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.     

Small-scale spatial patterns in N2-fixation and nutrient availability in an arctic hummock-hollow ecosystem

Atmospheric nitrogen that is fixed by associative cyanobacteria can be released into the surrounding soil environment providing a key source of N for arctic ecosystems. Yet, little is known about nitrogen fixation by Biological Soil Crusts (BSCs) within hummock-hollow complexes that are typical of many arctic environments. In this study, we examined spatial and temporal patterns in N₂-fixation, dinitrogenase reductase (nifH) gene abundance and release of N in a low arctic hummock-hollow ecosystem. The impacts of cyanobacteria on N status in soil were evaluated by assessing soil nitrogen in relation to the cyanobacterial associations found on Hummock and Hollow BSCs. In addition, potential P limitation of N₂-fixation by cyanobacteria was assessed for Hummock and Hollow BSCs. The tops of hummocks and the bottoms of hollows were areas of high N₂-fixation, whereas minimal N₂-fixation occurred on the sides of hummock-hollow complexes. Compared with Hummock BSCs, Hollow BSCs had a higher mean growing season N₂-fixation rate, a higher mean growing season nifH abundance, a higher mean total %N and δ¹⁵N values closer to that of atmospheric N₂. Soil N status was linked to rates of N₂-fixation by BSCs indicating that these N₂-fixing associations act as important point sources of soil N in this low arctic ecosystem. Over the course of a growing season temporal variation in N₂-fixation and nifH abundance were weakly linked suggesting that N₂-fixation was carried out by complex communities of diazotrophic microorganisms and that factors such as nutrient availability may limit N₂-fixation to a greater extent than nifH abundance.     

Field measurements of nitrogen fixation in leguminous trees used in agroforestry systems: Influence of 15N-labeling approaches and reference trees

Appropriate ¹⁵N-labeling methods are crucial for estimating N₂-fixation in trees used in agroforestry systems. A 4-year field experiment was conducted on an Alfisol in Southwestern Nigeria to compare the estimates of N₂ fixed in Leucaena leucocephala, using two non-N₂-fixing leguminous trees, Senna siamea and S. spectabilis, as reference plants and three different methods of introducing ¹⁵N into soil. The atom % ¹⁵N uptake pattern (as reflected in the leaves) was identical in both N₂- and non-N₂-fixing tree species irrespective of the ¹⁵N-application method. There was a significant decline in atom % ¹⁵N excess in the leaves of L. leucocephala (from 0.266 to 0.039), S. siamea (0.625 to 0.121), and S. spectabilis (from 0.683 to 0.118) from the first sampling 12 months after planting and the second sampling 18 months after sampling. From the second harvest in 1991 until the end of the experiment (fifth) harvest in 1993, however, the atom ¹⁵N % excess decline in leaves of the three species was less pronounced and depended on the method of ¹⁵N application. In those plants to which the tracer was applied once at planting, the ¹⁵N decline was steady between the second and the last prunings. In the split-application treatment, the atom ¹⁵N % excess increased slightly at the third pruning and decreased during the subsequent two prunings. The reference tree and the method of ¹⁵N application influenced the estimated proportion of N derived from atmospheric N₂ by L. leucocephala, calculated as 73 and 64%, corresponding to 119 and 98 kg N ha^-1 of N₂ fixed per 6 months, when S. spectabilis and S. siamea were used as reference trees, respectively. The approach by which ¹⁵N-labeled fertilizer was applied to the soil in three splits gave slightly higher estimates of N derived from the atmosphere but this was of little agronomic significance because total N₂ fixed was similar for all methods.     

Effect of nitrogen application on the A N value of soil

Pot experiments were conducted with two soils, from Rottenhaus and Seibersdorf in Austria, to ascertain whether the rate of fertilizer N application and the test crop would influence the amount of N available in the soil as assessed by the A-value method. ¹⁵N-labelled fertilizer was applied at rates of 10, 25, 40, 60, and 100 mg N kg^-1 soil, corresponding approximately to 20, 50, 80, 120 and 200 kg N ha^-1 respectively, and two crop species, barley (Hordeum vulgareL.) and non-nodulating soybean (Glycine max L.) were used to determine the soil A _N value under the various fertilizer regimes. The results showed that the Rottenhaus soil had a higher A _N value than the Seibersdorf soil, suggesting that the former was more fertile than the latter. The A _N values of both soils were significantly affected by the level of N application. When grown in the same soil, the two test crops showed significantly different fertilizer use efficiency and per cent N derived from fertilizer when the rate of N application exceeded 20 kg ha^-1. Thus, the A _N value as determined by the two test crops differed significantly for the same soil when the rate of N application was greater than 20 kg/ha. The difference was greater when the soil fertility level was high. The dependence of the A _N value on the level of N application and the species of crop seriously compromises the suitability of this method for determining plant-associated N₂ fixation. Hence, considerable caution is required when using this method to estimate plant-associated N₂ fixation.     

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     

Field evaluation of N2 fixation and N partitioning in climbing bean (Phaseolus vulgaris L.) using 15N

Summary The common bean (Phaseolus vulgaris L.) is generally regarded as a poor N₂ fixer. This study assessed the sources of N (fertilizer, soil, and fixed N), N partitioning and mobilization, and soil N balance under field conditions in an indeterminate-type climbing bean (P. vulgaris L. cv. Cipro) at the vegetative, early pod-filling, and physiological maturity stages, using the A-value approach. This involved the application of 10 and 100 kg N ha^-1 of ¹⁵N-labelled ammonium sulphate to the climbing bean and a reference crop, maize (Zea mays L.). At the late pod-filling stage (75 days after planting) the climbing bean had accumulated 119 kg N ha^-1, 84% being derived from fixation, 16% from soil, and only 0.2% from the ¹⁵N fertilizer. N₂ fixation was generally high at all stages of plant growth, but the maximum fixation (74% of the total N₂ fixed) occurred during the interval between early (55 days after planting) and late podfilling. The N₂ fixed between 55 and 75 days after planting bas a major source (88%) of the N demand of the developing pod, and only about 11% was contributed from the soil. There was essentially no mobilization of N from the shoots or roots for pod development. The cultivation of common bean cultivars that maintain a high N₂-fixing capacity especially during pod filling, satisfying almost all the N needs of the developing pod and thus requiring little or no mobilization of N from the shoots for pod development, may lead to a net positive soil N balance.     

Soybean nodulation and nitrogen fixation on soil amended with plant residues

Residues from some tree species may contain allelopathic chemicals that have the potential to inhibit plant growth and symbiotic N₂-fixing microorganisms. Soybean [Glycine max (L.) Merr] was grown in pots to compare nodulation and N₂-fixation responses of the following soil amendments: control soil, leaf compost, red oak (Quercus rubra L.) leaves, sugar maple (Acer saccharum Marsh) leaves, sycamore (Platanus occidentalis L.) leaves, black walnut (Juglans nigra L.) leaves, rye (Secale cereale L.) straw, and corn (Zea mays L.) stover. Freshly fallen leaves were collected from urban shade trees. Soil was amended with 20 g kg^-1 air-dried, ground plant materials. Nodulating and nonnodulating isolines of Clark soybean were grown to the R2 stage to determine N₂-fixation by the difference method. Although nodulation was not adversely affected, soybean grown on leaf-amended soil exhibited temporary N deficiency until nodulation. Nodule number was increased by more than 40% for soybean grown on amended soil, but nodule dry matter per plant generally was not changed compared with control soil. Nonnodulating plants were severely N deficient and stunted as a consequence of N immobilization. Nodulating soybean plants grown on leaf or crop residue amended soil were more dependent on symbiotically fixed N and had lower dry matter yields than the controls. When leaves were composted, the problem of N immobilization was avoided and dry matter yield was not reduced. No indication of an allelopathic inhibition on nodulation or N₂-fixation from heavy application of oak, maple, sycamore, or walnut leaves to soil was observed.     

Exploration of bacterial N2-fixation systems in association with soil-grown sugarcane,sweet potato,and paddy rice: a review and synthesis

N₂ fixation systems in the nonleguminous crops and bacteria associations have been intensively studied over the last 50 years. Their structure and regulation have been investigated to explore the enhancement of N acquisition in these ecosystems leading to crop-growth with minimum chemical fertilizers. Several lines of important evidence have been accumulated indicating that the magnitudes of associative (nonsymbiotic) N₂ fixation in sugarcane (Saccharum spp.), sweet potato (Ipomoea batatas L.), and paddy rice (Oryza sativa L.) are agronomically significant. In these three crops, unique bacterial N₂-fixation systems may function in addition to the low-level activity (due to the competition in carbon/energy use) of the commonly occurring rhizosphere-associated system by free-living bacteria such as Beijerinckia, Azotobacter, and Klebsiella. Active expressions of the dinitrogenase reductase-encoded gene (nifH) phylogenetically similar to those of Bradyrhizobium spp. and Azorhizobium sp. were abundantly found in the N₂-fixing sugarcane stems, sweet potato stems, and storage tubers. These rhizobia micro-aerobically fix N₂ in the carbon compounds-rich apoplasts. Gluconacetobacter diazotrophicus and Herbaspirillum spp. were previously isolated from inside the sugarcane stems, as the candidates of endophytic N₂ fixers. However, the current molecular and physiological investigations suggest that their major role is production of phytohormonal substances. In paddy rice fields, methane is produced from organic compounds in anoxia and oxidized by contacting with oxygen gas. An active N₂-fixation by methane-oxidizing methanotrophs such as Methylosinus sp. takes place in the root tissues (aerenchyma) and also in the surface soil. This methanotrophic N₂-fixation supports the sustainability of soil fertility although the N₂-fixation and soil fertility are affected by chemical fertilizers. Finally, we discuss the ecological implications of the newly identified rhizobia and methanotroph systems in the N nutrition in nonlegumes and N reservation in field environments.     

Heterotrophy-coordinated diazotrophy is associated with significant changes of rare taxa in soil microbiome

Soil heterotrophic respiration during decomposition of carbon (C)-rich organic matter plays a vital role in sustaining soil fertility. However, it remains poorly understood whether dinitrogen (N₂) fixation occurs in support of soil heterotrophic respiration. In this study, ¹⁵N₂-tracing indicated that strong N₂ fixation occurred during heterotrophic respiration of carbon-rich glucose. Soil organic ¹⁵N increased from 0.37 atom% to 2.50 atom% under aerobic conditions and to 4.23 atom% under anaerobic conditions, while the concomitant CO₂ flux increased by 12.0-fold under aerobic conditions and 5.18-fold under anaerobic conditions. Soil N₂ fixation was completely absent in soils replete with inorganic N, although soil N bioavailability did not alter soil respiration. High-throughput sequencing of the 16S rRNA gene further indicated that: i) under aerobic conditions, only 15.2% of soil microbiome responded positively to glucose addition, and these responses were significantly associated with soil respiration and N₂ fixation and ii) under anaerobic conditions, the percentage of responses was even lower at 5.70%. Intriguingly, more than 95% of these responses were originally rare with < 0.5% relative abundance in background soils, including typical N₂-fixing heterotrophs such as Azotobacter and Clostridium and well-recognized non-N₂-fixing heterotrophs such as Sporosarcina, Agromyces, and Sedimentibacter. These results suggest that only a small portion of the soil microbiome could respond quickly to the amendment of readily accessible organic C in a fluvo-aquic soil and highlighted that rare phylotypes might have played more important roles than previously appreciated in catalyzing soil C and nitrogen turnovers. Our study indicates that N₂ fixation could be closely associated with microbial turnover of soil organic C when available in excess.     

Biological nitrogen fixation in Azospirillum strain-maize genotype associations as evaluated by the 15N isotope dilution technique

Few studies of the inoculation of cereal crops with N₂-fixing bacteria have included more than one or two plant genotypes. In a recent study performed in Argentina using 12 different maize genotypes, it was found in 2 consecutive field experiments that several of them responded consistently, either negatively or positively, to inoculation with a mixture of strains of Azospirillum spp. The present study in post was performed to investigate the effect of inoculation of individual strains (and a mixture) of Azospirillum spp., and their nitrate reductase negative (NR-) mutants, on the growth of four of these maize genotypes. Two of these genotypes were grown in ¹⁵N-labelled soil with the aim of quantifying any contributions of biological N₂ fixation. Two genotypes (Morgan 318 and Dekalb 4D-70) produced similar increases in grain yield when they were inoculated with a mixture of Azospirillum spp. strains or fertilized with the equivalent of 100 kg N ha^-1. The other genotypes (Dekalb 2F-11 and CMS 22) showed little response to inoculation or N fertilization. The Morgan 318 and Dekalb 4D-70 genotypes showed a large increase in total N accumulation, suggesting that the response was due to increased N acquisition, but not due to bacterial nitrate reductase as the NR- mutants generally caused plant responses similar to those of the parent strains. Despite problems with the stabilization of the ¹⁵N enrichment in the soil, the ¹⁵N isotope dilution results indicated that there were very significant biological nitrogen fixation (BNF) contributions to the Dekalb 4D-70 and CMS 22 maize genotypes.Dedicated to Professor J.C.G. Ottow on the occasion of his 60th birthday     

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.     

Tree fallows: A comparison between five tropical tree species

The natural abundance of ¹⁵N and ¹³C, conventional soil analyses, and biomass production by maize were used to study the influence of five tropical tree species on soils and their fertility. The experiment was conducted in Morogoro, Tanzania, to compare Cassia (Senna) siamea, Eucalyptus camaldulensis, E. tereticornis (all non-N₂-fixing), Leucaena leucocephala, Prosopis chilensis (both N₂-fixing), and a grass fallow. Maize biomass production, which was correlated with N uptake (P=0.001), was higher on soils from plots with 5-year-old Leucaena and Prosopis spp. compared to the grass fallow, while other tree species had less favourable effects on maize growth. The per cent N was higher in soil and ¹⁵N of soil total N was lower under Prosopis sp. compared to soil under other tree species, which suggests an input from N₂ fixation by Prosopis sp. A transfer of fixed N to maize or to understorey grass species was, however, not indicated by the ¹⁵N natural abundance. Prosopis sp. contributed more C to the soil than the other four tree species; the difference in ¹³C between soils from Prosopis sp. plots and from grass fallow plots showed that the tree contributed 11% to the total C of the soil over a period of 8 years. The leaves of the N₂-fixing species had a low ratio of lignin+phenols to N, and maize growth was negatively correlated with this parameter. The Eucalyptus spp. had leaves with a high lignin+phenols to N ratio, contributed very little C to the soil, and lowered the soil pH.     

Nitrogen and phosphorus use in maize sole cropping and maize/cowpea mixed cropping systems on an Alfisol in the northern Guinea Savanna of Ghana

Summary The use of N and P by mixed and by sole cropping (crop rotation) of maize and cowpeas were compared in a field experiment on an Alfisol at the Nyankpala Agricultural Experiment Station in the northern Guinea Savanna of Ghana, using two levels of N (0 and 80 kg N ha^-1 year^-1 as urea) and P application (0 and 60 kg P ha^-1 year^-1 as Volta phosphate rock). Maize grain yields were significantly reduced in the mixed cropping system. This yield difference became smaller with the application of N and P fertilizer. The N and P concentrations in maize ear leaves at silking indicated that a deficiency in N and P contributed to the maize yield depression in mixed cropping. Competition for soil and fertilizer N between maize and cowpeas was suggested by: (1) A similarity in total N uptake between the two cropping systems; (2) efficient use of soil nitrate by the cowpeas; and (3) low N₂ fixation by the cowpeas, calculated with the aid of an extended-difference method. In general, N₂ fixation was low, with the highest values in the sole cropping (53 kg ha^-1) and a substantial reduction in the mixed cropping system. The application of N fertilizer further reduced N₂ fixation. This was substantiated by nodule counts. The lower N₂ fixation in the mixed cropping system was only partly explained by the lower density of cowpeas in this system. In addition, dry spells during the cropping season and shading by the maize component could have reduced the nodulation efficiency. No N transfer from the legume/rhizobium to the non-legume crop was observed. Impaired P nutrition in the mixed compared with the sole-cropped maize might have been due to less P mobility in the soil. This was indicated by lower soil moisture contents in the topsoil under mixed cropping, especially during the dry year of 1986. The results show that mixed cropping of maize and cowpeas did not lead to improved use of soil and fertilizer N and P or to an enhanced N₂ fixation. On the contrary, an annual rotation of maize and cowpeas was clearly superior.     

Nitrogenase activity associated with leaf,root, and rhizosphere soils in an Indian tropical forest

Summary N₂ fixation (acetylene reduction assay) by phylloplane microorganisms was measured in dominant and co-dominant plant species growing in a tropical rain forest. No significant acetylene reduction was recorded with intact leaf samples. Azotobacter sp., Beijerinckia sp., Derxia sp., and Klebsiella pneumoniae were isolated as phylloplane N₂-fixing bacteria. Azospirillum lipoferum was only isolated from soil samples containing the roots of Poaceae. Nitrogenase activity was recorded in culture derived from the roots and rhizosphere soil samples, although low acetylene reduction activity indicates that these associations did not provide large amounts of N to the systems studied.     

Effect of successive cuttings on uptake and partitioning of 15N among plant parts of Leucaena leucocephala

Summary We studied the effect of three successive cuttings on N uptake and fixation and N distribution in Leucaena leucocephala. Two isolines, uninoculated or inoculated with three different Rhizobium strains, were grown for 36 weeks and cut every 12 weeks. The soil was labelled with 50 ppm KNO₃ enriched with 10 atom % ¹⁵N excess soon after the first cutting. Except for the atom % ¹⁵N excess in branches of K28 at the second cutting, both the L. leucocephala isolines showed similar patterns of total N, fixed N₂, and N from fertilizer distribution in different parts of the plant at each cutting. The Rhizobium strain did not influence the partitioning of ¹⁵N among the different plant parts. Significant differences in ¹⁵N enrichment occurred in different parts. Live nodules of both isolines showed the lowest atom % ¹⁵N excess values (0.087), followed by leaves (0.492), branches (0.552), stems (0.591), and roots (0.857). The roots contained about 60% of the total plant N and about 70% of the total N derived from fertilizer over the successive cuttings. The total N₂ fixed in the roots was about 60% of that fixed in the whole plant, while the shoots contained only 20% of the fixed N₂. We conclude that N reserves in roots and nodules constitute another N source that must be taken into account when estimating fixed N₂ or the N balance after pruning or cutting plants. ¹⁵N enrichment declined up to about fivefold in the reference and the N₂-fixing plants over 24 weeks following the ¹⁵N application. The proportion and the amounts of N derived from fertilizer decreased, while the amount derived from N₂ fixation increased with time although its proportion remained constant.     

Amelioration of water stress effects on wetland rice by urea-N,plant growth regulators,and foliar spray of a diazotrophic bacterium Klebsiella sp.

Some of the measures suggested for amelioration of drought effects include application of N fertilizer and plant growth regulators (PGRs). Since N₂-fixing bacteria produce plant growth substances (PGRs), the effect of foliar application of an active strain of Klebsiella sp. (KUPOS) on IR-50 rice was examined using three foliar sprays applied at 10-day intervals. Irrigation once every 3 days was essential for plant growth. Application of KUPOS and 40 kg N ha^-1 improved grain yield of acutely water stressed plants from 330 kg ha^-1 to more than 1300 kg ha^-1 along with an improvement in several growth variables and yield determinants. Indole acetic acid, kinetin and GA₃, in a mixture of 10^-4 M of each, were less effective than KUPOS in alleviating stress effects. The adverse effects of water stress on respiration and photosynthesis as indicated by CO₂ exchange were also alleviated by these treatments. While uptake of K, Mg, Ca, Fe and Mo was increased, Na content decreased, accompanied by an increase in proline content. The order of effectiveness of the treatments was 40 kg N ha^-1 >KUPOS>PGRs.     

Effects of NPK fertilizer combinations on yield and nitrogen balance in sorghum or pigeonpea on a vertisol in the semi-arid tropics

A long-term experiment was carried out on a Vertisol from 1986 to 1992 to examine the combined effects of NPK fertilizers on yield using sorghum (Sorghum bicolor L. Moench cv. CSH 5) and short-duration pigeonpea (Cajanus cajan L. Millsp. cv. ICPL 87). The fertilizer treatments were as follows: 0 (no fertilization), N (150 kg N ha^-1 ), P (65.5 kg P₂O₅ ha^-1), K (124.5 kg K₂O ha^-1), and all possible combinations (NP, NK, PK, and NPK). In this study we continued this experiment during the period 1993 to 1994 and analyzed the crop yield response to fertilizers and the N balance. The amount of N derived from the atmosphere and fertilizer was estimated by the ¹⁵N natural abundance method and ^l5N isotope dilution method, respectively. A combined application of Nand P fertilizers gave the highest grain yield for the two crops under the 8th and 9th continuous croppings, unlike the application of K fertilizer. The values of total N for the two crops were significantly higher in the NP and NPK plots. These crops took up N mainly from soil. There was a significant positive relationship between the uptake of N_dff and N_dfs by each crop. Pigeonpea or sorghum took up more N from the soil in the N fertilizer plots than in the plots without N, suggesting that soil N fertility was enhanced and the amount of N supplied from soil increased in the plots with consecutive application of N fertilizer for 7 y. Even pigeonpea, which fixes atmospheric N inherently, needed N fertilizer to achieve high grain yield, suggesting that N fixation by the nodules was not always sufficient to meet the N requirements of the crop under these conditions. Although fertilizer N exerted a beneficial effect on plant growth and yield in the two crops, the values of fertilizer N recovery (FNR) by the two crops were considerably low. Therefore, it is suggested that the development of N fertilizer management which could maximize FNR of each crop should be promoted.     

Prediction of potentially mineralizable N from amidohydrolase activities in a manure-applied, corn residue-amended soil

Prediction of potentially mineralizable N as an important N pool from soil amidohydrolases was investigated. Composite soil samples were collected from plots of a field experiment in which 0, 50 and 100 Mg cow manure ha⁻¹ year⁻¹ had been applied for five consecutive years. The soils were treated with corn shoots or roots or remained untreated in a factorial combination with the manure treatments, with three replications. The mineralized inorganic N was measured periodically in 20-week incubations and potentially mineralizable N (N₀) was calculated based on a first-order kinetic model. Urease, l-glutaminase and l-asparaginase activities were measured before and after incubation. The values of N₀ ranged from 208.6 in the controls to 388.4 in soils that had received 50 Mg ha⁻¹ year⁻¹ of cow manure and were amended with corn shoots. Corn residue amendment in the manure treated soils, increased the values of N₀ or changed the N mineralization kinetic pattern from a first-order to a zero-order model. According to a relative sensitivity index, l-asparaginase was the most sensitive enzyme to the treatments. Multiple regression analysis revealed that 92% of N₀ variations can be described by the activities of urease and l-asparaginase and therefore the soil amidohydrolase activities have the potential to evaluate potentially mineralizable N.     

The effect of Azospirillum brasilense Sp7 and Azotobacter chroococcum on nitrogen balance in soil under cropping with oats (Avena sativa L.)

Summary Pot experiments with oats were carried out to study the effect of Azospirillum brasilense Sp 7 and Azotobacter chroococcum 94K on the yield of plants, the N content of soil and the ¹⁴N balance. The plants were grown on gray forest soil under irrigation with deionized water and application of ¹⁵N-labelled fertilizer at a rate of 4 mg N 100 g^-1 soil. Inoculation of plants with Azospirillum spp. and Azotobacter spp. failed to increase the plant yield. However, the increase in total N in the soil at the end of the experiment and the positive ¹⁴N balance in the soil-plant system due to increased nitrogenase activity in the rhizosphere were statistically significant. The amount of N accumulated in the soil was comparable with the rate of N applied as fertilizer.     

Nitrogen fixation in paddy soils

The 4 long-term experimental plots (Umbric haplaquept) with different fertilizer treatment at Cent. Agric. Exp. Sta. in Konosu City, Saitama Prefecture, were used for the sites of investigation. The 4 plots were NF (applied with no fertilizer), IF (applied with inorganic fertilizers), GM (applied with green manure and CaCO₂), and OM (applied with manure and inorganic fertilizers). Flooded water, floating weed, upper (0-2cm) and lower (2-10cm) parts of Apg horizon and rhizosphere were collected from each plot before flooding, during flooding, and after drainage. These samples were analyzed for N₂-fixing activity by acetylene reduction method, pH, Eh, and contents of Fe²⁺, NH₄ ⁺, chlorophyll-type compounds, and water-soluble carbohydrates.

The N₂-fixing activity of all samples showed almost the same pattern of change with time: very low before flooding, rapidly increased after flooding, the maximum value at the maximum tillering stage of rice plant, declined afterwards and reached a very low value after drainage.

Rough estimation of the “N₂-fixing capacity” of each part of the paddy field revealed that the most important site of the N₂ fixation was the reduced Apg horizon, that the importance of flooded water and/or the oxidized layer in the N₂ fixation was rather low except in infertile soil, and that the role of rhizosphere in the N₂ fixation could not be neglected also in Japan.

Reduced condition and content of easily decomposable organic substances were judged to be main factors which control the N₂-fixing activity in the flooded soil on the basis of correlations between the Nt-fixing activity and several analytical data of the paddy soils.