Comparison of 15N natural abundance and difference methods for estimating N2 fixation of soybeans grown in a tropical soil

Abstract

A study was carried out to compare the difference or N-yield method with the ¹⁵N natural abundance method for the estimation of the fractional contribution of biological N₂ fixation in the different plant parts of nodulating and non-nodulating isolines of soybeans. The results indicated that the δ¹⁵N values of most plant parts of soybeans were significantly lower (p<0.05) in the nodulating than in the non-nodulating isoline. However, in the case of the root+nodule component, the δ¹⁵N value was higher in the nodulating than in the non-nodulating isoline possibly due to isotopic discrimination of ¹⁵N over ¹⁴N which may have occurred in the nodules. Inoculation of soybeans with the Bradyrhizobium japonicum strain CB 1809 increased significantly (p<0.05) the δ¹⁵N value of the root+nodule component implying that the effectiveness of the soybean-rhizobium symbiosis had increased by inoculation.

Percentage of plant N derived from atmospheric N₂ fixation (%Ndfa) estimated by the ¹⁵N natural abundance method was highly correlated (r=0.762, p<0.01) with that by the difference or N-yield method and the differences between the two methods were not statistically significant. The agreement between the two methods was closer at maturity than at the early reproductive stage.

The %Ndfa obtained by the difference method ranged from 48.4 to 92.6% whereas the %Ndfa obtained by the ¹⁵N natural abundance method ranged from 43.2 to 92.4% in the different plant parts. Based on the ¹⁵N natural abundance method, approximately 15% of the N in pod, shoot, grain, and shell was derived from the soil but in the case of stover, this fraction was about 55%.     

Effect of soil physical properties on soybean nodulation and N2 fixation at the early growth stage in heavy soil field in Hachirougata Polder,Japan

We studied the effect of the soil physical properties on soybean nodulation and N₂ fixation in the heavy soil of an upland field (UF) and an upland field converted from a paddy field (UCPF) in the Hachirougata polder, Japan. Seeds of the soybean cultivar Ryuho were sown in each field with or without inoculation of Bradyrhizobium japonicum A1017. The soybean plants were sampled at 35 (V3) and 65 (Rl) d after sowing (DAS), and then nodulation and the percentage of N derived from N₂ fixation in the xylem sap were determined. The soil physical properties were different between UF and UCPF, especially the air permeability and soil water regime. Nodule growth was restricted in UCPF irrespective of rhizobial inoculation, though rhizobial infection was not inhibited by the unfavorable soil physical conditions. Soybean plant growth was closely related to the nodule mass and N₂ fixation activity, and the inoculation of a superior rhizobium strain was effective only at 35 DAS. These results indicate that soybean nodulation and N₂ fixation was considerably affected by the physical properties of heavy soil, and that it is important to maintain the N₂ fixation activity and inoculate the soybean plants with a superior rhizobium strain at a later growth stage in order to increase soybean production in heavy soil fields.     

15N标记的土壤估测某些豆科植物N2固定的温室实验

Pot experiments were carried out to estimate N2 fixation by vetch,milk vetch,sickle alfalfa and broadbean in pure stand using a ^15N-labelled soil.Winter wheat was used as the non-fixing control.The 15N-labelled soil used was prepared by growing corn-wheat-corn successively on a nearly organic-matter-free Xiashu loess supplemented with adequate amounts of (15NH4)2SO4,P,K and micronutrients,then incorporating these 15N-labelled plant materials into the soil after each havest,and allowing the plant materials to be decomposed aerobically for 410d after incorporation of the plant material of the thire crop.The 15N enrichment of wheat plant-N varied slightly with organs,with a maximum difference of 9.8%,Based on 15N enrichment of soil N inferred from the mean value of the 15N enrichment in different organs of wheat 79%-91% of total N in the tops and 67%-74% of total N in the roots of legumes studied were derived from atmosphere .Estimate by isotope dilution method was in good agreement with that by the conventional difference method provided values obtained by the latter were corrected for seed N,and also with that from the measurement of N accumulated in the tops of the legumes.     

Effect of crop residue C:N ratio on N2O emissions from Gray Lowland soil in Mikasa,Hokkaido, Japan

Abstract

We studied the effect of crop residues with various C:N ratios on N₂O emissions from soil. We set up five experimental plots with four types of crop residues, onion leaf (OL), soybean stem and leaf (SSL), rice straw (RS) and wheat straw (WS), and no residue (NR) on Gray Lowland soil in Mikasa, Hokkaido, Japan. The C:N ratios of these crop residues were 11.6, 14.5, 62.3, and 110, respectively. Based on the results of a questionnaire survey of farmer practices, we determined appropriate application rates: 108, 168, 110, 141 and 0 g C m^?2 and 9.3, 11.6, 1.76, 1.28 and 0 g N m^?2, respectively. We measured N₂O, CO₂ and NO fluxes using a closed chamber method. At the same time, we measured soil temperature at a depth of 5 cm, water-filled pore space (WFPS), and the concentrations of soil NH⁺ ₄-N, NO^? ₃-N and water-soluble organic carbon (WSOC). Significant peaks of N₂O and CO₂ emissions came from OL and SSL just after application, but there were no emissions from RS, WS or NR. There was a significant relationship between N₂O and CO₂ emissions in each treatment except WS, and correlations between CO₂ flux and temperature in RS, soil NH⁺ ₄-N and N₂O flux in SSL and NR, soil NH⁺ ₄-N and CO₂ flux in SSL, and WSOC and CO₂ flux in WS. The ratio of N₂O-N/NO-N increased to approximately 100 in OL and SSL as N₂O emissions increased. Cumulative N₂O and CO₂ emissions increased as the C:N ratio decreased, but not significantly. The ratio of N₂O emission to applied N ranged from ?0.43% to 0.86%, and was significantly correlated with C:N ratio (y = ?0.59 ln [x] + 2.30, r ² = 0.99, P < 0.01). The ratio of CO₂ emissions to applied C ranged from ?5.8% to 45% and was also correlated with C:N ratio, but not significantly (r ² = 0.78, P = 0.11).     

Nitrogen fixation and soil N level during maturation affect the contents of storage compounds of soybean seeds

We compared the concentrations and contents of protein and oil in mature seeds from nodulated and non-nodulated soybean plants grown on soils with four different N levels during maturation. We observed a positive correlation between the contents of protein and oil in seeds from nodulated plants. Seeds from nodulated plants grown on urea-treated soil showed higher protein and lower oil contents than those from plants grown on soil treated with coated slow release N fertilizer (LP-100). Contents of these compounds in seeds from nodulated plants grown on LP-100 soil were almost the same as those from non-nodulated plants on the same soil. These observations indicated that N economy in roots during seed maturation affects the contents of storage compounds. We suggested that the control of the N₂ fixation activity of soybean plants and management of soil N level during seed maturation are important to determine the contents of protein and oil in seeds.     

Atmospheric nitrogen fixation by photosynthetic microorganisms in a submerged Philippine soil

Photosynthetic nitrogen-fixing microorganisms help maintain the nitrogen level of soil in rice paddies when environmental factors favor the growth of microorganisms. Our studies showed that blue-green algae in particular have a significant role in nitrogen-fixation in light. The most active nitrogen-fixation by microorganisms occurred in the soil shortly after it had been submerged under light. The longer the submergence, the less nitrogen microorganisms were fixed. In a greenhouse experiment, the fixed nitrogen appeared not to be immediately available to the rice plant. The amount of nitrogen that can be fixed in the field by nitrogen-fixing microorganisms in paddy water was estimated using the acetylene reduction method during the rice-growing period. The amount of nitrogen fixation by these microorganisms is not sufficient to account for the amount of nitrogen uptake by rice during the rice-growing period.     

Evaluation of N2 fixation by applying 15N labeled plant material and ammonium sulfate

Effect of different ¹⁵N labeled sources on the estimation of N₂ fixation was investigated. The combination of ¹⁵N labeled ammonium sulfate, ¹⁵N labeled plant material, and ¹⁵N labeled ammonium sulfate with unlabeled plant material, was examined in pot experiments. Two cultivars of soybean (Glycine max) and one of mungbean (Vigna radiata) were used. No significant difference was observed among the treatments for the estimation of N₂ fixation. This was due to the homogeneity and stability of the ¹⁵N abundance in soil which resulted in a similar N uptake from the soil by the N² fixing and reference crops. The plant yield, total N uptake and amount of N₂ fixed were higher in the Yellow Soil than in the Andosol. The amount of N₂ fixed was strongly influenced by the plant growth and consequently it affected the plant yield. The slow decomposition of plant material in the Andosol resulted in a low yield in both the N₂ fixing and reference crops. Thus, the artificial decrease of the available N content in soil, by application of plant material, did not stimulate N, fixation but suppressed plant growth and N₂ fixation.     

Nitrous oxide production and consumption in serially diluted soil suspensions as related to in situ N2O emission in submerged soils

A simple method for characterizing soil microbial community composition relevant to N₂O production and consumption was proposed. Ten-fold series soil dilution was prepared. Nitrate or N₂O was provided as the sole electron acceptor. Nitrous oxide concentration in the headspace gas across the serially diluted soil suspensions was measured against controls. Results showed that the patterns of N₂O production and consumption across the soil suspensions provided useful information on the microbial community composition relevant to N₂O production and consumption in these soils. An independent method, to that proposed here, was also employed to characterize denitrifier community compositions of the same soils. Data indicated that information on the soil microbial community composition characterized by both methods were compatible or mutually supporting and apparently related to in situ N₂O emissions. Soil samples from manure (applied with animal manure plus chemical fertilizer) plots had higher denitrification rates than the samples from normal fertilizer (applied with chemical fertilizer only) plots. It was concluded that functional characteristics of soil microbial communities relevant to N₂O production and consumption could be characterized at ecological levels and may potentially affect N₂O emissions.     

Effects of nitrogen fertilization and irrigation on N2O emissions from a sandy soil in Germany

The aim of this study was to investigate the effect of supplemental irrigation on the amount of N₂O emissions on a sandy soil in north-east Germany. N₂O flux measurements were carried out over two vegetation periods from the emergence of plants to harvest. The level of N₂O emissions was low, which is typical for sandy soils in north-east Germany. In both periods, irrigation had no increasing effect on N₂O emissions. Relevant factors were the soil temperature and the soil water-filled pore space (WFPS), which were mainly influenced by weather conditions. This may indicate that nitrification was the main source of N₂O emissions. In conclusion, this study has confirmed that sandy soils under weather conditions of north-east Germany generally have a very low potential for N₂O emissions.     

Effects of CO2 concentration in rhizosphere on nodulation and N2 fixation of soybean and cowpea

Soybean (Glycine max L. Merr.) cvs. Akisengoku and Peking, and cowpea (Vigna unguiculata Walp.) cv. Kegonnotaki were inoculated with Bradyrhizobium japonicum AlO17, Shinorhizobium fredii USDAI93, and B. sp. Vigna MAFF03-03063, respectively and were cultured hydroponically with supply of CO₂-free air, 3dm³ m^-3 CO₂ air, or 25 dm³ m^-3 CO₂ air to study the effects of the CO₂ concentration in the rhizosphere on plant growth, nodulation, and nitrogen fixation. Increase of the CO₂ concentration in the rhizosphere led to the increase of the plant dry weight in the symbiosis between Peking and USDAI93, and that between Kegonnotaki and MAFF03-03063. On the other hand, dry matter accumulation in the symbiosis between Akisengoku and AI017 decreased under the supply of 25 dm³ m^-3 CO₂ air aimed at increasing the CO₂ concentration in the rhizosphere beyond the optimum CO₂ concentration for growth. Nodule mass and nodule number per plant were highest in Akisengoku, followed by Kegonnotaki and lowest in Peking. Also the increase of the CO₂ concentration in the rhizosphere led to the increase of the nodule mass and number in Kegonnotaki, while no changes were observed in Akisengoku and Peking. Biological nitrogen fixation (BNF) was highest in Akisengoku, followed by Kegonnotaki, and lowest or near zero in Peking. BNF in Akisengoku and Kegonnotaki showed a similar tendency to that of dry matter accumulation. BNF of Peking was especially low under the supply of CO₂-free air, and it increased with the increase of the CO₂ concentration in the rhizosphere. For the symbiosis of Bradyrhizobium strains with soybean and cowpea, the most suitable CO₂ concentration for N₂ fixation and plant growth was estimated to be about 10 dm³ m^-3, while for the symbiosis of S. fredii with soybean, the value was estimated to be above 30 dm³ m^-3.     

Effects of hairy vetch foliage application on nodulation and nitrogen fixation in soybean cultivated in three soil types

We investigated the effects of applying hairy vetch foliage on nodulation and atmospheric nitrogen (N₂) fixation in soybean cultivated in three soil types in pot experiments. Soybean plants were grown in Gley Lowland soil (GLS), Non-allophanic Andosol (NAS), and Sand-dune Regosol (SDR) with hairy vetch foliage application in a greenhouse for 45 days. In GLS, the nodule number was not influenced by the application, however, nodule dry weight and N₂ fixation activity tended to increase. In NAS and SDR, nodule formation was depressed by foliage application. Soybean plant growth was promoted in GLS and SDR but not in NAS. These promotive effects of hairy vetch foliage application on soybean plant growth in GLS were considered to be mainly caused by the increase in N₂ fixation activity of the nodules, whereas it was considered to be mainly caused by the increase in nitrogen uptake activity of the roots in SDR. The varying effects of hairy vetch foliage application on soybean nodulation may be due to soil chemical properties such as pH and cation exchange capacity, which are related to soil texture. Therefore, we conclude that it is important to use hairy vetch for soybean cultivation based on the different effects of hairy vetch on soybean plant growth in different soil types.     

Estimating N2 fixation by field-grown lupins (Lupinus angustifolius L.) using soil and plant 15N enrichment

Summary Biological N₂ fixation was estimated in a field experiment following the addition of NH₄Cl or KNO₃ to unconfined microplots (1.5 m²) at 2.5 g N m^-2 (10 atom% ¹⁵N). A model of total N and ¹⁵N accumulation in lupins and decreasing ¹⁵N enrichment in the KCl-extractable soil-N pool (0–0.15 m depth) was used to estimate the proportion of N in lupins derived from biological N₂ fixation. Estimates of N₂ fixation derived from the model were compared with ¹⁵N isotope-dilution estimates obtained using canola, annual ryegrass, and wheat as nonfixing reference plants. Biomass, total N accumulation, or ¹⁵N enrichment in the lupin and reference crops did not differ whether NH _inf4 ^sup+ or NO _inf3 ^sup- was added as the labelled inorganic-N source. The decrease in soil ¹⁵N enrichment was described by first-order kinetics, whereas total N and ¹⁵N accumulation in the lupins were described by logistical equations. Using these equations, the uptake of soil N by lupins was estimated and was then used to calculate fixed N₂. Estimates of N₂ fixation derived from the model increased from 0 at 50 days after sowing to a maximum of 0.79 at 190 days after sowing. Those based on the ¹⁵N enrichment of the NO _inf3 ^sup- pool were 10% higher than those based on the mineral-N pool. ¹⁵N isotope-dilution estimates of N₂ fixation ranged from 0.37 to 0.55 at 68 days after sowing and from 0.71 to 0.77 at 190 days after sowing. Reference plant-derived values of N₂ fixation were all higher than modelled estimates during the early states of growth, but were similar to modelled estimates at physiological maturity. The use of the model to estimate N₂ derived from the atmosphere has the intrinsic advantage that the need for a non-fixing reference plant is avoided.     

Nitrogen fixation in paddy soils

Several important features of the N. fixation in paddy fields which were reported previously were confirmed and some new additional results regarding the evaluation of the N₂ fixation in the rhizosphere were obtained by reinvestigation in the fields. In addition, rice plants were cultivated in the submerged soil in pots and various parts of the soil were analyzed for the N₂-fixing activity as well as several other properties. The results of the pot experiments were found to be fairly similar to those observed in the field investigations, indicating the validity of the submerged soil in a pot as a rather simulated model for the actual paddy field. By using this model system, the following facts were ascertained: (1) Water-percolation had almost no effect on the N₂-fixing activities of both the rhizosphere and the non-rhizosphere soils. (2) Suppressing effect of washing the root of rice plant on the N₂-fixing activity was slight in the seedling stage and marked in the tillering and flowering stages. (3) The N₂-fixing activity of a single rice root varied from tip to base.     

Nitrogen fixation by biological soil crusts and heterotrophic bacteria in an intact Mojave Desert ecosystem with elevated CO2 and added soil carbon

Fixation of N by biological soil crusts and free-living heterotrophic soil microbes provides a significant proportion of ecosystem N in arid lands. To gain a better understanding of how elevated CO₂ may affect N₂-fixation in aridland ecosystems, we measured C₂H₂ reduction as a proxy for nitrogenase activity in biological soil crusts for 2 yr, and in soils either with or without dextrose-C additions for 1 yr, in an intact Mojave Desert ecosystem exposed to elevated CO₂. We also measured crust and soil δ¹⁵N and total N to assess changes in N sources, and δ¹³C of crusts to determine a functional shift in crust species, with elevated CO₂. The mean rate of C₂H₂ reduction by biological soil crusts was 76.9±5.6 μmol C₂H₄ m⁻² h⁻¹. There was no significant CO₂ effect, but crusts from plant interspaces showed high variability in nitrogenase activity with elevated CO₂. Additions of dextrose-C had a positive effect on rates of C₂H₂ reduction in soil. There was no elevated CO₂ effect on soil nitrogenase activity. Plant cover affected soil response to C addition, with the largest response in plant interspaces. The mean rate of C₂H₂ reduction in soils either with or without C additions were 8.5±3.6 μmol C₂H₄ m⁻² h⁻¹ and 4.8±2.1 μmol m⁻² h⁻¹, respectively. Crust and soil δ¹⁵N and δ¹³C values were not affected by CO₂ treatment, but did show an effect of cover type. Crust and soil samples in plant interspaces had the lowest values for both measurements. Analysis of soil and crust [N] and δ¹⁵N data with the Rayleigh distillation model suggests that any plant community changes with elevated CO₂ and concomitant changes in litter composition likely will overwhelm any physiological changes in 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.     

Saccharidic compounds as soil redox effectors and their influence on potential N2O production

Cellulose, xylan, and glucose were compared in waterlogged soil as modifying factors of the redox potential (Eh), of the quantity of reducing equivalents, and of the soil capacity to produce N₂O and CO₂. During the study period (168 h) soils supplied with glucose and xylan showed a higher Eh decrease than the control soil and the soil treated with cellulose. In samples taken after 0, 24, 48, and 168 h, the soils supplied with C showed a higher number of reducing equivalents than the control soil did. These quantities were not correlated with Eh values, nor with N₂O production. N₂O production was increased compared with the control soil over the entire experimental period in the glucose-amended soils but only after 48 h in the xylan-amended soils and not until 168 h in the cellulose-treated soils. The CO₂:N₂O ratio was consistently higher than the theoretical value of 2, suggesting that denitrification and CO₂ production via fermentation occurred simultaneously. Moreover, this ratio was highly correlated with the Eh values. We conclude that more research is needed to explain the role of soil redox intensity (Eh) and capacity (quantity of redox species undergoing reduction) in the expression of soil denitrification-fermentation pathways.     

The effects on N2 fixation (C2H2 reduction), bacterial population and rice plant growth of two modes of straw application to a wetland rice field

Summary The effects of incorporation and surface application of straw to a wetland rice field on nitrogen fixation (C₂H₂ reduction), bacterial population and rice plant growth were studied. Rice straw (5 t ha^–1) was chopped (10- to 15-cm pieces) and applied to the field 2 weeks before transplanting IR42, a long-duration variety, and IR50, a short-duration variety. The acetylene-reducing activity (ARA) of IR42 and IR50 measured at heading stage for 3 consecutive days showed significantly higher ARA in IR42 as a result of the 2 straw application methods. Mostly up to 20 days after straw surface application and incorporation, the dark ARA in the soil, total and N₂-fixing heterotrophs, and photoorganotrophic purple nonsulphur bacteria (POPNS) in the soil and in association with degrading straw were stimulated. Higher bacterial populations were associated with straw on the surface than with straw incorporated. The POPNS counts, in particular, were increased hundreds fold in the surface-applied straw treatment. Straw applications also increased the root, shoot and total plant biomass at heading stage and the total dry matter yield at harvest in both varieties. The data show the potentials of straw as a source of substrate for the production of microbial biomass and for the non-symbiotic N₂ fixation to improve soil fertility and plant nutrition.     

Technique of soil sample pretreatment for analysis of 15N:14N isotope ratio

Abstract

The technique of simultaneous quantitative determination of mineral N soil forms (nitrates, exchangeable and non‐exchangeable ammonium, and total amount of these compounds) and sample pretreatment for the analysis of ¹⁵N:¹⁴N ratio is suggested. The technique is based on the selective association of NH₄ ⁺‐ions into indophenol complex and subsequent ethyl‐acetate extraction of this complex from solution. The mineralization of indophenol is carried out in alkaline medium with simultaneous NH₃ distillation into H₂SO₄ titrant. The application of given technique allows us to shorten significantly the time required for analysis and to increase the accuracy of analytical determination.     

Dinitrogen emissions and the N2:N2O emission ratio of a Rendzic Leptosol as influenced by pH and forest thinning

Reduction of nitrous oxide (N₂O) to dinitrogen (N₂) by denitrification in soils is of outstanding ecological significance since it is the prevailing natural process converting reactive nitrogen back into inert molecular dinitrogen. Furthermore, the extent to which N₂O is reduced to N₂ via denitrification is a major regulating factor affecting the magnitude of N₂O emission from soils. However, due to methodological problems in the past, extremely little information is available on N₂ emission and the N₂:N₂O emission ratio for soils of terrestrial ecosystems. In this study, we simultaneously determined N₂ and N₂O emissions from intact soil cores taken from a mountainous beech forest ecosystem. The soil cores were taken from plots with distinct differences in microclimate (warm-dry versus cool-moist) and silvicultural treatment (untreated control versus heavy thinning). Due to different microclimates, the plots showed pronounced differences in pH values (range: 6.3–7.3). N₂O emission from the soil cores was generally very low (2.0 ± 0.5–6.3 ± 3.8 μg N m⁻² h⁻¹ at the warm-dry site and 7.1 ± 3.1–57.4 ± 28.5 μg N m⁻² h⁻¹ at the cool-moist site), thus confirming results from field measurements. However, N₂ emission exceeded N₂O emission by a factor of 21 ± 6–220 ± 122 at the investigated plots. This illustrates that the dominant end product of denitrification at our plots and under the given environmental conditions is N₂ rather than N₂O. N₂ emission showed a huge variability (range: 161 ± 64–1070 ± 499 μg N m⁻² h⁻¹), so that potential effects of microclimate or silvicultural treatment on N₂ emission could not be identified with certainty. However, there was a significant effect of microclimate on the magnitude of N₂O emission as well as on the mean N₂:N₂O emission ratio. N₂:N₂O emission ratios were higher and N₂O emissions were lower for soil cores taken from the plots with warm-dry microclimate as compared to soil cores taken from the cool-moist microclimate plots. We hypothesize that the increase in the N₂:N₂O emission ratio at the warm-dry site was due to higher N₂O reductase activity provoked by the higher soil pH value of this site. Overall, the results of this study show that the N₂:N₂O emission ratio is crucial for understanding the regulation of N₂O fluxes of the investigated soil and that reliable estimates of N₂ emissions are an indispensable prerequisite for accurately calculating total N gas budgets for the investigated ecosystem and very likely for many other terrestrial upland ecosystems as well.     

Influences of organic debris and rice root on the nitrogen fixation in the submerged soil

Abstract

A series of experiments has been conducted on the N2 fixation in the paddy soils by the authors (1–4). The amount of organic substrates for microorganisms and the degree of reduction of the soil are found to be two major factors affecting the N₂-fixing activity of the heterotrophic microorganisms in the submerged soil. Organic debris, rice root and their neighboring soils are identified to be the important micro-sites for the heterotrophic N₂ fixers. The organic debris and the rice root are considered to play dual roles by supplying the organic substances; (1) increase of the population of the heterotrophic N₂ fixers—the amount of nitrogenase, (2) preparation of the reduced conditions favorable for the nitrogenase activity.

However, it is not yet clearly known which of these two roles of the organic substrates is more essential to the N₂-fixing activity in the paddy soil. In addition, it is expected that there must be some differences between the organic debris and rice root in their contribution to the N₂ fixation in the paddy soil.

An experiment was carried out to clarify these problems. Moist soil sample was collected from the plough layer of the paddy field at Central Agric. Exp. Sta. in Konosu City, Saitama Pref., passed through a 5 mm sieve and placed in pots (3 kg moist soil/pot). Ammonium sulfate, calcium superphosphate, and potassium chloride at the rate of 0.4-0.4-0.4 (N-P₂O₅-K₂0) g/pot were incorporated into the soil 7 days before transplanting. Split application of ammonium sulfate at the rates of 0.2 and 0.4 g N/pot were also incorporated at 30 and 41 days after transplanting respectively. These pots were divided into three series; planted (P-series), non-planted (N-series), and non-planted and applied with organic manure (O-series). In case of O-series, 60 g of fairly rotted organic manure was applied to each pot. Each pot of P-series was planted with two 4O-day-old seedlings of rice plant at 7 days after submergence. The Nseries was regarded as a control. Each series was not replicated in this preliminary experiment.