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.     

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.     

Estimating symbiotic N2 fixation in Robinia pseudoacacia

Estimating symbiotic di‐nitrogen (N₂) fixation is challenging, especially when working with woody N₂ fixers in field trials. Fortunately, isotope methods based on ¹⁵N natural abundance or on ¹⁵N artificial enrichment (dilution method) make it possible to estimate the proportion of nitrogen derived from the atmosphere (Ndfa) in N₂‐fixing species. These methods have been extensively used in the field for herbaceous species, much less for tree species such as alder and acacia, and rarely for black locust (Robinia pseudoacacia). The objectives of this study were to characterize the fixation potential of black locust in a plantation by using the two ¹⁵N isotope methods in the field, and to document values of isotope fractionation occurring during N₂ fixation (the B value). B values were estimated both by growing trees on an N‐free medium in controlled conditions (B_lab) and by making Ndfa calculated with the natural abundance method converge with Ndfa calculated with the ¹⁵N dilution method in the field (B_field). The two methods gave consistent estimates of the B value. B values ranging between –1.4 and –3.2‰ were found, varying with the age of the plant material. Up to 76% of the N in the black locust trees came from the atmosphere, representing more than 45 kg N ha^?1 over five years and confirming that black locust may be well adapted to N‐poor soils.     

Comparison of understorey biological nitrogen fixation and biomass production in grassed‐down conventional and organic apple orchards in Canterbury,New Zealand

Abstract

In a previous study, the understorey biomass production and biological nitrogen (N) fixation of a grassed‐down organic apple orchard were presented. The aim of this paper is to report the results of a similar study of two conventional orchards in a nearby location and to compare the present results with those of the organic orchard. Biological N₂ fixation was determined in the field using the ¹⁵N isotopic dilution technique and the experiments were conducted over a two‐year period. Present results showed that substantial amounts of N (112 to 143 kg N ha^‐1.2 years^‐1) were fixed in the understorey of the conventional orchard and these were not significantly different from those of the organic orchard. However, the N₂ fixation was sustained in the conventional, but not in the organic orchards in the second year, probably due to regular additions of fertilizers in the conventional orchards. In both orchards, N₂ fixation was better correlated with clover than total dry matter yield. Seasonal effects found were highest N₂ fixation and biomass production occurring during late spring and early summer and lowest during winter. Climatic factors were investigated in one of the conventional orchards and it was found that seasonal effects were related to a combination of temperature and moisture deficit effects.     

Short‐term N2O,CO2, NH3 fluxes,and N/C transfers in a Danish grass‐clover pasture after simulated urine deposition in autumn

Urine patches are significant hot‐spots of C and N transformations. To investigate the effects of urine composition on C and N turnover and gaseous emissions from a Danish pasture soil, a field plot study was carried out in September 2001. Cattle urine was amended with two levels of ¹³C‐ and ¹⁵N‐labeled urea, corresponding to 5.58 and 9.54 g urea‐N l^–1, to reflect two levels of protein intake. Urine was then added to a sandy‐loam pasture soil equivalent to a rate of 23.3 or 39.8 g urea‐N m^–2. Pools and isotopic labeling of nitrous oxide (N₂O) and CO₂ emissions, extractable urea, ammonium (NH₄⁺), and nitrate (NO₃^–), and plant uptake were monitored during a 14 d period, while ammonia (NH₃) losses were estimated in separate plots amended with unlabeled urine. Ammonia volatilization was estimated to account for 14% and 12% of the urea‐N applied in the low (UL) and high (UH) urea treatment, respectively. The recovery of urea‐derived N as NH₄⁺ increased during the first several days, but isotopic dilution was significant, possibly as a result of stress‐induced microbial metabolism. After a 2 d lag phase, nitrification proceeded at similar rates in UL and UH despite a significant difference in NH₄⁺ availability. Nitrous oxide fluxes were low, but generally increased during the 14 d period, as did the proportion derived from urea‐N. On day 14, the contribution from urea was 23% (UL) and 13% (UH treatment), respectively. Cumulative total losses of N₂O during the 14 d period corresponded to 0.021% (UL) and 0.015% (UH) of applied urea‐N. Nitrification was probably the source of N₂O. Emission of urea‐derived C as CO₂ was only detectable within the first 24 h. Urea‐derived C and N in above‐ground plant material was only significant at the first sampling, indicating that uptake of urine‐C and N via the leaves was small. Urine composition did not influence the potential for N₂O emissions from urine patches under the experimental conditions, but the importance of site conditions and season should be investigated further.     

Influence of Azolla (Azolla microphylla Kaulf.) compost on biogenic gas production,inorganic nitrogen and growth of upland kangkong (Ipomoea aquatica Forsk.) in a silt loam soil

Azolla microphylla Kaulf. (Azolla) biomass was composted to create a high nitrogen (N) organic matter amendment (Azolla compost). We examined the effect of this Azolla compost on carbon (C) and N mineralization and the production of biogenic gases, nitrous oxide (N₂O) and carbon dioxide (CO₂), in a soil incubation experiment. A pot experiment with upland kangkong (Ipomoea aquatica Forsk.) examined plant growth in silt loam soil treated with three levels of Azolla compost. The results showed that N₂O production from soil increased with urea amendment, but not with Azolla compost treatments. The Azolla-amended soil showed enhanced CO₂ production throughout the 4-week incubation. The Azolla-treated soils showed a 98% lower global warming potential compared to urea treatment over the 4-week incubation. However, Azolla-amended soil had higher nitrate (NO₃^–) levels compared to urea-fertilized soil at 1 week of incubation, and these were maintained until the fourth week. Soils amended with Azolla compost showed lower ammonium nitrogen (NH₄-N) levels than those in the urea-fertilized soils. The height and dry weight of upland kangkong fertilized with Azolla compost were similar to plants receiving urea fertilization. Therefore, the use of Azolla compost as a substitute for urea fertilizer would be beneficial for reducing the production of N₂O while maintaining plant growth.     

Evaluation of N2‐fixation measured by the 15N‐dilution and N‐difference methods in Nicaraguan and Ecuadorian Phaseolus vulgaris L. plants inoculated with Rhizobium leguminosarum biovar

Pot and field experiments were performed to assess N₂ fixation in Nicaraguan (R79 and R84) and Ecuadorian (Imba) common bean (Phaseolus vulgaris L.) cultivars, with the aim of improving their productivity by inoculating them with commercially produced Rhizobium phaseoli. With maize (Zea mays L.) as the non‐N₂‐fixing control, the percentage of N₂ fixed predicted by the ¹⁵N‐dilution method was significantly (P ≤ 0.05) higher than that predicted by the N‐difference method. However, the N₂ amounts predicted by the two methods were not significantly different. The correlation between the two methods was significant and positive (P ≤ 0.0001, n = 36). Compared with the native rhizobial strain, symbiotic associations of the bean cultivars with UMR1073, UMR1077 and UMR1899 rhizobial inoculants did not significantly (P ≤ 0.05) influence plant dry matter (DM) and N yields, the extent of N₂ fixation and uptake of soil and fertilizer N. Nevertheless, the UMR1077 and UMR1899 strains markedly increased the uptake of soil N by R84 plants, while decreasing N2 fixation. In contrast, the Imba‐UMR1899 association enhanced positive effects on all variables. About 60–70% of the total N taken up by the Imba plants was fixed N_2. The R79 and R84 plants fixed about 50% of their total N uptake. N₂ fixation rates were positively correlated with DM and total N yields, while being negatively correlated with soil N uptake (P ≤ 0.001, n = 36). Future research in Nicaragua should focus on selecting rhizobial strains suitable for indigenous common bean cultivars.     

Assimilation of ammonia by the azolla‐anabaena symbiosis

¹⁵N‐labeled ammonia was rapidly assimilated by Azolla caroliniana and incorporated into plant material even though sustained growth of the fern‐algae symbiosis cannot be maintained with ammonia as nitrogen source. During ammonia uptake, the nitrogenous pool of the fern rapidly increases and contains large amounts of free ammonia and glutamine. N₂ fixation activity of the algal symbiont declines during assimilation of ammonia, but it is restored to a high level upon transfer of plants to nitrogen‐free media, as the pool ammonia content decreases. During growth of the fern on N₂, the algal symbiont supplies ammonia in a manner permitting sustained growth of the plant. Exogenous ammonia, therefore, appears to interrupt regulation of inorganic nitrogen metabolism of the plant‐algal symbiosis.     

Effects of Multiple Reference Plants,Season, and Irrigation on Biological Nitrogen Fixation by Pasture Legumes using the Isotope Dilution Method

Abstract

The popular and widely used ¹⁵nitrogen (N)–isotope dilution method for estimating biological N fixation (BNF) of pasture and tree legumes relies largely on the ability to overcome the principal source of error due to the problem of selecting appropriate reference plants. A field experiment was conducted to evaluate the suitability of 12 non‐N₂‐fixing plants (i.e., nonlegumes) as reference plants for estimating the BNF of three pasture legumes (white clover, Trifolium repens L.; lucerne, Medicago sativa; and red clover, Trifolium pratense L.) in standard ryegrass–white clover (RWC) and multispecies pastures (MSP) under dry‐land and irrigation systems, over four seasons in Canterbury, New Zealand. The ¹⁵N‐isotope dilution method involving field ¹⁵N‐microplots was used to estimate BNF. Non‐N₂‐fixing plants were used either singly or in combination as reference plants to estimate the BNF of the three legumes. Results obtained showed that, on the whole, ¹⁵N‐enrichment values of legumes and nonlegumes varied significantly according to plant species, season, and irrigation. Grasses and herb species showed higher ¹⁵N‐enrichment than those of legumes. Highest ¹⁵N‐enrichment values of all plants occurred during late summer under dry‐land and irrigation conditions. Based on single or combined non‐N₂‐fixing plants as reference plants, the proportion of N derived from the atmosphere (% Ndfa) values were high (50 to 90%) and differed between most reference plants in the MSP pastures, especially chicory (Cichorium intybus), probably because it is different in phenology, rooting depth, and N‐uptake patterns compared to those of legumes. The percent Ndfa values of all plants studied also varied according to plant species, season, and irrigation in the MSP pastures. Estimated daily amounts of BNF varied according to pasture type, time of plant harvest, and irrigation, similar to those shown by percent Ndfa results as expected. Irrigation increased daily BNF more than 10‐fold, probably due to increased dry‐matter yield of pasture under irrigation compared to dry‐land conditions. Seasonal and irrigation effects were more important in affecting estimates of legume BNF than those due to the appropriate matching of N₂‐fixing and non‐N₂‐fixing reference plants.     

Nitrogen isotope analysis of free glycine in soil using isotope dilution mass spectrometry

To enable the estimation of production and consumption rates of free glycine in soils through ¹⁵N isotope dilution experiments, an isotope dilution mass spectrometric method was developed. The method, which enabled high precision N isotope ratio determination of glycine in soil extracts at δ¹⁵N levels up to 4000‰ and concentrations from approximately 2 μM, is based on the following steps: (i) addition of glycine spike to the soil extract, (ii) removal of humic substances and pre-concentration of glycine using solid phase extraction, (iii) derivatization of amino acids, (iv) separation of the derivatives using gas chromatography (GC), (v) their combustion to yield sample N₂ gas, and (vi) finally the use of N isotope ratio mass spectrometry (IRMS). Judging by uncertainty budget calculations, the precision obtained (SD=0.01-0.06 at% ¹⁵N) is sufficient for detecting differences in N isotopic ratios obtained in ¹⁵N isotope dilution experiments.     

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

Abstract

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.     

Estimation of cadmium load on Japanese farmland associated with the application of chemical fertilizers and livestock excreta

It is very important to use ¹⁵N labeled nitrogen gas (¹⁵N₂) in studies on biological nitrogen fixation. For example, ¹⁵N₂ is necessary for direct measurement of the amount of fixed nitrogen, and is useful for studies on the assimilation and transport of fixed nitrogen. However, ¹⁵N₂ is sometimes troublesome to deal with for the following reasons. Decline in the ¹⁵N content of ¹⁵N₂ gas may occur as a result of contamination with atmospheric N₂ during the storage period or application to nitrogen fixing organisms. Also, the ¹⁵N₂ gas provided commercially in a glass bottle or gas cylinder is technically and economically not convenient for experiments employing small amounts of ¹⁵N₂. Moreover, purification of ¹⁵N₂ gas is necessary for biological research, since contamination with the oxidized forms of nitrogen represses biological nitrogen fixation to a certain extent. A simple method for the preparation, purification and storage of ¹⁵N₂ gas for biological nitrogen fixation studies, was therefore devised.     

Alder-poplar associations: Determination of plant nitrogen sources by isotope techniques

Summary Three¹⁵N isotopic dilution methods (¹⁵N natural abundance, labelled mineral fertilizer, and organic matter) were used to determine the proportion of N derived from different available sources in seedlines ofAlnus glutinosa andPopulus nigra planted together or in monoculture under natural climatic conditions. The proportion of N derived from N₂ fixation in associated alders was appreciably higher than that determined in monoculture. The reduction of soil N uptake by associated alders contributed to an increase in total plant N and biomass production in associated poplars. When slightly N-labelled organic matter (alder leaf litter) was incorporated into the soil, 10–15% of its initial N content was recovered in poplar tissues, showing that this N source makes an important contribution to the N yield of associated non-fixing plants. There were no significant differences between the results obtained by¹⁵N natural abundance and those obtained by labelled fertilizer methods, suggesting that the ¹⁵N method could be used to evaluate annual N budgets in natural ecosystems.     

Dual isotope and isotopomer measurements for the understanding of N2O production and consumption during denitrification in an arable soil

The aim of our research was to obtain information on the isotopic fingerprint of nitrous oxide (N₂O) associated with its production and consumption during denitrification. An arable soil was preincubated at high moisture content and subsequently amended with glucose (400 kg C ha^?1) and KNO₃ (80 kg N ha^?1) and kept at 85% water‐filled pore space. Twelve replicate samples of the soil were incubated for 13 days under a helium‐oxygen atmosphere, simultaneously measuring gas fluxes (N₂O, N₂ and CO₂) and isotope signatures (δ¹⁸O‐N₂O, δ¹⁵N^bulk‐N₂O, δ¹⁵N^α, δ¹⁵N^β and ¹⁵N site preference) of emitted N₂O. The maximum N₂O flux (6.9 ± 1.8 kg N ha^?1 day^?1) occurred 3 days after amendment application, followed by the maximum N₂ flux on day 4 (6.6 ± 3.0 kg N ha^?1 day^?1). The δ¹⁵N^bulk was initially ?34.4‰ and increased to +4.5‰ during the periods of maximum N₂ flux, demonstrating fractionation during N₂O reduction, and then decreased. The δ¹⁸O‐N₂O also increased, peaking with the maximum N₂ flux and remaining stable afterwards. The site preference (SP) decreased from the initial +7.5 to ?2.1‰ when the N₂O flux peaked, and then simultaneously increased with the appearance of the N₂ peak to +8.6‰ and remained stable thereafter, even when the O₂ supply was removed. We suggest that this results from a non‐homogenous distribution of NO in the soil, possibly linked to the KNO₃ amendments to the soil, causing the creation of several NO pools, which affected differently the isotopic signature of N₂O and the N₂O and N₂ fluxes during the various stages of the process. The N₂O isotopologue values reflected the temporal patterns observed in N₂O and N₂ fluxes. A concurrent increase in ¹⁵N site preference and δ¹⁸O of N₂O was found to be indicative of N₂O reduction to N₂.     

Stimulation of N2‐fixation in chickpea by gamma irradiation as affected by different levels of ammonium sulfate fertilizer

Abstract

A pot experiment was conducted under natural climatic conditions to study the effect of low doses of gamma irradiation (0, 5, 10, and 20 Gy) on the performance of winter chickpea (Cicer arietinum L.) in the presence of increased supply of ¹⁵N labeled ammonium sulfate (0, 20, 50, and 100 kg N ha^‐1). Presowing seed irradiation produced a significant increase in dry matter production (up to 3 6%) and total nitrogen yield (up to 45%). The stimulative effect of irradiation was more pronounced with the application of NH₄ ⁺‐N fertilizer. Seed irradiation increased the amount of N₂‐fixation by 8–61% depending on the dose and level of NH₄ ⁺‐N fertilizer rate. A 10 Gy was found to be the optimal irradiation dose for enhancing N₂‐fixation. High levels of NH₄ ⁺‐N decreased the percentage and the amount of N₂‐fixation, but did not affect nodule formation. However, the presowing 10 Gy irradiation dose reduced the negative effect of ammonia‐N fertilizer on N₂‐fixation. Therefore, we recommend irradiating chickpea seeds with a 10 Gy dose before planting in soil containing high levels of mineral nitrogen to reduce its negative effect on N₂‐fixation.     

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.     

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.     

Site variability effect on field measurement of symbiotic nitrogen fixation using the 15N isotope dilution method

The sensitivity of the isotope dilution method involving the application of ¹⁵N-enriched fertilizers to estimate symbiotic N₂ fixation in legumes under field conditions is analysed with respect to the variability of atom% ¹⁵N excess in the fixing and non-fixing (reference) crops. Field data collected along a 96 m transect with 63 plots split into two strips, one sown with ryegrass and the other with alfalfa, showed that the sensitivity of the methodology increased with the level of fixation. The results further showed that the sensitivity of the ¹⁵N isotope dilution method for estimating N₂ fixed can be greatly improved by selecting fairly homogeneous sites, although use of a heterogenous site is quite practicable if the non-fixing reference crop is sited reasonably close to the test plots. Errors associated with mismatch between reference and fixing crops or heterogeneity of site become smaller as the amount of fixation increases. The data also revealed that relative comparisons of N₂ fixing abilities of legume treatments based on their ¹⁵N enrichments may not be valid for soils with highly variable N content. Under such circumstances, the use of a reference crop sited close to each legume treatment is necessary.     

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.     

N2 fixation and growth of legumes as affected by sulphur fertilization

The influence of three sulphur application rates in combination with two nitrogen application rates on N₂ fixation and growth of different legumes was investigated. N was applied as N-labelled ¹⁵NH₄ ¹⁵NO₃. The ¹⁵N isotope dilution technique was used to estimate N₂ fixation. At both N increments dry matter yield was highest with high S supply. Independently of the N supply, the high S application rate resulted in a significantly higher N accumulation, which was mainly caused by a higher N₂ fixation rate. With the grain legumes the weight of nodules was increased by the high S application rate. The higher number of nodules per pot with optimum S supply was the result of a better root growth. Rates of acetylene reduction correlated significantly with S supply.