Showing papers on "Nitrogen fixation published in 2020"

Effectiveness of nitrogen fixation in rhizobia.

Abstract: Biological nitrogen fixation in rhizobia occurs primarily in root or stem nodules and is induced by the bacteria present in legume plants. This symbiotic process has fascinated researchers for over a century, and the positive effects of legumes on soils and their food and feed value have been recognized for thousands of years. Symbiotic nitrogen fixation uses solar energy to reduce the inert N2 gas to ammonia at normal temperature and pressure, and is thus today, especially, important for sustainable food production. Increased productivity through improved effectiveness of the process is seen as a major research and development goal. The interaction between rhizobia and their legume hosts has thus been dissected at agronomic, plant physiological, microbiological and molecular levels to produce ample information about processes involved, but identification of major bottlenecks regarding efficiency of nitrogen fixation has proven to be complex. We review processes and results that contributed to the current understanding of this fascinating system, with focus on effectiveness of nitrogen fixation in rhizobia.

Current Progress in Nitrogen Fixing Plants and Microbiome Research.

Abstract: In agroecosystems, nitrogen is one of the major nutrients limiting plant growth. To meet the increased nitrogen demand in agriculture, synthetic fertilizers have been used extensively in the latter part of the twentieth century, which have led to environmental challenges such as nitrate pollution. Biological nitrogen fixation (BNF) in plants is an essential mechanism for sustainable agricultural production and healthy ecosystem functioning. BNF by legumes and associative, endosymbiotic, and endophytic nitrogen fixation in non-legumes play major roles in reducing the use of synthetic nitrogen fertilizer in agriculture, increased plant nutrient content, and soil health reclamation. This review discusses the process of nitrogen-fixation in plants, nodule formation, the genes involved in plant-rhizobia interaction, and nitrogen-fixing legume and non-legume plants. This review also elaborates on current research efforts involved in transferring nitrogen-fixing mechanisms from legumes to non-legumes, especially to economically important crops such as rice, maize, and wheat at the molecular level and relevant other techniques involving the manipulation of soil microbiome for plant benefits in the non-legume root environment.

Control of nitrogen fixation in bacteria that associate with cereals.

Abstract: Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous nitrogenase production confers an energy burden. Here, we engineer inducible nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.

Beneficial bacteria activate nutrients and promote wheat growth under conditions of reduced fertilizer application.

Abstract: Excessive application of chemical fertilizer has exerted a great threat to soil quality and the environment. The inoculation of plants with plant-growth-promoting rhizobacteria (PGPR) has emerged as a great prospect for ecosystem recovery. The aim of this work to isolate PGPRs and highlights the effect of bacterial inoculants on available N/P/K content in soil and on the growth of wheat under conditions of reduced fertilizer application. Thirty-nine PGPRs were isolated and tested for their growth-promoting potential. Thirteen isolates had nitrogen fixation ability, of which N9 (Azotobacter chroococcum) had the highest acetylene reduction activity of 156.26 nmol/gh. Eleven isolates had efficient phosphate solubilizing ability, of which P5 (Klebsiella variicola) released the most available phosphorus in liquid medium (231.68 mg/L). Fifteen isolates had efficient potassium solubilizing ability, of which K13 (Rhizobium larrymoorei) released the most available potassium in liquid medium (224.66 mg/L). In culture medium supplemented with tryptophan, P9 (Klebsiella pneumoniae) produced the greatest amount of IAA. Inoculation with the bacterial combination K14 + 176 + P9 + N8 + P5 increased the alkali-hydrolysed nitrogen, available phosphorus and available potassium in the soil by 49.46, 99.51 and 19.38%, respectively, and enhanced the N, P, and K content of wheat by 97.7, 96.4 and 42.1%, respectively. Moreover, reducing fertilizer application by 25% did not decrease the available nitrogen, phosphorus, and potassium in the soil and N/P/K content, plant height, and dry weight of wheat. The bacterial combination K14 + 176 + P9 + N8 + P5 is superior candidates for biofertilizers that may reduce chemical fertilizer application without influencing the normal growth of wheat.

Nitrogen Fixation with Water Vapor by Nonequilibrium Plasma: toward Sustainable Ammonia Production

Abstract: Ammonia is a crucial nutrient used for plant growth and as a building block in the pharmaceutical and chemical industry, produced via nitrogen fixation of the ubiquitous atmospheric N2. Current ind...

Biological nitrogen fixation by alternative nitrogenases in terrestrial ecosystems: a review

Abstract: Biological nitrogen fixation (BNF), a key reaction of the nitrogen cycle, is catalyzed by the enzyme nitrogenase. The best studied isoform of this metalloenzyme requires molybdenum (Mo) at its active center to reduce atmospheric dinitrogen (N2) into bioavailable ammonium. The Mo-dependent nitrogenase is found in all diazotrophs and is the only nitrogenase reported in diazotrophs that form N2-fixing symbioses with higher plants. In addition to the canonical Mo nitrogenase, two alternative nitrogenases, which use either vanadium (V) or iron (Fe) instead of Mo are known to fix nitrogen. They have been identified in ecologically important groups including free-living bacteria in soils and freshwaters and as symbionts of certain cryptogamic covers. Despite the discovery of these alternative isoforms more than 40 years ago, BNF is still believed to primarily rely on Mo. Here, we review existing studies on alternative nitrogenases in terrestrial settings, spanning inland forests to coastal ecosystems. These studies show frequent Mo limitation of BNF, ubiquitous distribution of alternative nitrogenase genes and significant contributions of alternative nitrogenases to N2 fixation in ecosystems ranging from the tropics to the subarctic. The effect of temperature on nitrogenase isoform activity and regulation is also discussed. We present recently developed methods for measuring alternative nitrogenase activity in the field and discuss the associated analytical challenges. Finally, we discuss how the enzymatic diversity of nitrogenase forces a re-examination of existing knowledge gaps and our understanding of BNF in nature.

Diversity of nitrogen-fixing rhizobacteria associated with sugarcane: A comprehensive study of plant-microbe interactions for growth enhancement in Saccharum spp

Abstract: Nitrogen is an essential element for sugarcane growth and development and is generally applied in the form of urea often much more than at recommended rates, causing serious soil degradation, particularly soil acidification, as well as groundwater and air pollution. In spite of the importance of nitrogen for plant growth, fewer reports are available to understand the application and biological role of N2 fixing bacteria to improve N2 nutrition in the sugarcane plant. In this study, a total of 350 different bacterial strains were isolated from rhizospheric soil samples of the sugarcane plants. Out of these, 22 isolates were selected based on plant growth promotion traits, biocontrol, and nitrogenase activity. The presence and activity of the nifH gene and the ability of nitrogen-fixation proved that all 22 selected strains have the ability to fix nitrogen. These strains were used to perform 16S rRNA and rpoB genes for their identification. The resulted amplicons were sequenced and phylogenetic analysis was constructed. Among the screened strains for nitrogen fixation, CY5 (Bacillus megaterium) and CA1 (Bacillus mycoides) were the most prominent. These two strains were examined for functional diversity using Biolog phenotyping, which confirmed the consumption of diverse carbon and nitrogen sources and tolerance to low pH and osmotic stress. The inoculated bacterial strains colonized the sugarcane rhizosphere successfully and were mostly located in root and leaf. The expression of the nifH gene in both sugarcane varieties (GT11 and GXB9) inoculated with CY5 and CA1 was confirmed. The gene expression studies showed enhanced expression of genes of various enzymes such as catalase, phenylalanine-ammonia-lyase, superoxide dismutase, chitinase and glucanase in bacterial-inoculated sugarcane plants. The results showed that a substantial number of Bacillus isolates have N-fixation and biocontrol property against two sugarcane pathogens Sporisorium scitamineum and Ceratocystis paradoxa. The increased activity of genes controlling free radical metabolism may at least in part accounts for the increased tolerance to pathogens. Nitrogen-fixation was confirmed in sugarcane inoculated with B. megaterium and B. mycoides strains using N-balance and 15N2 isotope dilution in different plant parts of sugarcane. This is the first report of Bacillus mycoides as a nitrogen-fixing rhizobacterium in sugarcane.

Effects of coinoculation of Rhizobium with plant growth promoting rhizobacteria on the nitrogen fixation and nutrient uptake of Trifolium repens in low phosphorus soil

Abstract: Low soil phosphorus (P) availability is a notable limitation for legume nodulation, nitrogen fixation and yield, and it is therefore important to improve legume growth under P-deficient conditions....

Chemolithoautotropic Diazotrophy Dominates the Nitrogen Fixation Process in Mine Tailings.

Abstract: Nutrient deficiency, especially bio-available nitrogen deficiency, often impedes the bioremediation efforts of mining generated tailings. Biological nitrogen fixation is a critical process necessary for the initial nitrogen buildup in tailings. Current knowledge regarding the diazotrophs that inhabit tailings is still in its infancy. Therefore, in this study, a comprehensive investigation combining geochemical characterization, sequence analyses, molecular techniques, and activity measurements was conducted to characterize the diazotrophic community residing in tailing environments. Significant differences between tailings and their adjacent soils in prokaryotic and diazotrophic communities were detected. Meanwhile, strong and significant correlations between the absolute abundance of the nitrogen fixation (nifH), carbon fixation (cbbL), sulfur oxidation (soxB), and arsenite oxidation (aioA) genes were observed in the tailings but not in the soils. The reconstructed nif-containing metagenome-assembled genomes (MAGs) suggest that the carbon fixation and sulfur oxidation pathways were important for potential diazotrophs inhabiting the tailings. Activity measurements further confirmed that diazotrophs inhabiting tailings preferentially use inorganic electron donors (e.g., elemental sulfur) compared to organic electron donors (e.g., sucrose), while diazotrophs inhabiting soils preferred organic carbon sources. Collectively, these findings suggest that chemolithoautotrophic diazotrophs may play essential roles in acquiring nutrients and facilitating ecological succession in tailings.

Complete Genome Sequence of Enterobacter roggenkampii ED5, a Nitrogen Fixing Plant Growth Promoting Endophytic Bacterium With Biocontrol and Stress Tolerance Properties, Isolated From Sugarcane Root.

Abstract: Sugarcane is the leading economic crop in China, requires huge quantities of nitrogen in the preliminary plant growth stages. However, the use of an enormous amount of nitrogen fertilizer increases the production price, and have detrimental results on the environment, causes severe soil and water pollution. In this study, a total of 175 endophytic strains were obtained from the sugarcane roots, belonging to five different species, i.e., Saccharum officinarum, Saccharum barberi, Saccharum robustum, Saccharum spontaneum, and Saccharum sinense. Among these, only 23 Enterobacter strains were chosen based on nitrogen fixation, PGP traits, hydrolytic enzymes production, and antifungal activities. Also, all selected strains were showed diverse growth range under different stress conditions, i.e., pH (5-10), temperature (20-45°C), and NaCl (7-12%) and 14 strains confirmed positive nifH, and 12 strains for acdS gene amplification, suggested that these strains could fix nitrogen along with stress tolerance properties. Out of 23 selected strains, Enterobacter roggenkampii ED5 was the most potent strain. Hence, this strain was further selected for comprehensive genome analysis, which includes a genome size of 4,702,851 bp and 56.05% of the average G + C content. Genome annotations estimated 4349 protein-coding with 83 tRNA and 25 rRNA genes. The CDSs number allocated to the KEGG, COG, and GO database were 2839, 4028, and 2949. We recognized a total set of genes that are possibly concerned with ACC deaminase activity, siderophores and plant hormones production, nitrogen and phosphate metabolism, symbiosis, root colonization, biofilm formation, sulfur assimilation and metabolism, along with resistance response toward a range of biotic and abiotic stresses. E. roggenkampii ED5 strain was also a proficient colonizer in sugarcane (variety GT11) and enhanced growth of sugarcane under the greenhouse. To the best of our knowledge, this is the first information on the whole-genome sequence study of endophytic E. roggenkampii ED5 bacterium associated with sugarcane root. And, our findings proposed that identification of predicted genes and metabolic pathways might describe this strain an eco-friendly bioresource to promote sugarcane growth by several mechanisms of actions under multi-stresses.

Genome-scale metabolic reconstruction of the symbiosis between a leguminous plant and a nitrogen-fixing bacterium.

Abstract: The mutualistic association between leguminous plants and endosymbiotic rhizobial bacteria is a paradigmatic example of a symbiosis driven by metabolic exchanges. Here, we report the reconstruction and modelling of a genome-scale metabolic network of Medicago truncatula (plant) nodulated by Sinorhizobium meliloti (bacterium). The reconstructed nodule tissue contains five spatially distinct developmental zones and encompasses the metabolism of both the plant and the bacterium. Flux balance analysis (FBA) suggests that the metabolic costs associated with symbiotic nitrogen fixation are primarily related to supporting nitrogenase activity, and increasing N2-fixation efficiency is associated with diminishing returns in terms of plant growth. Our analyses support that differentiating bacteroids have access to sugars as major carbon sources, ammonium is the main nitrogen export product of N2-fixing bacteria, and N2 fixation depends on proton transfer from the plant cytoplasm to the bacteria through acidification of the peribacteroid space. We expect that our model, called 'Virtual Nodule Environment' (ViNE), will contribute to a better understanding of the functioning of legume nodules, and may guide experimental studies and engineering of symbiotic nitrogen fixation.

Mycorrhizal networks facilitate the colonization of legume roots by a symbiotic nitrogen-fixing bacterium

Abstract: Arbuscular mycorrhizal fungi (AMF) absorb and translocate nutrients from soil to their host plants by means of a wide network of extraradical mycelium (ERM). Here, we assessed whether nitrogen-fixing rhizobia can be transferred to the host legume Glycine max by ERM produced by Glomus formosanum isolate CNPAB020 colonizing the grass Urochloa decumbens. An H-bridge experimental system was developed to evaluate the migration of ERM and of the GFP-tagged Bradyrhizobium diazoefficiens USDA 110 strain across an air gap compartment. Mycorrhizal colonization, nodule formation in legumes, and occurrence of the GFP-tagged strain in root nodules were assessed by optical and confocal laser scanning microscopy. In the presence of non-mycorrhizal U. decumbens, legume roots were neither AMF-colonized nor nodulated. In contrast, G. formosanum ERM crossing the discontinuous compartment connected mycorrhizal U. decumbens and G. max roots, which showed 30–42% mycorrhizal colonization and 7–11 nodules per plant. Fluorescent B. diazoefficiens cells were detected in 94% of G. max root nodules. Our findings reveal that, besides its main activity in nutrient transfer, ERM produced by AMF may facilitate bacterial translocation and the simultaneous associations of plants with beneficial fungi and bacteria, representing an important structure, functional to the establishment of symbiotic relationships.

Effects of Nitrogen Application on Nitrogen Fixation in Common Bean Production.

Abstract: The nitrogen fixing ability of common bean (Phaseolus vulgaris L.) in association with rhizobia is often characterized as poor compared to other legumes, and nitrogen fertilizers are commonly used in bean production to achieve high yields, which in general inhibits nitrogen fixation. In addition, plants cannot take up all the nitrogen applied to the soil as a fertilizer leading to runoff and groundwater contamination. The overall objective of this work is to reduce use of nitrogen fertilizer in common bean production. This would be a major advance in profitability for the common bean industry in Canada and would significantly improve the ecological footprint of the crop. In the current work, 22 bean genotypes [including recombinant inbred lines (RILs) from the Mist × Sanilac population and a non-nodulating mutant (R99)] were screened for their capacity to fix atmospheric nitrogen under four nitrogen regimes. The genotypes were evaluated in replicated field trials on N-poor soils over three years for the percent nitrogen derived from atmosphere (%Ndfa), yield, and a number of yield-related traits. Bean genotypes differed for all analyzed traits, and the level of nitrogen significantly affected most of the traits, including %Ndfa and yield in all three years. In contrast, application of rhizobia significantly affected only few traits, and the effect was inconsistent among the years. Nitrogen application reduced symbiotic nitrogen fixation (SNF) to various degrees in different bean genotypes. This variation suggests that SNF in common bean can be improved through breeding and selection for the ability of bean genotypes to fix nitrogen in the presence of reduced fertilizer levels. Moreover, genotypes like RIL_38, RIL_119, and RIL_131, being both high yielding and good nitrogen fixers, have potential for simultaneous improvement of both traits. However, breeding advancement might be slow due to an inconsistent correlation between these traits.

Molecular Basis of Root Nodule Symbiosis between Bradyrhizobium and 'Crack-Entry' Legume Groundnut (Arachis hypogaea L.).

Abstract: Nitrogen is one of the essential plant nutrients and a major factor limiting crop productivity. To meet the requirements of sustainable agriculture, there is a need to maximize biological nitrogen fixation in different crop species. Legumes are able to establish root nodule symbiosis (RNS) with nitrogen-fixing soil bacteria which are collectively called rhizobia. This mutualistic association is highly specific, and each rhizobia species/strain interacts with only a specific group of legumes, and vice versa. Nodulation involves multiple phases of interactions ranging from initial bacterial attachment and infection establishment to late nodule development, characterized by a complex molecular signalling between plants and rhizobia. Characteristically, legumes like groundnut display a bacterial invasion strategy popularly known as “crack-entry’’ mechanism, which is reported approximately in 25% of all legumes. This article accommodates critical discussions on the bacterial infection mode, dynamics of nodulation, components of symbiotic signalling pathway, and also the effects of abiotic stresses and phytohormone homeostasis related to the root nodule symbiosis of groundnut and Bradyrhizobium. These parameters can help to understand how groundnut RNS is programmed to recognize and establish symbiotic relationships with rhizobia, adjusting gene expression in response to various regulations. This review further attempts to emphasize the current understanding of advancements regarding RNS research in the groundnut and speculates on prospective improvement possibilities in addition to ways for expanding it to other crops towards achieving sustainable agriculture and overcoming environmental challenges.

Can naturally-occurring endophytic nitrogen-fixing bacteria of hybrid white spruce sustain boreal forest tree growth on extremely nutrient-poor soils?

Abstract: The West Chilcotin region in British Columbia, Canada is located in the Sub-Boreal zone characterized by dry and weakly developed soils lacking essential plant nutrients, particularly nitrogen. Yet, hybrid white spruce (Picea glauca x engelmannii) trees thrive on such nitrogen-limited soils, raising a crucial question regarding their nitrogen sources. The presence of endophytic diazotrophic bacteria (nitrogen-fixing bacteria living inside the plant tissues) was reported in these spruce trees, previously. But, can these bacteria actually sustain tree growth on nitrogen-limited soils of this region? To answer this question, we tested six endophytic diazotrophic bacteria under nitrogen-poor conditions in a year-long greenhouse trial with their original host (i.e. hybrid white spruce). In a different year-long trial, we also tested these bacteria with another host native to the West Chilcotin region (i.e. lodgepole pine) to examine their interaction with a foreign host. Endophytic colonization, seedling growth promotion and the amount of nitrogen fixed in planta by each bacterium were examined. We found that each bacterium colonized one or more tissues of the spruce and pine seedlings (102–107 colonies per gram fresh tissue) and fixed significant (P 50% of the nitrogen requirements of pine and spruce seedlings via nitrogen fixation, and enhanced seedling length and biomass by nearly 1.5-fold and 5-fold, respectively. Therefore, our results suggest that endophytic diazotrophic bacteria harboured by hybrid spruce trees can sustain their growth on nitrogen-limited soils via biological nitrogen fixation. These bacteria can also provide similar benefits to a foreign host - lodgepole pine, indicating their wide-range ecological applications in improving the N-supply of forest stands in this region and beyond. In particular, C. sordidicola LS-S2r holds strong potential to be possibly used as a biofertilizer in boreal forest stands, as an economical and eco-friendly alternative to chemical fertilizers.

Assessment of Nitrogen Fixation by Mungbean Genotypes in Different Soil Textures Using 15N Natural Abundance Method

Abstract: Ensuring food and nutritional security in light of high climate variability and a rapidly growing population remains a challenge. Mungbean (Vigna radiata (L.) Wilczek) is a short duration, drought-tolerant, and ureide-exporting legume crop capable of symbiotic atmospheric nitrogen fixation. Estimates of biological N2 fixation by mungbean in different soil textures have not been extensively studied. We conducted this study to evaluate plant growth and N2 fixation of five mungbean genotypes (Berken, 8735, IC 8972-1, STB#122, 223) inoculated with Bradyrhizobium spp. and grown on loamy sand and silt loam soils under glasshouse conditions. Mungbean dry matter yield, δ15N values, shoot content, amounts of N-fixed, and soil N uptake were all higher on the silt loam soil compared to the loamy sand soil, demonstrating the effects of soil properties on plant growth and N2 fixation potential. Among genotypes, IC 8972-1 produced the highest biomass (7.85 g plant−1), shoot N content (200 mg plant−1), and soil N uptake (155 mg plant−1) than other genotypes. The significant interaction between soil texture and genotypes for root dry matter and %Ndfa indicates the major role of legume root-nodule bacteria in symbiotic N2 fixation. This study demonstrated that N2 fixation in mungbean is affected by both genotypes and soil properties, illustrating the need to consider soil properties in order to maximize N contribution from mungbean to agricultural production systems.

What do we know about biological nitrogen fixation in insects? Evidence and implications for the insect and the ecosystem.

Abstract: Many insects feed on a low-nitrogen diet, and the origin of their nitrogen supply is poorly understood. It has been hypothesized that some insects rely on nitrogen-fixing bacteria (diazotrophs) to supplement their diets. Nitrogen fixation by diazotrophs has been extensively studied and convincingly demonstrated in termites, while evidence for the occurrence and role of nitrogen fixation in the diet of other insects is less conclusive. Here, we summarize the methods to detect nitrogen fixation in insects and review the available evidence for its occurrence (focusing on insects other than termites). We distinguish between three aspects of nitrogen fixation investigations: (i) detecting the presence of potential diazotrophs; (ii) detecting the activity of the nitrogen-fixing enzyme; and (iii) detecting the assimilation of fixed nitrogen into the insect tissues. We show that although evidence from investigations of the first aspect reveals ample opportunities for interactions with potential diazotrophs in a variety of insects, demonstrations of actual biological nitrogen fixation and the assimilation of fixed nitrogen are restricted to very few insect groups, including wood-feeding beetles, fruit flies, leafcutter ants, and a wood wasp. We then discuss potential implications for the insect's fitness and for the ecosystem as a whole. We suggest that combining these multiple approaches is crucial for the study of nitrogen fixation in insects, and argue that further demonstrations are desperately needed in order to determine the relative importance of diazotrophs for insect diet and fitness, as well as to evaluate their overall impact on the ecosystem.

Mechanisms in plant growth-promoting rhizobacteria that enhance legume-rhizobial symbioses.

Abstract: Nitrogen fixation is an important biological process in terrestrial ecosystems and for global crop production Legume nodulation and N2 fixation have been improved using nodule-enhancing rhizobacteria (NER) under both regular and stressed conditions The positive effect of NER on legume-rhizobia symbiosis can be facilitated by plant growth-promoting (PGP) mechanisms, some of which remain to be identified NER that produce aminocyclopropane-1-carboxylic acid deaminase and indole acetic acid enhance the legume-rhizobia symbiosis through (i) enhancing the nodule induction, (ii) improving the competitiveness of rhizobia for nodulation, (iii) prolonging functional nodules by suppressing nodule senescence and (iv) upregulating genes associated with legume-rhizobia symbiosis The means by which these processes enhance the legume-rhizobia symbiosis is the focus of this review A better understanding of the mechanisms by which PGP rhizobacteria operate, and how they can be altered, will provide opportunities to enhance legume-rhizobial interactions, to provide new advances in plant growth promotion and N2 fixation

Biological nitrogen fixation in maize: optimizing nitrogenase expression in a root-associated diazotroph.

Abstract: Plants depend upon beneficial interactions between roots and root-associated microorganisms for growth promotion, disease suppression, and nutrient availability. This includes the ability of free-living diazotrophic bacteria to supply nitrogen, an ecological role that has been long underappreciated in modern agriculture for efficient crop production systems. Long-term ecological studies in legume-rhizobia interactions have shown that elevated nitrogen inputs can lead to the evolution of less cooperative nitrogen-fixing mutualists. Here we describe how reprogramming the genetic regulation of nitrogen fixation and assimilation in a novel root-associated diazotroph can restore ammonium production in the presence of exogenous nitrogen inputs. We isolated a strain of the plant-associated proteobacterium Kosakonia sacchari from corn roots, characterized its nitrogen regulatory network, and targeted key nodes for gene editing to optimize nitrogen fixation in corn. While the wild-type strain exhibits repression of nitrogen fixation in conditions replete with bioavailable nitrogen, such as fertilized greenhouse and field experiments, remodeled strains show elevated levels in the rhizosphere of corn in the greenhouse and field even in the presence of exogenous nitrogen. Such strains could be used in commercial applications to supply fixed nitrogen to cereal crops.

Diazotrophic Anaeromyxobacter Isolates from Soils

Abstract: Biological nitrogen fixation is an essential reaction in a major pathway for supplying nitrogen to terrestrial environments. Previous culture-independent analyses based on soil DNA/RNA/protein sequencing could globally detect the nitrogenase genes/proteins of Anaeromyxobacter (in the class Deltaproteobacteria), commonly distributed in soil environments and predominant in paddy soils; this suggests the importance of Anaeromyxobacter in nitrogen fixation in soil environments. However, direct experimental evidence is lacking; there has been no research on the genetic background and ability of Anaeromyxobacter to fix nitrogen. Therefore, we verified the diazotrophy of Anaeromyxobacter based on both genomic and culture-dependent analyses using Anaeromyxobacter sp. strains PSR-1 and Red267 isolated from soils. Based on the comparison of nif gene clusters, strains PSR-1 and Red267 as well as strains Fw109-5, K, and diazotrophic Geobacter and Pelobacter in the class Deltaproteobacteria contain the minimum set of genes for nitrogenase (nifBHDKEN). These results imply that Anaeromyxobacter species have the ability to fix nitrogen. In fact, Anaeromyxobacter PSR-1 and Red267 exhibited N2-dependent growth and acetylene reduction activity (ARA) in vitro. Transcriptional activity of the nif gene was also detected when both strains were cultured with N2 gas as a sole nitrogen source, indicating that Anaeromyxobacter can fix and assimilate N2 gas by nitrogenase. In addition, PSR-1- or Red267-inoculated soil showed ARA activity and the growth of the inoculated strains on the basis of RNA-based analysis, demonstrating that Anaeromyxobacter can fix nitrogen in the paddy soil environment. Our study provides novel insights into the pivotal environmental function, i.e., nitrogen fixation, of Anaeromyxobacter, which is a common soil bacterium. IMPORTANCEAnaeromyxobacter is globally distributed in soil environments, especially predominant in paddy soils. Current studies based on environmental DNA/RNA analyses frequently detect gene fragments encoding nitrogenase of Anaeromyxobacter from various soil environments. Although the importance of Anaeromyxobacter as a diazotroph in nature has been suggested by culture-independent studies, there has been no solid evidence and validation from genomic and culture-based analyses that Anaeromyxobacter fixes nitrogen. This study demonstrates that Anaeromyxobacter harboring nitrogenase genes exhibits diazotrophic ability; moreover, N2-dependent growth was demonstrated in vitro and in the soil environment. Our findings indicate that nitrogen fixation is important for Anaeromyxobacter to survive under nitrogen-deficient environments and provide a novel insight into the environmental function of Anaeromyxobacter, which is a common bacterium in soils.

Nitrogen Stimulates Microcystis-Dominated Blooms More than Phosphorus in River Conditions That Favor Non-Nitrogen-Fixing Genera.

Abstract: Despite the implementation of intensive phosphorus reduction measures, periodic outbreaks of cyanobacterial blooms in large rivers remain a problem in Korea, raising the need for more effective solutions to reduce their occurrence. This study sought to evaluate whether phosphorus or nitrogen limitation is an effective approach to control cyanobacterial (Microcystis) blooms in river conditions that favor this non-nitrogen-fixing genus. These conditions include nutrient enrichment, high water temperature, and thermal stratification during summer. Mesocosm bioassays were conducted to investigate the limiting factors for cyanobacterial blooms in a river reach where severe Microcystis blooms occur annually. We evaluated the effect of five different nitrogen (3, 6, 9, 12, and 15 mg/L) and phosphorus (0.01, 0.02, 0.05, 0.1, and 0.2 mg/L) concentrations on algae growth. The results indicate that nitrogen treatments stimulated cyanobacteria (mostly Microcystis aeruginosa) more than phosphorus. Interestingly, phosphorus additions did not stimulate cyanobacteria, although it did stimulate Chlorophyceae and Bacillariophyceae. We conclude that phosphorus reduction might have suppressed the growth of Chlorophyceae and Bacillariophyceae more than that of cyanobacteria; therefore, nitrogen or at least both nitrogen and phosphorus control appears more effective than phosphorus reductions alone for reducing cyanobacteria in river conditions that are favorable for non-nitrogen-fixing genera.

Enhancement of soybean nodulation by seed treatment with non-thermal plasmas.

Abstract: Soybean (Glycine max (L.) Merrill) is one of the most important crops worldwide providing dietary protein and vegetable oil. Most of the nitrogen required by the crop is supplied through biological N2 fixation. Non-thermal plasma is a fast, economical, and environmental-friendly technology that can improve seed quality, plant growth, and crop yield. Soybean seeds were exposed to a dielectric barrier discharge plasma operating at atmospheric pressure air with superimposed flows of O2 or N2 as carrying gases. An arrangement of a thin phenolic sheet covered by polyester films was employed as an insulating barrier. We focused on the ability of plasma to improve soybean nodulation and biological nitrogen fixation. The total number of nodules and their weight were significantly higher in plants grown from treated seeds than in control. Plasma treatments incremented 1.6 fold the nitrogenase activity in nodules, while leghaemoglobin content was increased two times, indicating that nodules were fixing nitrogen more actively than control. Accordingly, the nitrogen content in nodules and the aerial part of plants increased by 64% and 23%, respectively. Our results were supported by biometrical parameters. The results suggested that different mechanisms are involved in soybean nodulation improvement. Therefore, the root contents of isoflavonoids, glutathione, auxin and cytokinin, and expansin (GmEXP1) gene expression were determined. We consider this emerging technology is a suitable pre-sowing seed treatment.

Biological nitrogen fixation detected under Antarctic sea ice

Abstract: Nitrogen fixation is the primary source of reactive nitrogen in the ocean. Most ecological models do not predict nitrogen fixation in the Antarctic Ocean because of the low availability of iron and high abundance of nitrogen. Here we extensively examined nitrogen fixation in the Antarctic Ocean, and found substantial nitrogen fixation (maximum: 44.4 nmol N l−1 d−1) near the Antarctic coast, especially around ice-covered regions. The nitrogenase gene (nifH) was detected at all coastal stations, including stations where no nitrogen fixation was found. At the stations where nitrogen fixation was detected, the nitrogen-fixing cyanobacterium UCYN-A (Candidatus ‘Atelocyanobacterium thalassa’) dominated nifH gene expression, and the nifH sequence was identical to that of the major oligotype in tropical and subtropical oceans. Our results suggest that marine nitrogen fixation is a ubiquitous process in the global ocean, and that UCYN-A is the keystone species for making it possible. Observational evidence of cyanobacterial activity in the Antarctic Ocean suggests that nitrogen fixation could be a ubiquitous process in the global ocean.

New insights into the distributions of nitrogen fixation and diazotrophs revealed by high-resolution sensing and sampling methods.

Abstract: Nitrogen availability limits marine productivity across large ocean regions Diazotrophs can supply new nitrogen to the marine environment via nitrogen (N2) fixation, relieving nitrogen limitation The distributions of diazotrophs and N2 fixation have been hypothesized to be generally controlled by temperature, phosphorus, and iron availability in the global ocean However, even in the North Atlantic where most research on diazotrophs and N2 fixation has taken place, environmental controls remain contentious Here we measure diazotroph composition, abundance, and activity at high resolution using newly developed underway sampling and sensing techniques We capture a diazotrophic community shift from Trichodesmium to UCYN-A between the oligotrophic, warm (25–29 °C) Sargasso Sea and relatively nutrient-enriched, cold (13–24 °C) subpolar and eastern American coastal waters Meanwhile, N2 fixation rates measured in this study are among the highest ever recorded globally and show significant increase with phosphorus availability across the transition from the Gulf Stream into subpolar and coastal waters despite colder temperatures and higher nitrate concentrations Transcriptional patterns in both Trichodesmium and UCYN-A indicate phosphorus stress in the subtropical gyre Over this iron-replete transect spanning the western North Atlantic, our results suggest that temperature is the major factor controlling the diazotrophic community structure while phosphorous drives N2 fixation rates Overall, the occurrence of record-high UCYN-A abundance and peak N2 fixation rates in the cold coastal region where nitrate concentrations are highest (~200 nM) challenges current paradigms on what drives the distribution of diazotrophs and N2 fixation

Microbiological strategies for enhancing biological nitrogen fixation in nonlegumes.

Abstract: In an agro-ecosystem, industrially produced nitrogenous fertilizers are the principal sources of nitrogen for plant growth; unfortunately these also serve as the leading sources of pollution. Hence, it becomes imperative to find pollution-free methods of providing nitrogen to crop plants. A diverse group of free-living, plant associative and symbiotic prokaryotes are able to perform biological nitrogen fixation (BNF). BNF is a two component process involving the nitrogen fixing diazotrophs and the host plant. Symbiotic nitrogen fixation is most efficient as it can fix nitrogen inside the nodule formed on the roots of the plant; delivering nitrogen directly to the host. However, most of the important crop plants are nonleguminous and are unable to form symbiotic associations. In this context, the plant associative and endophytic diazotrophs assume importance. BNF in nonlegumes can be encouraged either through the transfer of BNF traits from legumes or by elevating the nitrogen fixing capacity of the associative and endophytic diazotrophs. In this review we discuss mainly the microbiological strategies which may be used in nonleguminous crops for enhancement of BNF.

GmVTL1a is an iron transporter on the symbiosome membrane of soybean with an important role in nitrogen fixation.

Abstract: Legumes establish symbiotic relationships with soil bacteria (rhizobia), housed in nodules on roots. The plant supplies carbon substrates and other nutrients to the bacteria in exchange for fixed nitrogen. The exchange occurs across a plant-derived symbiosome membrane (SM), which encloses rhizobia to form a symbiosome. Iron supplied by the plant is crucial for rhizobial enzyme nitrogenase that catalyses nitrogen fixation, but the SM iron transporter has not been identified. We use yeast complementation, real-time PCR and proteomics to study putative soybean (Glycine max) iron transporters GmVTL1a and GmVTL1b and have characterized the role of GmVTL1a using complementation in plant mutants, hairy root transformation and microscopy. GmVTL1a and GmVTL1b are members of the vacuolar iron transporter family and homologous to Lotus japonicus SEN1 (LjSEN1), which is essential for nitrogen fixation. GmVTL1a expression is enhanced in nodule infected cells and both proteins are localized to the SM. GmVTL1a transports iron in yeast and restores nitrogen fixation when expressed in the Ljsen1 mutant. Three GmVTL1a amino acid substitutions that block nitrogen fixation in Ljsen1 plants reduce iron transport in yeast. We conclude GmVTL1a is responsible for transport of iron across the SM to bacteroids and plays a crucial role in the nitrogen-fixing symbiosis.