Real-time imaging of nitrogen fixation in an intact soybean plant with nodules using 13N-labeled nitrogen gas

Real-time images of nitrogen fixation in an intact nodule of hydroponically cultured soybean ( Glycine max [L] Merr.) were obtained. In the present study, we developed a rapid method to produce and purify ¹³N-labeled radioactive nitrogen gas (half life: 9.97 min). ¹³N was produced from a ¹⁶O (p, α) ¹³N nuclear reaction. The target chamber was filled with CO₂ and irradiated for 10 min with protons at an energy of 18.3 MeV and an electric current of 5 μA, which was delivered from a cyclotron. All CO₂ in the collected gas was absorbed and removed with powdered soda-lime in a syringe and replaced with helium gas. The resulting gas was injected into gas chromatography and separated and a 35 mL fraction, including the peak of [¹³N]-nitrogen gas, was collected by monitoring the chromatogram. The obtained gas was mixed with 10 mL of O₂ and 5 mL of N₂ and used in the tracer experiment. The tracer gas was fed into the underground part of intact nodulated soybean plants and serial images of the distribution of ¹³N were obtained non-invasively using a positron-emitting tracer imaging system (PETIS). The rates of nitrogen fixation of the six test plants were estimated to be 0.17 ± 0.10 μmol N₂ h⁻¹ from the PETIS image data. The decreasing rates of assimilated nitrogen were also estimated to be 0.012 ± 0.011 μmol N₂ h⁻¹. In conclusion, we successfully observed nitrogen fixation in soybean plants with nodules non-invasively and quantitatively using [¹³N]N₂ and PETIS.     

Effect of elevated carbon dioxide on growth and nitrogen fixation of two soybean cultivars in northern China

The effect of elevated carbon dioxide (CO₂) concentration on symbiotic nitrogen fixation in soybean under open-air conditions has not been reported. Two soybean cultivars (Glycine max (L.) Merr. cv. Zhonghuang 13 and cv. Zhonghuang 35) were grown to maturity under ambient (415?±?16?μmol?mol^?1) and elevated (550?±?17?μmol?mol^?1) [CO₂] at the free-air carbon dioxide enrichment experimental facility in northern China. Elevated [CO₂] increased above- and below-ground biomass by 16–18% and 11–20%, respectively, but had no significant effect on the tissue C/N ratio at maturity. Elevated [CO₂] increased the percentage of N derived from the atmosphere (%Ndfa, estimated by natural abundance) from 59% to 79% for Zhonghuang 13, and the amount of N fixed from 166 to 275?kg N ha^?1, but had no significant effect on either parameter for Zhonghuang 35. These results suggest that variation in N₂ fixation ability in response to elevated [CO₂] should be used as key trait for selecting cultivars for future climate with respect to meeting the higher N demand driven by a carbon-rich atmosphere.     

Effects of preceding compost application on the nitrogen budget in an upland soybean field converted from a rice paddy field on gray lowland soil in Akita,Japan

Abstract

The annual nitrogen (N) budget was measured in a soybean-cultivated upland field during the first year after conversion from a paddy field on gray lowland soil, which is typically found on the Sea of Japan side of northern Japan. Forage rice was cultivated on lysimeter fields for 4 consecutive years with applications of chemical fertilizer, immature compost, or mature compost (the control, immature compost, and mature compost plots, respectively), and then the fields were converted to upland fields for soybean (Glycine max [L.] Merrill cultivar Ryuho) cultivation. Input (seed, bulk N deposition, and symbiotic dinitrogen [N₂] fixation) and output (harvested grain, leached N via drainage water, and nitrous oxide emission) N flows were measured, and the field N budget was estimated from the difference between the input and output. The soybean plants in the immature and mature compost plots grew well and had higher yields (498–511 g m)^?2) compared to the control plot (410 g m)^?2). Total N accumulation in the soybean plants derived from N₂ fixation (g N m)^?2) in the mature compost plot (27.7) was higher than those in the control (18.1) and immature compost plots (19.9). Percentages of soybean N accumulation derived from N₂ fixation ranged from 53% to 74%. N derived from symbiotic N₂ fixation accounted for more than 90% of the total N input, whereas harvested grain accounted for approximately 85% of the total N output. N leaching mainly occurred during the fallow period, accounting for 13–15% of the total N output. The annual N budgets were negative (?10.0,?14.2, and ?6.4 g N m)^?2 year)^?1 for the control, immature compost, andmature compost plots, respectively). The Nloss from the immature compost plot was higher than that of the control plot, because the N output in harvested grain was higher, and the N input by N₂ fixation was similar between plots. While the N loss from the mature compost plot was lower than that of the control plot because the N output in harvested grain was higher, as was the case in the immature compost plot, the N input by N₂ fixation was also higher. Preceding compost application—whether immature or mature compost—to paddy fields increased the subsequent soybean yield during the first year after conversion. This result suggests that N loss and the following decrease in soil N availability in the field could be mitigated by increased N₂ fixation resulting from mature compost application with an appropriate application practice.     

Effects of changes in applied nitrogen concentrations on nodulation,nitrogen fixation and nitrogen accumulation during the soybean growth period

ABSTRACT

The specific mechanism by which nitrogen application affects nodulation and nitrogen fixation in legume crops remains uncertain. To further study the effects of nitrogen application on soybean nodulation and nitrogen accumulation, three consecutive tests were performed during the VC-V4, V4-R1 (10 days), and R1-R2 (10 days) growth periods of soybean. In a dual-root soybean system, seedlings on one side were watered with a nutrient solution containing NH₄⁺ or NO₃^? as the N source (N+ side), and those on the other side were watered with a nitrogen-free nutrient solution (N- side). During the VC-R2 period, on the N+ side, high nitrogen treatment inhibited nodule growth and nitrogenase activity (EC 1.18.6.1), and the inhibition was significantly increased with increasing high nitrogen supply time (10 days, 20 days). When the high nitrogen treatment time reached 20 days, the specific nitrogenase activity (C₂H₄ μmol^?1 g^?1 nodule dry mass h^?1) was similar to that in the low nitrogen treatment, indicating that the nitrogen fixation capacity per gram of dry mass nodules was almost the same. Therefore, it is assumed that long-term high nitrogen treatment mainly reduces nitrogen fixation by reducing the nodule number. The effect of nitrogen concentration on the roots on the N+ side was greater than that on the N- side. Taken together, these results indicate that nitrogen application affects a contact-dependent local inhibition of root nodule growth, nitrogenase activity, and nitrogen accumulation. The whole plant systematically regulates specific nitrogenase activity, and high nitrogen inhibition is recoverable.     

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.     

Incorporation of 15N into various nitrogenous compounds in intact soybean nodules after exposure to 15N2 gas

The intact nodules attached to the upper part of soybean roots were exposed to ¹⁵N₂ and the incorporation of ¹⁵N into various soluble nitrogen constituents was investigated. Results indicated that ammonia, a primary product of N₂ fixation, was located in more than two compartments. Ammonia reduced from N₂ gas seemed to be incorporated firstly into glutamine especially amido-group nitrogen. Newly fixed nitrogen was secondly incorporated into glutamic acid and alanine in this sequence. These results suggested that fixed ammonia was assimilated by glutamine synthetase/glutamate synthase pathway. Turn-over rate of allantoin plus allantoic acid and serine was relatively high, although apparently these compounds were not primary products of newly fixed ammonia. ¹⁵N content of allantoin was always higher than that of allantoic acid. ¹⁵N incorporation to aspartic acid and asparagine was relatively slow, especially in early period. In bacteroid fraction there is much amount of ammonia comparing with other compounds, while allantoin and asparagine were presented exclusively in cytosol. ¹⁵N was incorporated into nitrate within a few minutes especially in bacteroids.     

Modelling nitrogen fixation of pea ( Pisum sativum L.)

Abstract

Nitrogen fixation was simulated for a leafless variety (Delta) of pea (Pisum sativum L.) in central Sweden. It is assumed that N₂ fixation is basically proportional to root biomass, but limited by high root N or low substrate carbon concentrations. Input data on root carbon and nitrogen were estimated from observations of above-ground biomass and nitrogen. The simulated N₂ fixation was compared with estimated values from observations using the ¹⁵N labelling technique. Test data were taken from pea monocultures and pea-oat mixtures with varying pea biomass levels during 1999. Simulated within-season accumulated N₂ fixation correlated to the estimated N₂ fixation with a correlation coefficient (R ²) of 0.74. For seasonal simulations, the predictability was higher (R ²=0.93). Two alternative non-dynamic models, estimating seasonal N₂ fixation as proportional to above-ground biomass and above-ground N, respectively, gave lower predictability (R ²=0.83 and 0.80, respectively). The models were also applied to a second year (1998) and two other sites by comparison with accumulated N₂ fixation estimated by the Difference method. A halved specific N₂ fixation rate (expressed per unit of root biomass) in 1999, compared with 1998, corresponded to essentially dryer and warmer soil conditions during 1999. It was indicated that the variations in soil moisture were more important than soil temperature. It was concluded that the abiotic responses might be of great importance for modelling N₂ fixation rate under different soil conditions.     

13N-nitrate uptake sites and rhizobium-infectible region in a single root of common bean and soybean

The positron emitting tracer-imaging system (PETIS) was used to determine whether it was possible to obtain on image of ¹³N-distribution in common bean in which a single root was fed with a liquid medium containing nitrate at different concentrations with different ¹³N specific activities. The distribution of the images of the ¹³N atoms in the root could be obtained over a wide range of nitrate concentrations and ¹³N specific activities in the medium. As for nitrate stress on leguminous root nodulation, the positional relationship between the nitrate uptake sites and root hair just elongating area, where rhizobia capably initiate their infection, was studied in common bean and soybean. PETIS gave direct evidence that single roots of both common bean and soybean showed one or two dense ¹³N-distribution areas after 2 min pulse-feeding of ¹³N0₃ ^-. These areas remained stable over 30 min, and the first dense site, which was common in all the examined roots, extended over ca. 1 cm above the root apex. Microscopic observation revealed that this area covered both sites of rhizobium infection and of early nodule development in a common bean and soybean single root.     

Effects of deep placement of controlled-release nitrogen fertilizer on soybean growth and yield under sulfur deficiency

Sulfur (S) and Nitrogen (N) metabolisms in plants are interacted and it is known that S deficiency decrease N absorption and metabolism. In leguminous plants S deficiency also decreases N₂ fixation by rhizobia in the nodules. Deep placement of a controlled-release N fertilizer is a good method to provide nitrogen to soybean without inhibiting N₂ fixation; thus, it was hypothesized that this method is able to provide nitrogen effectively to sulfur-deficient soybean plants. In this study effects of deep placement of coated urea on S-N physicological interaction, growth and productivity in soybean plants were examined using pot experiments. Soybean plants were grown with sulfate concentrations of 30, 100, or 1000 μM, with or without deep placement of coated urea. Shoot weights at the developing stage were not affected by S deficiency. SPAD values of leaves during the flowering stage decreased with S deficiency and increased with the deep placement of coated urea. S deficiency decreased seed weight per plant at the harvesting stage, but this decrease was attenuated by the deep placement of coated urea. N and S content in shoots at the developing stage increased with the deep placement of coated urea, whereas in seeds, only the N content increased. N₂ fixation activity based on the relative ureide-N content in xylem sap indicated that the deep placement of coated urea did not inhibit N₂ fixation activity at the early flowering stage. Without deep placement of coated urea, the relative ureide-N content decreased under S deficiency at the seed filling stage. These results suggest that the deep placement of coated urea is an efficient method to supply N to support soybean yield under S deficiency.

Abbreviations: Deep+: with deep placement of coated urea; Deep–: without deep placement of coated urea     

Drought and nitrogen source effects on nitrogen nutrition,seed growth,and yield in soybean

Drought in soybean [Glycine max (L.) Merr.] decreases yield‐related processes and N₂ fixation is more sensitive to drought than are many other of these processes. Therefore, application of nitrogen (N) fertilizer may increase drought tolerance over those plants primarily dependent on N₂ fixation. In a field experiment, NH₄NO₃ applications (+N) to drought‐stressed soybean resulted in biomass and N accumulation rates similar to those rates for an irrigated treatment without N fertilizer (‐N). In contrast, biomass and N accumulation rates were decreased for the ‐N treatment. N fertilization increased seed growth rate and decreased seed fill duration for irrigated and drought treatments. In the drought treatment, N application increased seed number per unit area, which resulted in higher yields. In a greenhouse experiment, fertilization with either KN0₃ or NH₄C1 increased biomass and N accumulation rates during drought over those of plants dependent solely on N₂ fixation. It was concluded that application of N fertilizer to soybean increases drought tolerance because of the extreme sensitivity of N₂ fixation to drought.     

Visualization of 15O-water flow in tomato and rice in the light and dark using a positron-emitting tracer imaging system (PETIS)

Abstract

¹⁵P-water flow from the roots to the top in tomato (Lycopersicon esculentum Mill.) and rice (Oryza sativa L.) plants was visualized with time using a positron-emitting tracer imaging system (PETIS). The ¹⁵O-water flow was switched on by light and completely stopped in the dark. The flow rate in the stem of tomato and the shoot of rice at a light intensity of 500 μmol·m^?2·s^?1 was 1.9 and 0.4 cm min^?1, respectively.     

Quantitative analysis of the initial transport of fixed nitrogen in nodulated soybean plants using 15N as a tracer

The quantitative analysis of the initial transport of fixed isotope 15-nitrogen (¹⁵N) in intact nodulated soybean plants (Glycine max [L.] Merr. cv. Williams) was investigated at the vegetative stage (36 days after planting, DAP) and pod-filling stage (91 DAP) by the ¹⁵N pulse-chase experiment. The nodulated roots were exposed to N₂ gas labeled with a stable isotope ¹⁵N for 1 h, followed by 0, 1, 3 and 7 h of exposure with normal air. Plant roots and shoots were separated into three sections (basal, middle and distal parts) with the same length of the main stem or primary root. Approximately 80 and 92% of fixed N was distributed in the basal part of the nodulated roots at the vegetative and pod-filling stages by the end of 1 h of ¹⁵N₂ exposure, respectively. In addition, about 90% of fixed ¹⁵N was retained in the nodules and 10% was exported to root and shoot after 1 h of ¹⁵N₂ exposure at 91 DAP. The percentage distribution of ¹⁵N in the nodules at the pod-filling stage decreased from 90% to 7% during the 7 h of the chase period, and increased in the roots (14%), stems (54%), leaves (12%), pods (10%) and seeds (4%). The ¹⁵N distribution was negligible in the distal root segment, suggesting that N fixation activity was negligible and recycling fixed N from the shoot to the roots was very low in the initially short time of the experiment.     

Growth and Nitrogen Fixation in Soybean as Affected by Phosphorus Fertilizer and Sheep Manure using 15N Isotopic Dilution

A field experiment was conducted to study the effect of adding different phosphorus (P) fertilizer levels [0, 40, and 80 kg phosphorus pentoxide (P₂O₅) ha^?1 (abbreviated as P0, P1, and P2, respectively)] and rates of sheep manure (M) [0, 20, and 40 ton ha^?1 (abbreviated as M0, M1, and M2, respectively)] on growth and nitrogen (N₂) fixation of soybean (Glycine max L.). Sorghum bicolor L. was employed as a reference crop to evaluate N₂ fixation using the ¹⁵N-isotpic dilution technique. Results showed that addition of P fertilizer or sheep manure had positive effects on dry-matter production, N accumulation, and seed yield. Such effects were more pronounced when adding sheep manure and P together than adding separately. Solely P fertilizer had a small impact on N₂ fixation. A tangible increase in the amounts of N₂ fixed due to manure addition occurred. The efficient use of N fertilizer (%NUE) increased significantly as the result of adding a high level of P fertilizer. However, a drastic decrease in %NUE was observed when sheep manure was added solely or in combination with P fertilizer. From productivity and ecological standpoints, P2M1 and P2M2 surpassed the other treatments in showing greater grain yield and greater N₂ fixation. However, considering the high cost of sheep manure, P2M1 was the optimal treatment for improving growth and N₂ fixation in soybean plants with minimal manure consumption. In conclusion, the integrated use of manure and P fertilizer could be considered a useful agricultural practice for improving the performance of soybean plants grown in an Aridisol. Their beneficial effects were mainly attributed to the enhancement of N₂ fixation through root growth and soil property improvements besides being a source of P and other nutrients that are essential for N₂-fixation process.     

Growth and Nitrogen Fixation in Dhaincha/Sorghum and Dhaincha/Sunflower Intercropping Systems Using 15Nitrogen and 13Carbon Natural Abundance Techniques

A field experiment on dhaincha, sunflower, and sorghum plants grown in monocropping and intercropping systems was conducted to evaluate growth and nitrogen (N₂) fixation using ¹³carbon (C) and ¹⁵N natural abundance techniques. Intercropping of sesbania/sorghum showed a greater efficiency than monocropping in producing dry matter during the entire growth period, whereas the efficiency of producing dry matter in the sesbania/sunflower intercropping was similar to that in the monocropping system. Moreover, sorghum plants (C4) were more competitive than sesbania (C3) for soil N uptake, whereas sesbania seemed to be more competitive than its associated sunflower (C3). Nitrogen uptake in the mixed stand of sesbania/sorghum was improved as a result of the increase in soil N uptake by the component sorghum and the greater root nodule activity of component sesbania without affecting the amount of N₂ fixed. The Δ ¹³C in plant materials was affected by plant species and the cropping system.     

Dynamics of nodulation,nitrogen fixation,nitrogen use and biomass yield over time in pot-grown Leucaena leucocephala (Lam.) de Wit

The dynamics of nodulation, N₂-fixation and N use in Leucaena leucocephala cv. K28 over time was investigated in a screenhouse at 4, 8, 12 and 16 months after planting (MAP) using the ¹⁵N-labelling method. Leucaena had a consistently increasing pattern of nodulation, dry biomass and nitrogen yield. A sharp rise in nodulation was observed between 12 and 16 MAP, whereas for biomass, N accumulation and N₂-fixation, and N₂-fixation, an upward surge occurred between 4 and 12 months. Nodulation, N accumulation, N₂-fixation and biomass yield all peaked at 16 MAP. Along with the steady increase in N₂-fixation throughout the 16-month growth period, the % N derived from the atmosphere rose from 17.9% to 61.5%, 70.1% and 74%, equivalent to 191, 1623, 2395 and 3385 mg N₂ fixed plant^-1 at 4, 8, 12 and 16 MAP, respectively. Nitrogen assimilation from soil and fertilizer decreased inversely to the increase in symbiotic nitrogen fixation with time.     

Nitrogen accumulation in soybean [Glycine max (L.) Merr.] is increased by manure compost application in drained paddy fields as a result of increased soil nitrogen mineralization

The application of manure compost is an effective way to increase soybean [Glycine max (L.) Merr.] yield and nitrogen (N) fertility in drained paddy fields. We investigated changes in soil N mineralization during soybean cultivation using reaction kinetics analysis to determine the contribution of increased N mineralization after manure compost application (at a rate of 0 to 6?kg?m^?2) on N accumulation and seed yield of soybean under drained paddy field conditions. The seed yield and N accumulation decreased markedly in the second and third year of the experiment, but soil N mineralization increased in both years. No decrease in soil N mineralization occurred even after two soybean crops. Soil N availability was not the main cause of decreased soybean yield in the second and third years. The differences in plant aboveground N content between plots with and without manure compost was similar to the increase in N mineralization caused by manure compost application in the second and third years. The application of 6?kg?m^?2 of manure compost increased the amount of ureide-N and nitrate-N in soybean in the third year. Our results suggest that manure compost application increases soil N mineralization and soybean N₂ fixation, resulting in increased N accumulation and seed yield. However, the soybean yield remained less than 300?g?m^?2 in the second and third years (i.e., below the yield in the first year) at all levels of manure compost application due to the remarkable decrease of N accumulation in the second and the third crops.     

Effect of low static nitrate concentrations on mineral nitrogen uptake,nodulation, and nitrogen fixation in field pea

Abstract

Combined nitrogen [nitrate (NO₃‐), ammonium (NH₄+), and urea] will inhibit all components of symbiotic nitrogen (N₂) fixation if present in sufficient concentrations. It is generally accepted that nitrate is particularly inhibitory to nodule growth and nitrogenase activity, and somewhat less inhibitory to the infection process. This project examined whether providing low (0.1 ‐ 0.5 mM) static concentrations of NO₃‐ to pea (Pisum sativum L. cv. Express), seedlings could avoid the period of N hunger experienced prior to the establishment of N₂ fixation, without delaying or reducing the symbiotic N₂ fixation. All concentrations of NO₃ ^? tested significantly inhibited all measured components of N₂ fixation. The nodulation process as measured by nodule number was inhibited to a similar degree as the other parameters. A concentration dependent response was evident, with 0.1 mM NO₃ ^? causing less inhibition than the 0.2 or 0.5 mM concentrations. Our results indicate the within the concentrations of 0.1 mM and 0.5 mM NO₃ ^?, it is not possible to stimulate the growth of pea plants without inhibiting nodulation and N₂ fixation.     

Time-course of nitrogen fixation in two bambara groundnut (Vigna subterranea L. Verdc.) cultivars

TheA-value method, involving the application of a higher¹⁵N rate to a reference non-N₂-fixing plant, was used to assess the magnitude of N₂ fixation in two bambara groundnut cultivars at four growth stages [vegetative, 0–47 days after planting (DAP); early pod-filling, 47–99 DAP; mid-pod-filling, 99–120 DAP; physiological maturity, 120–148 DAP). The cultivars were Ex-Ada, a bunchy type, and CS-88-11, a slightly spreading type. They were grown on a loamy sand. Uninoculated Ex-Ada and CS-88-11 were used as reference plants to measure the N₂ fixed in the inoculated bambara groundnuts. In this greenhouse study, soil was the major source of N in bambara groundnuts during vegetative growth, and during this period it accounted for over 80% of the N accumulaed in the plants. However, N₂ fixation became the major source of plant N during reproductive growth. There were significant differences between the two cultivars in the ability to fix N₂, and at physiological maturity, almost 75% of the N in CS-88-11 was derived from the atmosphere compared to 55% in Ex-Ada. Also, the total N fixed in CS-88-11 at physiological maturity was almost double that in Ex-Ada. Our data indicate that the higher N₂ fixation in CS-88-11 was due to two factors, a higher intensity of N₂ fixation and a longer active period of N₂ fixation. The results also suggest that bambara groundnut genotypes could be selected for higher N₂ fixation in farining systems.     

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     

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.