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.     

Landscape-scale estimates of dinitrogen fixation by Pisum sativum by nitrogen-15 natural abundance and enriched isotope dilution

Topography and slope position influence the soil and environmental factors that affect N₂ fixation by legumes. The present study was conducted to (1) estimate N₂ fixation by field peas in a gently rolling farm field using the natural ¹⁵N abundance and the ¹⁵N-enriched isotope dilution techniques and (2) identify soil and environmental factors that influence N₂ fixation at the landscape scale. Whereas soil available water capacity, available NH _inf4 ^sup+ , total crop yield, and percent N derived from N₂ fixation (% Ndfa) estimated using enriched N were significantly affected by landform patterns, soil NO _inf3 ^sup- levels, seed yield, and the % Ndfa estimated using natural abundance did not follow landform patterns. The % Ndfa using natural abundance was correlated with NH _inf4 ^sup+ but not with available soil water, pH, electrical conductivity, NO _inf3 ^sup- , or particle size. Estimates of the % Ndfa using enriched ¹⁵N ranged from 0 to 92.8%. The highest median value (68.6%) for % Ndfa using enriched N occurred on the divergent footslopes, with the lowest value (28.1%) on the convergent shoulders. Estimates of % Ndfa using natural abundance ranged from 13.2% to 96.9%. Smaller fluctuations during the growing season in the ¹⁵N of the available N pool may have resulted in less variability for % Ndfa using natural abundance compared to enriched ¹⁵N. Despite similar mean values for % Ndfa using natural abundance (44.5) and enriched ¹⁵N (49.6), no significant correlation between the two estimates was found. These results suggest that although topography may exert gross controls on N₂ fixation, large variations in N₂ fixation at the microsite level may preclude correlations between individual estimates and limit detection of landscape scale patterns of N₂ fixation.Contribution No. R754 of the Saskatchewan Center of Soil Research     

Evaluation of isotopic dilution method for measuring N2 fixation in Azolla: Comparison with other methods 1

An isotopic dilution method that overcomes the drawbacks of commonly used methods for measuring N₂ fixation by aquatic N‐fixers such as Azolla pinnata‐Anabaena azollae association (Azolla) is presented. The method was compared with ¹⁵N₂ gas (while maintaining CO₂) and the difference methods of measuring N₂ fixation. The isotopic dilution method was used for two conditions: a. For ¹⁵N‐free growth medium, Azolla was pre‐enriched with ¹⁵N, and N₂ fixation was determined by measuring the dilution of ¹⁵N in the tissue. b. For the growth medium containing N, N₂ fixation was determined by providing ¹⁵N enriched ammonium sulfate in the growth medium and measuring ¹⁵N to ¹⁴N ratio in the tissue. An airtight chamber, necessary for ¹⁵N₂ gas and acetylene reduction methods, was not representative of the growing environment of Azolla. Temperature in the airtight chamber was far from uniform and CO₂ was rapidly depleted. The isotopic dilution method is simpler, relatively inexpensive, subject to fewer errors and applicable to more diverse conditions, and yet was as accurate as ¹⁵N₂‐gas method.     

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.     

Early nitrogen fixation and utilization in albizia lebbeck,leucaena leucocephala,and gliricidia sepium using nitrogen (15n) labelling

Abstract

High nitrogen (N₂)‐fixing potential is a desirable characteristic for any candidate hedgerow tree. Thus a study was conducted to evaluate Albizia lebbeck as a N₂‐fixing tree in comparison to Gliricidia sepium and Leucaena leucocephala currently used in alley cropping. Nitrogen fixation and utilization were assessed in a screenhouse at four months after planting by the ¹⁵N dilution technique using Senna siamea as the non N₂‐fixing reference. A. lebbeck accumulated significantly more N than L. leucocephala, but G. sepium was intermediate. This superiority in N yield was mainly due to its abundant nodule dry weight production which accounted for up to 10.8% of its total N. This was equivalent to 2.5 and 6 fold that of Gliricidia and Leucaena nodules, respectively. A. lebbeck had bigger but significantly (P<0.05) lower number of nodules per plant than G. sepium, but it did not differ from Leucaena. Albizia was the best N₂ fixer with 44% Ndfa equivalent to 533 mg N per plant. G. sepium followed with 28% Ndfa and L. leucocephala with 18% Ndfa corresponding to 321 and 191 mg N fixed, respectively. However, the relatively higher N₂ fixation in Albizia was not translated into higher N or dry matter yields. As A. lebbeck fixed more N, it depended less on soil N (49.8%) than did Leucaena (72.5% Ndfs) and Gliricidia (63.9% Ndfs) and less on fertilizer N as well. Thus A. lebbeck appears to be a potential hedgerow species for alley cropping purpose.     

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

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

Dual inoculation with strains of Bradyrhizobium japonicum and Azospirillum brasilense to improve growth and biological nitrogen fixation of soybean (Glycine max L.)

The effects of inoculation with Bradyrhizobium japonicum and Azospirillum brasilense strains on the growth of soybean were evaluated with regard to the estimation of N₂ fixation using the ¹⁵N isotope dilution technique. Inoculation, in general, increased the dry mass of soybean as well as nitrogen content. Dual inoculation with a mixture of B. japonicum and A. brasilense strains was superior over single inoculation with B. japonicum. Nitrogen fixed (Ndfa) varied according to inoculant and soil conditions. Percentages of nitrogen derived from air (% Ndfa) using a non-nodulating isoline were 72% and 76% for B. japonicum and B. japonicum plus A. brasilense, respectively, in non-sterile soil. A similar but higher trend was recorded in sterilized soil, in which the percentages of N₂ fixed were 81% and 86% for single and dual inoculation, respectively. The correlation coefficient between N₂ fixed and N uptake (r=0.94) and dry mass (r=0.89) was significant. Application of special bacterial inoculants in agricultural systems of Egypt seems to be a promising technology and could be used for improving soybean growth as well as soil fertility, thus minimizing environmental pollution. Received: 10 January 1996     

Medium‐ and short‐term available organic matter,microbial biomass,and enzyme activities in soils under Pinus sylvestris L. and Robinia pseudoacacia L. in a sandy soil in NE Saxony,Germany

Total, mobile, and easily available C and N fractions, microbial biomass, and enzyme activities in a sandy soil under pine (Pinus sylvestris L.) and black locust (Robinia pseudoacacia L.) stands were investigated in a field study near Riesa, NE Germany. Samples of the organic layers (Oi and Oe‐Oa) and the mineral soil (0–5, 5–10, 10–20, and 10–30 cm) were taken in fall 1999 and analyzed for their contents of organic C and total N, hot‐water‐extractable organic C and N (HWC and HWN), KCl‐extractable organic C and N (C_org(KCl) and N_org(KCl)), NH ‐N and NO ‐N, microbial‐biomass C and N, and activities of β‐glucosidase and L‐asparaginase. With exception of the HWC, all investigated C and N pools showed a clear response to tilling, which was most pronounced in the Oi horizon. Compared to soils under pine, those under black locust had higher contents of medium‐ and short‐term available C (HWC, C_org(KCl)) and N (HWN, N_org(KCl)), mineral N (NH ‐N, NO ‐N), microbial‐biomass C and N, and enzyme activities in the uppermost horizons of the soil. The strong depth gradient found for all studied parameters was most pronounced in soils under black locust. Microbial‐biomass C and N and enzyme activities were closely related to the amounts of readily mineralizable organic C (HWC and C_org(KCl)). However, the presented results implicate a faster C and N turnover in the top‐soil layers under black locust caused by higher N‐input rates by symbiotic N₂ fixation.     

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

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

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.     

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.     

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.     

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

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

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.     

种植模式对苜蓿固氮和固氮产物转移的影响

田间小区试验研究了不同种植模式下苜蓿的共生固氮贡献,并利用~(15)N同位素示踪技术评估了苜蓿的％Ndfa和Ndfa,以及与之混作生长的牛尾草植株中来自苜蓿固氮产物的转移量。研究表明,豆科与禾本科牧草混作对发挥草地的优势有一定影响,混作条播在干草产量、全氮产量、％Ndfa和Ndfa等方面均优于间作与混作撒播模式,且高于单作苜蓿与牛尾草的平均值。用~(15)N同位素稀释法与~(15)N天然丰度法评估苜蓿的％Ndfa与Ndfa值时,无明显差异(P<0.05),前者还能准确测出混种牛尾革植株中的固氮产物转移量,后者则大大低估,甚至不能测出固氮产物转移。     

Adaptation of green manure legumes to adverse conditions in rice lowlands

Poor adoption of sustainable pre-rice green manure technology by lowland farmers is frequently associated with unreliable legume performance under adverse environmental conditions such as marginal soils, short photoperiod, and unfavorable hydrology. A series of field and microplot experiments were conducted at the International Rice Research Institute (IRRI) in 1991 and 1992 to screen and evaluate 12 promising flood-tolerant legumes for adaptation (N accumulation and biological N₂ fixation) to a range of environmental stresses, frequently encountered in rice lowlands. Legumes belonging to the genera Sesbania and Aeschynomene were grown for 8 weeks at 10×10 cm spacing: (1) in a fertile control soil and in four marginally productive irrigated lowland rice soils (sandy Entisol, P-deficient Inceptisol, acid Ultisol, and saline Mollisol); (2) during short- (11.7h) and long-day (12.3 h) seasons in a favorable irrigated lowland soil; and (3) in an aerobic soil (drought-prone rain-fed lowland) and a deep-flood-prone lowland soil (1 week seedling submergence). A large variability in N accumulation was obsersed among legume species and across different environments, ranging from less than 1 to over 70 mg N plant^-1. The nitrogen derived from the atmosphere (Ndfa) accounted on average for 82% of total N accumulation. Sesbania virgata was least affected by unfavorable soil conditions but its Ndfa was the lowest among the tested species (less than 60%). Stem nodule formation did not convey a significant advantage to legumes grown under adverse soil conditions. However, flooding reduced N₂ fixation less in stem-nodulating than in solely root-nodulating species. Most species drastically reduced N accumulation under short-day conditions. Aeschynomene afraspera and S. speciosa were least affected by photoperiod. The considerable genetic variability in the germplasm screened allows the selection of potentially appropriate legumes to most conditions studied, thus increasing N accumulation in green manures.     

Quantification of N2 fixation and annual N benefit from N2 fixation in soybean accrued to the soil under soybean‐wheat continuous rotation

A computational exercise was undertaken to quantify the percent N derived from atmosphere %Ndfa) in soybean and consequent N benefit from biological N₂‐fixation process annually accrued to the soil by the soybean crop using average annual N‐input/‐output balance sheet from a 7 yr old soybean‐wheat continuous rotational experiment on a Typic Haplustert. The experiment was conducted with 16 treatments comprised of combinations of four annual rates of farmyard manure (FYM ? 0, 4, 8, and 16 t ha^–1) and four annual rates of fertilizer N (? 0, 72.5, 145, and 230 kg N ha^–1) applications. The estimated N contributed through residual biomass of soybean (RBN_S) consisting of leaf fall, root, nodules, and rhizodeposition varied in the ranges of 7.02–16.94, 11.65–28.83, 3.31–8.91, and 11.3–23.8 kg N ha^–1 yr^–1, respectively. A linear relationship was observed between RBN_S and harvested biomass N (HBN_S) of soybean in the form of RBN_S = 0.461 × HBN_S – 20.67 (r = 0.989, P < 0.01), indicating that for each 100 kg N assimilated by the harvested biomass of soybean, 25.4 kg N was added to the soil through residual biomass. The Ndfa values ranged between 13% and 81% depending upon the annual rates of application of fertilizer N and FYM. As per the main effects, the %Ndfa declined from 76.4 to 26.0 with the increase in annual fertilizer‐N application from 0 to 230 kg N ha^–1, whereas %Ndfa increased from 40.8 to 65.8 with the increase in FYM rates from 0 to 16 t ha^–1, respectively. The N benefit from biological N₂ fixation accrued to the soil through residual biomass of soybean ranged from 7.6 to 53.7 kg N ha^–1 yr^–1. The treatments having %Ndfa values higher than 78 showed considerable annual contribution of N from N₂ fixation to the soil which were sufficient enough to offset the quantity of N removed from the soil (i.e., native soil N / FYM‐N / fertilizer‐N) with harvested biomass of soybean.     

Field evaluation of lentil cultivars inoculated with Rhizobium leguminosarum bv. viciae strains for nitrogen fixation using nitrogen-15 isotope dilution

 A ¹⁵N isotope dilution technique was applied to quantify the extent of N₂ fixation in lentil (Lens culinaris Medik.) cultivars as influenced by Rhizobium leguminosarum bv. viciae strains in a field experiment in Pakistan. The experiment was conducted on a soil with a very small indigenous rhizobial population and where N was a limiting factor for crop production. Significant variations in number of nodules, dry weight of nodules, biomass yield, grain yield, total N yield, proportion of plant N derived from N₂ fixation (P_fix) and amount of N derived from the atmosphere (N_dfa) were observed among combined treatments of four rhizobial strains and six lentil varieties. In a field previously labelled with ¹⁵N, to which a basal dose of 75 kg P₂O₅ ha^–1 was applied as single super phosphate, N_dfa ranged from 15 to 24 kg N ha^–1 when calculated according to rhizobial strain and from 4 to 38 kg N ha^–1 when calculated according to lentil variety. Lc 26 was the most effective strain and fixed 243% more N than the indigenous population in the uninoculated control. In treatments with the lentil variety PL-406, N_dfa was 38 kg N ha^–1, which was 850% higher than with the lentil variety Precoz/F6-20-1×M-85. Generally, the varieties with greater P_fix produced a higher dry matter yield. Received: 26 May 1999     

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.     

Microbial activity,organic C accumulation and <Superscript>13</Superscript>C abundance in soils under alley cropping systems after 9 years of recultivation of quaternary deposits

The impact of alley cropping on post-lignite mine soils developing from quaternary deposits after 9 years of recultivation was evaluated on the basis of microbial indicators, organic C and total N contents, and the isotope characteristics of soil C. Soils were sampled at the 0 to 3, 3 to 10, and 10 to 30 cm depths under black locust (Robinia pseudoacacia L.), poplar (Populus spp.), the transition zone and in the middle of alley under rye (Secale cereale). There was no significant effect of vegetation on microbial properties presumably, due to the high variability, whereas organic C and total N contents at the 0- to 3-cm layer were significantly higher under black locust and poplar than in the transition zone and rye field. Organic C total N contents, and basal respiration, microbial biomass, and microbial quotient decreased with soil depth. Soil organic C and total N contents were more than doubled after 9 years of recultivation, with annual C and N accretion rate of 162 g C _org m⁻² year⁻¹ and 6 g N _t m⁻² year⁻¹. Microbial properties indicated that the soils are in early stages of development; the C isotope characteristics confirmed that the sequestered C was predominantly from C3 plants of the alley cropping.