% of the bacteria. The microbial community composition of the NRE in the root nodules differed from that in the rhizosphere soil and the bulk soil of S. davidii. Moreover, we found that the biogeographic patterns and assembly process of the rhizobia and non-rhizobia communities differed in the root nodule, the rhizosphere soil and the bulk soil. Furthermore, the correlation analysis between the soil's physical and chemical properties and the bacteria showed that available phosphorus was the predominant factor affecting the bacterial diversity within the rhizosphere soil. Finally, our results revealed that the microbial network diagram of co-occurrence patterns showed more complexes in the soil than in the root nodules. This indicates that only specific microorganisms could colonize and thrive in the rhizosphere through the selection and filtering effects of roots. In conclusion, there are significant differences in bacterial community composition in the nodules, rhizosphere and bulk soil in the hilly and gully region of the Loess Plateau, which is the result of environmental filtration. Our study improves the understanding of the biogeographic patterns and diversity of bacterial microbiota inhabiting root nodules and can help quantify and define the root nodule assemblage process of S. davidii.

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Rhizobia are a diazotrophic group of bacteria that are usually isolated form the nodules in roots, stem of leguminous plants and are able to form nodules in the host plant owing to the presence of symbiotic genes. The rhizobial community is highly diverse, and therefore, the taxonomy and genera-wise classification of rhizobia has been constantly changing since the last three decades. This is mainly due to technical advancements, and shifts in definitions, resulting in a changing paradigm of rhizobia taxonomy. Initially, the taxonomic definitions at the species and sub species level were based on phylogenetic analysis of 16S rRNA sequence, followed by polyphasic approach to have phenotypic, biochemical, and genetic analysis including multilocus sequence analysis. Rhizobia mainly belonging to α- and ß-proteobacteria, and recently new additions from γ-proteobacteria had been classified. Nowadays rhizobial taxonomy has been replaced by genome-based taxonomy that allows gaining more insights of genomic characteristics. These omics-technologies provide genome specific information that considers nodulation and symbiotic genes, along with molecular markers as taxonomic traits. Taxonomy based on complete genome sequence (genotaxonomy), average nucleotide identity, is now being considered as primary approach, resulting in an ongoing paradigm shift in rhizobial taxonomy. Also, pairwise whole-genome comparisons, phylogenomic analyses offer correlations between DNA and DNA re-association values that have delineated biologically important species. This review elaborates the present classification and taxonomy of rhizobia, vis-a-vis development of technical advancements, parameters and controversies associated with it, and describe the updated information on evolutionary lineages of rhizobia.

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It is currently assumed that around 100 million years ago, the common ancestor to the Fabales, Fagales, Rosales and Cucurbitales in Gondwana, developed a root nodule symbiosis with a nitrogen-fixing bacterium. The symbiotic trait evolved first in Frankia cluster-2; thus, strains belonging to this cluster are the best extant representatives of this original symbiont. Most cluster-2 strains could not be cultured to date, except for Frankia coriariae, and therefore many aspects of the symbiosis are still elusive. Based on phylogenetics of cluster-2 metagenome-assembled genomes (MAGs), it has been shown that the genomes of strains originating in Eurasia are highly conserved. These MAGs are more closely related to Frankia cluster-2 in North America than to the single genome available thus far from the southern hemisphere, i.e., from Papua New Guinea.To unravel more biodiversity within Frankia cluster-2 and predict routes of dispersal from Gondwana, we sequenced and analysed the MAGs of Frankia cluster-2 from Coriaria japonica and Coriaria intermedia growing in Japan, Taiwan and the Philippines. Phylogenetic analyses indicate there is a clear split within Frankia cluster-2, separating a continental from an island lineage. Presumably, these lineages already diverged in Gondwana.Based on fossil data on the host plants, we propose that these two lineages dispersed via at least two routes. While the continental lineage reached Eurasia together with their host plants via the Indian subcontinent, the island lineage spread towards Japan with an unknown host plant.

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Drought dramatically affects crop productivity worldwide. For legumes this effect is especially pronounced, as their symbiotic association with rhizobia is highly-sensitive to dehydration. This might be attributed to the oxidative stress, which ultimately accompanies plants' response to water deficit. Indeed, enhanced formation of reactive oxygen species in root nodules might result in up-regulation of lipid peroxidation and overproduction of reactive carbonyl compounds (RCCs), which readily modify biomolecules and disrupt cell functions. Thus, the knowledge of the nodule carbonyl metabolome dynamics is critically important for understanding the drought-related losses of nitrogen fixation efficiency and plant productivity. Therefore, here we provide, to the best of our knowledge, for the first time a comprehensive overview of the pea root nodule carbonyl metabolome and address its alterations in response to polyethylene glycol-induced osmotic stress as the first step to examine the changes of RCC patterns in drought treated plants. RCCs were extracted from the nodules and derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). The relative quantification of CHH-derivatives by liquid chromatography-high resolution mass spectrometry with a post-run correction for derivative stability revealed in total 194 features with intensities above 1 × 105 counts, 19 of which were down- and three were upregulated. The upregulation of glyceraldehyde could accompany non-enzymatic conversion of glyceraldehyde-3-phosphate to methylglyoxal. The accumulation of 4,5-dioxovaleric acid could be the reason for down-regulation of porphyrin metabolism, suppression of leghemoglobin synthesis, inhibition of nitrogenase and degradation of legume-rhizobial symbiosis in response to polyethylene glycol (PEG)-induced osmotic stress effect. This effect needs to be confirmed with soil-based drought models.

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We recently showed that yellow lupine is highly sensitive to soil water deficits since this stressor disrupts nodule structure and functioning, and at the same time triggers flower separation through abscission zone (AZ) activation in the upper part of the plant. Both processes require specific transformations including cell wall remodeling. However, knowledge about the involvement of particular cell wall elements in nodulation and abscission in agronomically important, nitrogen-fixing crops, especially under stressful conditions, is still scarce. Here, we used immuno-fluorescence techniques to visualize dynamic changes in cell wall compounds taking place in the root nodules and flower AZ of Lupinus luteus following drought. The reaction of nodules and the flower AZ to drought includes the upregulation of extensins, galactans, arabinans, xylogalacturonan, and xyloglucans. Additionally, modifications in the localization of high- and low-methylated homogalacturonans and arabinogalactan proteins were detected in nodules. Collectively, we determined for the first time the drought-associated modification of cell wall components responsible for their remodeling in root nodules and the flower AZ of L. luteus. The involvement of these particular molecules and their possible interaction in response to stress is also deeply discussed herein.

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Bacteriocins are narrow-spectrum antibiotics of bacterial origin that can affect competition in resource-limited environments, such as the rhizosphere. Therefore, bacteriocins may be good candidates for manipulation to generate more competitive inocula for soybean. In this study, Bradyrhizobium japonicum FN1, along with other Bradyrhizobia in our culture collection, was screened for bacteriocin-like activity. Five distinct inhibitory effects were observed. FN1 genes putatively involved in bacteriocin production were computationally identified. These genes were mutagenized, and the subsequent strains were screened for loss of inhibitory activity. Mutant strain BRJ-48, with an insert in bjfn1_01204, displayed a loss of ability to inhibit an indicator strain. This loss can be complemented by the introduction of a plasmid expressing bjfn1_01204 in trans. The strain carrying the mutation did not affect competition in broth cultures but was less competitive for nodule occupancy. Annotation suggests that bjfn1_01204 encodes a carboxymuconolactone decarboxylase; however, the direct contribution of how this enzyme contributes to inhibiting the tester strain remains unknown.

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