Showing papers on "Nitrogen fixation published in 2014"

Mechanism of Nitrogen Fixation by Nitrogenase: The Next Stage

Abstract: Brian M. Hoffman,* Dmitriy Lukoyanov, Zhi-Yong Yang,† Dennis R. Dean,*,‡ and Lance C. Seefeldt*,† †Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, Utah 84322, United States ‡Department of Biochemistry, Virginia Tech, 900 West Campus Drive, Blacksburg, Virginia 24061, United States Departments of Chemistry and Molecular Biosciences, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208, United States

Isolation and characterization of PGPR and their effect on growth, yield and nutrient content in wheat (Triticum aestivum L.)

Abstract: The aims of our study were to enhance growth, yield and micronutrient status of wheat crop by various combinations of microbial strains (Bacillus megaterium, Arthrobacter chlorophenolicus and Enterobacter sp.) under pot and field experiments. Microbial strains were isolated from soils of different cropping systems and characterized by biochemical and molecular methods. Microbial strains (B. megaterium BHU1 and A. chlorophenolicus BHU3) showed positive result for nitrogen fixation and phosphate solubilization, while Enterobacter sp. BHU5 gave positive result in nitrogen fixation only. However, A. chlorophenolicus and Enterobacter sp. showed HCN production while B. megaterium and Enterobacter sp. gave siderophore. Maximum N2-fixation and IAA production were observed with 15.0 mg N g−1 carbon by A. chlorophenolicus and 26.4 µg ml−1 at tryptophan 100 µg ml−1 by Enterobacter sp, respectively. Triple combination of strains B. megaterium, A. chlorophenolicus and Enterobacter significantly increased 17.5%, 79.8%, 78.6% and 26.7% plant height, grain yield, straw yield and test weight under pot condition and also 29.4%, 27.5%, 29.5% and 17.6% under field condition, respectively. Similarly these treatment combinations showed maximum nutrient acquisition and content of micronutrient viz. Fe, Cu, Mn and Zn in grain of wheat under both conditions. The results showed that the combined application of indigenous PGPR, B. megaterium, A. chlorophenolicus and Enterobacter can be used as efficient microbial consortium for wheat production.

Synthetic biology approaches to engineering the nitrogen symbiosis in cereals

Abstract: Nitrogen is abundant in the earth’s atmosphere but, unlike carbon, cannot be directly assimilated by plants. The limitation this places on plant productivity has been circumvented in contemporary agriculture through the production and application of chemical fertilizers. The chemical reduction of nitrogen for this purpose consumes large amounts of energy and the reactive nitrogen released into the environment as a result of fertilizer application leads to greenhouse gas emissions, as well as widespread eutrophication of aquatic ecosystems. The environmental impacts are intensified by injudicious use of fertilizers in many parts of the world. Simultaneously, limitations in the production and supply of chemical fertilizers in other regions are leading to low agricultural productivity and malnutrition. Nitrogen can be directly fixed from the atmosphere by some bacteria and Archaea, which possess the enzyme nitrogenase. Some plant species, most notably legumes, have evolved close symbiotic associations with nitrogen-fixing bacteria. Engineering cereal crops with the capability to fix their own nitrogen could one day address the problems created by the over- and under-use of nitrogen fertilizers in agriculture. This could be achieved either by expression of a functional nitrogenase enzyme in the cells of the cereal crop or through transferring the capability to form a symbiotic association with nitrogen-fixing bacteria. While potentially transformative, these biotechnological approaches are challenging; however, with recent advances in synthetic biology they are viable long-term goals. This review discusses the possibility of these biotechnological solutions to the nitrogen problem, focusing on engineering the nitrogen symbiosis in cereals.

Phytoremediation of Heavy and Transition Metals Aided by Legume-Rhizobia Symbiosis

Abstract: Legumes are important for nitrogen cycling in the environment and agriculture due to the ability of nitrogen fixation by rhizobia. In this review, we introduce an important and potential role of legume-rhizobia symbiosis in aiding phytoremediation of some metal contaminated soils as various legumes have been found to be the dominant plant species in metal contaminated areas. Resistant rhizobia used for phytoremediation could act on metals directly by chelation, precipitation, transformation, biosorption and accumulation. Moreover, the plant growth promoting (PGP) traits of rhizobia including nitrogen fixation, phosphorus solubilization, phytohormone synthesis, siderophore release, and production of ACC deaminase and the volatile compounds of acetoin and 2, 3-butanediol may facilitate legume growth while lessening metal toxicity. The benefits of using legumes inoculated with naturally resistant rhizobia or recombinant rhizobia with enhanced resistance, as well as co-inoculation with other plant growth promoting bacteria (PGPB) are discussed. However, the legume-rhizobia symbiosis appears to be sensitive to metals, and the effect of metal toxicity on the interaction between legumes and rhizobia is not clear. Therefore, to obtain the maximum benefits from legumes assisted by rhizobia for phytoremediation of metals, it is critical to have a good understanding of interactions between PGP traits, the symbiotic plant-rhizobia relationship and metals.

Biological nitrogen fixation and biomass accumulation within poplar clones as a result of inoculations with diazotrophic endophyte consortia

Abstract: Sustainable production of biomass for bioenergy relies on low-input crop production. Inoculation of bioenergy crops with plant growth-promoting endophytes has the potential to reduce fertilizer inputs through the enhancement of biological nitrogen fixation (BNF). Endophytes isolated from native poplar growing in nutrient-poor conditions were selected for a series of glasshouse and field trials designed to test the overall hypothesis that naturally occurring diazotrophic endophytes impart growth promotion of the host plants. Endophyte inoculations contributed to increased biomass over uninoculated control plants. This growth promotion was more pronounced with multi-strain consortia than with single-strain inocula. Biological nitrogen fixation was estimated through (15)N isotope dilution to be 65% nitrogen derived from air (Ndfa). Phenotypic plasticity in biomass allocation and branch production observed as a result of endophyte inoculations may be useful in bioenergy crop breeding and engineering programs.

Nitrogen signalling in plant interactions with associative and endophytic diazotrophic bacteria

Abstract: Some beneficial plant-interacting bacteria can biologically fix N2 to plant-available ammonium. Biological nitrogen fixation (BNF) is an important source of nitrogen (N) input in agriculture and represents a promising substitute for chemical N fertilizers. Diazotrophic bacteria have the ability to develop different types of root associations with different plant species. Among the highest rates of BNF are those measured in legumes nodulated by endosymbionts, an already very well documented model of plant-diazotrophic bacterial association. However, it has also been shown that economically important crops, especially monocots, can obtain a substantial part of their N needs from BNF by interacting with associative and endophytic diazotrophic bacteria, that either live near the root surface or endophytically colonize intercellular spaces and vascular tissues of host plants. One of the best reported outcomes of this association is the promotion of plant growth by direct and indirect mechanisms. Besides fixing N, these bacteria can also produce plant growth hormones, and some species are reported to improve nutrient uptake and increase plant tolerance against biotic and abiotic stresses. Thus, this particular type of plant-bacteria association consists of a natural beneficial system to be explored; however, the regulatory mechanisms involved are still not clear. Plant N status might act as a key signal, regulating and integrating various metabolic processes that occur during association with diazotrophic bacteria. This review will focus on the recent progress in understanding plant association with associative and endophytic diazotrophic bacteria, particularly on the knowledge of the N networks involved in BNF and in the promotion of plant growth.

Nitrogen Fertilization Has a Stronger Effect on Soil Nitrogen-Fixing Bacterial Communities than Elevated Atmospheric CO2

Abstract: Biological nitrogen fixation is the primary supply of N to most ecosystems, yet there is considerable uncertainty about how N-fixing bacteria will respond to global change factors such as increasing atmospheric CO2 and N deposition. Using the nifH gene as a molecular marker, we studied how the community structure of N-fixing soil bacteria from temperate pine, aspen, and sweet gum stands and a brackish tidal marsh responded to multiyear elevated CO2 conditions. We also examined how N availability, specifically, N fertilization, interacted with elevated CO2 to affect these communities in the temperate pine forest. Based on data from Sanger sequencing and quantitative PCR, the soil nifH composition in the three forest systems was dominated by species in the Geobacteraceae and, to a lesser extent, Alphaproteobacteria. The N-fixing-bacterial-community structure was subtly altered after 10 or more years of elevated atmospheric CO2, and the observed shifts differed in each biome. In the pine forest, N fertilization had a stronger effect on nifH community structure than elevated CO2 and suppressed the diversity and abundance of N-fixing bacteria under elevated atmospheric CO2 conditions. These results indicate that N-fixing bacteria have complex, interacting responses that will be important for understanding ecosystem productivity in a changing climate.

Facets of diazotrophy in the oxygen minimum zone waters off Peru.

Abstract: Nitrogen fixation, the biological reduction of dinitrogen gas (N2) to ammonium (NH4+), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N2 fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N2 fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N2 fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N2 fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO2− and PO43− are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N2 fixation may increase in the future.

A review on possible elicitor molecules of cyanobacteria: their role in improving plant growth and providing tolerance against biotic or abiotic stress.

Abstract: Cyanobacteria are prominent inhabitants of many agricultural soils, where they potentially contribute towards biological nitrogen fixation, help in phosphate solubilization and mineral release to improve soil fertility and crop productivity. However, beside naturally fertilizing and balancing mineral nutrition in the soil, many cyanobacteria are known to release various kinds of biologically active substances like proteins, vitamins, carbohydrates, amino acids, polysaccharides and phytohormones that function as elicitor molecules to promote plant growth and help them to fight against biotic and abiotic stress. These metabolites produced by the cyanobacteria affect the gene expression of the host plants and thereby bring about qualitative and quantitative changes in the phytochemical composition of the plants. Experiments carried out with live inoculum or with the extracts of cyanobacterial strains on several plant species, such as rice, wheat, maize, cotton etc., have demonstrated the synthesis of signalling metabolites. Thus, in view of its beneficial effect, this paper reviews the role of cyanobacteria in triggering the growth and development of plants and hence its utilization in agriculture.

Metaproteomic identification of diazotrophic methanotrophs and their localization in root tissues of field-grown rice plants

Abstract: In a previous study by our group, CH4 oxidation and N2 fixation were simultaneously activated in the roots of wild-type rice plants in a paddy field with no N input; both processes are likely controlled by a rice gene for microbial symbiosis. The present study examined which microorganisms in rice roots were responsible for CH4 oxidation and N2 fixation under the field conditions. Metaproteomic analysis of root-associated bacteria from field-grown rice (Oryza sativa Nipponbare) revealed that nitrogenase complex-containing nitrogenase reductase (NifH) and the alpha subunit (NifD) and beta subunit (NifK) of dinitrogenase were mainly derived from type II methanotrophic bacteria of the family Methylocystaceae, including Methylosinus spp. Minor nitrogenase proteins such as Methylocella, Bradyrhizobium, Rhodopseudomonas, and Anaeromyxobacter were also detected. Methane monooxygenase proteins (PmoCBA and MmoXYZCBG) were detected in the same bacterial group of the Methylocystaceae. Because these results indicated that Methylocystaceae members mediate both CH4 oxidation and N2 fixation, we examined their localization in rice tissues by using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). The methanotrophs were localized around the epidermal cells and vascular cylinder in the root tissues of the field-grown rice plants. Our metaproteomics and CARD-FISH results suggest that CH4 oxidation and N2 fixation are performed mainly by type II methanotrophs of the Methylocystaceae, including Methylosinus spp., inhabiting the vascular bundles and epidermal cells of rice roots.

Nitrogen-fixing Rhizobium-legume symbiosis: are polyploidy and host peptide-governed symbiont differentiation general principles of endosymbiosis?

Abstract: The symbiosis between rhizobia soil bacteria and legumes is facultative and initiated by nitrogen starvation of the host plant. Exchange of signal molecules between the partners leads to the formation of root nodules where bacteria are converted to nitrogen-fixing bacteroids. In this mutualistic symbiosis, the bacteria provide nitrogen sources for plant growth in return for photosynthates from the host. Depending on the host plant the symbiotic fate of bacteria can either be reversible or irreversible. In Medicago plants the bacteria undergo a host-directed multistep differentiation process culminating in the formation of elongated and branched polyploid bacteria with definitive loss of cell division ability. The plant factors are nodule-specific symbiotic peptides. About 500 of them are cysteine-rich NCR peptides produced in the infected plant cells. NCRs are targeted to the endosymbionts and the concerted action of different sets of peptides governs different stages of endosymbiont maturation. This review focuses on symbiotic plant cell development and terminal bacteroid differentiation and demonstrates the crucial roles of symbiotic peptides by showing an example of multi-target mechanism exerted by one of these symbiotic peptides.

Possible contribution of alternative nitrogenases to nitrogen fixation by asymbiotic N2-fixing bacteria in soils

Abstract: The relative contributions of the alternative (V- and Fe-only) and canonical (Mo-) nitrogenases to N 2 fixation in natural environments are still largely unknown. Here we combine chemical and molecular approaches to examine their role in terrestrial ecosystems. Pure-culture studies show that while the R ratio (defined as R = acetylene reduction rate/N 2 fixation rate by nitrogenase) is highly variable, low R ratios (0.5 R R ratios and a large increase in N 2 fixation rates in response to vanadium amendments, indicating V-nitrogenase activity. Using RT-PCR, we were able to demonstrate expression of the V-nitrogenase, together with the Mo-nitrogenase, in these soils. An analysis of published R ratios showed that the contribution of alternative nitrogenases to asymbiotic N 2 fixation in soils may be more prevalent than previously recognized.

Interactions among nitrogen fixation and soil phosphorus acquisition strategies in lowland tropical rain forests

Abstract: Paradoxically, symbiotic dinitrogen (N2 ) fixers are abundant in nitrogen (N)-rich, phosphorus (P)-poor lowland tropical rain forests. One hypothesis to explain this pattern states that N2 fixers have an advantage in acquiring soil P by producing more N-rich enzymes (phosphatases) that mineralise organic P than non-N2 fixers. We assessed soil and root phosphatase activity between fixers and non-fixers in two lowland tropical rain forest sites, but also addressed the hypothesis that arbuscular mycorrhizal (AM) colonisation (another P acquisition strategy) is greater on fixers than non-fixers. Root phosphatase activity and AM colonisation were higher for fixers than non-fixers, and strong correlations between AM colonisation and N2 fixation at both sites suggest that the N-P interactions mediated by fixers may generally apply across tropical forests. We suggest that phosphatase enzymes and AM fungi enhance the capacity of N2 fixers to acquire soil P, thus contributing to their high abundance in tropical forests.

Molecular characterization and identification of plant growth promoting endophytic bacteria isolated from the root nodules of pea ( Pisum sativum L.)

Abstract: Root nodule accommodates various non-nodulating bacteria at varying densities. Present study was planned to identify and characterize the non-nodulating bacteria from the pea plant. Ten fast growing bacteria were isolated from the root nodules of cultivated pea plants. These bacterial isolates were unable to nodulate pea plants in nodulation assay, which indicate the non-rhizobial nature of these bacteria. Bacterial isolates were tested in vitro for plant growth promoting properties including indole acetic acid (IAA) production, nitrogen fixation, phosphate solubilization, root colonization and biofilm formation. Six isolates were able to produce IAA at varying level from 0.86 to 16.16 μg ml(-1), with the isolate MSP9 being most efficient. Only two isolates, MSP2 and MSP10, were able to fix nitrogen. All isolates were able to solubilize inorganic phosphorus ranging from 5.57 to 11.73 μg ml(-1), except MSP4. Bacterial isolates showed considerably better potential for colonization on pea roots. Isolates MSP9 and MSP10 were most efficient in biofilm formation on polyvinyl chloride, which indicated their potential to withstand various biotic and abiotic stresses, whereas the remaining isolates showed a very poor biofilm formation ability. The most efficient plant growth promoting agents, MSP9 and MSP10, were phylogenetically identified by 16S rRNA gene sequence analysis as Ochrobactrum and Enterobacter, respectively, with 99% similarity. It is suggested the potential endophytic bacterial strains, Ochrobactrum sp. MSP9 and Enterobacter sp. MSP10, can be used as biofertilizers for various legume and non-legume crops after studying their interaction with the host crop and field evaluation.

Fixating on metals: new insights into the role of metals in nodulation and symbiotic nitrogen fixation

Abstract: Symbiotic nitrogen fixation is one of the most promising and immediate alternatives to the overuse of polluting nitrogen fertilizers for improving plant nutrition. At the core of this process are a number of metalloproteins that catalyze and provide energy for the conversion of atmospheric nitrogen to ammonia, eliminate free radicals produced by this process, and create the microaerobic conditions required by these reactions. In legumes, metal cofactors are provided to endosymbiotic rhizobia within root nodule cortical cells. However, low metal bioavailability is prevalent in most soils types, resulting in widespread plant metal deficiency and decreased nitrogen fixation capabilities. As a result, renewed efforts have been undertaken to identify the mechanisms governing metal delivery from soil to the rhizobia, and to determine how metals are used in the nodule and how they are recycled once the nodule is no longer functional. This effort is being aided by improved legume molecular biology tools (genome projects, mutant collections, and transformation methods), in addition to state-of-the-art metal visualization systems.

Salt-tolerant and plant growth-promoting bacteria isolated from Zn/Cd contaminated soil: identification and effect on rice under saline conditions

Abstract: The bacteria of PDMCd0501, PDMCd2007, and PDMZnCd2003 were isolated from a Zn/Cd contaminated soil. They were classified as salt-tolerant bacteria in this experiment. The bacteria had indole-3-acetic acids (IAA) production, nitrogen fixation, and phosphate solubilization, under 8% (w/v) NaCl condition. Biochemical test (API 20E) and 16S rDNA sequencing identified PDMCd2007 and PDMCd0501 as Serratia sp. and PDMZnCd2003 was Pseudomonas sp. The effect of Pseudomonas sp. PDMZnCd2003 on the germination and seedlings of Oryza sativa L.cv. RD6 was determined under a salinity of 0–16 dS/m. The salinity levels of 4–16 dS/m affected to decrease germination and seedlings of rice. Comparison between uninoculated and inoculated system, however, Pseudomonas sp. PDMZnCd2003 had a negative impact on the rice growth. This unexpected effect was a case that should be concerned and studied further before application as a plant growth-promoting bacteria (PGPB).

A new species of Burkholderia isolated from sugarcane roots promotes plant growth.

Abstract: Sugarcane is a globally important food, biofuel and biomaterials crop. High nitrogen (N) fertilizer rates aimed at increasing yield often result in environmental damage because of excess and inefficient application. Inoculation with diazotrophic bacteria is an attractive option for reducing N fertilizer needs. However, the efficacy of bacterial inoculants is variable, and their effective formulation remains a knowledge frontier. Here, we take a new approach to investigating diazotrophic bacteria associated with roots using culture-independent microbial community profiling of a commercial sugarcane variety (Q208(A) ) in a field setting. We first identified bacteria that were markedly enriched in the rhizosphere to guide isolation and then tested putative diazotrophs for the ability to colonize axenic sugarcane plantlets (Q208(A) ) and promote growth in suboptimal N supply. One isolate readily colonized roots, fixed N2 and stimulated growth of plantlets, and was classified as a new species, Burkholderia australis sp. nov. Draft genome sequencing of the isolate confirmed the presence of nitrogen fixation. We propose that culture-independent identification and isolation of bacteria that are enriched in rhizosphere and roots, followed by systematic testing and confirming their growth-promoting capacity, is a necessary step towards designing effective microbial inoculants.

Phenotypic and molecular characterisation of efficient nitrogen-fixing Azotobacter strains from rice fields for crop improvement

Abstract: Biological nitrogen fixation (BNF) is highly effective in the field and potentially useful to reduce adverse effects chemical fertilisers. Here, Azotobacter species were selected via phenotypic, biochemical and molecular characterisations from different rice fields. Acetylene reduction assay of Azotobacter spp. showed that Azotobacter vinelandii (Az3) fixed higher amount of nitrogen (121.09 nmol C2H4 mg-1 bacteria h-1). Likewise, its plant growth functions, viz. siderophore, hydrogen cyanide, salicylic acid, IAA, GA3, zeatin, NH3, phosphorus solubilisation, ACC deaminase and iron tolerance, were also higher. The profile of gDNA, plasmid DNA and cellular protein profile depicted inter-generic and inter-specific diversity among the isolates of A. vinelandii. The PCR-amplified genes nifH, nifD and nifK of 0.87, 1.4 and 1.5 kb , respectively, were ascertained by Southern blot hybridisation in isolates of A. vinelandii. The 16S rRNA sequence from A. vinelandii (Az3) was novel, and its accession number (JQ796077) was received from NCBI data base. Biofertiliser formulation of novel A. vinelandii isolates along with commercial one was evaluated in rice (Oriza sativa L. var. Khandagiri) fields. The present finding revealed that treatment T4 (Az3) (A. vinelandii) are highly efficient to improved growth and yield of rice crop.

Nitrogen Fixing Cyanobacteria: Future Prospect

Abstract: Cyanobacteria are often called "blue-green algae", this name is convenient for talking about organisms in water that make their own food, but does not reflect any relationship between the cyanobacteria and other organisms called algae. Cyanobacteria are relatives to bacteria, not eukaryotes, and it is only the chloroplast in eukaryotic algae to which cya‐ nobacteria are related. Some cyanobacteria are aquatic and photosynthetic, that is, they live in water, and can manufacture their own food. They are quite small and usually uni‐ cellular, though they often grow in colonies large enough to see. In fact, it may surprise you then to know that the cyanobacteria are still around; they are one of the largest and most important groups of bacteria on earth (Berry et al, 2008). The great contribution of cyanobacteria is the origin of plants chloroplast with which plants make food for them‐ selves is actually a cyanobacterium living within the plant's cells. Sometime in the late Proterozoic or in the early Cambrian, cyanobacteria began to take up residence within certain eukaryote cells, making food for the eukaryote host in return for a home. This event is known as endosymbiosis, and is also the origin of eukaryotic mitochondrion (Is‐ sa et al., 2002). Majority of cyanobacteria are aerobic photoautotrophs, their life processes require only water, carbon dioxide, inorganic substances and light. Photosynthesis is their principal mode of energy metabolism. In the natural environment, however, it is known that some species are able to survive long periods in complete darkness. Furthermore, cer‐ tain cyanobacteria show a distinct ability for heterotrophic nutrition (Fay, 1965). Cyano‐ bacteria might be the first plants to colonies bare areas of rock and soil. Adaptations, such as ultraviolet absorbing sheath pigments, increase their fitness in the relatively exposed land environment. Many species are capable of living in soil and other terrestrial habi‐ tats, where they are important in the functional processes of ecosystems and cycling of nutrient elements (Whitton, 1992). The prominent habitats of cyanobacteria are limnic and

Abundance and diversity of soybean-nodulating rhizobia in black soil are impacted by land use and crop management.

Abstract: To investigate the effects of land use and crop management on soybean rhizobial communities, 280 nodule isolates were trapped from 7 fields with different land use and culture histories. Besides the known Bradyrhizobium japonicum, three novel genospecies were isolated from these fields. Grassland (GL) maintained a higher diversity of soybean bradyrhizobia than the other cultivation systems. Two genospecies (Bradyrhizobium spp. I and III) were distributed widely in all treatments, while Bradyrhizobium sp. II was found only in GL treatment. Cultivation with soybeans increased the rhizobial abundance and diversity, except for the soybean monoculture (S-S) treatment. In monoculture systems, soybeans favored Bradyrhizobium sp. I, while maize and wheat favored Bradyrhizobium sp. III. Fertilization decreased the rhizobial diversity indexes but did not change the species composition. The organic carbon (OC) and available phosphorus (AP) contents and pH were the main soil parameters positively correlated with the distribution of Bradyrhizobium spp. I and II and Bradyrhizobium japonicum and negatively correlated with Bradyrhizobium sp. III. These results revealed that different land uses and crop management could not only alter the diversity and abundance of soybean rhizobia, but also change interactions between rhizobia and legume or nonlegume plants, which offered novel information about the biogeography of rhizobia.

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Abstract: All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium- or iron-dependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

Diazotroph community structure and abundance in wheat–fallow and wheat–pea crop rotations

Abstract: Biological input of nitrogen (N) from the atmosphere by free-living diazotrophs can help alleviate fertilizer use in agricultural systems. In this study, we investigated the effect of N fertilizer and winter pea ( Pisum sativum L.) crop on the community structure and abundance of free-living diazotrophs in a two year study of dryland winter wheat ( Triticum aestivum L.) no-till production system in Eastern Oregon, USA. Based on quantification of the nifH gene, diazotroph abundance was strongly influenced by plant species and the crop year in which the soil samples were collected. A greater amount of nifH copies was recovered in 2012 compared to 2011 either as copies per gram soil or normalized to the abundance of bacterial 16S rRNA genes. The quantity of genes was greater under pea than wheat in 2012 although no difference was observed in the preceding year. The nifH gene abundance was positively correlated to ammonium concentration in 2011 and bacterial abundance in 2012. Nitrogen application did not influence diazotroph abundance in the top 0–5 cm; however the abundance was reduced by application at the lower 5–10 cm depth under wheat crop. The diazotroph community structure appeared to be influenced more by N fertilization rather than plant species with the exception of wheat in 2012. Changes in the community structure over the two years were greater for fertilized than unfertilized soil. Collectively, these data suggest that year-to-year variability had a greater influence on diazotroph communities rather than specific parameters of plant species, fertilization, total N, total organic C, or soil pH. Multi-year studies are necessary to define the specific drivers of diazotroph abundance, community structure and function.

Physiological and genetic characterization of rice nitrogen fixer PGPR isolated from rhizosphere soils of different crops

Abstract: We aimed to identify plant growth-promoting rhizobacteria that could be used to develop a biofertilizer for rice. To obtain plant growth-promoting rhizobacteria, rhizosphere soils from different crops (rice, wheat, oats, crabgrass, maize, ryegrass, and sweet potato) were inoculated to rice plants. In total, 166 different bacteria were isolated and their plant growth-promoting traits were evaluated in terms of colony morphology, indole-3-acetic acid production, acetylene reduction activity, and phosphate solubilization activity. Moreover, genetic analysis was carried out to evaluate their phylogenetic relationships based on 16S rRNA sequence data. Strains of Bacillus altitudinis, Pseudomonas monteilii, and Pseudomonas mandelii formed associations with rice plants and fixed nitrogen. A strain of Rhizobium daejeonense showed nitrogen fixation activity in an in vitro assay and in vivo. Strains of B. altitudinis and R. daejeonense derived from rice rhizosphere soil, strains of P. monteilii and Enterobacter cloacae derived from wheat rhizosphere soil, and a strain of Bacillus pumilus derived from maize rhizosphere soil significantly promoted rice plant growth. These methods are effective to identify candidate species that could be developed as biofertilizers for target crops.

Diversity of Nitrogen-Fixing Bacteria Associated with Switchgrass in the Native Tallgrass Prairie of Northern Oklahoma

Abstract: Switchgrass (Panicum virgatum L.) is a perennial C4 grass native to North America that is being developed as a feedstock for cellulosic ethanol production. Industrial nitrogen fertilizers enhance switchgrass biomass production but add to production and environmental costs. A potential sustainable alternative source of nitrogen is biological nitrogen fixation. As a step in this direction, we studied the diversity of nitrogen-fixing bacteria (NFB) associated with native switchgrass plants from the tallgrass prairie of northern Oklahoma (United States), using a culture-independent approach. DNA sequences from the nitrogenase structural gene, nifH, revealed over 20 putative diazotrophs from the alpha-, beta-, delta-, and gammaproteobacteria and the firmicutes associated with roots and shoots of switchgrass. Alphaproteobacteria, especially rhizobia, predominated. Sequences derived from nifH RNA indicated expression of this gene in several bacteria of the alpha-, beta-, delta-, and gammaproteobacterial groups associated with roots. Prominent among these were Rhizobium and Methylobacterium species of the alphaproteobacteria, Burkholderia and Azoarcus species of the betaproteobacteria, and Desulfuromonas and Geobacter species of the deltaproteobacteria.

Efficiency of partner choice and sanctions in Lotus is not altered by nitrogen fertilization

Abstract: Eukaryotic hosts must exhibit control mechanisms to select against ineffective bacterial symbionts. Hosts can minimize infection by less-effective symbionts (partner choice) and can divest of uncooperative bacteria after infection (sanctions). Yet, such host-control traits are predicted to be context dependent, especially if they are costly for hosts to express or maintain. Legumes form symbiosis with rhizobia that vary in symbiotic effectiveness (nitrogen fixation) and can enforce partner choice as well as sanctions. In nature, legumes acquire fixed nitrogen from both rhizobia and soils, and nitrogen deposition is rapidly enriching soils globally. If soil nitrogen is abundant, we predict host control to be downregulated, potentially allowing invasion of ineffective symbionts. We experimentally manipulated soil nitrogen to examine context dependence in host control. We co-inoculated Lotus strigosus from nitrogen depauperate soils with pairs of Bradyrhizobium strains that vary in symbiotic effectiveness and fertilized plants with either zero nitrogen or growth maximizing nitrogen. We found efficient partner choice and sanctions regardless of nitrogen fertilization, symbiotic partner combination or growth season. Strikingly, host control was efficient even when L. strigosus gained no significant benefit from rhizobial infection, suggesting that these traits are resilient to short-term changes in extrinsic nitrogen, whether natural or anthropogenic.

Influence of Arbuscular Mycorrhizae on Biomass Production and Nitrogen Fixation of Berseem Clover Plants Subjected to Water Stress

Abstract: Several studies, performed mainly in pots, have shown that arbuscular mycorrhizal symbiosis can mitigate the negative effects of water stress on plant growth. No information is available about the effects of arbuscular mycorrhizal symbiosis on berseem clover growth and nitrogen (N) fixation under conditions of water shortage. A field experiment was conducted in a hilly area of inner Sicily, Italy, to determine whether symbiosis with AM fungi can mitigate the detrimental effects of drought stress (which in the Mediterranean often occurs during the late period of the growing season) on forage yield and symbiotic N2 fixation of berseem clover. Soil was either left under water stress (i.e., rain-fed conditions) or the crop was well-watered. Mycorrhization treatments consisted of inoculation of berseem clover seeds with arbuscular mycorrhizal spores or suppression of arbuscular mycorrhizal symbiosis by means of fungicide treatments. Nitrogen biological fixation was assessed using the 15N-isotope dilution technique. Arbuscular mycorrhizal symbiosis was able to mitigate the negative effect of water stress on berseem clover grown in a typical semiarid Mediterranean environment. In fact, under water stress conditions, arbuscular mycorrhizal symbiosis resulted in increases in total biomass, N content, and N fixation, whereas no effect of crop mycorrhization was observed in the well-watered treatment.

Azotobacter vinelandii siderophore can provide nitrogen to support the culture of the green algae Neochloris oleoabundans and Scenedesmus sp. BA032.

Abstract: Microalgae are viewed as a potential future agricultural and biofuel feedstock and also provide an ideal biological means of carbon sequestration based on rapid growth rates and high biomass yields. Any potential improvement using high-yield microalgae to fix carbon will require additional fertilizer inputs to provide the necessary nitrogen required for protein and nucleotide biosynthesis. The free-living diazotroph Azotobacter vinelandii can fix nitrogen under aerobic conditions in the presence of reduced carbon sources such as sucrose or glycerol and is also known to produce a variety of siderophores to scavenge different metals from the environment. In this study, we identified two strains of green algae, Neochloris oleoabundans and Scenedesmus sp. BA032, that are able to utilize the A. vinelandii siderophore azotobactin as a source of nitrogen to support growth. When grown in a co-culture, S. sp. BA032 and N. oleoabundans obtained the nitrogen required for growth through the association with A. vinelandii. These results, indicating a commensalistic relationship, provide a proof of concept for developing a mutualistic or symbiotic relationship between these two species using siderophores as a nitrogen shuttle and might further indicate an additional fate of siderophores in the environment.