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Showing papers on "Nitrogen fixation published in 2010"


Journal ArticleDOI
TL;DR: The results show that almost 80% of African countries are confronted with nitrogen scarcity or nitrogen stress problems, which, along with poverty, cause food insecurity and malnutrition.
Abstract: Crop production is the single largest cause of human alteration of the global nitrogen cycle. We present a comprehensive assessment of global nitrogen flows in cropland for the year 2000 with a spatial resolution of 5 arc-minutes. We calculated a total nitrogen input (IN) of 136.60 trillion grams (Tg) of N per year, of which almost half is contributed by mineral nitrogen fertilizers, and a total nitrogen output (OUT) of 148.14 Tg of N per year, of which 55% is uptake by harvested crops and crop residues. We present high-resolution maps quantifying the spatial distribution of nitrogen IN and OUT flows, soil nitrogen balance, and surface nitrogen balance. The high-resolution data are aggregated at the national level on a per capita basis to assess nitrogen stress levels. The results show that almost 80% of African countries are confronted with nitrogen scarcity or nitrogen stress problems, which, along with poverty, cause food insecurity and malnutrition. The assessment also shows a global average nitrogen recovery rate of 59%, indicating that nearly two-fifths of nitrogen inputs are lost in ecosystems. More effective management of nitrogen is essential to reduce the deleterious environmental consequences.

498 citations


Journal ArticleDOI
TL;DR: The results suggest that the successional stages of the ecosystem are strongly linked to bacterial functional group abundance, and that the legacy of agricultural practices can be sustained over decades.
Abstract: Nitrogen fixing and denitrifying bacteria, respectively, control bulk inputs and outputs of nitrogen in soils, thereby mediating nitrogen-based greenhouse gas emissions in an ecosystem. Molecular techniques were used to evaluate the relative abundances of nitrogen fixing, denitrifying and two numerically dominant ribotypes (based on the > or =97% sequence similarity at the 16S rRNA gene) of bacteria in plots representing 10 agricultural and other land-use practices at the Kellogg biological station long-term ecological research site. Quantification of nitrogen-related functional genes (nitrite reductase, nirS; nitrous oxide reductase, nosZ; and nitrogenase, nifH) as well as two dominant 16S ribotypes (belonging to the phyla Acidobacteria, Thermomicrobia) allowed us to evaluate the hypothesis that microbial community differences are linked to greenhouse gas emissions under different land management practices. Our results suggest that the successional stages of the ecosystem are strongly linked to bacterial functional group abundance, and that the legacy of agricultural practices can be sustained over decades. We also link greenhouse gas emissions with specific compositional responses in the soil bacterial community and assess the use of denitrifying gene abundances as proxies for determining nitrous oxide emissions from soils.

275 citations


Journal ArticleDOI
TL;DR: The current state of knowledge of symbiotic relationships between prokaryotes involved in nitrogen fixation and other nitrogen transformations in eukaryotic hosts in the marine environment is reviewed.

201 citations


Journal ArticleDOI
TL;DR: This review presents an overview of approaches that have been used for the molecular study of ammonia oxidizers and discusses their application in different environments.
Abstract: The oxidation of ammonia plays a significant role in the transformation of fixed nitrogen in the global nitrogen cycle. Autotrophic ammonia oxidation is known in three groups of microorganisms. Aerobic ammonia-oxidizing bacteria and archaea convert ammonia into nitrite during nitrification. Anaerobic ammonia-oxidizing bacteria (anammox) oxidize ammonia using nitrite as electron acceptor and producing atmospheric dinitrogen. The isolation and cultivation of all three groups in the laboratory are quite problematic due to their slow growth rates, poor growth yields, unpredictable lag phases, and sensitivity to certain organic compounds. Culture-independent approaches have contributed importantly to our understanding of the diversity and distribution of these microorganisms in the environment. In this review, we present an overview of approaches that have been used for the molecular study of ammonia oxidizers and discuss their application in different environments.

161 citations


Journal ArticleDOI
TL;DR: Marine waters are generally considered to be nitrogen (N) limited and are therefore favourable environments for diazotrophs, i.e. organisms converting atmospheric N-2 into ammonium or nitrogen oxid ...
Abstract: Marine waters are generally considered to be nitrogen (N) limited and are therefore favourable environments for diazotrophs, i.e. organisms converting atmospheric N-2 into ammonium or nitrogen oxid ...

155 citations


Journal ArticleDOI
TL;DR: This study strongly suggests that Micromonospora populations are natural inhabitants of nitrogen-fixing root nodules of Lupinus angustifolius, and that some of them may represent new species.
Abstract: Our current knowledge of plant-microbe interactions indicate that populations inhabiting a host plant are not restricted to a single microbial species but comprise several genera and species. No one knows if communities inside plants interact, and it has been speculated that beneficial effects are the result of their combined activities. During an ecological study of nitrogen-fixing bacterial communities from Lupinus angustifolius collected in Spain, significant numbers of orange-pigmented actinomycete colonies were isolated from surface-sterilized root nodules. The isolates were analysed by BOX-PCR fingerprinting revealing an unexpectedly high genetic variation. Selected strains were chosen for 16S rRNA gene sequencing and phylogenetic analyses confirmed that all strains isolated belonged to the genus Micromonospora and that some of them may represent new species. To determine the possibility that the isolates fixed atmospheric nitrogen, chosen strains were grown in nitrogen-free media, obtaining in some cases, significant growth when compared with the controls. These strains were further screened for the presence of the nifH gene encoding dinitrogenase reductase, a key enzyme in nitrogen fixation. The partial nifH-like gene sequences obtained showed a 99% similarity with the sequence of the nifH gene from Frankia alni ACN14a, an actinobacterium that induces nodulation and fixes nitrogen in symbiosis with Alnus. In addition, in situ hybridization was performed to determine if these microorganisms inhabit the inside of the nodules. This study strongly suggests that Micromonospora populations are natural inhabitants of nitrogen-fixing root nodules.

134 citations


Journal ArticleDOI
TL;DR: In this paper, a dynamic five-box ecosystem model is presented showing that non-Redfield utilization of dissolved nitrogen and phosphorus by non-nitrogen-fixing phytoplankton controls the magnitude and distribution of nitrogen fixation.
Abstract: The ratio of nitrogen to phosphorus in phytoplankton varies greatly with taxa and growth conditions. An ecosystem model suggests that the relative abundance of fast- and slow-growing phytoplankton controls the amount of new nitrogen added to the ocean. The elemental stoichiometry of sea water and particulate organic matter is remarkably similar. This observation led Redfield to hypothesize that the oceanic ratio of nitrate to phosphate is controlled by the remineralization of phytoplankton biomass1. The Redfield ratio is used universally to quantitatively link the marine nitrogen and phosphorus cycles in numerous biogeochemical applications2,3,4. Yet, empirical and theoretical studies show that the ratio of nitrogen to phosphorus in phytoplankton varies greatly with taxa5,6 and growth conditions7,8,9. Here we present a dynamic five-box ecosystem model showing that non-Redfield utilization of dissolved nitrogen and phosphorus by non-nitrogen-fixing phytoplankton controls the magnitude and distribution of nitrogen fixation. In our simulations, systems dominated by rapidly growing phytoplankton with low nitrogen to phosphorus uptake ratios reduce the phosphorus available for nitrogen fixation. In contrast, in systems dominated by slow-growing phytoplankton with high nitrogen to phosphorus uptake ratios nitrogen deficits are enhanced, and nitrogen fixation is promoted. We show that estimates of nitrogen fixation are up to fourfold too high when non-Redfield uptake stoichiometries are ignored. We suggest that the relative abundance of fast- and slow-growing phytoplankton controls the amount of new nitrogen added to the ocean.

129 citations


Journal ArticleDOI
TL;DR: This study presents isolation and identification of three plant growth promoting bacteria that were identified as Enterobacter cloacae (CR1), Pseudomonas putida (CR7) and Stenotrophomonas maltophilia (CR3) and reveals broad spectrum activity against most Fusarium species.
Abstract: Fertilizer costs are a major component of corn production. The use of biofertilizers may be one way of reducing production costs. In this study we present isolation and identification of three plant growth promoting bacteria that were identified as Enterobacter cloacae (CR1), Pseudomonas putida (CR7) and Stenotrophomonas maltophilia (CR3). All bacterial strains produced IAA in the presence of 100 mg l −1 of tryptophan and antifungal metabolites to several soilborne pathogens. S. maltophilia and E. cloacae had broad spectrum activity against most Fusarium species. The only strain that was positive for nitrogen fixation was E. cloacae and it, and P. putida , were also positive for phosphate solubilization. These bacteria and the corn isolate Sphingobacterium canadense CR11, and known plant growth promoting bacterium Burkholderia phytofirmans E24 were used to inoculate corn seed to examine growth promotion of two lines of corn, varieties 39D82 and 39M27 under greenhouse conditions. When grown in sterilized sand varieties 39M27 and 39D82 showed significant increases in total dry weights of root and shoot of 10–20% and 13–28% and 17–32% and 21–31% respectively. Plants of the two varieties grown in soil collected from a corn field had respective increases in dry weights of root and shoot of 10–30% and 12–35% and 11–19% and 10–18%. In sand, a bacterial mixture was highly effective whereas in soil individual bacteria namely P. putida CR7 and E. cloacae CR1 gave the best results with 39M27 and 39D82 respectively. These isolates and another corn isolate, Azospirillum zeae N7, were tested in a sandy soil with a 55 and 110 kg ha −1 of nitrogen fertility at the Delhi research Station of Agriculture and Agri-Food Canada over two years. Although out of seven bacterial treatments, no treatment provided a statistically significant yield increase over control plots but S. canadense CR11 and A. zeae N7 provided statistically significant yield increase as compared to other bacteria. The 110 kg rate of nitrogen provided significant yield increase compared to the 55 kg rate in both years.

124 citations


Journal ArticleDOI
TL;DR: Compared with zones of average activity, the N fixation ‘hotspots’ in unfertilized plots were characterized by marked differences in N-fixer community composition and had substantially higher overall diversity, suggesting that the rain forest litter layer maintains high N fixation rates and unique N- fixing organisms.
Abstract: The role of biodiversity in ecosystem function receives substantial attention, yet despite the diversity and functional relevance of microorganisms, relationships between microbial community structure and ecosystem processes remain largely unknown. We used tropical rain forest fertilization plots to directly compare the relative abundance, composition and diversity of free-living nitrogen (N)-fixer communities to in situ leaf litter N fixation rates. N fixation rates varied greatly within the landscape, and ‘hotspots’ of high N fixation activity were observed in both control and phosphorus (P)-fertilized plots. Compared with zones of average activity, the N fixation ‘hotspots’ in unfertilized plots were characterized by marked differences in N-fixer community composition and had substantially higher overall diversity. P additions increased the efficiency of N-fixer communities, resulting in elevated rates of fixation per nifH gene. Furthermore, P fertilization increased N fixation rates and N-fixer abundance, eliminated a highly novel group of N-fixers, and increased N-fixer diversity. Yet the relationships between diversity and function were not simple, and coupling rate measurements to indicators of community structure revealed a biological dynamism not apparent from process measurements alone. Taken together, these data suggest that the rain forest litter layer maintains high N fixation rates and unique N-fixing organisms and that, as observed in plant community ecology, structural shifts in N-fixing communities may partially explain significant differences in system-scale N fixation rates.

115 citations


Journal ArticleDOI
TL;DR: Terminal bacteroid differentiation among legume species has evolved independently multiple times, perhaps due to the increased host fitness benefits observed in this study.
Abstract: Symbiotic rhizobia differentiate physiologically and morphologically into nitrogen-fixing bacteroids inside legume host nodules. The differentiation is apparently terminal in some legume species, such as peas (Pisum sativum) and peanuts (Arachis hypogaea), likely due to extreme cell swelling induced by the host. In other legume species, such as beans (Phaseolus vulgaris) and cowpeas (Vigna unguiculata), differentiation into bacteroids, which are similar in size and shape to free-living rhizobia, is reversible. Bacteroid modification by plants may affect the effectiveness of the symbiosis. Here, we compare symbiotic efficiency of rhizobia in two different hosts where the rhizobia differentiate into swollen nonreproductive bacteroids in one host and remain nonswollen and reproductive in the other. Two such dual-host strains were tested: Rhizobium leguminosarum A34 in peas and beans and Bradyrhizobium sp. 32H1 in peanuts and cowpeas. In both comparisons, swollen bacteroids conferred more net host benefit by two measures: return on nodule construction cost (plant growth per gram nodule growth) and nitrogen fixation efficiency (H2 production by nitrogenase per CO2 respired). Terminal bacteroid differentiation among legume species has evolved independently multiple times, perhaps due to the increased host fitness benefits observed in this study.

111 citations


Journal ArticleDOI
TL;DR: This is believed to be the first report on the physiology of N(2) fixation within the phylum Verrucomicrobia and the activity of the nitrogenase was not inhibited by ammonium concentrations up to 94 mM.
Abstract: The ability to utilize atmospheric nitrogen (N2) as a sole nitrogen source is an important trait for prokaryotes. Knowledge of N2 fixation by methanotrophs is needed to understand their role in nitrogen cycling in different environments. The verrucomicrobial methanotroph ‘Methylacidiphilum fumariolicum’ strain SolV was investigated for its ability to fix N2. Physiological studies were combined with nitrogenase activity measurements and phylogenetic analysis of the nifDHK genes, encoding the subunits of the nitrogenase. ‘M. fumariolicum’ SolV was able to fix N2 at low oxygen (O2) concentration (0.5 %, v/v) in chemostat cultures. This low oxygen concentration was also required for an optimal nitrogenase activity [47.4 nmol ethylene h−1 (mg cell dry weight)−1]. Based on acetylene reduction assay and growth experiments, the nitrogenase of strain SolV seems to be extremely oxygen sensitive compared to most proteobacterial methanotrophs. The activity of the nitrogenase was not inhibited by ammonium concentrations up to 94 mM. This is believed to be the first report on the physiology of N2 fixation within the phylum Verrucomicrobia.

Journal ArticleDOI
TL;DR: In this paper, a gradient of endogenous soil N levels resulting from different management legacies and soil textures was used to investigate the effects of soil organic matter dynamics and N availability on soybean (Glycine max) N2 fixation.
Abstract: Legume-based cropping systems have the potential to internally regulate N cycling due to the suppressive effect of soil N availability on biological nitrogen fixation. We used a gradient of endogenous soil N levels resulting from different management legacies and soil textures to investigate the effects of soil organic matter dynamics and N availability on soybean (Glycine max) N2 fixation. Soybean N2 fixation was estimated on 13 grain farm fields in central New York State by the 15N natural abundance method using a non-nodulating soybean reference. A range of soil N fractions were measured to span the continuum from labile to more recalcitrant N pools. Soybean reliance on N2 fixation ranged from 36% to 82% and total N2 fixed in aboveground biomass ranged from 40 to 224 kg N ha−1. Soil N pools were consistently inversely correlated with % N from fixation and the correlation was statistically significant for inorganic N and occluded particulate organic matter N. However, we also found that soil N uptake by N2-fixing soybeans relative to the non-nodulating isoline increased as soil N decreased, suggesting that N2 fixation increased soil N scavenging in low fertility fields. We found weak evidence for internal regulation of N2 fixation, because the inhibitory effects of soil N availability were secondary to the environmental and site characteristics, such as soil texture and corresponding soil characteristics that vary with texture, which affected soybean biomass, total N2 fixation, and net N balance.

Journal ArticleDOI
TL;DR: Genes involved in particular in transcriptional regulation, signaling processes, protein drug export, protein secretion, lipopolysaccharide, and peptidoglycan biosynthesis that may play a role in symbiosis were identified and it was shown that this Frankia symbiotic transcriptome was highly similar among phylogenetically distant plant families Betulaceae and Myricaceae.
Abstract: The actinobacteria Frankia spp. are able to induce the formation of nodules on the roots of a large spectrum of actinorhizal plants, where they convert dinitrogen to ammonia in exchange for plant photosynthates. In the present study, transcriptional analyses were performed on nitrogen-replete free-living Frankia alni cells and on Alnus glutinosa nodule bacteria, using whole-genome microarrays. Distribution of nodule-induced genes on the genome was found to be mostly over regions with high synteny between three Frankia spp. genomes, while nodule-repressed genes, which were mostly hypothetical and not conserved, were spread around the genome. Genes known to be related to nitrogen fixation were highly induced, nif (nitrogenase), hup2 (hydrogenase uptake), suf (sulfur-iron cluster), and shc (hopanoids synthesis). The expression of genes involved in ammonium assimilation and transport was strongly modified, suggesting that bacteria ammonium assimilation was limited. Genes involved in particular in transcriptional regulation, signaling processes, protein drug export, protein secretion, lipopolysaccharide, and peptidoglycan biosynthesis that may play a role in symbiosis were also identified. We also showed that this Frankia symbiotic transcriptome was highly similar among phylogenetically distant plant families Betulaceae and Myricaceae. Finally, comparison with rhizobia transcriptome suggested that F. alni is metabolically more active in symbiosis than rhizobia.

Journal ArticleDOI
TL;DR: Results indicating that strains Pseudomonas sp.
Abstract: The objectives of this work were to phenotypically and genetically characterize alfalfa rhizosphere bacteria and to evaluate the effect of single or mixed inoculation upon nodulation and biological nitrogen fixation. Thirty-two strains showed tricalcium phosphate solubilization ability, and two of them caused bigger or equal solubilization halos than the control strain P. putida SP22. The comparison of the 16S ribosomal DNA sequences indicated that these strains are phylogenetically related to Bacillus spp. and Pseudomonas spp. A beneficial effect of both isolates on alfalfa growth was observed in coinoculation assays. Pseudomonas sp. FM7d caused a significant increase in root and shoot dry weight, length, and surface area of roots, number, and symbiotic properties of alfalfa plants. The plants coinoculated with Sinorhizobium meliloti B399 and the Bacillus sp. M7c showed significant increases in the measured parameters. Our results indicating that strains Pseudomonas sp. FM7d and Bacillus sp. M7c can be considered for the formulation of new inoculants.

Journal ArticleDOI
TL;DR: Overall, soil physicochemistry explained more variation in NFG abundance than livestock presence or plant invasion, particularly for chiA and bacterial amoA, with significant relationships between the abundance of all five NFGs and total nitrogen or nitrate.
Abstract: A diverse soil microbial community is involved in nitrogen cycling, and these microbes can be affected by land management practices and weed invasion. We surveyed 20 woodlands with a history of livestock grazing, with livestock recently excluded from 10 sites. We investigated whether soil nutrients were lower when grazing was excluded and higher when exotic grasses dominated the understory. Second, using quantitative real-time PCR, we investigated whether microbial nitrogen functional gene (NFG) abundance was altered with soil nutrient enrichment, livestock exclusion, and exotic grass invasion. The target genes were chiA (decomposition-ammonification), nifH (nitrogen fixation), nirK and narG (denitrification), and bacterial amoA (nitrification). Woodland soils were enriched in phosphorus and nitrogen compared to reference condition sites, but soil nutrients were not lower following livestock exclusion. Total nitrogen and nifH were negatively correlated in grazed woodlands, suggesting that aboveground herbivory reduces the capacity for belowground nitrogen fixation. Woodlands dominated by exotic grasses had higher levels of nitrate, narG, and nirK than those dominated by native grasses. We hypothesize that the increase in potential for denitrification was due to increases in soil nitrate, rather than changes in plant composition. Overall, soil physicochemistry explained more variation in NFG abundance than livestock presence or plant invasion, particularly for chiA and bacterial amoA, with significant relationships between the abundance of all five NFGs and total nitrogen or nitrate. All woodlands investigated had a history of anthropogenic disturbance and nutrification, and soil nutrient levels and the abundance of NFGs are likely to be related to long-term land management practices.

Journal ArticleDOI
TL;DR: It is demonstrated that glyphosate can influence the symbiotics N2 fixation by lowering nickel content available to the symbiotic microorganisms.

Journal ArticleDOI
TL;DR: It is concluded that transcriptional regulation of nif gene expression in A1501 is mediated by the nif- specific and ntr gene regulatory systems, and many genes of unknown function may play some essential roles in controlling the expression or activity of nitrogenase.
Abstract: Biological nitrogen fixation is highly controlled at the transcriptional level by regulatory networks that respond to the availability of fixed nitrogen. In many diazotrophs, addition of excess ammonium in the growth medium results in immediate repression of nif gene transcription. Although the regulatory cascades that control the transcription of the nif genes in proteobacteria have been well investigated, there are limited data on the kinetics of ammonium-dependent repression of nitrogen fixation. Here we report a global transcriptional profiling analysis of nitrogen fixation and ammonium repression in Pseudomonas stutzeri A1501, a root-associated and nitrogen-fixing bacterium. A total of 166 genes, including those coding for the global nitrogen regulation (Ntr) and Nif-specific regulatory proteins, were upregulated under nitrogen fixation conditions but rapidly downregulated as early as 10 min after ammonium shock. Among these nitrogen fixation-inducible genes, 95 have orthologs in each of Azoarcus sp. BH72 and Azotobacter vinelandii AvoP. In particular, a 49-kb expression island containing nif and other associated genes was markedly downregulated by ammonium shock. Further functional characterization of pnfA, a new NifA-σ54-dependent gene chromosomally linked to nifHDK, is reported. This gene encodes a protein product with an amino acid sequence similar to that of five hypothetical proteins found only in diazotrophic strains. No noticeable differences in the transcription of nifHDK were detected between the wild type strain and pnfA mutant. However, the mutant strain exhibited a significant decrease in nitrogenase activity under microaerobic conditions and lost its ability to use nitrate as a terminal electron acceptor for the support of nitrogen fixation under anaerobic conditions. Based on our results, we conclude that transcriptional regulation of nif gene expression in A1501 is mediated by the nif- specific and ntr gene regulatory systems. Furthermore, microarray and mutational analyses revealed that many genes of unknown function may play some essential roles in controlling the expression or activity of nitrogenase. The findings presented here establish the foundation for further studies on the physiological function of nitrogen fixation-inducible genes.

Book ChapterDOI
10 Sep 2010
TL;DR: The effects of abiotic stresses, such as drought, phosphate deficiency and aluminium toxicity, on symbiotic nitrogen fixation and provide perspectives on molecular approaches to the analysis of stress responses in legumes are discussed.
Abstract: Legumes are important components of the nitrogen cycle on land. Agricultural systems have traditionally relied much on legumes for nitrogen input because many species are able to establish symbioses with diazotrophic bacteria (rhizobia) and thus trade metabolites and reduced compounds. Photosynthates produced in the leaves are allocated to the root nodule to supply the bacteroids with carbon, in exchange for reduced nitrogen (ammonia) produced by the rhizobia from atmospheric nitrogen. Despite its major significance to plant breeding and sustainable agriculture, the impact of abiotic stresses on nodule development and stability and on symbiotic nitrogen fixation remains poorly understood, particularly at the molecular level. However, the study of model legume species and the development of a plethora of resources, particularly the elucidation of the genome sequences of three legume species, are now revealing many traits of agricultural importance in legumes as well as other aspects that are not easily studied in other plant models, such as Arabidopsis or rice. In this chapter, we will discuss the effects of abiotic stresses, such as drought, phosphate deficiency and aluminium toxicity, on symbiotic nitrogen fixation and provide perspectives on molecular approaches to the analysis of stress responses in legumes.

Journal ArticleDOI
TL;DR: High degree of phenotypic and genotypic diversity is present in S. meliloti and S. medicae populations from marginal soils affected by salt and drought, in arid and semi-arid regions of Morocco.
Abstract: Background: Sinorhizobium meliloti and S. medicae are symbiotic nitrogen fixing bacteria in root nodules of forage legume alfalfa (Medicago sativa L.). In Morocco, alfalfa is usually grown in marginal soils of arid and semi-arid regions frequently affected by drought, extremes of temperature and soil pH, soil salinity and heavy metals, which affect biological nitrogen fixing ability of rhizobia and productivity of the host. This study examines phenotypic diversity for tolerance to the above stresses and genotypic diversity at Repetitive Extragenic Pallindromic DNA regions of Sinorhizobium nodulating alfalfa, sampled from marginal soils of arid and semi-arid regions of Morocco. Results: RsaI digestion of PCR amplified 16S rDNA of the 157 sampled isolates, assigned 136 isolates as S. meliloti and the rest as S. medicae. Further phenotyping of these alfalfa rhizobia for tolerance to the environmental stresses revealed a large degree of variation: 55.41%, 82.16%, 57.96% and 3.18% of the total isolates were tolerant to NaCl (>513 mM), water stress (-1.5 MPa), high temperature (40°C) and low pH (3.5), respectively. Sixty-seven isolates of S. meliloti and thirteen isolates of S. medicae that were tolerant to salinity were also tolerant to water stress. Most of the isolates of the two species showed tolerance to heavy metals (Cd, Mn and Zn) and antibiotics (chloramphenicol, spectinomycin, streptomycin and tetracycline). The phenotypic clusters observed by the cluster analysis clearly showed adaptations of the S. meliloti and S. medicae strains to the multiple stresses. Genotyping with rep-PCR revealed higher genetic diversity within these phenotypic clusters and classified all the 157 isolates into 148 genotypes. No relationship between genotypic profiles and the phenotypes was observed. The Analysis of Molecular Variance revealed that largest proportion of significant (P < 0.01) genetic variation was distributed within regions (89%) than among regions (11%). Conclusion: High degree of phenotypic and genotypic diversity is present in S. meliloti and S. medicae populations from marginal soils affected by salt and drought, in arid and semi-arid regions of Morocco. Some of the tolerant strains have a potential for exploitation in salt and drought affected areas for biological nitrogen fixation in alfalfa.

Journal ArticleDOI
TL;DR: PGPR strains have potential to enhance the symbiotic potential of rhizobia, as a significant increase in the proportion of nitrogen derived from atmosphere is demonstrated.
Abstract: Nitrogen (N) fixation by legume-Rhizobium symbiosis is important to agricultural productivity and is therefore of great economic interest. Growing evidence indicates that soil beneficial bacteria can positively affect symbiotic performance of rhizobia. The effect of co-inoculation with plant growth-promoting rhizobacteria (PGPR) and Rhizobium, on nodulation, nitrogen fixation, and yield of common bean (Phaseolus vulgaris L.) cultivars was investigated in two consecutive years under field conditions. The PGPR strains Pseudomonas fluorescens P-93 and Azospirillum lipoferum S-21 as well as two highly effective Rhizobium strains were used in this study. Common bean seeds of three cultivars were inoculated with Rhizobium singly or in a combination with PGPR to evaluate their effect on nodulation and nitrogen fixation. A significant variation of plant growth in response to inoculation with Rhizobium strains was observed. Treatment with PGPR significantly increased nodule number and dry weight, shoot dry weight,...

Journal ArticleDOI
TL;DR: It is shown that M. truncatula is able to select rhizobia based on recognition signals, both before and after the nitrogen fixation step, but no sanction mechanism, described as a decrease in Rhizobia fitness inside the nodules, was detected.
Abstract: In nitrogen-fixing symbiosis, plant sanctions against ineffective bacteria have been demonstrated in previous studies performed on soybean and yellow bush lupin, both developing determinate nodules with Bradyrhizobium sp. strains. In this study, we focused on the widely studied symbiotic association Medicago truncatula‐Sinorhizobium meliloti, which forms indeterminate nodules. Using two strains isolated from the same soil and displaying different nitrogen fixation phenotypes on the same fixed plant line, we analysed the existence of both partner choice and plant sanctions by performing split-root experiments. By measuring different parameters such as the nodule number, the nodule biomass per nodule and the number of viable rhizobia per nodule, we showed that M. truncatula is able to select rhizobia based on recognition signals, both before and after the nitrogen fixation step. However, no sanction mechanism, described as a decrease in rhizobia fitness inside the nodules, was detected. Consequently, even if partner choice seems to be widespread among legumes, sanction of non-effective rhizobia might not be universal.

Journal ArticleDOI
TL;DR: N2 fixation was drastically reduced in S-deficient plants as a consequence of a low nodule development, but also due to low nitrogenase and leghaemoglobin production, which is likely to be due to down-regulation by a N-feedback mechanism.
Abstract: The role of S in legume growth, N uptake, and N2 fixation was investigated using white clover (Trifolium repens L.) as a model species. We examined whether the effect of sulphate addition on N fixation resulted from a stimulation of host plant growth, a specific effect of S on nodulation, or a specific effect of S on nodule metabolism. Clones of white clover, inoculated with Rhizobium leguminosarum, were grown for 140 d in a hydroponic system with three levels of sulphate concentration (0 mM, 0.095 mM, and 0.380 mM). Nodule morphological and biochemical traits, such as root length, nodule biomass and volume, nodule protein contents (nitrogenase and leghaemoglobin obtained by an immunological approach), and root amino acid concentrations, were used to analyse the effect of sulphate availability on N2 fixation. The application of sulphate increased whole plant dry mass, root length, and nodule biomass, expressed on a root-length basis. N uptake proved less sensitive than N2 fixation to the effects of S-deficiency, and decreased as a consequence of the lower root length observed in S-deficient plants. N2 fixation was drastically reduced in S-deficient plants as a consequence of a low nodule development, but also due to low nitrogenase and leghaemoglobin production. This effect is likely to be due to down-regulation by a N-feedback mechanism, as, under severe S-deficiency, the high concentration of whole plant N and the accumulation of N-rich amino acids (such as asparagine) indicated that the assimilation of N exceeded the amount required for plant growth.

Journal ArticleDOI
TL;DR: The amount of amino acids was strongly increased in both phloem exudates and nodules of the treatments with downregulated nodule activity, and a possible role of asparagine in an N-feedback regulation of nitrogen fixation in M. truncatula is discussed.
Abstract: The objective of this study was to assess whether a whole plant N-feedback regulation impact on nitrogen fixation in Medicago truncatula would manifest itself in shifts of the composition of the amino acid flow from shoots to nodules. Detected shifts in the phloem amino acid composition were supposed to be mimicked through artificial phloem feeding and concomitant measurement of nodule activity. The amino acid composition of the phloem exudates was analyzed from plants grown under the influence of treatments (limiting P supply or application of combined nitrogen) known to reduce nodule nitrogen fixation activity. Plants in nutrient solution were supplied with sufficient (9 microM) control, limiting (1 microM) phosphorus or 3 mM NH(4)NO(3) (downregulated nodule activity). Low phosphorus and the application of NH(4)NO(3) reduced per plant and specific nitrogenase activity (H(2) evolution). At day 64 of growth, phloem exudates were collected from cuts of the shoot base. The amount of amino acids was strongly increased in both phloem exudates and nodules of the treatments with downregulated nodule activity. The increase in the downregulated treatments was almost exclusively the result of a higher proportion of asparagine in both phloem exudates and nodules. Leaf labeling with (15)N showed that nitrogen from the leaves is retranslocated to nodules. An artificial phloem feeding with asparagine resulted in an increased concentration of asparagine in nodules and a decreased nodule activity. A possible role of asparagine in an N-feedback regulation of nitrogen fixation in M. truncatula is discussed.

Journal ArticleDOI
TL;DR: Dinitrogen-fixing organisms in cyanobacterial mats were studied in two shallow coral reef ecosystems: La Reunion Island, southwestern Indian Ocean, Sesoko (Okinawa) Island, and northwestern Pacific Ocean by microscopy and molecular tools and showed few differences in taxonomic composition.
Abstract: Dinitrogen-fixing organisms in cyanobacterial mats were studied in two shallow coral reef ecosystems: La Reunion Island, southwestern Indian Ocean, Sesoko (Okinawa) Island, and northwestern Pacific Ocean. Rapidly expanding benthic miniblooms, frequently dominated by a single cyanobacterial taxon, were identified by microscopy and molecular tools. In addition, nitrogenase activity by these blooms was measured in situ. Dinitrogen fixation and its contribution to mat primary production were calculated using 15N2 and 13C methods. Dinitrogen-fixing cyanobacteria from mats in La Reunion and Sesoko showed few differences in taxonomic composition. Anabaena sp. among heterocystous and Hydrocoleum majus and Symploca hydnoides among nonheterocystous cyanobacteria occurred in microbial mats of both sites. Oscillatoria bonnemaisonii and Leptolyngbya spp. occurred only in La Reunion, whereas Hydrocoleum coccineum dominated in Sesoko. Other mats dominated by Hydrocoleum lyngbyaceum, Phormidium laysanense, and Trichocoleus tenerrimus occurred at lower frequencies. The 24-h nitrogenase activity, as measured by acetylene reduction, varied between 11 and 324 nmoles C2H2 reduced µg−1 Chl a. The highest values were achieved by heterocystous Anabaena sp. performed mostly during the day. Highest values for nonheterocystous cyanobacteria were achieved by H. coccineum mostly during the night. Daily nitrogen fixation varied from nine (Leptolyngbya) to 238 nmoles N2 µg−1 Chl day−1 (H. coccineum). Primary production rates ranged from 1,321 (S. hydnoides) to 9,933 nmoles C µg−1 Chl day−1 (H. coccineum). Dinitrogen fixation satisfied between 5% and 21% of the nitrogen required for primary production.

Journal ArticleDOI
TL;DR: The data suggest that due to Azospirillum inoculation the increase of total N accumulated in rice plants could be a tool to help farmers to improve production and maintain high input of plant residues, providing more organic matter to the soil and guaranteeing sustainability of the system.
Abstract: The response of rice plants to the application of inoculant containing two Azospirillum brasilense strains was studied under field conditions. The experiment was performed as three treatments with four replicates in randomized complete blocks arranged as plots of 60 m2 in an area on a Vertic Argiudol soil type in the province of Entre Rios, Argentina. The bacterial rhizosphere community and also the diazotrophic isolates obtained from control and inoculated rice plants were analyzed in relation to their physiology and biological nitrogen fixation (BNF). The MPN of diazotrophs in the rhizosphere varied during the ontogenic cycle. The patterns of distribution of the microbial physiological activities obtained by principal component analysis of community-level physiological profiles (CLPP) showed differences in the utilization of carbon sources by the rhizosphere communities among treatments. Although the analyses of DGGE 16S and nifH profiles have not indicated that the inoculation influenced the genetic diversity of bacterial communities among treatments, they revealed that the banding profiles were altered in different parts of the rice plant by each Azospirillum inoculation treatment. These observations suggest that physiological responses of plant tissues to the inoculation may have occurred. According to agronomic parameters of each treatment, the Azospirillum inoculation increased aerial biomass at the tillering and grain-filling stages. Although the N content accumulated in rice plants increased by 16 and 50 kg ha−1, the BNF contribution could not be estimated under our experimental conditions by the 15N balance technique. Based on this field inoculation experiment to rice plants, it is noteworthy that our data suggest that due to Azospirillum inoculation the increase of total N accumulated in rice plants could be a tool to help farmers to improve production and maintain high input of plant residues, providing more organic matter to the soil and guaranteeing sustainability of the system.

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TL;DR: The BNF of tree legumes was estimated in four areas of the Brazilian dry forest (caatinga), analyzing potentially fixing species (target species) and non fixingspecies (reference species), using the 15N natural abundance methodology.

Journal ArticleDOI
TL;DR: In this paper, the authors reconstructed major parts of the nitrogen cycle in the rhizosphere soil/root system of Leucanthemopsis alpina (L.) Heathwood: as well as the corresponding bulk soil by quantifying functional genes of nitrogen fixation (nifH), nitrogen mineralisation (chiA, aprA), nitrification (amoA AOB, amoA AOA) and denitrification (nirS, nirK and nosZ) in a 10-year and a 120-year ice-free soil of the Damma glacier
Abstract: Glacier forefields are an ideal playground to investigate the role of development stages of soils on the formation of plant-microbe interactions as within the last decades, many alpine glaciers retreated, whereby releasing and exposing parent material for soil development. Especially the status of macronutrients like nitrogen differs between soils of different development stages in these environments and may influence plant growth significantly. Thus, in this study, we reconstructed major parts of the nitrogen cycle in the rhizosphere soil/root system of Leucanthemopsis alpina (L.) HEYWOOD: as well as the corresponding bulk soil by quantifying functional genes of nitrogen fixation (nifH), nitrogen mineralisation (chiA, aprA), nitrification (amoA AOB, amoA AOA) and denitrification (nirS, nirK and nosZ) in a 10-year and a 120-year ice-free soil of the Damma glacier forefield. We linked the results to the ammonium and nitrate concentrations of the soils as well as to the nitrogen and carbon status of the plants. The experiment was performed in a greenhouse simulating the climatic conditions of the glacier forefield. Samples were taken after 7 and 13 weeks of plant growth. Highest nifH gene abundance in connection with lowest nitrogen content of L. alpina was observed in the 10-year soil after 7 weeks of plant growth, demonstrating the important role of associative nitrogen fixation for plant development in this soil. In contrast, in the 120-year soil copy numbers of genes involved in denitrification, mainly nosZ were increased after 13 weeks of plant growth, indicating an overall increased microbial activity status as well as higher concentrations of nitrate in this soil.

01 Jan 2010
TL;DR: This study reconstructed major parts of the nitrogen cycle in the rhizosphere soil/root system of Leucanthemopsis alpina (L.) Heywood as well as the corresponding bulk soil by quantifying functional genes of nitrogen fixation, nitrogen mineralisation, nitrification and denitrification in a 10-year and a 120-year ice-free soil of the Damma glacier forefield.
Abstract: Glacier forefields are an ideal playground to investigate the role of development stages of soils on the formation of plant-microbe interactions as within the last decades, many alpine glaciers retreated, whereby releasing and exposing parent material for soil development. Espe- cially the status of macronutrients like nitrogen differs between soils of different development stages in these environments and may influence plant growth significantly. Thus, in this study, we reconstructed major parts of the nitrogen cycle in the rhizosphere soil/root system of Leucanthemopsis alpina (L.) heywood as well as the corresponding bulk soil by quantifying functional genes of nitrogen fixation (nifli), nitrogen mineralisation (chiA, aprA), nitrification (amoA AOB, amoA AOA) and denitri- fication (nirS, nirK and nosZ) in a 10-year and a 120-year ice-free soil of the Damma glacier forefield. We linked the results to the ammonium and nitrate concentrations of the

Journal ArticleDOI
TL;DR: Despite the very high rates of N2 fixation associated with cyanolichens, large cover of mosses per unit area at the landscape scale compensates for their lower fixation rates, thereby probably making them the primary regional atmospheric nitrogen sink.
Abstract: Dinitrogen fixation by cyanobacteria is of particular importance for the nutrient economy of cold biomes, constituting the main pathway for new N supplies to tundra ecosystems. It is prevalent in cyanobacterial colonies on bryophytes and in obligate associations within cyanolichens. Recent studies, applying interspecific variation in plant functional traits to upscale species effects on ecosystems, have all but neglected cryptogams and their association with cyanobacteria. Here we looked for species-specific patterns that determine cryptogam-mediated rates of N2 fixation in the Subarctic. We hypothesised a contrast in N2 fixation rates (1) between the structurally and physiologically different lichens and bryophytes, and (2) within bryophytes based on their respective plant functional types. Throughout the survey we supplied 15N-labelled N2 gas to quantify fixation rates for monospecific moss, liverwort and lichen turfs. We sampled fifteen species in a design that captures spatial and temporal variations during the growing season in Abisko region, Sweden. We measured N2 fixation potential of each turf in a common environment and in its field sampling site, in order to embrace both comparativeness and realism. Cyanolichens and bryophytes differed significantly in their cyanobacterial N2 fixation capacity, which was not driven by microhabitat characteristics, but rather by morphology and physiology. Cyanolichens were much more prominent fixers than bryophytes per unit dry weight, but not per unit area due to their low specific thallus weight. Mosses did not exhibit consistent differences in N2 fixation rates across species and functional types. Liverworts did not fix detectable amounts of N2. Despite the very high rates of N2 fixation associated with cyanolichens, large cover of mosses per unit area at the landscape scale compensates for their lower fixation rates, thereby probably making them the primary regional atmospheric nitrogen sink.

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TL;DR: Surprisingly, effects on the transcript level were less pronounced, which might indicate that parts of the investigated functional groups were tolerant or resistant against SDZ or, as in the case of nifH and clover, have been protected by the nodules.
Abstract: The antibiotic sulfadiazine (SDZ) can enter the environment by application of manure from antibiotic-treated animals to arable soil. Because antibiotics are explicitly designed to target microorganisms, they likely affect microbes in the soil ecosystem, compromising important soil functions and disturbing processes in nutrient cycles. In a greenhouse experiment, we investigated the impact of sulfadiazine-contaminated pig manure on functional microbial communities involved in key processes of the nitrogen cycle in the root-rhizosphere complexes (RRCs) of maize (Zea mays) and clover (Trifolium alexandrinum). At both the gene and transcript level, we performed real-time PCR using nifH, amoA (in both ammonia-oxidizing bacteria and archaea), nirK, nirS, and nosZ as molecular markers for nitrogen fixation, nitrification, and denitrification. Sampling was performed 10, 20, and 30 days after the application. SDZ affected the abundance pattern of all investigated genes in the RRCs of both plant species (with stronger effects in the RRC of clover) 20 and 30 days after the addition. Surprisingly, effects on the transcript level were less pronounced, which might indicate that parts of the investigated functional groups were tolerant or resistant against SDZ or, as in the case of nifH and clover, have been protected by the nodules.