Contribution Of Biological Nitrogen Fixation Research Articles

Rooting and growth of pre-germinated sugarcane seedlings inoculated with diazotrophic bacteria

Renovation of Brazilian sugarcane plantations using the pre-sprouted seedling technique (PSS) was undertaken to increase sanitation and productivity while reducing the cost of sugarcane planting. The objective of this study was to evaluate growth, nutrient acquisition, and contribution of biological nitrogen fixation (BNF) in two sugarcane cultivars RB867515 and IACSP95-5000, inoculated with five strains/species of diazotrophic bacteria, applied together and individually, for 60 days during growth. The cultivars were grown in a greenhouse in boxes filled with a sterile substrate followed by growth in tubes filled with a commercial substrate in the second phase; finally the plants were transferred to pots containing soil enriched with 15N and grown outdoors. The treatments used were control (no inoculation); mixed inoculation with the five strains, Gluconacetobacter diazotrophicus (PAL5T), Herbaspirillum rubrisubalbicans (HCC103), Herbaspirillum seropedicae (HRC54), Nitrospirillum amazonense (CBAmC) and Paraburkholderia tropica (PPe4T); and individual inoculation with each strain. Increases of up to 50% in the root dry mass was observed following mixed inoculation of the seedlings in the germination phase, with a significant increase in root initiation, volume, and area, especially of the fine roots. Inoculation of seedlings of both cultivars led to increased biomass in tubes of non-sterile substrate. In soil, a higher nitrogen accumulation was also observed in the cvar. IACSP95-5000 in all treatments except plants inoculated with N. amazonense. For the cvar. RB867515 shoots, the opposite was observed were the highest N accumulation was observed for N. amazonense, H. seropedicae followed by the mixture. In this experiment using soil labelled with 15N there was a response on N uptake caused by all bacteria used in one cvar but only by 2 of the 5 strains in other cvar. Using the 15N enrichment of the plants the results showed that the control and inoculated plants all derived 60–70% of N from the atmosphere, and inoculation had no effect on BNF contribution. For P and K levels, RB867515 showed no effect of inoculation, but the other cvar. did respond to inoculation.

Role contribution of biological nitrogen fixation to future terrestrial net land carbon accumulation under warming condition at centennial scale

Future changes in net land carbon (C) accumulation in the terrestrial ecosystem remain highly uncertain. This uncertainty is mainly due to nitrogen (N) availability not being included in model simulations. N availability is ultimately determined by the balance of N inputs and outputs for the terrestrial ecosystem. Here, as the largest N input pathway, the influence of biological N fixation (BNF) combined with increasing atmospheric carbon dioxide (CO2) concentration, climate change, and atmospheric N deposition on future terrestrial C sequestration is investigated to the year 2100 using the Community Atmosphere–Biosphere–Land Exchange (CABLE) global land surface model. Projections show a net land C accumulation increase of ∼20% by 2100 for the terrestrial ecosystem, relative to the pre-industrial (1901–1910) level. BNF will increase terrestrial C sequestration by ∼104 Pg C by the end of this century, accounting for ∼28% of the total increase in net land C accumulation due to increasing CO2, climate change, BNF, and atmospheric N deposition. Therefore, the findings highlight the fundamental importance of BNF in determining the size of future net terrestrial C accumulation, although there are still unresolved questions regarding BNF and its role in forecasting climate change. Furthermore, a climate warming-induced increase in soil N mineralization of ∼431 Tg N per °C is estimated, resulting in increased N uptake (∼17 Tg N yr−1) from soil to vegetation by the end of this century, relative to the pre-industrial period, which could offset soil carbon loss due to warming. This highlights the effect of warming-induced N mineralization on land net C accumulation. Thus, BNF variations and warming-stimulated soil mineralization should be included in model simulations of land C carbon sequestration to prevent the effect of N limitation on land C loss being overestimated.

Rhizobium Inoculation of Cowpea in Brazilian Cerrado Increases Yields and Nitrogen Fixation

Core Ideas Cowpea cropping after soybean is increasing in the Cerrado region of Brazil and can benefit from BNF.Soybean bradyrhizobia can abundantly nodulate cowpea but are often inefficient.Inoculated elite cowpea Bradyrhizobium strains increased grain yields.Nitrogen fixation inputs were increased by inoculation with selected cowpea bradyrhizobia.Nitrogen‐15 natural abundance showed inoculation increased N2 fixation by over 25 kg N ha–1 crop–1. Cowpea (Vigna unguiculata Walp.) is increasingly planted as a second season crop after soybean [Glycine max (L.) Merr.] in the Brazilian Cerrado region. Experiments were conducted at Sinop and Primavera do Leste in Mato Grosso State to investigate the impact of cowpea inoculation with recommended rhizobium strains on biological nitrogen fixation (BNF) and grain yield. Plants were either non‐inoculated, with or without 70 kg N fertilizer ha−, or inoculated with five rhizobium strains, separately or as a mixture. Nodulation and shoot dry matter (DM) were evaluated at 35 d after emergence (DAE). Dry matter and N accumulation were evaluated and BNF contributions quantified using 15N natural abundance at 50 DAE. Nodule number and dry weight were high regardless of inoculation treatment. However, several of the inoculation treatments at Sinop and Primavera do Leste promoted significant increases in total N at 50 DAE. At both sites, the 15N abundance results indicated that inoculation significantly increased BNF contributions from 36 to 64 kg ha− at Sinop and from 48 to 75 kg N ha− at Primavera. Grain yield was significantly increased from approximately 1200 to 1500 kg ha− by strains BR 3262 and INPA 03‐11B at the Primavera do Leste, and from approximately 850 to 1400 kg ha− by strain BR 3262 at Sinop. The results indicate inoculation of cowpea with elite rhizobium strains can increase BNF contributions and grain yields in the Brazilian Cerrado region, even when there are abundant populations of soybean rhizobium present in the soil.

Agronomic efficiency of Rhizobium strains from the Amazon region in common bean

ABSTRACT To maximize the contribution of biological nitrogen fixation in common bean, it is necessary to use bacterial strains that are more adapted, competitive, and efficient in the symbiotic process. In this regard, the aim of this study was to evaluate the agronomic efficiency (AE) of three bacterial strains isolated from acid soils with high Al content from the Amazon region in an Argissolo Vermelho Distrófico típico soil (Typic Rhodustults - USDA Classification) from the municipality of Formiga, MG, Brazil. We compared their AE to that of the reference strain CIAT 899T and of two controls without inoculation (one without and another with 80 kg ha-1 of N-urea). The results indicated that inoculation with the strains UFLA 02-100 and UFLA 02-127 provides grain yield equivalent to inoculation with the reference strain and to the control with mineral N. Thus, both have potential for recommendation as inoculants for common bean.

Metabolic features involved in drought stress tolerance mechanisms in peanut nodules and their contribution to biological nitrogen fixation

Legumes belong to the most important crops worldwide. They increase soil fertility due their ability to establish symbiotic associations with soil microorganisms, known as rhizobia, capable of fixing nitrogen from the atmosphere. However, they are frequently exposed to abiotic stress conditions in particular drought. Such adverse conditions impair the biological nitrogen fixation (BNF) and depend largely on the legume. Therefore, two peanut cultivars with contrasting tolerance to drought, namely the more tolerant EC-98 and the sensitive Granoleico, were investigated to elucidate the relative contribution of BNF to the tolerance to drought. The tolerant cultivar EC-98 sustained growth and BNF similar to the control condition despite the reduced water potential and photosynthesis, suggesting the functioning of distinct metabolic pathways that contributed to enhance the tolerance. The biochemical and metabolomics approaches revealed that nodules from the tolerant cultivar accumulated trehalose, proline and gamma-aminobutyric acid (GABA), metabolites with known function in protecting against drought stress. The amide metabolism was severely affected in nodules from the sensitive cultivar Granoleico as revealed by the low content of asparagine and glutamine in the drought stressed plants. The sensitive cultivar upon rehydration was unable to re-establish a metabolism similar to well-watered plants. This was evidenced by the low level of metabolites and, transcripts and specific activities of enzymes from the carbon (sucrose synthase) and nitrogen (glutamine synthetase) metabolism which decreased below the values of control plants. Therefore, the increased content of metabolites with protective functions under drought stress likely is crucial for the full restoration upon rehydration. Smaller changes of drought stress-related metabolites in nodule are another trait that contributes to the effective control of BNF in the tolerant peanut cultivar (EC-98).

Chemical soil attributes after wheat cropping under nitrogen fertilization and inoculation with Azospirillum brasilense

Azospirillum brasilense plays an important role in biological nitrogen fixation (BNF) in grasses. However, further studies are needed to define how much mineral N can be applied while simultaneously maintaining BNF contribution and maximizing crop yield and to determine the impact of these practices on soil fertility. Thus, we aimed to investigate the effect of inoculation with A. brasilense, in conjunction with varying N doses and sources in a Cerrado soil, on soil chemical attributes after two years of irrigated wheat production. The experiment was initiated in Selvíria - MS under no-tillage production in an Oxisol in 2014 and 2015. The experimental design was a randomized block design with four replications, and treatments were arranged in a 2 x 5 x 2 factorial arrangement as follows: two N sources (urea and Super N - urea with inhibitor of the enzyme urease NBPT (N - (n-butyl thiophosphoric triamide))), five N rates (0, 50, 100, 150 and 200 kg ha-1), and with or without seed inoculation with A. brasilense. The increase in N rates did not influence the chemical soil attributes. Super N acidified the soil more compared to urea. A. brasilense inoculation reduced the effect of soil acidification in intensive irrigated wheat cultivation; however, the base extraction was higher, resulting in a lower soil CEC after cultivation with inoculation. Therefore, the cultivation of wheat inoculated with A. brasilense was not harmful to soil fertility because it did not reduce the base saturation and organic matter content (P, K, Ca, Mg, and S).

Contribution of a mixed inoculant containing strains of Burkholderia spp. and Herbaspirillum ssp. to the growth of three sorghum genotypes under increased nitrogen fertilization levels

Sorghum bicolor is a C4 plant that is normally associated with diazotrophs. Inoculation with selected bacteria can improve plant growth, biological nitrogen fixation (BNF) and nutrient uptake. Therefore, the objective of the current study was to evaluate the ability of twenty-two diazotroph strains to improve sorghum growth in a greenhouse. Then, a mixture of the best performing strains was applied in the field under different levels of N-fertilizer applied to three sorghum genotypes to evaluate crop productivity, N content, BNF contribution and the efficiency of the N fertilizer used under field conditions. Two Herbaspirillum and two Burkholderia species were selected under greenhouse conditions using a sterile substrate of three sorghum genotypes: grain sorghum lineage 9920044, grass BRS655 and sweet sorghum BR501. Four diazotrophs were selected for the field trial. Burkholderia kururiensis (16 and 109), B. tropica (PPe6) and Herbaspirillum seropedicae (ZMS176) were cultivated and mixed at planting in a Typic Hapladult soil using peat inoculant. Crop yield, N-content and BNF quantification were determined using the natural abundance of 15N, and the use efficiency of the applied N was assessed using urea labelled with 15N. The grain yield showed a 24% enhancement in the inoculated grain sorghum compared with the control without nitrogen. In addition, the aerial biomass was 13% higher in the grass genotype compared with the control. The sweet sorghum did not respond to any treatment. BNF quantification reached 31.4% in the grain, 22.7% in the grass and 19.4% in the sweet sorghum genotypes. Grain sorghum inoculation increased BNF by 4% (9.5kgha−1N fixed). Inoculation increased fertilizer efficiency recovery by 28.9%. Inoculation using a mixture containing the four diazotrophs improved the sorghum productivity by enhancing the dry mass, N-content, N from BNF and N use efficiency in the grain and grass genotypes. Under zero nitrogen fertilizer application, 22% of the N that accumulated in the biomass was derived from the air. New inoculants using a mixture of different bacterial species/strains can improve the knowledge and contribution of BNF in agricultural systems.

Biological nitrogen fixation in C4 grasses of different growth strategies of South America natural grasslands

In South American natural grasslands coexist species of C4 grasses with a higher growth strategies and species with low growth strategies. Biological nitrogen fixation (BNF) is very important for these grasses because the soil is nutrient poor. The understanding of this process may help explain the vegetation dynamics and improve the management of these grasslands. The objective of this work was to evaluate the association of four native grasses with three genus of diazotrophic bacteria under two levels of fertilization, and to determine the contribution of BNF. The four most abundant C4 grasses of South America natural grasslands (Axonopus affinis and Paspalum notatum, high growth strategies; Andropogon lateralis and Aristida laevis, low growth strategies) were cultivated in a greenhouse in a randomized block design with four replications. In a first experiment, two fertilization treatments were tested: 50mgPkg−1 soil+100mgNkg−1 soil (N+P) and a control (without N and P). The number of diazotrophic bacteria for the Azotobacter, Azospirillum and Herbaspirillum genus in the rhizosphere, rhizoplane and endorhizoplane were quantified. In a second experiment, BNF contribution was determined through natural 15N abundance technique. The results of this study point out that the genus of bacteria Azotobacter, Azospirillum and Herbaspirillum colonize the root system of these native grasses, contributing to the nitrogen nutrition of these species. Colonization increased with time, but N and P fertilization reduced colonization. Our results also showed that the N-fixation potential differ among species with different growth strategies. Aristida laevis (low growth strategy species) is the grass with the greater BNF contribution. P. notatum (high growth strategy species) showed great ability to absorb N from soil, with low contribution of BNF, being species with great response to fertilization.

The plant growth-promoting effect of the nitrogen-fixing endophyte Pseudomonas stutzeri A15.

The use of plant growth-promoting rhizobacteria as a sustainable alternative for chemical nitrogen fertilizers has been explored for many economically important crops. For one such strain isolated from rice rhizosphere and endosphere, nitrogen-fixing Pseudomonas stutzeri A15, unequivocal evidence of the plant growth-promoting effect and the potential contribution of biological nitrogen fixation (BNF) is still lacking. In this study, we investigated the effect of P. stutzeri A15 inoculation on the growth of rice seedlings in greenhouse conditions. P. stutzeri A15 induced significant growth promotion compared to uninoculated rice seedlings. Furthermore, inoculation with strain A15 performed significantly better than chemical nitrogen fertilization, clearly pointing to the potential of this bacterium as biofertilizer. To assess the contribution of BNF to the plant growth-promoting effect, rice seedlings were also inoculated with a nitrogen fixation-deficient mutant. Our results suggest that BNF (at best) only partially contributes to the stimulation of plant growth.

A Comparative Nitrogen Balance and Productivity Analysis of Legume and Non-legume Supported Cropping Systems: The Potential Role of Biological Nitrogen Fixation.

The potential of biological nitrogen fixation (BNF) to provide sufficient N for production has encouraged re-appraisal of cropping systems that deploy legumes. It has been argued that legume-derived N can maintain productivity as an alternative to the application of mineral fertilizer, although few studies have systematically evaluated the effect of optimizing the balance between legumes and non N-fixing crops to optimize production. In addition, the shortage, or even absence in some regions, of measurements of BNF in crops and forages severely limits the ability to design and evaluate new legume–based agroecosystems. To provide an indication of the magnitude of BNF in European agriculture, a soil-surface N-balance approach was applied to historical data from 8 experimental cropping systems that compared legume and non-legume crop types (e.g., grains, forages and intercrops) across pedoclimatic regions of Europe. Mean BNF for different legume types ranged from 32 to 115 kg ha−1 annually. Output in terms of total biomass (grain, forage, etc.) was 30% greater in non-legumes, which used N to produce dry matter more efficiently than legumes, whereas output of N was greater from legumes. When examined over the crop sequence, the contribution of BNF to the N-balance increased to reach a maximum when the legume fraction was around 0.5 (legume crops were present in half the years). BNF was lower when the legume fraction increased to 0.6–0.8, not because of any feature of the legume, but because the cropping systems in this range were dominated by mixtures of legume and non-legume forages to which inorganic N as fertilizer was normally applied. Forage (e.g., grass and clover), as opposed to grain crops in this range maintained high outputs of biomass and N. In conclusion, BNF through grain and forage legumes has the potential to generate major benefit in terms of reducing or dispensing with the need for mineral N without loss of total output.

Soybean Seed Treatment with Nickel Improves Biological Nitrogen Fixation and Urease Activity

Nickel (Ni) is an essential micronutrient required for plants’ metabolism due to its role as a structural component of urease and hydrogenase, which in turn perform nitrogen (N) metabolism in many legume species. Seed treatment with cobalt, molybdenum and Bradyrhizobium strains has been widely practiced to improve crops. Additionally, seed treatment together with Ni fertilization of soybean might improve the efficiency of biological nitrogen fixation (BNF), boosting grain dry matter yield and N content. The objective of this study was to evaluate the effect of soybean seed treatment with Ni rates (0, 45, 90,135, 180, 360 and 540 mg kg-1) on biological nitrogen fixation (BNF), directly by the 15N natural abundance method (δ15N‰) and by measurement of urease [E.C. 3.5.1.5] activity, as well as indirectly by nitrogenase (N-ase) activity [E.C. 1.18.6.1]. Soybean plants (cultivar BMX Potencia RR) were grown in a sandy soil up to the R7 developmental stage (grain maturity), at which point the nutrient content in the leaves, chlorophyll content, urease and N-ase activities, Ni and N content in the grains, nodulation (at R1 - flowering stage), as well as the contribution of biological nitrogen fixation (δ15N ‰), were evaluated. The proportion of N derived from N2 fixation varied from 77 to 99% using the natural 15N abundance method and non-nodulating Panicum miliaceum and Phalaris canariensis as references. A Ni rate of 45 mg kg-1 increased BNF by 12% compared to the control. The increased N uptake in the grains was closely correlated with chlorophyll content in the leaves, urease and N-ase activities, as well as with nodulation. Grain dry matter yield and aerial part dry matter yield increased, respectively, by 84% and 51% in relation to the control plants at 45 mg kg-1 Ni via seed treatment. Despite, Ni concentration was increased with Ni-seed treatment, Ni rates higher than 135 mg kg-1 promoted negative effects on plant growth and yield. In these experimental conditions, seed treatment with low Ni rates caused higher dry matter yield of plants and grains.

Grain legumes differ in nitrogen accumulation and remobilisation during seed filling

ABSTRACTIn grain legumes, the N requirements of growing seeds are generally greater than biological nitrogen fixation (BNF) and soil N uptake during seed filling, so that the N previously accumulated in the vegetative tissues needs to be redistributed in order to provide N to the seeds. Chickpea, field bean, pea, and white lupin were harvested at flowering and maturity to compare the relative contribution of BNF, soil N uptake, and N remobilisation to seed N. From flowering to maturity, shoot dry weight increased in all crops by approximately 50%, root did not appreciably change, and nodule decreased by 18%. The amount of plant N increased in all crops, however in field bean (17 g m−2) it was about twice that in chickpea, pea, and lupin. The increase was entirely due to seeds, whose N content at maturity was 26 g m−2 in field bean and 16 g m−2 in chickpea, pea, and lupin. The seed N content at maturity was higher than total N accumulation during grain filling in all crops, and endogenous N previously accumulated in vegetative parts was remobilised to fulfil the N demand of filling seeds. Nitrogen remobilisation ranged from 7 g m−2 in chickpea to 9 g m−2 in field bean, and was crucial in providing N to the seeds of chickpea, pea, and lupin (half of seed N content) but it was less important in field bean (one-third). All the vegetative organs of the plants underwent N remobilisation: shoots contributed to the N supply of seeds from 58% to 85%, roots from 11% to 37%, and nodules less than 8%. Improving grain legume yield requires either reduced N remobilisation or enhanced N supply, thus, a useful strategy is to select cultivars with high post-anthesis N2 fixation or add mineral N at flowering.

Rhizobia Strain and Legume Genome Interaction Effects on Nitrogen Fixation and Yield of Grain Legume: A Review

Though molecular nitrogen represents nearly 80% of the earth’s atmosphere, it is chemically inert and cannot be directly assimilated by plants. Only limited numbers of prokaryotes are able to convert the N 2 molecule into a usable form of N through a process known as biological nitrogen fixation. Rhizobia are soil bacteria able to form nodules and establish symbiosis with the roots or the stems of leguminous plants. Nitrogen fixation in legume provides important economic advantages for crop production by reducing the cost of N fertilizer. This review covers contribution of biological nitrogen fixation in agriculture, rhizobia and host-legume related factors influencing symbiotic performance. It highlights the rhizobial strain and host-legume interaction effects on N 2 fixation, soil residual nitrogen, and nitrogen and phosphorus uptake of the plant. The review aims to elucidate the approach for selection of the best rhizobia strain-legume variety combination for maximum nitrogen fixation and yield of grain legume. Variation in nodulation and nitrogen fixation frequently occur in a bacteria strain-legume cultivar specific manner. Genotype of both the host and the competing rhizobia strains have been shown to influence inoculant performance .

Diazotrophic bacteria associated with sisal (Agave sisalana Perrine ex Engelm): potential for plant growth promotion

In the semi-arid region of Bahia State, Brazil, sisal (Agave sisalana Perrine ex Engelm) has been cultivated for fibre production for several decades without the use of chemical fertilizers. The contribution of biological nitrogen fixation (BNF) to this crop and the occurrence of diazotrophic rhizosphere, epiphytic or endophytic bacteria have not been investigated. The aims of this work were to study the occurrence and diversity of diazotrophic bacteria in rhizosphere soil, root and leaf tissues of sisal plants from this region, as well as to test their potential for plant growth promotion. Burk’s nitrogen free semi-solid medium was used for isolation and the MPN method was used for quantification of diazotrophic bacteria. BOX-A1R PCR and 16S rRNA sequence analyses were performed to study the diversity of bacterial isolates harboring the nifH-gene. Six isolates were selected for growth promotion of cucumber plants. A high diversity of BNF bacterial isolates was observed in sisal plants and rhizosphere soil. Bacterial populations were higher in roots, followed by soil and sisal leaves. Burkholderia, Leifsonia and Paenibacillus were the predominant genera. All isolates tested were able to promote cucumber growth. This study showed that sisal-associated diazotrophic bacteria are diverse and may be further exploited to promote plant growth.

Recent Advances in Biological Nitrogen Fixation in Agricultural Systems

The environmental concerns due to the increasing amount of the reactive forms of nitrogen in atmosphere, originating from the manufacture and use of chemical fertilizers have resulted in a re-focus on the importance of biological nitrogen fixation (BNF), particularly by legumes. The worldwide contribution of BNF is estimated at 105 Tg yr. Nitrogen fixation is catalysed by the enzyme nitrogenase. Advances have included discovery of alternative nitrogenases, elucidation of more details on the mechanism of action, discovery of novel nitrogenases, existence of nitrogenase siblings in eukaryotes etc. There are fresh efforts to engineer cereal plants to nodulate and fix nitrogen. Discovery of common signalling mechanisms between mycorrhization and nodulation, discovery of cereal endophytes and advances in plastid physiology have given a fresh impetus to such approaches. However, the contribution of agronomically significant quantities of N through endophytes still requires more rigorous evidences. Advances in molecular biology have included a greater understanding of the native diversity of rhizobia and together with advances in plant breeding for selection of high nodulating genotypes, presents an attractive opportunity to maximize BNF. Recent advances include the discovery of novel rhizobial species, emphasis on study of climate change and adaptation to various abiotic stresses like high temperature, drought and salinity. This review focuses on major developments in BNF in last two decades with emphasis on rhizobia. The beneficial effects of applying organics and of rhizobial inoculation practices are discussed to bring out the strategies to improve the contribution of BNF in agriculture.

Short communication. Biological fixation of nitrogen and N balance in soybean crops in the pampas region

Biological nitrogen fixation (BNF) is of key importance in the N balance of soybean (Glycine max) crops. A number of authors have suggested that a negative balance may occur under high yield conditions. Few studies have measured the contribution of BNF to soil N in the pampas region. The aims of the present study were to compare three BNF determination methods – two isotopic methods using sorghum or a non-nodulating soybean isoline as a reference crop, and one involving the calculation of the difference in N content between the nodulating and non-nodulating soybean isolines – and to estimate the N balance in soybean crops raised under conventional tillage and no tillage practices. The study was performed in 2004-2005; a complete randomised block design was used with three replicates (plot dimensions 3 × 7 m). The different methodologies estimated BNF to account for 45-58% of total plant N, equivalent to 94 to 123 kg N ha-1. Depending on the methodology for estimating the BNF the soil N balance varied between –7 and 22 kg N ha-1. With an average grain yield of 1,618 kg ha-1 and a BNF accounting for approximately 50% of total plant N (i.e.,115 kg N ha-1), the soil N balance was slightly positive (14 kg ha-1) and independent of the tillage practice. The tillage systems had no effect (P < 0.05) on the mass or number of nodules, shoot biomass production at the R1 or R6 growth stages, the N content, BNF, or grain yield. Since the present results were obtained using non-commercial soybean isolines, further research is required to determine the soil N balance when high yielding soybean crops are raised.

Contribution of nitrogen fixation to first year Miscanthus × giganteus

AbstractMany characteristics make Miscanthus × giganteus an appealing bioenergy feedstock in temperate North America, but the degree to which this plant species interacts with nitrogen‐fixing bacteria remains understudied. Demonstration of associative nitrogen fixation in Miscanthus would support management with minimal fertilizer inputs that is demanded of long‐term biofuel sustainability. As a first step, we investigate the role of biological nitrogen fixation in nutrition of immature Miscanthus and temporal dynamics of plant‐associated nitrogen fixers. The contribution of biological nitrogen fixation to plant nitrogen acquisition in first year Miscanthus × giganteus was estimated using a yield‐dependent 15N isotope dilution model. Temporal changes in plant‐associated diazotroph relative abundance and community composition were analyzed with quantitative PCR and terminal restriction fragment length polymorphism of the nifH gene in rhizome and rhizosphere DNA extracts. We estimate 16% of new plant nitrogen was derived by nitrogen fixation during the growing season, despite non‐limiting soil nitrogen. Diazotroph communities from rhizome and rhizosphere changed with plant development and endophytic nitrogen fixers had significantly higher relative abundance and altered community composition at sampling dates in July and August. This study provides evidence for a small, but measurable, benefit of associative nitrogen fixation to first year Miscanthus × giganteus that underscores the potential and need for selection of breeding lines that maximize this trait.

Possible contribution of Bradyrhizobium on nitrogen fixation in sweet potatoes

Sweet potato (Ipomoea batatas) is known for its ability to grow under nitrogen-limited conditions. To clarify the possible contribution of biological nitrogen fixation, we tried to isolate and identify diazotrophic bacteria from sweet potatoes. By using cultivation technique, we isolated putative endophytes, which possess nifH genes, from surface-sterilized sweet potatoes. Their nitrogen-fixing abilities were demonstrated by the acetylene reduction assay in a semi-solid malate medium and sweet potato extracts. We also examined the colonization of an isolated strain (AT1) in sweet potatoes and their influence on growth and nitrogen fixation in plants as assessed by an acetylene reduction assay and 15N-isotope dilution technique. The isolates were identified as strains of Bradyrhizobium sp. AT1, Paenibacillus sp. AS2 and Pseudomonas sp. T16 based on their 16S rRNA gene sequences. They showed acetylene reduction activity (ARA) in the semi-solid malate medium. Among them, B. sp. AT1 showed ARA in sweet potato extracts under micro-aerobic conditions whereas both P. sp. AS2 and P. sp. T16 showed no ARA. The inoculation of B. sp. AT1 to the sweet potatoes resulted in increases in the fresh weights and detection of ARA in the inoculated plants. Moreover, the reduction of 15N atom % was observed in the inoculated plants compared to uninoculated controls. B. sp. AT1 actively expresses nitrogenase activity in sweet potatoes and may contribute to the nitrogen nutrition of host plants.

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

Background and aims 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.

Quantitative Proteomic Analysis of the Interaction Between the Endophytic Plant-Growth-Promoting Bacterium Gluconacetobacter diazotrophicus and Sugarcane

Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium that colonizes sugarcane. In order to investigate molecular aspects of the G. diazotrophicus-sugarcane interaction, we performed a quantitative mass spectrometry-based proteomic analysis by (15)N metabolic labeling of bacteria, root samples, and co-cultures. Overall, more than 400 proteins were analyzed and 78 were differentially expressed between the plant-bacterium interaction model and control cultures. A comparative analysis of the G. diazotrophicus in interaction with two distinct genotypes of sugarcane, SP70-1143 and Chunee, revealed proteins with fundamental roles in cellular recognition. G. diazotrophicus presented proteins involved in adaptation to atypical conditions and signaling systems during the interaction with both genotypes. However, SP70-1143 and Chunee, sugarcane genotypes with high and low contribution of biological nitrogen fixation, showed divergent responses in contact with G. diazotrophicus. The SP70-1143 genotype overexpressed proteins from signaling cascades and one from a lipid metabolism pathway, whereas Chunee differentially synthesized proteins involved in chromatin remodeling and protein degradation pathways. In addition, we have identified 30 bacterial proteins in the roots of the plant samples; from those, nine were specifically induced by plant signals. This is the first quantitative proteomic analysis of a bacterium-plant interaction, which generated insights into early signaling of the G. diazotrophicus-sugarcane interaction.