Showing papers on "Nitrogen fixation published in 2012"

Contribution of cryptogamic covers to the global cycles of carbon and nitrogen

Abstract: Many terrestrial surfaces are covered by photoautotrophic communities, which are capable of synthesizing their own food from inorganic substances using sunlight. According to an analysis of previously published data, these communities account for nearly half of the biological nitrogen fixation on land.

Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes

Abstract: The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes. Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions. Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes.

Nitrogen isotopes link mycorrhizal fungi and plants to nitrogen dynamics

Abstract: Contents Summary 367 I. Introduction 367 II. Background on isotopes 368 III. Patterns of soil δ15N 370 IV. Patterns of fungal δ15N 372 V. Biochemical basis for the influence of fungi on δ15N patterns in plant–soil systems 373 VI. Patterns of δ15N in plant and fungal culture studies 374 VII. Mycoheterotrophic and parasitic plants 375 VIII. Patterns of foliar δ15N in autotrophic plants 376 IX. Controls over plant δ15N 377 X. Conclusions and research needs 378 Acknowledgements 379 References 379 Summary In this review, we synthesize field and culture studies of the 15N/14N (expressed as δ15N) of autotrophic plants, mycoheterotrophic plants, parasitic plants, soil, and mycorrhizal fungi to assess the major controls of isotopic patterns. One major control for plants and fungi is the partitioning of nitrogen (N) into either 15N-depleted chitin, ammonia, or transfer compounds or 15N-enriched proteinaceous N. For example, parasitic plants and autotrophic hosts are similar in δ15N (with no partitioning between chitin and protein), mycoheterotrophic plants are higher in δ15N than their fungal hosts, presumably with preferential assimilation of fungal protein, and autotrophic, mycorrhizal plants are lower in 15N than their fungal symbionts, with saprotrophic fungi intermediate, because mycorrhizal fungi transfer 15N-depleted ammonia or amino acids to plants. Similarly, nodules of N2-fixing bacteria transferring ammonia are often higher in δ15N than their plant hosts. N losses via denitrification greatly influence bulk soil δ15N, whereas δ15N patterns within soil profiles are influenced both by vertical patterns of N losses and by N transfers within the soil–plant system. Climate correlates poorly with soil δ15N; climate may primarily influence δ15N patterns in soils and plants by determining the primary loss mechanisms and which types of mycorrhizal fungi and associated vegetation dominate across climatic gradients.

Heterotrophic organisms dominate nitrogen fixation in the South Pacific Gyre

Abstract: Oceanic subtropical gyres are considered biological deserts because of the extremely low availability of nutrients and thus minimum productivities. The major source of nutrient nitrogen in these ecosystems is N2-fixation. The South Pacific Gyre (SPG) is the largest ocean gyre in the world, but measurements of N2-fixation therein, or identification of microorganisms involved, are scarce. In the 2006/2007 austral summer, we investigated nitrogen and carbon assimilation at 11 stations throughout the SPG. In the ultra-oligotrophic waters of the SPG, the chlorophyll maxima reached as deep as 200 m. Surface primary production seemed limited by nitrogen, as dissolved inorganic carbon uptake was stimulated upon additions of 15N-labeled ammonium and leucine in our incubation experiments. N2-fixation was detectable throughout the upper 200 m at most stations, with rates ranging from 0.001 to 0.19 nM N h−1. N2-fixation in the SPG may account for the production of 8–20% of global oceanic new nitrogen. Interestingly, comparable 15N2-fixation rates were measured under light and dark conditions. Meanwhile, phylogenetic analyses for the functional gene biomarker nifH and its transcripts could not detect any common photoautotrophic diazotrophs, such as, Trichodesmium, but a prevalence of γ-proteobacteria and the unicellular photoheterotrophic Group A cyanobacteria. The dominance of these likely heterotrophic diazotrophs was further verified by quantitative PCR. Hence, our combined results show that the ultra-oligotrophic SPG harbors a hitherto unknown heterotrophic diazotrophic community, clearly distinct from other oceanic gyres previously visited.

Biological nitrogen fixation and phosphate solubilization by bacteria isolated from tropical soils

Abstract: In addition to fixing atmospheric nitrogen, some bacterial isolates can also solubilize insoluble phosphates, further contributing to plant growth. The objectives of this study were the following: isolate, select, and identify nodulating bacteria in the cowpea that are efficient not only in biological nitrogen fixation (BNF) but also in the solubilization of insoluble inorganic phosphates; identify and quantify the organic acids produced; and establish the relationship between those acids and the solubilizing capacity. The bacteria were captured from two soils containing high concentrations of insoluble phosphorus from the cities of Lavras and Patos de Minas, using the cowpea [Vigna unguiculata (L.) Walp.] as bait. We obtained 78 strains, which were characterized according to their cultural attributes in culture medium 79 with the strains UFLA 03-84, INPA 03-11B, and BR3267 (approved by the Ministry of Livestock and Supply Agriculture—MAPA, as inoculants for the cowpea) and Burkholderia cepacia (LMG1222T), which was used as a positive control for phosphate solubilization. Strains that were selected for their efficiency in both processes were identified by 16S rDNA sequence analysis. We evaluated the symbiotic efficiency (BNF) in a greenhouse and the solubilization efficiency of CaHPO4, Al(H2PO4)3, and FePO4.2H2O in solid and liquid GELP media. Strains that excelled at the solubilization of these phosphate sources were also evaluated for the production of the following organic acids: oxalic, citric, gluconic, lactic, succinic, and propionic. The presence of Acinetobacter, Bacillus, Firmicutes, Microbacterium, Paenibacillus, and Rhizobium was detected by 16S rDNA sequencing and analysis. Bacterial strains obtained from cowpea nodules varied greatly in the efficiency of their BNF and phosphate solubilization processes, especially in the strains UFLA 03-09, UFLA 03-10, UFLA 03-12, and UFLA 03-13, which were more efficient in both processes. More strains were able to solubilize insoluble inorganic calcium and iron phosphates in liquid medium than in solid medium. The production of organic acids was related to the solubilization of CaHPO4 and FePO4.2H2O for some strains, and the type and concentration of the acid influenced this process. These are the first results obtained with bacterial isolates from tropical soils in which the production of organic acids was detected and quantified to examine the solubilization of insoluble inorganic phosphates.

The contribution of nitrogen fixation to sugarcane (Saccharum officinarum L.), and the identification and characterization of part of the associated diazotrophic bacterial community

Abstract: Rhizospheric, epiphytic and endophytic bacteria are associated with several non-legumes, colonizing their surface and inner tissues. Many of these bacteria are beneficial to their hosts, and are collectively termed plant growth-promoting rhizobacteria (PGPR). Recent interest has focused particularly upon PGPR that are endophytic (i.e. PGPE), and which have been reported to be associated with important crops such as rice, wheat and sugarcane. Different mechanisms are involved in bacteria-induced plant growth promotion (PGP), including biological nitrogen fixation (BNF), mineral solubilization, production of phytohormones and pathogen biocontrol. In Uruguay, sugarcane (Saccharum officinarum L.) is considered a strategic multipurpose crop, used for bioenergy, feed, sugar and bioethanol production. The aim of this work was to estimate the BNF contribution to Uruguayan sugarcane cultivars, as well as to identify and characterize the (culturable) putatively endophytic diazotrophic bacteria associated with these varieties. Results using the 15N-dilution technique have shown that these sugarcane varieties obtain significant inputs of N from BNF (34.8–58.8% Ndfa). In parallel, a collection of 598 isolates of potentially endophytic diazotrophs was obtained from surface-sterilized stems using standard isolation techniques, and nifH + isolates from these were the subject of further studies. The bacteria were shown to belong to several genera, including Pseudomonas, Stenotrophomonas, Xanthomonas, Acinetobacter, Rhanella, Enterobacter, Pantoea, Shinella, Agrobacterium and Achromobacter. Additionally, some PGP features were studied in 35 selected isolates. The data obtained in this study represent the initial steps in a program aimed at determining the mechanisms of PGP of non-legume crops in Uruguay (such as sugarcane) with potentially beneficial plant-associated bacteria.

Influence of co-inoculation of bacteria-cyanobacteria on crop yield and C–N sequestration in soil under rice crop

Abstract: The performance of three selected bacterial strains—PR3, PR7 and PR10 (Providencia sp., Brevundimonas sp., Ochrobacterium sp.) and three cyanobacterial strains CR1, CR2 and CR3 (Anabaena sp., Calothrix sp., Anabaena sp.), and their combinations was evaluated in a pot experiment with rice variety Pusa-1460, comprising 51 treatments along with recommended fertilizer controls. Highest yield enhancement of 19.02% was recorded in T12 (CR2), over control, while significant enhancement in nitrogen fixing potential was recorded in treatments involving combination of bacterial-cyanobacterial strains—T37 (PR3 + CR1 + CR3) and T21 (PR7 + CR1). Organic carbon was significantly increased in all microbe-inoculated treatments, which could be correlated with microbial biomass carbon values and activities of all the enzymes tested in our study. Also, panicle weight and plant biomass were highly correlated with soil microbial carbon. Comparative evaluation revealed the superior performance of strains CR2, CR1 (both Anabaena sp.) and PR10 (Ochrobacterium sp.) in increasing the growth and grain yield of rice and improving soil health, besides N (nitrogen) savings of 40–80 kg ha−1. The study for the first time illustrated the positive effects of co-inoculation of bacterial and cyanobacterial strains for integrated nutrient management of rice crop.

Never too many? How legumes control nodule numbers.

Abstract: Restricted availability of nitrogen compounds in soils is often a major limiting factor for plant growth and productivity. Legumes circumvent this problem by establishing a symbiosis with soil-borne bacteria, called rhizobia that fix nitrogen for the plant. Nitrogen fixation and nutrient exchange take place in specialized root organs, the nodules, which are formed by a coordinated and controlled process that combines bacterial infection and organ formation. Because nodule formation and nitrogen fixation are energy-consuming processes, legumes develop the minimal number of nodules required to ensure optimal growth. To this end, several mechanisms have evolved that adapt nodule formation and nitrogen fixation to the plant's needs and environmental conditions, such as nitrate availability in the soil. In this review, we give an updated view on the mechanisms that control nodulation.

Molybdenum and phosphorus interact to constrain asymbiotic nitrogen fixation in tropical forests.

Abstract: Biological di-nitrogen fixation (N2) is the dominant natural source of new nitrogen to land ecosystems. Phosphorus (P) is thought to limit N2 fixation in many tropical soils, yet both molybdenum (Mo) and P are crucial for the nitrogenase reaction (which catalyzes N2 conversion to ammonia) and cell growth. We have limited understanding of how and when fixation is constrained by these nutrients in nature. Here we show in tropical forests of lowland Panama that the limiting element on asymbiotic N2 fixation shifts along a broad landscape gradient in soil P, where Mo limits fixation in P-rich soils while Mo and P co-limit in P-poor soils. In no circumstance did P alone limit fixation. We provide and experimentally test a mechanism that explains how Mo and P can interact to constrain asymbiotic N2 fixation. Fixation is uniformly favored in surface organic soil horizons - a niche characterized by exceedingly low levels of available Mo relative to P. We show that soil organic matter acts to reduce molybdate over phosphate bioavailability, which, in turn, promotes Mo limitation in sites where P is sufficient. Our findings show that asymbiotic N2 fixation is constrained by the relative availability and dynamics of Mo and P in soils. This conceptual framework can explain shifts in limitation status across broad landscape gradients in soil fertility and implies that fixation depends on Mo and P in ways that are more complex than previously thought.

Genetic and Symbiotic Diversity of Nitrogen-Fixing Bacteria Isolated from Agricultural Soils in the Western Amazon by Using Cowpea as the Trap Plant

Abstract: Cowpea is a legume of great agronomic importance that establishes symbiotic relationships with nitrogen-fixing bacteria. However, little is known about the genetic and symbiotic diversity of these bacteria in distinct ecosystems. Our study evaluated the genetic diversity and symbiotic efficiencies of 119 bacterial strains isolated from agriculture soils in the western Amazon using cowpea as a trap plant. These strains were clustered into 11 cultural groups according to growth rate and pH. The 57 nonnodulating strains were predominantly fast growing and acidifying, indicating a high incidence of endophytic strains in the nodules. The other 62 strains, authenticated as nodulating bacteria, exhibited various symbiotic efficiencies, with 68% of strains promoting a significant increase in shoot dry matter of cowpea compared with the control with no inoculation and low levels of mineral nitrogen. Fifty genotypes with 70% similarity and 21 genotypes with 30% similarity were obtained through repetitive DNA sequence (BOX element)-based PCR (BOX-PCR) clustering. The 16S rRNA gene sequencing of strains representative of BOX-PCR clusters showed a predominance of bacteria from the genus Bradyrhizobium but with high species diversity. Rhizobium, Burkholderia, and Achromobacter species were also identified. These results support observations of cowpea promiscuity and demonstrate the high symbiotic and genetic diversity of rhizobia species in areas under cultivation in the western Amazon.

What determines the efficiency of N2-fixing rhizobium-legume symbioses?

Abstract: Biological nitrogen fixation is vital to nutrient cycling in the biosphere and is the major route by which atmospheric dinitrogen (N2) is reduced to ammonia. The largest single contribution to biological N2 fixation is carried out by rhizobia, which include a large group of both alpha and beta-proteobacteria, almost exclusively in association with legumes. Rhizobia must compete to infect roots of legumes and initiate a signaling dialog with host plants that leads to nodule formation. The most common form of infection involves the growth of rhizobia down infection threads which are laid down by the host plant. Legumes form either indeterminate or determinate types of nodules, with these groups differing widely in nodule morphology and often in the developmental program by which rhizobia form N2 fixing bacteroids. In particular, indeterminate legumes from the inverted repeat-lacking clade (IRLC) (e.g., peas, vetch, alfalfa, medics) produce a cocktail of antimicrobial peptides which cause endoreduplication of the bacterial genome and force rhizobia into a nongrowing state. Bacteroids often become dependent on the plant for provision of key cofactors, such as homocitrate needed for nitrogenase activity or for branched chain amino acids. This has led to the suggestion that bacteroids at least from the IRLC can be considered as ammoniaplasts, where they are effectively facultative plant organelles. A low O2 tension is critical both to induction of genes needed for N2 fixation and to the subsequent exchange of nutrient between plants and bacteroids. To achieve high rates of N2 fixation, the legume host and Rhizobium must be closely matched not only for infection, but for optimum development, nutrient exchange, and N2 fixation. In this review, we consider the multiple steps of selection and bacteroid development and how these alter the overall efficiency of N2 fixation.

What determines the efficiency of N(2)-fixing Rhizobium-legume symbioses?

Abstract: Biological nitrogen fixation is vital to nutrient cycling in the biosphere and is the major route by which atmospheric dinitrogen (N 2 ) is reduced to ammonia. The largest single contribution to biological N 2 fixation is carried out by rhizobia, which include a large group of both alpha and beta-proteobacteria, almost exclusively in association with legumes. Rhizobia must compete to infect roots of legumes and initiate a signaling dialog with host plants that leads to nodule formation. The most common form of infection involves the growth of rhizobia down infection threads which are laid down by the host plant. Legumes form either indeterminate or determinate types of nodules, with these groups differing widely in nodule morphology and often in the developmental program by which rhizobia form N 2 fixing bacteroids. In particular, indeterminate legumes from the inverted repeat-lacking clade (IRLC) (e.g., peas, vetch, alfalfa, medics) produce a cocktail of antimicrobial peptides which cause endoreduplication of the bacterial genome and force rhizobia into a nongrowing state. Bacteroids often become dependent on the plant for provision of key cofactors, such as homocitrate needed for nitrogenase activity or for branched chain amino acids. This has led to the suggestion that bacteroids at least from the IRLC can be considered as ammoniaplasts, where they are effectively facultative plant organelles. A low O 2 tension is critical both to induction of genes needed for N 2 fixation and to the subsequent exchange of nutrient between plants and bacteroids. To achieve high rates of N 2 fixation, the legume host and Rhizobium must be closely matched not only for infection, but for optimum development, nutrient exchange, and N 2 fixation. In this review, we consider the multiple steps of selection and bacteroid development and how these alter the overall efficiency of N 2 fixation.

Nitrogen fixation in annual and perennial legume-grass mixtures across a fertility gradient

Abstract: Background and aims The selection of legume species and species mixtures influences agroecosystem nitrogen (N) and carbon cycling. We utilized a fertility gradient to investigate the effects of plant species interactions on biological N fixation of an annual and perennial legume in response to shifting soil resource availability.

Two MicroRNAs Linked to Nodule Infection and Nitrogen-Fixing Ability in the Legume Lotus japonicus

Abstract: Legumes overcome nitrogen shortage by developing root nodules in which symbiotic bacteria fix atmospheric nitrogen in exchange for host-derived carbohydrates and mineral nutrients. Nodule development involves the distinct processes of nodule organogenesis, bacterial infection, and the onset of nitrogen fixation. These entail profound, dynamic gene expression changes, notably contributed to by microRNAs (miRNAs). Here, we used deep-sequencing, candidate-based expression studies and a selection of Lotus japonicus mutants uncoupling different symbiosis stages to identify miRNAs involved in symbiotic nitrogen fixation. Induction of a noncanonical miR171 isoform, which targets the key nodulation transcription factor Nodulation Signaling Pathway2, correlates with bacterial infection in nodules. A second candidate, miR397, is systemically induced in the presence of active, nitrogen-fixing nodules but not in that of noninfected or inactive nodule organs. It is involved in nitrogen fixation-related copper homeostasis and targets a member of the laccase copper protein family. These findings thus identify two miRNAs specifically responding to symbiotic infection and nodule function in legumes.

The Global Nitrogen Cycle

Abstract: The geobiology of nitrogen is dominated by large inert reservoirs and small biological fluxes. The largest reservoirs are nitrogen gas (in the atmosphere and dissolved in the ocean) and sedimentary nitrogen (sequestered in continental crust). Because most organisms cannot utilize gaseous nitrogen, we distinguish between fixed nitrogen compounds (which contain no N–N bonds) that are biologically available, and the dinitrogen gases (N2 and N2O) that are largely inaccessible to organisms. Fluxes into and out of the fixed nitrogen pools are biologically controlled, and microbes control the rates of transformations and the distribution of nitrogen among inorganic and organic pools. Possibly more than any other biologically important element, the global nitrogen cycle has been perturbed by anthropogenic activities. The rate of industrial nitrogen fixation now approximately equals the natural rate, resulting in a twoto threefold increase in the total inventory of fixed N on the surface of the Earth through agricultural fertilizer applications (Galloway et al., 2004). Nitrogen oxides enter the atmosphere via fossil fuel combustion and catalyse the atmospheric chemistry of ozone through pathways that were either nonexistent or insignificant prior to humans. Because of the relative biological inaccessibility of nitrogen, ecosystems have responded to the increased flux of fixed N with changes in the rates and fates of production, and in some cases, large changes in ecosystem chemistry and health. The microbiology of nitrogen transformations has been intensely studied for over a century, but important new discoveries have been made in only the last decade. These involve the discovery of previously suspected, but unknown processes, as well as the discovery of new and diverse microbes involved in many of the nitrogen cycle fluxes.

Molecular characterisation of the diazotrophic bacterial community in uninoculated and inoculated field-grown sugarcane (Saccharum sp.)

Abstract: To identify active diazotrophs in sugarcane, 16S rRNA and nifH transcript analyses were applied. This should help to better understand the basis of the biological nitrogen fixation (BNF) activity of a high nitrogen fixing sugarcane variety. A field experiment using the sugarcane variety RB 867515 was conducted in Seropedica, RJ, Brazil, receiving the following treatments: unfertilised and fertilised controls without inoculation, unfertilised with inoculation. The five-strain mixture developed by EMBRAPA-CNPAB was used as inoculum. Root and leaf sheath samples were harvested in the third year of cultivation to analyse the 16S rRNA and nifH transcript diversity. In addition to nifH expression from Gluconacetobacter spp. and Burkholderia spp., a wide diversity of nifH sequences from previously uncharacterised Ideonella/Herbaspirillum related phylotypes in sugarcane shoots as well as Bradyrhizobium sp. and Rhizobium sp. in roots was found. These results were confirmed using 16S cDNA analysis. From the inoculated bacteria, only nifH transcripts from G. diazotrophicus and B. tropica were detected in leaf sheaths and roots. Known as well as yet uncultivated diazotrophs were found active in sugarcane roots and stems using molecular analyses. Two strains of the inoculum mix were identified at the late summer harvest.

Plant Growth-Promoting Nitrogen-Fixing Enterobacteria Are in Association with Sugarcane Plants Growing in Guangxi, China

Abstract: Guangxi is the major sugarcane- and sugar-producing area in China and produces about 60% of China’s sugarcane and sugar. The present sugarcane mean yields are between 70 and 80 Mg ha−1. The cost of sugarcane production in Guangxi is much higher than in Brazil. One of the major factors of the high cost is high N-fertilization. Over 60% of the sugarcane fields are applied with urea at over 600 kg ha−1 yr−1 (32). In Brazil, the present sugarcane mean yields are also between 70 and 80 Mg ha−1, but N-fertilizer mean applications are between 60 and 70 kg N ha−1 yr−1 (50). 15N isotope assays have demonstrated that some Brazilian sugarcane varieties are able to obtain considerable nitrogen from biological nitrogen fixation (BNF; 25, 49, 50). A number of nitrogen-fixing bacteria have been isolated from the rhizosphere and interior of sugarcane plants, and have shown potential to fix N2 associated with sugarcane plants (5, 13, 35, 43). BNF may help farmers to maintain sugarcane yields under reduced N-fertilization and develop environmentally benign sugarcane production in Guangxi. At present, little is known about the diversity and predominant population of nitrogen-fixing bacteria associated with the sugarcane plants growing in Guangxi. Recently, some nitrogen-fixing bacteria have been isolated from sugarcane plants grown in Guangxi (26, 28, 44, 52, 53) using NFb, JNFb and LGI-P media that were respectively used to isolate Azospirillum (10), Herbaspirillum (6) and Gluconacetobacter diazotrophicus (8); however, nitrogen-fixing bacteria belonging to the genera Azospirillum, Herbaspirillum, and Gluconacetobacter, which are predominantly associated with sugarcane plants in Brazil, have not been isolated. The diversity of nitrogen-fixing bacteria associated with sugarcane plants grown with high N-fertilization in Guangxi may be different from in Brazil. The ROC22 cultivar is the main sugarcane cultivar, growing in over 60% of sugarcane-planting areas in Guangxi. It is sensitive to low nitrogen stress and requires at least 150 kg ha−1 urea fertilization at the seedling stage for tillering and elongation of the plant cane crops (23). The recommended dose of urea fertilization for plant cane crops at the seedling stage is 180 kg ha−1, 30% of urea fertilization for a season (46). Recent studies have shown that nitrogen-fixing bacterial strains isolated from other sugarcane cultivars are able to provide nitrogen to micropropagated ROC22 sugarcane seedlings via BNF and promote sugarcane growth (26, 29); however, neither indigenous nitrogen-fixing bacteria associated with ROC22 sugarcane plants nor their associative BNF under recommended N-fertilization have been investigated. Here, we attempted to isolate a large number of nitrogen-fixing bacteria associated with ROC22 sugarcane plants, investigate their diversity and predominant affiliation, and evaluate their potential for plant-growth promotion and associative BNF under the recommended N-fertilization. We initially obtained 196 fast-growing isolates from rhizosphere soil and roots of ROC22 sugarcane plants grown in 14 production areas. Nitrogen-fixing isolates were screened using the acetylene reduction assay (ARA) and PCR amplification of the nifH gene encoding the iron protein of nitrogenase (55). We found that enterobacteria were predominant among the obtained nitrogen-fixing bacteria by analyzing their 16S rRNA gene (rrs) sequences. We further screened their plant growth-promoting activities, including the production of indole acetic acids (IAA) and siderophores, phosphate solubilization and ACC (1-aminocyclopropane-1-carboxylic acid) deamination. Finally, we chose two Enterobacter spp. strains isolated from the same ROC22 plant to inoculate micropropagated ROC22 sugarcane seedlings and investigate their plant growth-promoting and associative BNF activities under the recommended N-fertilization for ROC22 crops using the 15N isotope dilution technique.

Soybean ureide transporters play a critical role in nodule development, function and nitrogen export.

Abstract: Legumes can access atmospheric nitrogen through a symbiotic relationship with nitrogen-fixing bacteroids that reside in root nodules. In soybean, the products of fixation are the ureides allantoin and allantoic acid, which are also the dominant long-distance transport forms of nitrogen from nodules to the shoot. Movement of nitrogen assimilates out of the nodules occurs via the nodule vasculature; however, the molecular mechanisms for ureide export and the importance of nitrogen transport processes for nodule physiology have not been resolved. Here, we demonstrate the function of two soybean proteins - GmUPS1-1 (XP_003516366) and GmUPS1-2 (XP_003518768) - in allantoin and allantoic acid transport out of the nodule. Localization studies revealed the presence of both transporters in the plasma membrane, and expression in nodule cortex cells and vascular endodermis. Functional analysis in soybean showed that repression of GmUPS1-1 and GmUPS1-2 in nodules leads to an accumulation of ureides and decreased nitrogen partitioning to roots and shoot. It was further demonstrated that nodule development, nitrogen fixation and nodule metabolism were negatively affected in RNAi UPS1 plants. Together, we conclude that export of ureides from nodules is mediated by UPS1 proteins, and that activity of the transporters is not only essential for shoot nitrogen supply but also for nodule development and function.

Nitrogen fixation and identification of potential diazotrophs in the Canadian Arctic

Abstract: [1] Global gaseous nitrogen (N2) fixation rates may be underestimated and data is lacking from many regions without conspicuous diazotrophic cyanobacteria, such as cold oceans. We estimated N2 fixation rates at diverse sites in the Canadian Arctic, including the mouth of the Mackenzie River, the offshore Beaufort Sea, Lancaster Sound, Baffin Bay and a river influenced fjord. We also identified potential diazotrophic communities using a targeted survey of the nifH gene. Nitrogen fixation rates ranged from 0.02 nmol N L−1 d−1 in Baffin Bay to 4.45 nmol N L−1 d−1 in the Mackenzie River plume. Sequences recovered from the nifH gene survey belonged mainly to Cluster III, a group of nifH sequences associated with diverse microorganisms, with some α- andγ-proteobacterianifH genes at most sites. Cyanobacteria nifH genes with best matches to Nostocales, which are common in Arctic freshwaters, were recovered from the marine Beaufort Sea. The geographic pattern of N2 fixation rates and nifHgene identities suggest that the Mackenzie River is the source of a diazotrophic community that contributes new nitrogen to the nitrogen-depleted surface waters of the Beaufort Sea. This first record of N2 fixation at high latitudes refines our understanding of the global nitrogen budget.

Review: Nitrogen Fixing Microorganisms

Abstract: Microorganisms employed to enhance the availability of nutrients, viz., nitrogen (by fixing atmosphere N ), to the crops are called biofertilizers. In recent years, biofertilizers have emerged as an important 2 component for biological nitrogen fixation. It offers an economically attractive and ecologically sound route for augmenting nutrient supply. Plant growth promoting species are commonly used to improve crop yield. In addition to their agricultural usefulness, there are potential benefits in environmental applications. Thus various species of Rhizobium for legumes, blue - green algae (BGA) or cynaobacteria and Azolla (a fern containing symbiotic N - fixing BGA, i.e., Azolla Anabaena Azollae) for wet land rice and Azotobacter / Azospirillum for 2 several crops can play significant role in agriculture.

Endophytic and rhizospheric enterobacteria isolated from sugar cane have different potentials for producing plant growth-promoting substances

Abstract: Endophytic and rhizospheric environments differ in many respects, leading to the presence of different bacterial communities at each site. However, microorganisms such as enterobacteria can be found both within plants and in the surrounding soil. Bacteria must present differences in the traits that affect such environments in order to successfully colonise them. The present study compared the plant growth-promoting potential of diazotrophic enterobacteria isolated from the rhizosphere and from within surface-disinfected plants. A total of 46 diazotrophic enterobacterial strains (21 rhizospheric and 25 putatively endophytic) belonging to the Klebsiella and Enterobacter genera, which are prevalent in sugar cane plantations, were isolated from the rhizosphere and from surface-disinfected plants. Their ability to synthesise amino acids using combined nitrogen obtained from nitrogen fixation, and their ability to synthesise indole-3-acetic acid (IAA) were determined by high performance liquid chromatography. Endogenous ethylene production by the bacteria was measured using gas chromatography, and biocontrol of phytopathogenic fungi was determined qualitatively using a dual culture technique. The putative endophytes released significantly higher amounts of amino acids than the rhizospheric bacteria, whilst the latter produced higher quantities of ethylene and were more actively antagonistic to fungi. Both types of bacteria released similar amounts of IAA. Endophytic and rhizospheric bacteria differ in their capacity to release plant growth-promoting substances, which may be a reflection of their adaptations and an indication of their potential impact on their natural environment.

The integral membrane protein SEN1 is required for symbiotic nitrogen fixation in lotus japonicus nodules

Abstract: Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.

A Medicago truncatula tobacco retrotransposon insertion mutant collection with defects in nodule development and symbiotic nitrogen fixation.

Abstract: A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod−), 51 mutants with totally ineffective nodules (Nod+ Fix−), 17 mutants with partially ineffective nodules (Nod+ Fix+/−), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/− Fix−), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/− Fix+), and 11 supernodulating mutants (Nod++Fix+/−). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN’T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod− lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging.

The evolution of nitrogen fixation in cyanobacteria

Abstract: Motivation: Fixed nitrogen is an essential requirement for the biosynthesis of cellular nitrogenous compounds. Some cyanobacteria can fix nitrogen, contributing significantly to the nitrogen cycle, agriculture and biogeochemical history of Earth. The rate and position on the species phylogeny of gains and losses of this ability, as well as of the underlying nif genes, are controversial. Results: We use probabilistic models of trait evolution to investigate the presence and absence of cyanobacterial nitrogen-fixing ability. We estimate rates of change on the species phylogeny, pinpoint probable changes and reconstruct the state and nif gene complement of the ancestor. Our results are consistent with a nitrogen-fixing cyanobacterial ancestor, repeated loss of nitrogen fixation and vertical descent, with little horizontal transfer of the genes involved. Contact: db60@st-andrews.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Phylogenetic perspectives of nitrogen-fixing actinobacteria

Abstract: It was assumed for a long time that the ability to catalyze atmospheric nitrogen (diazotrophy) has a narrow distribution among actinobacteria being limited to the genus Frankia. Recently, the number of nitrogen fixation (nifH) genes identified in other non-Frankia actinobacteria has dramatically increased and has opened investigation on the origin and emergence of diazotrophy among actinobacteria. During the last decade, Mycobacterium flavum, Corynebacterium autotrophicum and a fluorescent Arthrobacter sp. have been reported to have nitrogenase activity, but these studies have not been further verified. Additional reports of nitrogen fixation by Agromyces, Microbacterium, Corynebacterium and Micromonospora isolated from root nodules of leguminous and actinorhizal plants have increased. For several actinobacteria, nitrogen fixation was demonstrated by the ability to grow on nitrogen-free medium, acetylene reduction activity, 15N isotope dilution analysis and identification of a nifH gene via PCR amplification. Moreover, the analyses of draft genome sequences of actinobacteria including Slackia exigua, Rothia mucilaginosa and Gordonibacter pamelaeae have also revealed the presence of nifH-like sequences. Whether these nifH sequences are associated with effective nitrogen fixation in these actinobacteria taxa has not yet been demonstrated. These genes may be vertically or horizontally transferred and be silent sequences. These ideas merit further investigation. This minireview presents a phylogenetic comparison of nitrogen fixation gene (nifH) with the aim of elucidating the processes underlying the evolutionary history of this catalytic ability among actinobacteria.

Recent developments in synthetic nitrogen fixation

Abstract: Within the background of biological nitrogen fixation mediated by the enzyme nitrogenase, transition-metal complexes capable of binding and activating dinitrogen towards protonation and reduction have been synthesised. Of particular interest in this research area has been the transition-metal mediated conversion of dinitrogen to ammonia (stoichiometric and catalytic) and the elucidation of the corresponding mechanistic pathways. The present review summarises recent studies in this field. To this end, the available complexes binding N2 and/or its intermediates on the pathway to ammonia are subdivided into systems containing early transition metals, Mo & W, iron and late transition metals. The implications of the findings obtained on low-molecular weight compounds with respect to the functionalisation of N2 and the reduction of N2 to ammonia on the FeMoco of nitrogenase are considered.

Effective factors on biological nitrogen fixation

Abstract: Although relationships among plant, biological N2 fixation, and response to soil and environmental conditions have received considerable coverage in the scientific literature, a comprehensive summary and interpretation of these interactions with specific emphasis are lacking. Fluctuations in pH, nutrient availability, temperature, and water status, among other factors, greatly influence the growth, survival, and metabolic activity of nitrogen fixation bacteria. The subsequent inhibition of nitrogenase would result in O2 accumulation in the infected zones, inducing the decrease in nodule permeability. Poor nodulation of legumes in arid soils is likely due to decreases in population levels of rhizobia during the dry season. Fixation, therefore, also tends to decrease with legume age, mainly because of the concomitant increase in soil N. Calcium deficiency, with or without the confounding influence of low pH also affects attachment of rhizobia to root hairs. Rhizobia may have different tolerances to soil acidity factors than the host plant. Relatively, high-root temperature has also been shown to influence infection, N2- fixation ability, and legume growth. Also, root nodulation by the bacteria can be dependent on the formation of mycorrhiza. Key words: Legume, nitrogen fixation, rhizobia, root, stress.