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Three strains of Gram-negative bacterium, Rhizobium, were developed by gamma (γ)-irradiation random mutagenesis. The developed strains were evaluated for their augmented features for symbiotic association, nitrogen fixation, and crop yield of three leguminous plants-chickpea, field-pea, and lentil-in agricultural fields of the northern Indian state of Haryana. Crops treated with developed mutants exhibited significant improvement in plant features and the yield of crops when compared to the control-uninoculated crops and crops grown with indigenous or commercial crop-specific strains of Rhizobium. This improvement was attributed to generated mutants, MbPrRz1 (on chickpea), MbPrRz2 (on lentil), and MbPrRz3 (on field-pea). Additionally, the cocultured symbiotic response of MbPrRz1 and MbPrRz2 mutants was found to be more pronounced on all three crops. The statistical analysis using Pearson's correlation coefficients revealed that nodulation and plant biomass were the most related parameters of crop yield. Among the effectiveness of developed mutants, MbPrRz1 yielded the best results for all three tested crops. Moreover, the developed mutants enhanced macro- and micronutrients of the experimental fields when compared with fields harboring the indigenous rhizobial community. These developed mutants were further genetically characterized, predominantly expressing nitrogen fixation marker, nifH, and appeared to belong to Mesorhizobium ciceri (MbPrRz1) and Rhizobium leguminosarum (both MbPrRz2 and MbPrRz3). In summary, this study highlights the potential of developed Rhizobium mutants as effective biofertilizers for sustainable agriculture, showcasing their ability to enhance symbiotic relationships, crop yield, and soil fertility., (© 2024 The Author(s). Journal of Basic Microbiology published by Wiley‐VCH GmbH.)

We evaluated the effect of three different Bradyrhizobium strains inoculated in two soybean genotypes (R01-581F, drought-tolerant, and NA5858RR, drought-sensitive) submitted to drought in two trials conducted simultaneously under greenhouse. The strains (SEMIA 587, SEMIA 5019 (both B. elkanii), and SEMIA 5080 (B. diazoefficiens)) were inoculated individually in each genotype and then submitted to water restriction (or kept well-watered, control) between 45 and 62 days after emergence. No deep changes in plant physiological variables were observed under the moderate water restriction imposed during the first 10 days. Nevertheless, photosynthesis and transpiration decreased after the severe water restriction imposed for further 7 days. Water restriction reduced growth (- 30%) and the number of nodules (- 47% and - 58% for R01-581F and NA5858RR, respectively) of both genotypes, with a negative effect on N-metabolism. The genotype R01-581F inoculated with SEMIA 5019 strain had higher photosynthetic rates compared with NA5858RR, regardless of the Bradyrhizobium strain. On average, R01-581F showed better performance under drought than NA5858RR, with higher number of nodules (51 vs. 38 nodules per plant, respectively) and less accumulation of ureides in petioles (15 μmol g -1 vs. 34 μmol g -1 , respectively). Moreover, plants inoculated with SEMIA 5080 had higher glutamine synthetase activity under severe water restriction, especially in the drought-tolerant R01-518F, suggesting maintenance of N metabolism under drought. The Bradyrhizobium strain affects the host plant responses to drought in which the strain SEMIA 5080 improves the drought tolerance of R01-518F genotype.

Biological nitrogen fixation emerging from the symbiosis between bacteria and crop plants holds promise to increase the sustainability of agriculture. One of the biggest hurdles for the engineering of nitrogen-fixing organisms is an incomplete knowledge of metabolic interactions between microbe and plant. In contrast to the previously assumed supply of only succinate, we describe here the CATCH-N cycle as a novel metabolic pathway that co-catabolizes plant-provided arginine and succinate to drive the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. Using systems biology, isotope labeling studies and transposon sequencing in conjunction with biochemical characterization, we uncovered highly redundant network components of the CATCH-N cycle including transaminases that interlink the co-catabolism of arginine and succinate. The CATCH-N cycle uses N 2 as an additional sink for reductant and therefore delivers up to 25% higher yields of nitrogen than classical arginine catabolism-two alanines and three ammonium ions are secreted for each input of arginine and succinate. We argue that the CATCH-N cycle has evolved as part of a synergistic interaction to sustain bacterial metabolism in the microoxic and highly acid environment of symbiosomes. Thus, the CATCH-N cycle entangles the metabolism of both partners to promote symbiosis. Our results provide a theoretical framework and metabolic blueprint for the rational design of plants and plant-associated organisms with new properties to improve nitrogen fixation., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Pigeon pea ( Cajanus cajan (L.) Millspaugh) is cultivated widely in semiarid agricultural regions in over 90 countries around the world. This important legume can enter into symbiotic associations with a wide range of rhizobia including Bradyrhizobium and fast-growing rhizobia. In comparison with other major legumes such as soybean and common bean, only limited information is available on the symbiotic interaction of pigeon pea with rhizobia. In this study, we investigated the ability of two classical soybean symbionts- S. fredii USDA191 and B. diazoefficiens USDA110-and their type 3 secretion system (T3SS) mutants, to nodulate pigeon pea. Both S. fredii USDA191 and a T3SS mutant S. fredii RCB26 formed nitrogen-fixing nodules on pigeon pea. Inoculation of pigeon pea roots with B. diazoefficiens USDA110 and B. diazoefficiens Δ136 (a T3SS mutant) resulted in the formation of Fix- and Fix+ nodules, respectively. Light and transmission electron microscopy of Fix- nodules initiated by B. diazoefficiens USDA110 revealed the complete absence of rhizobia within these nodules. In contrast, Fix+ nodules formed by B. diazoefficiens Δ136 revealed a central region that was completely filled with rhizobia. Ultrastructural investigation revealed the presence of numerous bacteroids surrounded by peribacteroid membranes in the infected cells. Analysis of nodule proteins by one- and two-dimensional gel electrophoresis revealed that leghemoglobin was absent in B. diazoefficiens USDA110 nodules, while it was abundantly present in B. diazoefficiens Δ136 nodules. Results of competitive nodulation assays indicated that B. diazoefficiens Δ136 had greater competitiveness for nodulation on pigeon pea than did the wild type strain. Our results suggest that this T3SS mutant of B. diazoefficiens , due to its greater competitiveness and ability to form Fix+ nodules, could be exploited as a potential inoculant to boost pigeon pea productivity.

Plants, especially perennials, growing in drylands and seasonally dry ecosystems are uniquely adapted to dry conditions. Legume shrubs and trees, capable of symbiotic dinitrogen (N 2 ) fixation, often dominate in drylands. However, the strategies that allow symbiotic fixation in these ecosystems, and their influence on the nitrogen cycle, are largely unresolved. We evaluated the climatic, biogeochemical and ontogenetic factors influencing nitrogen fixation in an abundant Mediterranean legume shrub, Calicotome villosa. We measured nodulation, fixation rate, nitrogen allocation and soil biogeochemistry in three field sites over a full year. A controlled experiment evaluated differences in plant regulation of fixation as a function of soil nutrient availability and seedling and adult developmental stages. We found a strong seasonal pattern, shifting between high fixation rates during the rainy season at flowering and seed-set times to almost none in the rainless season. Under controlled conditions, plants downregulated fixation in response to soil nitrogen availability, but this response was stronger in seedlings than in adult shrubs. Finally, we did not find elevated soil nitrogen under N 2 -fixing shrubs. We conclude that seasonal nitrogen fixation, regulation of fixation, and nitrogen conservation are key adaptations influencing the dominance of dryland legumes in the community, with broader consequences on the ecosystem nitrogen cycle., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Plants able to establish a nitrogen-fixing root nodule symbiosis with the actinobacterium Frankia are called actinorhizal. These interactions lead to the formation of new root organs, called actinorhizal nodules, where the bacteria are hosted intracellularly and fix atmospheric nitrogen thus providing the plant with an almost unlimited source of nitrogen for its nutrition. Like other symbiotic interactions, actinorhizal nodulation involves elaborate signalling between both partners of the symbiosis, leading to specific recognition between the plant and its compatible microbial partner, its accommodation inside plant cells and the development of functional root nodules. Actinorhizal nodulation shares many features with rhizobial nodulation but our knowledge on the molecular mechanisms involved in actinorhizal nodulation remains very scarce. However recent technical achievements for several actinorhizal species are allowing major discoveries in this field. In this review, we provide an outline on signalling molecules involved at different stages of actinorhizal nodule formation and the corresponding signalling pathways and gene networks.

Biological nitrogen fixation, conducted by soil diazotrophs, is the primary nitrogen source for natural grasslands. However, the diazotrophs in grassland soils are still far from fully investigated. Particularly, their regional-scale distribution patterns have never been systematically examined. Here, soils (0-5 cm) were sampled from 54 grasslands on the Tibetan Plateau to examine the diazotroph abundance, diversity, and community composition, as well as their distribution patterns and driving factors. The diazotroph abundance was expressed as nifH gene copies, measured using real-time PCR. The diversity and community composition of diazotrophs were analyzed through MiSeq sequencing of nifH genes. The results showed that Cyanobacteria (47.94%) and Proteobacteria (45.20%) dominated the soil diazotroph communities. Most Cyanobacteria were classified as Nostocales which are main components of biological crusts. Rhizobiales, most of which were identified as potential symbiotic diazotrophs, were also abundant in approximately half of the soil samples. The soil diazotroph abundance, diversity, and community composition followed the distribution patterns in line with mean annual precipitation. Moreover, they also showed significant correlations with prokaryotic abundance, plant biomass, vegetation cover, soil pH values, and soil nutrient contents. Among these environmental factors, the soil moisture, organic carbon, available phosphorus, and inorganic nitrogen contents could be the main drivers of diazotroph distribution due to their strong correlations with diazotroph indices. These findings suggest that autotrophic and symbiotic diazotrophs are the predominant nitrogen fixers in Tibetan grassland soils, and highlight the key roles of water and nutrient availability in determining the soil diazotroph distribution on the Tibetan Plateau., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Indigenous soybean rhizobial strains were isolated from root nodules sampled from farmers' fields in Mozambique to determine their identity, distribution and symbiotic relationships. Plant infection assays revealed variable nodulation and symbiotic effectiveness among the 43 bacterial isolates tested. Strains from Ruace generally promoted greater whole-plant growth than the others. 16S rRNA-RFLP analysis of genomic DNA extracted from the rhizobial isolates produced different banding patterns, a clear indication of high bacterial diversity. However, the multilocus sequence analysis (MLSA) data showed alignment of the isolates with B. elkanii species. The 16S rRNA sequences of representative soybean isolates selected from each 16S rRNA-RFLP cluster showed their relatedness to B. elkanii, as well as to other Bradyrhizobium species. But a concatenated phylogeny of two housekeeping genes (glnII and gyrB) identified the soybean nodulating isolates as Bradyrhizobium, with very close relatedness to B. elkanii. The nifH and nodC sequences also showed that the majority of the test soybean isolates were closely related to B. elkanii, albeit the inconsistency with some isolates. Taken together, these findings suggest that the B. elkanii group are the preferred dominant microsymbiont of soybean grown in Mozambican soils. Furthermore, the distribution of soybean rhizobia in the agricultural soils of Mozambique was found to be markedly influenced by soil pH, followed by the concentrations of plant-available P and Mn. This study suggested that the identified isolates TUTMJM5, TUTMIITA5A and TUTLBC2B can be used as inoculants for increased soybean production in Mozambique., (Copyright © 2018 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Erythrina velutina ("mulungu") is a legume tree from Caatinga that associates with rhizobia but the diversity and symbiotic ability of "mulungu" rhizobia are poorly understood. The aim of this study was to characterize "mulungu" rhizobia from Caatinga. Bacteria were obteined from Serra Talhada and Caruaru in Caatinga under natural regeneration. The bacteria were evaluated to the amplification of nifH and nodC and to metabolic characteristics. Ten selected bacteria identified by 16S rRNA sequences. They were tested in vitro to NaCl and temperature tolerance, auxin production and calcium phosphate solubilization. The symbiotic ability were assessed in an greenhouse experiment. A total of 32 bacteria were obtained and 17 amplified both symbiotic genes. The bacteria showed a high variable metabolic profile. Bradyrhizobium (6), Rhizobium (3) and Paraburkholderia (1) were identified, differing from their geographic origin. The isolates grew up to 45°C to 0.51molL -1 of NaCl. Bacteria which produced more auxin in the medium with l-tryptophan and two Rhizobium and one Bradyrhizobium were phosphate solubilizers. All bacteria nodulated and ESA 90 (Rhizobium sp.) plus ESA 96 (Paraburkholderia sp.) were more efficient symbiotically. Diverse and efficient rhizobia inhabit the soils of Caatinga dry forests, with the bacterial differentiation by the sampling sites., (Copyright © 2018 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION ( NIN ) and RHIZOBIUM-DIRECTED POLAR GROWTH ( RPG ). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN , RPG , and NOD FACTOR PERCEPTION Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Background: Polyploidy is known to significantly alter plant genomes, phenotypes and interactions with the abiotic environment, yet the impacts of polyploidy on plant-biotic interactions are less well known. A particularly important plant-biotic interaction is the legume-rhizobia mutualism, in which rhizobia fix atmospheric nitrogen in exchange for carbon provided by legume hosts. This mutualism regulates nutrient cycles in natural ecosystems and provides nitrogen to agricultural environments. Despite the ecological, evolutionary and agricultural importance of plant polyploidy and the legume-rhizobia mutualism, it is not yet fully understood whether plant polyploidy directly alters mutualism traits or the consequences on plant growth., Scope: The aim was to propose a conceptual framework to understand how polyploidy might directly enhance the quantity and quality of rhizobial symbionts hosted by legume plants, resulting in increased host access to fixed nitrogen (N). Mechanistic hypotheses have been devised to examine how polyploidy can directly alter traits that impact the quantity (e.g. nodule number, nodule size, terminal bacteroid differentiation) and quality of symbionts (e.g. nodule environment, partner choice, host sanctions). To evaluate these hypotheses, an exhaustive review of studies testing the effects of plant polyploidy on the mutualism was conducted. In doing so, overall trends were synthesized, highlighting the limited understanding of the mechanisms that underlie variation in results achieved thus far, revealing striking gaps in knowledge and uncovering areas ripe for future research., Conclusions: Plant polyploidy can immediately alter nodule size, N fixation rate and the identity of rhizobial symbionts hosted by polyploid legumes, but many of the mechanistic hypotheses proposed here, such as bacteroid number and enhancements of the nodule environment, remain unexplored. Although current evidence supports a role of plant polyploidy in enhancing key aspects of the legume-rhizobia mutualism, the underlying mechanisms and effects on host benefit from the mutualism remain unresolved., (© The Author 2017. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The strain BR 3351 T (Bradyrhizobium manausense) was obtained from nodules of cowpea (Vigna unguiculata L. Walp) growing in soil collected from Amazon rainforest. Furthermore, it was observed that the strain has high capacity to fix nitrogen symbiotically in symbioses with cowpea. We report here the draft genome sequence of strain BR 3351 T . The information presented will be important for comparative analysis of nodulation and nitrogen fixation for diazotrophic bacteria. A draft genome with 9,145,311bp and 62.9% of GC content was assembled in 127 scaffolds using 100bp pair-end Illumina MiSeq system. The RAST annotation identified 8603 coding sequences, 51 RNAs genes, classified in 504 subsystems., (Published by Elsevier Editora Ltda.)

The family Leguminosae comprises approximately 20,000 species that mostly form symbioses with arbuscular mycorrhizal fungi (AMF) and nitrogen-fixing bacteria (NFB). This study is aimed at investigating and confirming the dependence on nodulation and biological nitrogen fixation in the specie Piptadenia gonoacantha (Mart.) Macbr., which belongs to the Piptadenia group. Two consecutive experiments were performed in a greenhouse. The experiments were fully randomized with six replicates and a factorial scheme. For the treatments, the two AMF species and three NFB strains were combined to nodulate P. gonoacantha in addition to the control treatments. The results indicate this species' capacity for nodulation without the AMF; however, the AMF+NFB combinations yielded a considerable gain in P. gonoacantha shoot weight compared with the treatments that only included inoculating with bacteria or AMF. The results also confirm that the treatment effects among the AMF+NFB combinations produced different shoot dry weight/root dry weight ratios. We conclude that AMF is not necessary for nodulation and that this dependence improves species development because plant growth increases upon co-inoculation., (Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

The strain BR 3262 was isolated from nodule of cowpea (Vigna unguiculata L. Walp) growing in soil of the Atlantic Forest area in Brazil and it is reported as an efficient nitrogen fixing bacterium associated to cowpea. Firstly, this strain was assigned as Bradyrhizobium elkanii, however, recently a more detailed genetic and molecular characterization has indicated it could be a Bradyrhizobium pachyrhizi species. We report here the draft genome sequence of B. pachyrhizi strain BR 3262, an elite bacterium used as inoculant for cowpea. The whole genome with 116 scaffolds, 8,965,178bp and 63.8% of C+G content for BR 3262 was obtained using Illumina MiSeq sequencing technology. Annotation was added by the RAST prokaryotic genome annotation service and shown 8369 coding sequences, 52 RNAs genes, classified in 504 subsystems., (Published by Elsevier Editora Ltda.)

Background: Common bean (Phaseolus vulgaris L.) is the most important legume cropped worldwide for food production and its agronomic performance can be greatly improved if the benefits from symbiotic nitrogen fixation are maximized. The legume is known for its high promiscuity in nodulating with several Rhizobium species, but those belonging to the Rhizobium tropici "group" are the most successful and efficient in fixing nitrogen in tropical acid soils. Rhizobium leucaenae belongs to this group, which is abundant in the Brazilian "Cerrados" soils and frequently submitted to several environmental stresses. Here we present the first high-quality genome drafts of R. leucaenae, including the type strain CFN 299(T) and the very efficient strain CPAO 29.8. Our main objective was to identify features that explain the successful capacity of R. leucaenae in nodulating common bean under stressful environmental conditions., Results: The genomes of R. leucaenae strains CFN 299(T) and CPAO 29.8 were estimated at 6.7-6.8 Mbp; 7015 and 6899 coding sequences (CDS) were predicted, respectively, 6264 of which are common to both strains. The genomes of both strains present a large number of CDS that may confer tolerance of high temperatures, acid soils, salinity and water deficiency. Types I, II, IV-pili, IV and V secretion systems were present in both strains and might help soil and host colonization as well as the symbiotic performance under stressful conditions. The symbiotic plasmid of CPAO 29.8 is highly similar to already described tropici pSyms, including five copies of nodD and three of nodA genes. R. leucaenae CFN 299(T) is capable of synthesizing Nod factors in the absence of flavonoids when submitted to osmotic stress, indicating that under abiotic stress the regulation of nod genes might be different., Conclusion: A detailed study of the genes putatively related to stress tolerance in R. leucaenae highlighted an intricate pattern comprising a variety of mechanisms that are probably orchestrated to tolerate the stressful conditions to which the strains are submitted on a daily basis. The capacity to synthesize Nod factors under abiotic stress might follow the same regulatory pathways as in CIAT 899(T) and may help both to improve bacterial survival and to expand host range to guarantee the perpetuation of the symbiosis.

The symbiosis between α nitrogen (N2)-fixing Proteobacteria (family Rhizobiaceae) and legumes belonging to the Fabaceae (a single phylogenetic group comprising three subfamilies: Caesalpinioideae, Mimosoideae and Papilionoideae) results in the formation of a novel root structure called a nodule, where atmospheric N2 is fixed into NH3(+). In the determinate type of nodules harbored by Rhizobium-nodulated Fabaceae species, newly synthesized NH3(+) is finally converted into allantoin (C4H6N4O3) and allantoic acid (C4H8N4O4) (ureides) through complex pathways involving at least 20 different enzymes that act synchronously in two types of nodule cells with contrasting ultrastructure, including the tree nodule cell organelles. Newly synthesized ureides are loaded into the network of nodule-root xylem vessels and transported to aerial organs by the transpirational water current. Once inside the leaves, ureides undergo an enzymatically driven reverse process to yield NH4(+) that is used for growth. This supports the role of ureides as key nitrogen (N)-compounds for the growth and yield of legumes nodulated by Rhizobium that grow in soils with a low N content. Thus, a concrete understanding of the mechanisms underlying ureide biogenesis and catabolism in legumes may help agrobiologists to achieve greater agricultural discoveries. In this review we focus on the transmembranal and transorganellar symplastic and apoplastic movement of N-precursors within the nodules, as well as on the occurrence, localization and properties of enzymes and genes involved in the biogenesis and catabolism of ureides. The synthesis and transport of ureides are not unique events in Rhizobium-nodulated N2-fixing legumes. Thus, a brief description of the synthesis and catabolism of ureides in non-legumes was included for comparison. The establishment of the symbiosis, nodule organogenesis and the plant's control of nodule number, synthesis and translocation of ureides via feed-back inhibition mechanisms are also reviewed., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Millions of small scale legume farmers lack access to rhizobia bacterial inoculants that improve crop protein and yield, and minimize fossil-fuel based nitrogen fertilizers, through biological nitrogen fixation (BNF). BNF converts atmospheric nitrogen gas into ammonia, required to synthesize chlorophyll and amino acids. BNF is catalyzed by rhizobia that inhabit nodule organs in legume roots (e.g., soybean, cowpea, chickpeas, lentil, fava, peas, beans). Rhizobia inoculant access in remote communities is limited by centralized facilities to grow bacteria, inadequate transportation networks and refrigeration. Recently, we proposed that rhizobia inoculants can be diffused by farmers themselves, simply, by crushing nodules onto seeds, and demonstrated its efficacy under field conditions. A concern was whether nodules remain viable between growing seasons. Here we provide preliminary evidence that bean nodules, dried and stored at room temperature after 6 months, retain nodulation potential. We discuss: (1) the feasibility, constraints and risks of nodule crushing; (2) scaling up strategies; (3) entrepreneurship that benefits women farmers (e.g., noduleselling microenterprises); and (4) empowering farmers to directly select, evolve and indigenize rhizobia for the first time. Finally, we prioritize research questions and encourage the formation of a global participatory research network, with the goal of decentralizing and democratizing rhizobia inoculants. [ABSTRACT FROM AUTHOR]

The curriculum for undergraduate Agricultural Engineering does not usually include much laboratory practice. In order to address this, here we propose the design of a practical class on legume-rhizobia symbiosis, using an interdisciplinary approach that takes elements from botany, agriculture, and microbiology. The students perform an assay to stimulate nodulation in chickpea seedlings through inoculation with Mesorhizobium strains recommended as commercial inoculants, and evaluate improvement in plant growth as a result of nitrogen fixation. The experiment could serve as a springboard to discuss the importance of plant-bacteria interactions in the cultivation of a regionally relevant crop. It may also encourage the use of abilities of different kinds, including the appropriate manipulation of lab materials, the active engagement of scientific thought, the application of knowledge to specific real world scenarios and collaborative teamwork, all of which will be of paramount importance throughout the students' careers. The class can be easily adapted for other degrees connected to environmental microbiology, as well as for other symbiotic pairs that may include significant crops from other regions around the world. [ABSTRACT FROM AUTHOR]

Legume crops establish symbiosis with nitrogen-fixing rhizobia for biological nitrogen fixation (BNF), a process that provides a prominent natural nitrogen source in agroecosystems; and efficient nodulation and nitrogen fixation processes require a large amount of phosphorus (P). Here, a role of GmPAP4, a nodule-localized purple acid phosphatase, in BNF and seed yield was functionally characterized in whole transgenic soybean (Glycine max) plants under a P-limited condition. GmPAP4 was specifically expressed in the infection zones of soybean nodules and its expression was greatly induced in low P stress. Altered expression of GmPAP4 significantly affected soybean nodulation, BNF, and yield under the P-deficient condition. Nodule number, nodule fresh weight, nodule nitrogenase, APase activities, and nodule total P content were significantly increased in GmPAP4 overexpression (OE) lines. Structural characteristics revealed by toluidine blue staining showed that overexpression of GmPAP4 resulted in a larger infection area than wild-type (WT) control. Moreover, the plant biomass and N and P content of shoot and root in GmPAP4 OE lines were also greatly improved, resulting in increased soybean yield in the P-deficient condition. Taken together, our results demonstrated that GmPAP4, a purple acid phosphatase, increased P utilization efficiency in nodules under a P-deficient condition and, subsequently, enhanced symbiotic BNF and seed yield of soybean. [ABSTRACT FROM AUTHOR]

This editorial published in the journal Frontiers in Agronomy discusses the importance of maximizing nitrogen fixation in legumes for sustainable agriculture intensification. It addresses the challenges faced by agricultural systems in providing food for a growing population while maintaining soil and water quality and mitigating climate change. The editorial explores the potential of biological nitrogen fixation in legumes as an alternative strategy to increase nitrogen availability and reduce the need for synthetic fertilizers. It also presents various research topics related to the symbiotic relationship between legumes and rhizobia and its application in sustainable agricultural systems. The article concludes by emphasizing the importance of inoculating legumes with efficient rhizobia strains in areas where inefficient strains are likely to be present. The authors also discuss the use of elite microorganisms to improve the growth of alfalfa crops in acid soils and suggest that more research is needed to fully understand plant-microbe interactions and develop effective strategies for replacing synthetic nitrogen fertilizers. [Extracted from the article]

Millions of small scale legume farmers lack access to rhizobia bacterial inoculants that improve crop protein and yield, and minimize fossil-fuel based nitrogen fertilizers, through biological nitrogen fixation (BNF). BNF converts atmospheric nitrogen gas into ammonia, required to synthesize chlorophyll and amino acids. BNF is catalyzed by rhizobia that inhabit nodule organs in legume roots (e.g., soybean, cowpea, chickpeas, lentil, fava, peas, beans). Rhizobia inoculant access in remote communities is limited by centralized facilities to grow bacteria, inadequate transportation networks and refrigeration. Recently, we proposed that rhizobia inoculants can be diffused by farmers themselves, simply, by crushing nodules onto seeds, and demonstrated its efficacy under field conditions. A concern was whether nodules remain viable between growing seasons. Here we provide preliminary evidence that bean nodules, dried and stored at room temperature after 6 months, retain nodulation potential. We discuss: (1) the feasibility, constraints and risks of nodule crushing; (2) scaling up strategies; (3) entrepreneurship that benefits women farmers (e.g., nodule-selling microenterprises); and (4) empowering farmers to directly select, evolve and indigenize rhizobia for the first time. Finally, we prioritize research questions and encourage the formation of a global participatory research network, with the goal of decentralizing and democratizing rhizobia inoculants.

Plants perceive N-acetyl-d-glucosamine-containing oligosaccharides that play a role in the interaction with bacteria and fungi, through cell-surface receptors containing a tight bundle of three LysM domains in their extracellular region. However, the identification of LysM domains of receptor-like kinases (RLK)/receptor-like proteins (RLP) using sequence based methods has led to some ambiguity, as some proteins have been annotated with only one or two LysM domains. This missing annotation was likely produced by the failure of the LysM hidden Markov model (HMM) from the Pfam database to correctly identify some LysM domains in proteins of plant origin. In this work, we provide improved HMMs for LysM domain detection in plants, that were built from the structural alignment of manually curated LysM domain structures from the Protein Data Bank and AlphaFold Protein Structure Database. Furthermore, we evaluated different sets of ligand-specific HMMs that were able to correctly classify a limited set of fully characterised RLK/Ps by their ligand specificity. In contrast, the phylogenetic analysis of the extracellular region of RLK/Ps, or of their individual LysM domains, was unable to discriminate these proteins by their ligand specificity. The HMMs reported here will allow a more sensitive detection of plant proteins containing LysM domains and help improve their characterisation. [ABSTRACT FROM AUTHOR]

Abstract Background Bradyrhizobium japonicum strain SEMIA 5079 (= CPAC 15) is a nitrogen-fixing symbiont of soybean broadly used in commercial inoculants in Brazil. Its genome has about 50% of hypothetical (HP) protein-coding genes, many in the symbiosis island, raising questions about their putative role on the biological nitrogen fixation (BNF) process. This study aimed to infer functional roles to 15 HP genes localized in the symbiosis island of SEMIA 5079, and to analyze their expression in the presence of a nod-gene inducer. Results A workflow of bioinformatics tools/databases was established and allowed the functional annotation of the HP genes. Most were enzymes, including transferases in the biosynthetic pathways of cobalamin, amino acids and secondary metabolites that may help in saprophytic ability and stress tolerance, and hydrolases, that may be important for competitiveness, plant infection, and stress tolerance. Putative roles for other enzymes and transporters identified are discussed. Some HP proteins were specific to the genus Bradyrhizobium, others to specific host legumes, and the analysis of orthologues helped to predict roles in BNF. Conclusions All 15 HP genes were induced by genistein and high induction was confirmed in five of them, suggesting major roles in the BNF process.

Simple Summary: Partially efficient and inefficient rhizobia strains can decrease biological nitrogen fixation in legumes, especially when not inoculated. This work shows the high nodulation competitiveness in Trifolium repens of two strains with intermediate nitrogen fixation capacity. The genome sequence of one of them resulted in the first identification of Rhizobium redzepovicii as a T. repens symbiont. The analysis of symbiotic genes of different Rhizobium sp. capable of nodulating T. repens evidences the horizontal transfer of genes from efficient to inefficient strains, leading to a strain with intermediate efficiency. For the first time, sequencing, and analysis of the genome of the rhizobium used as clover inoculant in Uruguay confirmed that it is a strain belonging to R. leguminosarum phylogenetically close to bv. viciae. The search for hrrP-like genes in different Rhizobium species shows their presence in the chromosome and plasmids. This is interesting because hrrP encodes a metallopeptidase of the M16A family whose activity is involved in symbiotic efficiency. Our results highlight the need for farmers to make an effort in seed inoculation to improve clover yields and mitigate the effect of parasitic and low-efficiency strains present in soils. In Uruguayan soils, populations of native and naturalized rhizobia nodulate white clover. These populations include efficient rhizobia but also parasitic strains, which compete for nodule occupancy and hinder optimal nitrogen fixation by the grassland. Nodulation competitiveness assays using gusA-tagged strains proved a high nodule occupancy by the inoculant strain U204, but this was lower than the strains with intermediate efficiencies, U268 and U1116. Clover biomass production only decreased when the parasitic strain UP3 was in a 99:1 ratio with U204, but not when UP3 was at equal or lower numbers than U204. Based on phylogenetic analyses, strains with different efficiencies did not cluster together, and U1116 grouped with the parasitic strains. Our results suggest symbiotic gene transfer from an effective strain to U1116, thereby improving its symbiotic efficiency. Genome sequencing of U268 and U204 strains allowed us to assign them to species Rhizobium redzepovicii, the first report of this species nodulating clover, and Rhizobium leguminosarun, respectively. We also report the presence of hrrP- and sapA-like genes in the genomes of WSM597, U204, and U268 strains, which are related to symbiotic efficiency in rhizobia. Interestingly, we report here chromosomally located hrrP-like genes. [ABSTRACT FROM AUTHOR]

Aims To isolate and characterize non-rhizobial nodule-associated bacteria (NAB) from cowpea root-nodules regarding their performance of plant-growth-promoting mechanisms and their ability to enhance cowpea growth and symbiosis when co-inoculated with bradyrhizobia. Methods and Results Sixteen NAB were isolated, identified, and in vitro evaluated for plant growth promotion traits. The ability to promote cowpea growth was analyzed when co-inoculated with Bradyrhizobium pachyrhizi BR 3262 in sterile and non-sterile substrates. The 16S rRNA gene sequences analysis revealed that NAB belonged to the genera Chryseobacterium (4), Bacillus (3), Microbacterium (3), Agrobacterium (1), Escherichia (1), Delftia (1), Pelomonas (1), Sphingomonas (1), and Staphylococcus (1). All strains produced different amounts of auxin siderophores and formed biofilms. Twelve out of the 16 strains carried the nifH , a gene associated with nitrogen fixation. Co-inoculation of NAB (ESA 424 and ESA 29) with Bradyrhizobium pachyrhizi BR 3262 significantly promoted cowpea growth, especially after simultaneous inoculation with the three strains. Conclusions NAB are efficient cowpea growth promoters and can improve the efficiency of the symbiosis between cowpea and the N2-fixing microsymbiont B. pachyrhizi BR 3262, mainly under a specific triple microbial association. [ABSTRACT FROM AUTHOR]

Background: Bradyrhizobium japonicum strain SEMIA 5079 (= CPAC 15) is a nitrogen-fixing symbiont of soybean broadly used in commercial inoculants in Brazil. Its genome has about 50% of hypothetical (HP) protein-coding genes, many in the symbiosis island, raising questions about their putative role on the biological nitrogen fixation (BNF) process. This study aimed to infer functional roles to 15 HP genes localized in the symbiosis island of SEMIA 5079, and to analyze their expression in the presence of a nod-gene inducer. Results: A workflow of bioinformatics tools/databases was established and allowed the functional annotation of the HP genes. Most were enzymes, including transferases in the biosynthetic pathways of cobalamin, amino acids and secondary metabolites that may help in saprophytic ability and stress tolerance, and hydrolases, that may be important for competitiveness, plant infection, and stress tolerance. Putative roles for other enzymes and transporters identified are discussed. Some HP proteins were specific to the genus Bradyrhizobium, others to specific host legumes, and the analysis of orthologues helped to predict roles in BNF. Conclusions: All 15 HP genes were induced by genistein and high induction was confirmed in five of them, suggesting major roles in the BNF process. [ABSTRACT FROM AUTHOR]

Biological nitrogen fixation by legume-rhizobacterial symbiosis in temperate grasslands is an important source of soil nitrogen. The aim of the present study was to characterize the dependence of different accessions of T. fragiferum, a rare crop wild relative legume species, from their native rhizobia as well as additional nitrogen fertilization in controlled conditions. Asymbiotically cultivated, mineral-fertilized T. fragiferum plants gradually showed signs of nitrogen deficiency, appearing as a decrease in leaf chlorophyll concentration, leaf senescence, and a decrease in growth rate. The addition of nitrogen, and the inoculation with native rhizobia, or both treatments significantly prevented the onset of these symptoms, leading to both increase in plant shoot biomass as well as an increase in tissue concentration of N. The actual degree of each type of response was genotype-specific. Accessions showed a relatively similar degree of dependence on nitrogen (70–95% increase in shoot dry mass) but the increase in shoot dry mass by inoculation with native rhizobia ranged from 27 to 85%. In general, there was no correlation between growth stimulation and an increase in tissue N concentration by the treatments. The addition of N or rhizobial inoculant affected mineral nutrition at the level of both macronutrient and micronutrient concentration in different plant parts. In conclusion, native rhizobial strains associated with geographically isolated accessions of T. fragiferum at the northern range of distribution of the species represent a valuable resource for further studies aimed at the identification of salinity-tolerant N2-fixing bacteria for the needs of sustainable agriculture, as well as in a view of understanding ecosystem functioning at the level of plant-microorganism interactions. [ABSTRACT FROM AUTHOR]

Biological nitrogen fixation (BNF) is what makes soybean economically viable. However, the launch of cultivars, with a high-yield potential and abiotic factors that reduce BNF, raised doubts about the need for nitrogen (N) fertilizers for soybeans. The objective of the research was to evaluate the effects of nitrogen supply, in two application times in furrow starter fertilization and top-dressing. The experimental design was in the 3 × 6 factorial scheme. The treatments consisted of three rates of N at in furrow starter fertilization (0, 10, and 20 kg ha−1 of N), and six rates of N in top-dressing, applied at the soybean R1 stage (0, 30, 60, 90, 120, and 150 kg ha−1 of N). The highest rate in furrow starter fertilization of 20 kg ha−1 of N resulted in the increase in the leaf area index (LAI) and specific leaf area (SLA), which are attributes that maximize the absorption of light energy, thus being able to positively influence yield. However, the BNF demonstrated that the strains of the selected Bradyrhizobium are efficient and capable of supplying the required N because the supplementation of nitrogen in furrow and/or top-dressing did not result in significant differences in the yield in relation to inoculated soybean. [ABSTRACT FROM AUTHOR]

1. Soil microbial community coalescence, where entire microbial communities mix and interact under new conditions, is a widespread phenomenon whose applicability for targeted root microbiome assembly has not been studied. Whether soil mixing can lead to predictable outcomes for community assembly and functioning of specific functional groups, for example, N2-fixing rhizobia, remains unknown. 2. Using a legume shrub adapted to nutrient-poor soils, we tested the effects of community coalescence on plant nutrition and growth, and its influence on the rhizobial root nodule symbiosis. We grew seedlings of rooibos [Aspalathus linearis (Burm.f.) Dahlg.] in individual rhizosphere soils collected from cultivated and wild rooibos plants and their mixtures, and included a fertilization treatment. Portions of the taxonomic gyrB and the symbiotic nodA gene makers were sequenced to characterize rhizobial communities present in rooibos root nodules under the different soil conditions. 3. Overall, community coalescence by soil mixing had positive effects on plant nutrition and growth, and interacted with fertilizer addition to concurrently change rhizobial taxonomic evenness and promote higher relative N2 fixation. We identified particular rhizobia preferentially associated with rooibos plants that showed higher N fractions from N2 fixation, raised in mixed fertilized soils. 4. These findings indicate that soil bacterial community coalescence and fertilization can have synergistic effects on plant performance, while promoting the assembly of alternative symbiotic rhizobial communities that provide improved nutritional benefits to host plants. 5. Synthesis and applications. The combination of soil mixing and fertilizer addition may be an important, but hitherto overlooked measure to improve the functioning of rhizobium symbioses in legume crops. Microbial community coalescence should gain recognition as a potentially effective mechanism to improve the functioning of plant microbiomes. [ABSTRACT FROM AUTHOR]

High abundance of legumes in drylands suggests that symbiotic nitrogen fixation provides an advantage in water-limited environments. However, the interactive effect of nitrogen availability and water scarcity on the nitrogen fixation strategies of dryland legumes remain largely unexplained. We conducted two experiments to test the effects of nitrogen availability and drought on symbiotic nitrogen fixation in two drought-adapted Acacia tree species. Seedlings were grown under deficient and sufficient levels of nitrogen and with and without an imposed drought to test the effect of resource availability on nitrogen fixation and plant growth. We found that seedlings that grew in extreme deficiency of nitrogen reached a similar biomass as seedling that grew with a sufficient supply of nitrogen, showing a high nitrogen-use efficiency. Nitrogen fixation was strongly downregulated (reduction of 90% in biomass allocation to nodules) when plants received sufficient nitrogen supply. Under nitrogen deficiency, drought had a slight negative effect on nodule biomass and total biomass. Under sufficient nitrogen, drought reduced nitrogen availability enough to induce an increase in symbiotic nitrogen fixation in the risk-averse A. raddiana but not in the risk-taking A. tortilis. We conclude that strong regulation of nitrogen fixation together with low nitrogen demand, and flexible strategies of carbon and nitrogen allocation, increase the chance of legume tree survival and establishment in dry and unpredictable environments. • The combination of water and nutrient limitation is globally common. • We analyzed growth and symbiotic nitrogen fixation in woody legumes under low water and nitrogen availabilities. • Strong downregulation strategy of N fixation was found when nitrogen was sufficient. • Low nitrogen demand allowed similar seedling growth with sufficient or deficient availability of nitrogen. • Regulation of nitrogen supply and demand sustained growth and survival under the combined stress of nitrogen and water. [ABSTRACT FROM AUTHOR]

Biological nitrogen fixation by Rhizobium -legume symbioses represents an environmentally friendly and inexpensive alternative to the use of chemical nitrogen fertilizers in legume crops. Rhizobial inoculants, applied frequently as biofertilizers, play an important role in sustainable agriculture. However, inoculants often fail to compete for nodule occupancy against native rhizobia with inferior nitrogen-fixing abilities, resulting in low yields. Strains with excellent performance under controlled conditions are typically selected as inoculants, but the rates of nodule occupancy compared to native strains are rarely investigated. Lack of persistence in the field after agricultural cycles, usually due to the transfer of symbiotic genes from the inoculant strain to naturalized populations, also limits the suitability of commercial inoculants. When rhizobial inoculants are based on native strains with a high nitrogen fixation ability, they often have superior performance in the field due to their genetic adaptations to the local environment. Therefore, knowledge from laboratory studies assessing competition and understanding how diverse strains of rhizobia behave, together with assays done under field conditions, may allow us to exploit the effectiveness of native populations selected as elite strains and to breed specific host cultivar-rhizobial strain combinations. Here, we review current knowledge at the molecular level on competition for nodulation and the advances in molecular tools for assessing competitiveness. We then describe ongoing approaches for inoculant development based on native strains and emphasize future perspectives and applications using a multidisciplinary approach to ensure optimal performance of both symbiotic partners. [ABSTRACT FROM AUTHOR]

The goal of this study was to evaluate the efficiency of Bradyrhizobium brasilense strains native to the soil of the semiarid region in northeastern Brazil in symbiosis with soybeans in two contrasting soils in southwestern Piauí. A pot experiment was conducted using a randomized block design in a 10 × 2 factorial scheme. There were 10 nitrogen (N) sources: six native Bradyrhizobium strains [UFLA06-13, UFLA06-15, UFLA06-19, UFLA06-21, UFLA06-22 (B. brasilense), and UFLA06-24 (Bradyrhizobium sp.)], two controls with strain SEMIA 5019 (B. elkanii) and a commercial inoculant [SEMIA 5079 (B. japonicum) + SEMIA 5080 (B. diazoefficiens)] recommended for soybeans, and two controls without the application of an inoculant (one with and the other without the application of mineral N). The second experimental factor corresponded to the use of two soils (Oxisol and Quartzarenic Neosol). All strains showed increased nodulation and shoot nitrogen content in soybean plants in both soils. Most strains promoted higher nitrogen fixation when inoculated in the Oxisol. UFLA06-19, UFLA06-22, and UFLA06-24 were efficient in nitrogen accumulation in the shoots of soybeans in the Oxisol. This is the first report regarding the efficiency of B. brasilense strains in symbiosis with soybeans under different soil conditions. [ABSTRACT FROM AUTHOR]

ABSTRACT The present study aimed to evaluate the effect of inoculation with nitrogen-fixing bacteria (NFB) and arbuscular mycorrhizal fungi (AMF) on the development of Tachigali vulgaris seedlings under nursery conditions. The seedlings were produced in 1 kg bags on a substrate of sand and vermiculite (1:1), following a completely randomized experimental design in a 3 (NFB) x 2 (with and without AMF) factorial scheme with 3 additional control treatments consisting of: seedlings inoculated only with AMF (mycorrhizal control), non-inoculated seedlings fertilized with N (nitrogenized control) and without N (absolute control). The following variables were evaluated: height, stem diameter (SD), shoot dry mass (SDM), root dry mass (RDM) and nodule dry mass (NDM), P accumulation in the shoot, and root mycorrhizal colonization (RMC). Efficiency and effectiveness were calculated to evaluate the plant response to double inoculation. The treatments showed a significant effect for all variables, except for mycorrhizal colonization, nodule number, and dry mass, with the nitrogen treatment having the highest growth values. Plants submitted to double inoculation showed a higher accumulation of dry matter, height and SD, reaching a 124% higher RDM regarding the absolute control, 90% more SDM, and 207% more NDM regarding the seedlings inoculated only with rhizobia. The positive effect of double inoculation occurred regardless of the strain used. The results indicate that the joint inoculation of NFB and AMF was beneficial for the species, promoting its growth.

Seed inoculation with Azospirillum brasilense might be an interesting alternative to reduce the nitrogen demand applied to plants and to enhance the effect of these fertilizers. This study aimed to evaluate the components of maize crops and its response to inoculation with A. brasilense associated with sowing and top cover nitrogen fertilization. A completely randomized block experimental design was used, and the tests were carried out in two crop years. The treatments in the first crop year were: BF + CF: {nitrogen base fertilization (BF) + cover fertilization (CF)}; BF + BI: {BF + A. brasilense bacterium inoculation (BI)}; BF + BI + CF; BI; BI + CF and NFI: {no fertilization or inoculation}. The same treatments were tested in the second crop year, and the CF was added. The tests were carried out in the field, in the municipality of Abelardo Luz - SC - Brazil. The climate is humid mesothermal with hot summers and cold winters (Cfa). The following parameters were evaluated: plant height, leaf and grain nitrogen, yield, thousand-grains weight, and grain moisture. The inoculation of A. brasilense associated with cover fertilization could be an alternative to reduce the demand for N, including an increase in productivity. The use of A. brasilense without N fertilization provided an increase in crop yield. [ABSTRACT FROM AUTHOR]

The global contribution of biological nitrogen fixation (BNF) to soybean production ranks Brazil as the leading country. In 2013/2014, a new technology based on soybean co-inoculation with Bradyrhizobium spp. and Azospirillum brasilense strains Ab-V5 and Ab-V6 was released, and here we report a five-year set of extension activities in Paraná state, southern Brazil, to demonstrate the benefits of co-inoculation. In total, 273 technical reference units (TRUs) were installed in two main soybean macro-regions, all showing naturalized populations of soybean bradyrhizobia. In each TRU, side-by-side plots consisting of soybean seeds non-inoculated or co-inoculated with Bradyrhizobium spp. and A. brasilense were installed, and 260 TRUs were evaluated for nodulation and 242 for grain yield. Consistent nodulation increases, of up to 350% were observed in every cropping season, with an average significant (p < 0.01) increase of 35% in the five-year period. Similarly, grain yield increased up to 80% due to co-inoculation, with an average significant (p < 0.01) increase of 8%, corresponding to 273 kg ha−1. The increases were confirmed in the two macro-regions and different levels of grain yield, from low (<3000 kg ha−1) to high (>4000 kg ha−1). In the five-year period, 3299 small farmers were reached (farms of ∼50 ha) and the average profit due to co-inoculation was estimated at US$ 111.5 ha−1 per cropping season. In addition, we discuss environmental benefits associated with the mitigation of greenhouse gases emissions, estimated at 350 kg ha−1 of CO 2 -e, in addition to soil N enrichment. [Display omitted] • Soybean can benefit from co-inoculation with Bradyrhizobium spp. and A, brasilense. • The technology was demonstrated to 3299 farmers in Brazil, for five cropping seasons. • Increases in nodulation, grain yield, and US$ 111.5 ha−1 in profit were confirmed. • Extension activities with farmers are needed for the adoption of microbial inoculants. • Soybean co-inoculation resulted in economic and environmental benefits. [ABSTRACT FROM AUTHOR]

To quantify the BNF contribution to legumes using the 15N natural abundance technique, it is important to know the abundance of 15N of the plants grown entirely dependent on BNF (value 'B'). The aim of the study was to determine the 15N natural abundance of N2 fixed by different Bradyrhizobium strains in symbiosis with one soybean cultivar. Treatments consisted of soybean plants cultivated with and without inoculation with ten Bradyrhizobium strains, in five replicates planted in Leonard jars in a sand/vermiculite mixture. Plants were harvested after 46 days. The 'B' values of the aerial tissue ('Bs') ranged from -2.6 to -3.9‰. There was a tendency for the 'Bs' values of plants inoculated with strains of B. elkanii to be more negative than plants inoculated with other strains. All 'B' values of the whole plant were less than 1 unit of d15N (‰) different from zero, suggesting that the symbioses have little tendency to show significant isotopic fractionation during N2 fixation, but there is considerable depletion in 15N of the N translocated to the shoot tissue. [ABSTRACT FROM AUTHOR]

Abstract There is growing demand to produce both food and renewable energy in a sustainable manner, while avoiding competition between food and energy production. In our study, we investigated the potential of harnessing biogas production into nutrient recycling in an integrated system of organic food production and food processing. We used the case of Agroecological Symbiosis (AES) at Palopuro, which is a combination of three farms, a biogas plant, and a bakery, as a case to explore how biogas production using feedstocks from the farms can be used to improve nutrient cycling, and to calculate how much energy could be produced from the within-system feedstocks. The current system (CS) used in organic farms, and the integrated farm and food processing AES system, were analyzed using Substance Flow analysis. In the AES, annual nitrogen (N) and phosphorus (P) surpluses were projected to be reduced from 95 kg ha−1 to 36 kg ha−1 and from 3.4 kg ha−1 to −0.5 kg ha−1 respectively, compared to the CS. Biogas produced from green manure leys as the major feedstock, produced 2809 MWh a−1. This was 70% more than the energy consumed (1650 MWh a−1) in the systemand thus the AES system turned out to be a net energy producer. Results demonstrated the potential of biogas production to enhance the transition to bioenergy, nutrient recycling, and crop productivity in renewable localized farming and food systems. Graphical abstract Unlabelled Image Highlights • Increased food production by enhancing nutrient recycling within the system • Substantial reductions in nitrogen and phosphorus losses • 70% more energy produced than consumed. • Substantial climate change mitigation through reduction in fossil fuel use [ABSTRACT FROM AUTHOR]

Rhizobium tropici CIAT 899 is a strain known by its ability to nodulate a broad range of legume species, to synthesize a variety of Nod factors, its tolerance of abiotic stresses, and its high capacity to fix atmospheric N2, especially in symbiosis with common bean (Phaseolus vulgaris L.). Genes putatively related to the synthesis of indole acetic acid (IAA) have been found in the symbiotic plasmid of CIAT 899, in the vicinity of the regulatory nodulation gene nodD5, and, in this study, we obtained mutants for two of these genes, y4wF and tidC (R. tropiciindole-3-pyruvic acid decarboxylase), and investigated their expression in the absence and presence of tryptophan (TRP) and apigenin (API). In general, mutations of both genes increased exopolysaccharide (EPS) synthesis and did not affect swimming or surface motility; mutations also delayed nodule formation, but increased competitiveness. We found that the indole-3-acetamide (IAM) pathway was active in CIAT 899 and not affected by the mutations, and—noteworthy—that API was required to activate the tryptamine (TAM) and the indol-3-pyruvic acid (IPyA) pathways in all strains, particularly in the mutants. High up-regulation of y4wF and tidC genes was observed in both the wild-type and the mutant strains in the presence of API. The results obtained revealed an intriguing relationship between IAA metabolism and nod-gene-inducing activity in R. tropici CIAT 899. We discuss the IAA pathways, and, based on our results, we attribute functions to the y4wF and tidC genes of R. tropici. [ABSTRACT FROM AUTHOR]

Background and aims: To evaluate the influence of soil type on the symbiosis between Mimosa spp. and rhizobia.Methods: A greenhouse experiment was carried out with trap plants using seeds of six species of Mimosa and soils from three different locations in central Brazil: Posse, Brasília and Cavalcante. Plant dry biomass and number of nodules were measured after four months. Symbiotic bacteria were isolated from nodules and their molecular identification was performed. Three housekeeping genes (16S rRNA, recA and gyrB) plus the nodC and nifH symbiotic genes were used to determine the identity of the symbionts and to reconstruct the phylogenetic relationships among the isolated nitrogen-fixing bacteria.Results: Rhizobia from the Betaproteobacterial genus Paraburkholderia (former Burkholderia) and the Alphaproteobacterial genus Rhizobium were isolated from different species of Mimosa. As in previous studies, the phylogenies of their symbiosis-essential genes, nodC and nifH, were broadly congruent with their core housekeeping genes (16S rRNA, recA and gyrB), which suggests limited or no horizontal gene transfer. Edaphic factors such as pH and fertility influenced the occurrence of these unrelated rhizobial types in the nodules on these Mimosa spp.Conclusions: Mimosa species have the ability to associate with different types of rhizobia (α- and β-proteobacteria), suggesting low specificity between host and bacterium in experimental conditions. Soil factors such as pH, nitrogen and fertility seem to favour the predominance of certain types of rhizobia, thus influencing the establishment of symbiotic relationships. [ABSTRACT FROM AUTHOR]

Lima bean ( Phaseolus lunatus L.) is an important legume species that establishes symbiosis with rhizobia, mainly of the Bradyrhizobium genus. The aim of this study was to evaluate the efficiency of rhizobia of the genus Bradyrhizobium in symbiosis with lima bean, in both Leonard jars and in pots with a Latossolo Amarelo distrófico (Oxisol). In the experiment in Leonard jars, 17 strains isolated from nodules of the three legume subfamilies, Papilionoideae ( Vigna unguiculata, Pterocarpus sp., Macroptilium atropurpureum, Swartzia sp., and Glycine max), Mimosoideae ( Inga sp.), and Caesalpinioideae ( Campsiandra surinamensis) and two uninoculated controls, one with a low concentration (5.25 mg L) and another with a high concentration (52.5 mg L) of mineral nitrogen (N) were evaluated. The six strains that exhibited the highest efficiency in Leonard jars, isolated from nodules of Vigna unguiculata (UFLA 03-144, UFLA 03-84, and UFLA 03-150), Campsiandra surinamensis (INPA 104A), Inga sp. (INPA 54B), and Swartzia sp. (INPA 86A), were compared to two uninoculated controls, one without and another with 300 mg N dm (NHNO) applied to pots with samples of an Oxisol in the presence and absence of liming. In this experiment, liming did not affect nodulation and plant growth; the INPA 54B and INPA 86A strains stood out in terms of shoot dry matter production and provided increases of approximately 48% in shoot N accumulation compared to the native rhizobia populations. Our study is the first to indicate Bradyrhizobium strains isolated from the three legume subfamilies are able to promote lima bean growth via biological nitrogen fixation in soil conditions. [ABSTRACT FROM AUTHOR]

Soybean inoculation with effective rhizobial strains makes unnecessary the use of N-fertilizers in the tropics. A frequently reported problem is the failure of the inoculant strains to overcome the competition imposed by indigenous rhizobial populations. The screening of indigenous rhizobia, already adapted to local conditions, searching for highly effective strains for use as inoculants represents a promising strategy in overcoming inoculation failure. The objective of this study was to isolate and characterize indigenous rhizobia and to identify strains that hold potential to be included in inoculant formulations for soybean production, with both promiscuous and non-promiscuous soybean cultivars, in Mozambican agro-climatic conditions. A total of 105 isolates obtained from nodules of promiscuous soybean grown at 15 sites were screened for N 2 -fixation effectiveness in the greenhouse along with five commercial strains. Eighty-seven isolates confirmed the ability to form effective nodules on soybean and were used for genetic characterization by rep-PCR (BOX) and sequencing of the 16S rRNA gene, and also for symbiotic effectiveness. BOX-PCR fingerprinting revealed remarkable genetic diversity, with 41 clusters formed, considering a similarity level of 65%. The 16S rRNA analysis assigned the isolates to the genera Bradyrhizobium (75%) and Agrobacterium/Rhizobium (25%). Great variability in symbiotic effectiveness was detected among the indigenous rhizobia from Mozambique, with ten isolates performing better than the commercial strain B. diazoefficiens USDA 110, the best reference strain, and 51 isolates with lower performance than all reference strains. Thirteen of the best isolates from the first greenhouse trial were evaluated, along with the five commercial strains, in two promiscuous (TGx 1963-3F and TGx 1835-10E) and one non-promiscuous (BRS 284) soybean cultivars in a second greenhouse trial. In general the promiscous soybeans responded better to inoculation. The 13 isolates were also characterized for tolerance to acidity and alkalinity (pH 3.5 and 9.0, respectively), salinity (0.1, 0.3 and 0.5 mol L −1 of NaCl) and high temperatures (35, 40 and 45 °C) in vitro . Five isolates, three (Moz 4, Moz 19 and Moz 22) belonging to the superclade B. elkanii and two (Moz 27 and Moz 61) assigned to the superclade B. japonicum, consistently showed high symbiotic effectiveness, suggesting that the inoculation with indigenous rhizobia adapted to local conditions represents a possible strategy for increasing soybean yields in Mozambique. Phylogenetic position of the five elite isolates was confirmed by the MLSA with four protein-coding housekeeping genes, dnaK , glnII , gyrB and recA. [ABSTRACT FROM AUTHOR]

Bradyrhizobium comprises most tropical symbiotic nitrogen-fixing strains, but the correlation between symbiotic and core genes with host specificity is still unclear. In this study, the phylogenies of the nodY/K and nifH genes of 45 Bradyrhizobium strains isolated from legumes of economic and environmental importance in Brazil ( Arachis hypogaea , Acacia auriculiformis , Glycine max , Lespedeza striata , Lupinus albus , Stylosanthes sp. and Vigna unguiculata ) were compared to 16S rRNA gene phylogeny and genetic diversity by rep-PCR. In the 16S rRNA tree, strains were distributed into two superclades— B. japonicum and B. elkanii —with several strains being very similar within each clade. The rep-PCR analysis also revealed high intra-species diversity. Clustering of strains in the nodY/K and nifH trees was identical: 39 strains isolated from soybean grouped with Bradyrhizobium type species symbionts of soybean, whereas five others occupied isolated positions. Only one strain isolated from Stylosanthes sp. showed similar nodY/K and nifH sequences to soybean strains, and it also nodulated soybean. Twenty-one representative strains of the 16S rRNA phylogram were selected and taxonomically classified using a concatenated glnII - recA phylogeny; nodC sequences were also compared and revealed the same clusters as observed in the nodY/K and nifH phylograms. The analyses of symbiotic genes indicated that a large group of strains from the B. elkanii superclade comprised the novel symbiovar sojae, whereas for another group, including B. pachyrhizi , the symbiovar pachyrhizi could be proposed. Other potential new symbiovars were also detected. The co-evolution hypotheses is discussed and it is suggested that nodY/K analysis would be useful for investigating the symbiotic diversity of the genus Bradyrhizobium . [ABSTRACT FROM AUTHOR]

Aims: Anthropogenic nitrogen deposition can provide legumes with a cheap source of nitrogen relative to symbiotic nitrogen fixation, leading to the potential breakdown of this critical symbiosis. Here, the effects of nitrogen deposition were tested on a native symbiosis between legumes and rhizobia. Methods: Deposition rates, soil nitrogen concentration, and plant nitrogen isotopic composition were quantified along a predicted deposition gradient in California. Acmispon strigosus seedlings were exposed to fertilization spanning nitrogen concentrations observed in the plant's California range. Both wild and experimental plants from pristine and nitrogen polluted sites were tested using rhizobial strains that varied in nitrogen fixation. Results: Deposition intensity was tightly correlated with nitrogen concentration in soils. The growth benefits of rhizobial nodulation were dramatically reduced by even modest levels of mineral nitrogen, and all Acmispon lines failed to form root nodules at high nitrogen concentrations. Conclusions: Our dataset suggests that anthropogenic deposition has greatly increased soil nitrogen concentrations in Southern California leading to significantly reduced benefits of rhizobial symbiosis. If nitrogen deposition increases continue, plant host mortality and a total collapse of the symbiosis could result. [ABSTRACT FROM AUTHOR]