Confirmation of nitrogen fixation in two tropical grasses by 15N2 incorporation

Intact soil cores containing plants of Paspalum notatum or Digitaria decumbens were selected with the acetylene reduction method, and then exposed to ¹⁵N₁₅ to confirm nitrogen fixation in tropical grass-bacteria associations. In a preliminary experiment with P. notatum¹⁵N₂ incorporation was slow but progressive during 24 h in roots but translocation to rhizomes and leaves ceased after 17h. With improved assay chambers, enrichments of 0.151 and 0.563 ¹⁵N atom % excess were obtained in roots of D. decumbens cv transvala and P. notatum systems respectively, after 3 days. Enrichments in rhizomes were similar to those of roots; however in the leaves only 8% of root enrichment was observed. The addition of sucrose to the soil doubled N₂-fixation in roots in both grass species studied, but did not result in increased incorporation into the leaves of P. notatum.     

Nitrogen fixation by Azospirillium brasilense in soil and the rhizosphere under controlled environmental conditions

Summary Acetylene reduction activity by Azospirillum brasilense, either free-living in soils or associated with wheat roots, was determined in a sterilised root environment at controlled levels of O₂ tension and with different concentrations of mineral N. In an unplanted, inoculated soil nitrogenase activity remained low, at approximately 40 nmol C₂H₄ h^-1 per 2kg fresh soil, increasing to 300 nmol C₂H₄ h^-1 when malic acid was added as a C source via a dialyse tubing system. The N₂ fixation by A. brasilense in the rhizosphere of an actively growing plant was much less sensitive to the repressing influence of free O₂ than the free-living bacteria were. An optimum nitrogenase activity was observed at 10 kPa O₂, with a relatively high level of activity remaining even at an O₂ concentration of 20 kPa. Both NO _inf3 ^sup- and NH _inf4 ^sup+ repressed nitrogenase activity, which was less pronounced in the presence than in the absence of plants. The highest survival rates of inoculated A. brasilense and the highest rates of acetylene reduction were found in plants treated with azospirilli immediately after seedling emergence. Plants inoculated at a later stage of growth showed a lower bacterial density in the rhizosphere and, as a consequence, a lower N₂-fixing potential. Subsequent inoculations with A. brasilense during plant development did not increase root colonisation and did not stimulate the associated acetylene reduction. By using the ¹⁵N dilution method, the affect of inoculation with A. brasilense in terms of plant N was calculated as 0.067 mg N₂ fixed per plant, i.e., 3.3% of the N in the root and 1.6% in the plant shoot were of atmospheric origin. This ¹⁵N dilution was comparable to that seen in plants inoculated with non-N₂-fixing Psudomonas fluorescens.     

Use of 15N isotope dilution for quantification of N2 fixation associated with roots of kallar grass (Leptochloa fusca (L.))

Summary Leptochloa fusca (L.) Kunth (kallar grass) has previously been found to exhibit high rates of nitrogen fixation. A series of experiments to determine the level of biological nitrogen fixation using ¹⁵N isotopic dilution were carried out in nutrient solution and saline soil. In the nutrient solution, E. coli inoculated plants were taken as non-nitrogen-fixing control. It was observed that nearly 60%–80% of the plant N was derived from atmospheric fixation. Estimations based on the N difference method gave much lower values (18%–35%). In experiments with saline soil which was initially sterilized with chloroform fumigation, a mixed culture of N₂-fixing rhizospheric isolates from kallar grass roots was inoculated and planted to kallar grass. Uninoculated treatments were regarded as controls. The soil was previously labelled with ¹⁵N by adding cellulose and (¹⁵NH₄)₂SO₄. The results of these studies showed fixation values of 6%–32% when estimated by ¹⁵N dilution, whereas by the N difference method 54% of the plant N was estimated to be derived from fixation. This discrepancy is due to the increase in root proliferation due to inoculation, which results in greater uptake of soil N. The distribution of ¹⁵N in different fractions of the soil-N indicted isotopic dilution due to bacterial fixation of atmospheric N₂.     

Estimating biological N2 fixation by a tropical legume tree using the non-nodulating phenophase as the reference in the 15N natural abundance method

The non-nodulating phenophase of a legume tree was tested as a non-N₂-fixing reference in application of the ¹⁵N natural abundance method for estimating the N₂ fixation. We applied this method to study the effects of three pruning intensities, complete pruning every 6 months (T-6), ca. 50% pruning every 3 months (P-3) and intact control (C), on N₂ fixation in Erythrina lanceolata (Papilionaceae) planted as shade and support trees for vanilla (Vanilla planifolia) in a subhumid tropical site in Quepos, Costa Rica. We measured nodulation and N₂ fixation for 12 months. The trees under the C regime nodulated abundantly during the rainy season vegetative growth but did not nodulate during the dry season and flowering. A linear regression (r²=0.76) was observed between the nodule biomass and δ¹⁵N values under the C regime, suggesting quite a stable specific N₂ fixation rate. The stable δ¹⁵N values throughout the year in the non-N₂-fixing Morus nigra (Moraceae) growing on the same soil indicated that the temporal variation in the plant available soil ¹⁵N was low. We used the intercept term of the regression (δ¹⁵N=3.5 when nodule biomass was 0) as the non-N₂-fixing δ¹⁵N value when estimating the percentage of N fixed from atmosphere out of tree total N. The percentage varied from 0 during the driest period to 53% during rainy season. Pruning reduced rainy season nodulation under the T-6 and P-3 regimes almost to nil, and the δ¹⁵N values were high. Our results suggest that the conditions for using the non-nodulating phenophase as the non-N₂-fixing reference required in the ¹⁵N natural abundance method were fulfilled. The C trees showed a clear phenological cycle in N₂ fixation, while pruning severely disturbed the N₂ fixation under the T-6 and P-3 regimes, indicating that E. lanceolata is better suited for agroforestry systems in which the trees are not managed by periodic prunings.     

Influence of pruning frequency of Albizia lebbeck, Gliricidia sepium and Leucaena leucocephala on nodulation and potential nitrogen fixation

The influence of four pruning frequencies on biomass, nodulation and N₂ fixation was investigated on Albizia lebbeck, Gliricidia sepium and Leucaena leucocephala grown in the screenhouse for 16 months, using acetylene reduction and ¹⁵N dilution methods. Frequent prunings at 4-month intervals had no deleterious effect on symbiotic N₂ fixation, which increased in Gliricidia and Leucaena in particular. Nodulation and nitrogenase activity varied inconsistently within species, and were not influenced by pruning frequency. Cumulative assessment of pruning effect showed higher biomass, N yield and N₂-fixing capacity of the woody species than at last harvest, and appeared to have more practical relevance. Across species, cumulative total dry matter, N yields, and both percentage and absolute amount of N₂ derived from atmosphere increased with pruning frequency, except when trees were pruned 3 times. Of the three species, G. sepium had the lowest biomass production, N₂ fixation and N accumulation. Received: 25 October 1995     

Nitrogenase activity on the roots of tropical forage grasses

Nitrogen fixation in the rhizospheres of field grown tropical forage grasses was studied by the acetylene reduction method. Values varied considerably between sites but indicate the possible economic importance of several of the species studied. Maximal nitrogenase activity measured (nmoles C₂H₄g^?1 dry roots h^?1) was 754 for Pennisetum purpureum, 750 for Brachiaria mutica, 341 for Digitaria decumbens, 299 for Panicum maximum, 283 for Paspalum notatum, 269 for Cynodon dactylon, 41 for Melinis minutiflora and 29 for Hyparrhenia rufa. Nitrogenase activity varied considerably with season and was maximal during active vegetative growth of two of the grasses. Significant differences between Paspalum notatum ecotypes and cultivars. in Azotohacter paspali occurrence and nitrogen fixation, indicate the possibility of plant breeding to enhance nitrogen fixation in grass rhizospherc associations. Other research lines of agronomic importance are fertilizer effects. In intact soil plant cores with the Paspalum system 10 parts/10⁶ NH_4⁺J-N inhibited nitrogenase activity within 2 h and 10 parts/10⁶ NO^?₃-N within 4 h. but after 1 week these effects were negligible. In the field, nitrogenase activity on roots of P. purpureum and D. decumbens, assayed 2 weeks after top dressings of 20 kg N ha^?1 as NH₄NO₃. was not affected even after eight such dressings.     

Estimating N2 fixation by Sesbania rostrata and S. cannabina (syn. S. aculeata) in lowland rice soil by the 15N dilution method

Summary A field experiment in concrete-based plots was conducted to estimate the contribution of N derived from air (Ndfa) or biological N₂ fixation in Sesbania rostrata and S. cannabina (syn. S. aculeata), using various references, by the ¹⁵N dilution method. The two Sesbania species as N₂-fixing reference plants and four aquatic weed species as non-N₂-fixing references were grown for 65 days after sowing in two consecutive crops, in the dry and the wet seasons, under flooded conditions. Soil previously labeled with ¹⁵N at 0.26 atom % ¹⁵N excess in mineralizable N was further labeled by ammonium sulfate with 3 and 6 atom % ¹⁵N excess. The results showed that ¹⁵N enrichment of soil NH ₄ ⁺ -N dropped exponentially in the first crop to half the original level in 50 days while in the second crop, it declined gradually to half the level in 130 days. The decline in ¹⁵N enrichment, in both N₂-fixing and non-fixing species, was also steeper in the first crop than in the second crop. Variations in ¹⁵N enrichment among non-fixing species were smaller in the second crop. The ratio of the uptake of soil N to that of fertilizer N in N₂-fixing and non-fixing species was estimated by the technique of varying the ¹⁵N level. In the second crop, this ratio in non-fixing species was higher than that in N₂-fixing species. Comparable estimates of % Ndfa were obtained by using ¹⁵N enrichment of various non-fixing species. There was also good agreement between the estimates obtained by using ¹⁵N enrichment of non-fixing species and those by using soil NH ₄ ⁺ -N, particularly in the second crop. By 25 days after sowing, the first crop of both Sesbania spp. had obtained 50% of total N from the atmosphere and the second crop had obtained 75%. The contribution from air increased with the age of the plant and ranged from 70% to 95% in 45–55 days. S. rostrata fixed substantially higher amounts of N₂ due to its higher biomass production compared with S. cannabina. Mathematical considerations in applying the ¹⁵N dilution method are discussed with reference to these results.     

A comparison of nitrogen fixation in genotypes of groundnut (Arachis hypogaea L.) using 15N-isotope dilution

Summary Nitrogen fixation in seven groundnut genotypes was measured by ¹⁵N-isotope dilution using a non-nodulating cultivar of groundnut as the nonfixing reference plant. Nitrogen fixation varied between 100 kg N ha^–1 in genotype J-11 and 153 kg N ha^–1 in Robut 33-1. The amount of plant-available soil N was small, so that 86%–92% of plant nitrogen was derived from N₂-fixation. Thus differences in N₂-fixation between genotypes closely reflected differences in their total N accumulation.ICRISAT Journal Article no. 600     

Evaluation of 15N-isotope dilution for measurement of nitrogen fixation in chickpea (Cicer arietinum L.)

Summary N accumulation, nodulation, and acetylene reduction activity were measured at frequent intervals during the growth of two chickpea genotypes, and N₂ fixation was estimated by an isotope-dilution method, using safflower as a non-N₂-fixing reference. Safflower was more efficient at N uptake than both the chickpea genotypes for at least the first 50 days and thus could not be used as an accurate reference control. We recommend that further work should employ non-nodulatiog genotypes of chickpea as reference plants and use slow-release forms of 15N fertilizer. Direct genotype comparison by isotope dilution estimated that genotype K 850 fixed 16–18 kg ha^–1 more N than G 130, and this difference was supported by the greater nodule mass and acetylene reduction activity in the K 850 cultivar. Inoculation with an ineffective chickpea Rhizobium sp. led to 69% nodulation on cultivar K 850 but only 33% on G 130. While nodule weight, N uptake, and acetylene reduction activity decreased with inoculation in K 850, the isotope dilutions were similar for both inoculation treatments. The lack of a significant effect on N₂ fixation was ascribed to the partial success of inoculant establishment.Published as Journal Article No. JA 692 of the International Crops Research Institute for the Semi Arid Tropics, Patancheru, A.P. 502324, India     

Enrichment of natural 15N abundance in frankia-infected nodules

Abstract

The enrichment of ¹⁵N in the nodules of some N₂-fixing leguminous plants is an interesting finding (Shearer et al. 1982). The extent of ¹⁵N enrichment differed depending on the plant species (Shearer et al. 1982; Yoneyama 1987) and bacterial strains (Steele et al. 1983), and in soybeans it was apparently related to the nitrogen fixation efficiency (Shearer et al. 1984)     

Determining Symbiotic Nitrogen Fixation in Dry Bean Cultivars Using Ureide Method and Isotope Dilution Techniques

Symbiotic nitrogen fixation (SNF) is an environmentally safe source of nitrogen (N) to the crop plants. In total, 12 dry bean (Phaseolus vulgaris L.) cultivars from pinto, navy, black, and kidney market classes were inoculated with rhizobia and grown in a greenhouse. SNF was estimated using isotope dilution technique and ‘ureide’ method. The amount of SNF ranged between 33 and 68 mg N plant^–1 when determined using ¹⁵N isotope dilution and followed the order: pinto > navy > black > kidney. Percent N derived from atmosphere (%Ndfa) significantly varied between 49% and 90% at V₃ and between 71% and 98% at R₂ stages. The outcomes of the experiment suggested that dry bean cultivars from different market classes have variable N₂ fixation ability, and fertilizer N required should be calculated according to their SNF potentials and N need of a specific market class or cultivar. Stable isotope dilution should be used as the standard procedure to estimate the SNF in dry bean.     

Assessment of the relative uptake of added and indigenous soil nitrogen by nodulated legumes and reference plants in the 15N dilution measurement of N2 fixation: Derivation of method

Current methods for measuring N₂ fixation by nodulated legumes involve the addition of small amounts of ¹⁵N-labelled plant-available N compounds to soil so that plant N derived from the soil may be identified. All such methods assume that the proportions of added N and indigenous soil N assimilated by N₂-fixing and non-fixing plants grown in the same soil are the same, irrespective of the amount of soil N assimilated. The development of a method for assessing these proportions is described.Nodulated legumes and reference plants are grown in soils receiving none or one rate of addition of labelled N compound containing several (two or more) concentrations of ¹⁵N. The proportions of added and indigenous N assimilated, are determined from the intercepts and slopes of regression lines relating isotopic composition of plant N to that of added N, together with some other readilly-obtainable plant N measurements.     

Effects of root-associated N2-fixing cyanobacteria on the growth and nitrogen content of wheat (Triticum vulgare L.) seedlings

Summary Following co-cultivation of wheat with N₂-fixing cyanobacterial isolates capable of forming associations, Nostoc 2S6B, 2S9B or Anabaena C5, for 15 days in the presence or absence of combined N a large stimulation of root length was observed without any increase in root dry weight. Increases in the N concentrations of both roots and shoots occurred following co-cultivation with most cyanobacteria tested. The increase in plant N concentrations appeared to be dependent on the wheat cultivar and the cyanobacterial isolate used. Nostoc isolates had similar nitrogenase activities when associated with roots and when grown in shake-flask cultures. The nitrogenase activity of roots colonized by Anabaena C5 or Nostoc 2S6B was higher following removal of loosely associated cyanobacteria.     

Symbiotic nitrogen fixation in the alpine community of a lichen heath of the Northwestern Caucasus Region (the Teberda Reserve)

The symbiotic fixation of atmospheric nitrogen by leguminous plants in the alpine community of a lichen heath at the Teberda State Biosphere Reserve is well adapted to low soil temperature characteristic for the altitude of 2800 m a.s.l. For the determination of the N fixation by isotopic methods (the method of the natural ¹⁵N abundance and the method of isotopic ¹⁵N dilution), Trifolium polyphyllum was taken as the control plant. This plant was used as it does not form symbiosis with the nitrogen-fixing bacteria in the highlands of the Northern Caucasus Region. The contribution of the N fixation to the N nutrition of different leguminous plant species as determined by the natural ¹⁵N abundance method amounted to 28–73% at δ¹⁵N₀ = 0‰ and 46–117% at δ¹⁵N₀ = −1‰; for the determination of the N fixation by the method of the isotopic label’s dilution, it was 34–97%. The best correlation of the results obtained by these two isotopic methods was observed for the natural fractionation of the N isotopes in the course of the N fixation in the range of −0.5 to −0.7‰. The determination of the nitrogenase activity of the roots by the acetylene method confirmed the absence of N fixation in T. polyphyllum and its different contribution to the N nutrition of different species of leguminous plants.     

Host-rhizobia interaction for effective inoculation: evaluation of the potential use of the ureide assay to monitor the symbiotic performance of tepary bean (Phaseolus acutifolius A. Gray)

Domesticated and wild-type tepary beans (Phaseolus acutifolius A. Gray) were grown with or without inoculation with rhizobia in pots under bacteriologically controlled conditions in a temperature-controlled glasshouse. Seeds were inoculated with a mixture of seven strains isolated from nodules collected from domesticated field-grown tepary bean in Arizona, USA, or with a commercial inoculant strain for Phaseolus vulgaris (CC511). Different degrees of plant reliance upon N₂ fixation for growth were generated by supplying the inoculated plants throughout growth with nutrients containing a range of concentrations of ¹⁵N-labeled NO₃ (0, 1, 2, 5 or 10 mM). An uninoculated treatment that received 10 mM ¹⁵N-labeled NO₃ was included to provide data for plants solely dependent upon NO₃ for growth. Six weeks after sowing, shoots were harvested for dry matter determination and subsequent ¹⁵N analysis, root-bleeding xylem sap was collected, and nodulation assessed. With regard to shoot biomass production, domesticated lines were more responsive to inoculation, but less responsive to applied N than wild types. All inoculated plants were nodulated, but the field isolates from tepary bean were more effective in N₂ fixation than strain CC511. It was concluded that tepary bean requires a specific inoculant to benefit from fixation of atmospheric N₂. Xylem sap samples were analysed for ureides (allantoin and allantoic acid), amino acid content (α-amino-N), and NO₃ concentration. The amount of ureide-N present in xylem sap was expressed as a percentage of total solute N, described as the relative abundance of ureide-N (RUN), for each N treatment and was compared to the proportion of plant N derived from N₂ fixation (%Ndfa) calculated using a ¹⁵N dilution technique. The RUN values ranged from 8% for saps collected from uninoculated plants provided with 10 mM NO₃ in the nutrient solution (%Ndfa=0) to 86-91% for nodulated plants grown in the absence of externally supplied NO₃ (%Ndfa=100). These data indicated that ureides were the principal product of N₂ fixation exported from the nodules to the shoot in xylem sap. Since RUN values were closely related to %Ndfa, it was proposed that N-solute analysis of xylem sap could provide a valuable analytical tool to monitor the symbiotic performance of tepary bean.     

Nitrogen Accumulation in Soybean Following Defoliation

The effects of defoliation on soybean [Glycine max (L.) Merr.] growth and yield have been well studied, but relatively little is known about its nitrogen (N) accumulation after defoliation. An experiment was conducted to examine soybean recovery and N accumulation following defoliation. The indeterminate cultivar (‘Tousan 69’) was planted in a greenhouse, and two defoliation treatments (no defoliation and 67% defoliation) were imposed at the R₂ stage when plants had at least one flower in the two uppermost nodes. At 0, 15, 30 and 45 days after defoliation (DAD), plants were destructively sampled to measure dry mass production, nitrogen accumulation and nitrogen fixation. Seed yield and N concentration also were measured at maturity. Neither the seed yield nor its N concentration was affected by defoliation. Although defoliation temporarily reduced soybean dry weight and N accumulation during 15 DAD, defoliated plants completely recovered their dry weight and N accumulation 30 DAD. There was little difference in N concentration between defoliated and non defoliated plants, indicating that N acquisition was restored during the recovery process. Recovery of N accumulation in defoliated plant was due to complete recovery of N₂-fixing ability and maybe related to improvement in N absorption after defoliation.     

Nitrogen fixation by nodulated soybean under tropical field conditions estimated by the 15N isotope dilution technique

The contribution of biological N₂ fixation to the N nutrition of nodulated soybean was estimated using the ¹⁵N isotope dilution technique and a non-nodulating soybean isoline as a non-fixing control plant. The plants were grown in the field in concrete cylinders (60 cm dia) and harvested at seven stages of plant growth. Labelled N was added to the soil either as labelled organic matter before planting or in seven small additions (2kg N ha^?1) of (NH₄)₂SO₄ during the growing period.There was good agreement between isotope dilution estimates of nitrogen fixation for the two labelling methods. Acetylene reduction assays on intact root systems greatly underestimated N₂ fixing activity. The difference in total N between nodulated and non-nodulated plants generally gave higher estimates compared with the isotope technique. The data indicate that this was because nodulated plants recovered more N from the soil than the non-nodulated plants. After 92 days of growth, the soybean derived approximately 250kg N ha^?1 from biological N₂ fixation.     

The fate of markerAzospirillum lipoferum inoculated into rice and its effect on growth,yield and N2 fixation of plants studied by acetylene reduction,15N2 feeding and15N dilution techniques

Summary A spontaneous mutant ofAzospirillum lipoferum, resistant to streptomycin and rifampicin, was inoculated into the soil immediately before and 10 days after transplanting of rice (Oryza sativa L.). Two rice varieties with high and low nitrogen-fixing supporting traits, Hua-chou-chi-mo-mor (Hua) and OS4, were used for the plant bacterial interaction study. The effect of inoculation on growth and grain and dry matter yields was evaluated in relation to nitrogen fixation, by in situ acetylene reduction assay,¹⁵N₂ feeding and¹⁵N dilution techniques. A survey of the population of marker bacteria at maximum tillering, booting and heading revealed poor effectivety. The population of nativeAzospirillum followed no definite pattern. Acetylene-reducing activity (ARA) did not differ due to inoculation at two early stages but decreased in the inoculated plants at heading. In contrast, inoculation increased tiller number, plant height of Hua and early reproductive growth of both varieties. Grain yield of both varieties significantly increased along with the dry matter. Total N also increased in inoculated plants, which was less compared with dry matter increase.¹⁵N₂ feeding of OS4 at heading showed more¹⁵N₂ incorporation in the control than in the inoculated plants. The ARA,¹⁵N and N balance studies did not provide clear evidence that the promotion of growth and nitrogen uptake was due to higher N₂ fixation.     

Short- and long-term effects of ammonium on photodependent nitrogen fixation in wetland rice fields of Spain

Short- and long-term experiments were conducted in the rice fields of Valencia, Spain, to determine the ecological significance of ammonium on nitrogen fixation. A significant inhibition of nitrogenase activity by ammonium, at concentrations higher than 0.5mM, was observed after 8h of incubation in short-term experiments done with a bloom of the N₂-fixing cyanobacterium Anabaena sp. In a second set of short-term experiments for in situ assays of nitrogenase activity in the field, a significant correlation between nitrogenase activity and the number of N₂-fixing cyanobacteria in soil was found. No significant inhibition of nitrogenase activity by ammonium at concentrations up to 2mM was observed in these assays after 24h of incubation. This lack of inhibition was probably due to the rapid decrease in ammonium content in the flood water. Only 5% of the ammonium initially added remained in the water 24h later. In the long-term experiments, nitrogenase activity was assayed in plots fertilized with 0, 70 and 140kgNha^–1, over the cultivation cycle, for 5 years. A partial inhibition of nitrogenase activity by deep-placed N fertilizers was observed. Differences were only significant in 2 years. Mean results from 5 years only showed significant differences between plots fertilized with 0 and 140kgNha^–1. The partial inhibition of nitrogenase activity by ammonium increased over the cultivation cycle. Inhibition was only significant in September, at the end of the cultivation cycle. Received: 28 January 1996     

Paddy System with a Hybrid Rice Enhances Cyanobacteria Nostoc and Increases N2 Fixation

Biological nitrogen(N) fixation(BNF) plays a significant role in maintaining soil fertility in paddy field ecosystems. Rice variety influences BNF, but how different rice varieties regulate BNF and associated diazotroph communities has not been quantified. Airtight,field-based ~(15)N_2-labelling growth chamber experiments were used to assess the BNF capacity of different rice varieties. In addition,both the 16 S rRNA and nifH genes were sequenced to assess the influence of different rice varieties on bacterial and diazotrophic communities in paddy soils. After subjecting a rice-soil system to 74 d of continuous airtight, field-based ~(15)N_2 labelling in pots in a growth chamber, the amounts of fixed N were 22.3 and 38.9 kg ha~(-1) in inbred japonica(W23) and hybrid indica(IIY) rice cultivars planted in the rice-soil systems, respectively, and only 1%–2.5% of the fixed N was allocated to the rice plants and weeds. A greater abundance of diazotrophs was found in the surface soil(0–1 cm) under IIY than under W23. Sequencing of the 16 S rRNA gene showed significantly greater abundances of the cyanobacterial genera Nostoc, Anabaena, and Cylindrospermum under IIY than under W23.Sequencing of the nifH gene also showed a significantly greater abundance of Nostoc under IIY than under W23. These results indicate that the hybrid rice cultivar(IIY) promoted BNF to a greater extent than the inbred rice cultivar(W23) and that the increase in BNF might have been due to the enhanced heterocystous cyanobacteria Nostoc.