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     

The use of nitrogen-15 natural abundance in white clover (Trifolium repens L.) to determine nitrogen fixation under different management practices

 The ¹⁵N natural abundance (δ¹⁵N) of white clover (Trifolium repens L.) grown in pasture under different management practices was determined. Plants were split into leaflets, petioles and stolons and the ¹⁵N signature of each tissue was measured. The δ¹⁵N of leaflet tissue from plants of two non-N₂-fixing species (Lolium perenne L. and Ranunculus repens L.), growing in close proximity to the sampled T. repens, was also measured. By using T. repens plants grown in the absence of mineral N to provide reference material, the proportion of N derived from N₂ fixation (%Ndfa) in pasture plants was calculated. Within a plot, variation was present in the δ¹⁵N between the tissues of T. repens. Variation was also present between the same tissues under different management practices. The %Ndfa in the leaf material of T. repens varied from 34% to 100% between the plots. The use of different reference species did not affect the estimate of %Ndfa. Received: 14 December 1998     

Genetic variability in dinitrogen fixation between cowpea [Vigna unguiculata (L.) Walp] cultivars determined using the nitrogen-15 isotope dilution technique

 N fixed in 16 cultivars of cowpea [Vigna unguiculata (L.) Walp] inoculated with effective Bradyrhizobium strains collected from the West African MIRCEN culture collection was measured by ¹⁵N isotope dilution technique. In all plant parts, significant differences in the percentage of N derived from the atmosphere (%Ndfa) and the amount of Ndfa occurred between the cultivars. Ndoute variety exhibited the highest %Ndfa (74.33% in shoots; 60.90% in roots) and accumulated more fixed N (960 mg N plant^–1 and 38 mg N plant^–1 in shoots and roots, respectively). Therefore this cultivar should be selected as the highest N-fixing cowpea cultivar. It also should be used in a breeding programme to contribute to the development of cultivars that could stimulate an intensive use of cowpea in many different cropping systems in Africa with a view to maintaining soil fertility. Received: 14 June 1999     

Nodulation and N2 fixation of faba bean (Vicia faba L.) after soil amendment with crop residues

The effect of prior soil amendment with different N sources at 50 mg N (kg soil)^—1 on nodulation and N₂ fixation of faba bean (Vicia faba L. cv. Troy) using wheat (Triticum aestivum L. cv. Star) as reference crop was assessed in a pot experiment. Four treatments viz legume manure (LEGM) as clover shoots, cereal manure (CEREM) as barley straw, N fertilizer (FERT‐N) as Ca(NO₃)₂, and no‐manure control (NOMAN) were investigated consecutively at 45, 70, and 90 days after sowing (DAS). Faba bean nodulated profusely, with an increase on average from 629 nodules per pot at 45 DAS to nearly 2.3‐ and 3.3‐fold at 70 and 90 DAS, respectively. Low nodule numbers and nodule dry matter occurred under FERT‐N and CEREM, whereas high values were found for NOMAN and LEGM. Soil amendment affected percent N₂ fixation in relation to N source and plant age. Highest percent N₂ fixation (≥ 90 %) was found under the lowest N‐supplying amendments, no‐manure, and cereal manure, respectively. FERT‐N depressed N₂ fixation particularly at 45 DAS when N₂ fixation was reduced to as low as 23 %. The rise in N₂ fixation thereafter suggests that faba bean adjusted after depletion of mineral N in the soil. N₂ fixation was also decreased after cereal straw application, even though N concentration in faba bean plants was high. The results indicate that plant residues, both with high and low N concentration, applied to soil to raise its fertility may interfere with N₂ fixation of faba bean.     

The use of nitrogen-15 natural abundance and nitrogen yield of non-nodulating isolines to estimate nitrogen fixation by soybeans (Glycine max L.) across three elevations

Summary Dissimilarities in soil N uptake between N₂-fixing and reference non-N₂-fixing plants can lead to inaccurate N₂ fixation estimates by N difference and ¹⁵N enrichment methods. The natural ¹⁵N abundance ( ¹⁵N) method relies on a stabilized soil ¹⁵N pool and may provide reliable estimates of N₂ fixation. Estimates based on the ¹⁵N and differences in N yield of nodulating and non-nodulating isolines of soybean were compared in this study. Five soybeans from maturity groups 00, IV, VI, and VIII and their respective non-nodulating isolines were grown at three elevations differing in ambient temperature and soil N availability. Despite large differences in phenological development and N yield between the non-nodulating isolines, the ¹⁵N values measured on seeds were relatively constant within a site. The ¹⁵N method consistently produced lower N₂ fixation estimates than the N difference method, but only in three of the 15 observations did they differ significantly. The average crop N derived from N₂ fixation across sites and maturity groups was 81% by N difference compared to 71% by ¹⁵N. The magnitude of difference between the two methods increased with increasing proportions of N derived from N₂ fixation. These differences between the two methods were not related to differences in total N across sites or genotypes. The low N₂ fixation estimates based on ¹⁵N might indicate that the nodulating isolines had assimilated more soil N than the non-nodulating ones. A lower variance indicated that the estimates by N difference using non-nodulating isolines were more precise than those by ¹⁵N. Since the differences between the estimates were large only at high N₂ fixation levels (low soil N availability), either method may be used in most situations when a non-nodulating isoline is used as the reference plant. The ¹⁵N method may have a comparative advantage over N difference and ¹⁵N enrichment methods in the absence of a suitable non-N₂-fixing reference plant such as a non-nodulating isoline.     

Estimating biological N2 fixation by a tropical legume tree using the non-nodulating phenophase as the reference in the 15N natural abundance method

The non-nodulating phenophase of a legume tree was tested as a non-N₂-fixing reference in application of the ¹⁵N natural abundance method for estimating the N₂ fixation. We applied this method to study the effects of three pruning intensities, complete pruning every 6 months (T-6), ca. 50% pruning every 3 months (P-3) and intact control (C), on N₂ fixation in Erythrina lanceolata (Papilionaceae) planted as shade and support trees for vanilla (Vanilla planifolia) in a subhumid tropical site in Quepos, Costa Rica. We measured nodulation and N₂ fixation for 12 months. The trees under the C regime nodulated abundantly during the rainy season vegetative growth but did not nodulate during the dry season and flowering. A linear regression (r²=0.76) was observed between the nodule biomass and δ¹⁵N values under the C regime, suggesting quite a stable specific N₂ fixation rate. The stable δ¹⁵N values throughout the year in the non-N₂-fixing Morus nigra (Moraceae) growing on the same soil indicated that the temporal variation in the plant available soil ¹⁵N was low. We used the intercept term of the regression (δ¹⁵N=3.5 when nodule biomass was 0) as the non-N₂-fixing δ¹⁵N value when estimating the percentage of N fixed from atmosphere out of tree total N. The percentage varied from 0 during the driest period to 53% during rainy season. Pruning reduced rainy season nodulation under the T-6 and P-3 regimes almost to nil, and the δ¹⁵N values were high. Our results suggest that the conditions for using the non-nodulating phenophase as the non-N₂-fixing reference required in the ¹⁵N natural abundance method were fulfilled. The C trees showed a clear phenological cycle in N₂ fixation, while pruning severely disturbed the N₂ fixation under the T-6 and P-3 regimes, indicating that E. lanceolata is better suited for agroforestry systems in which the trees are not managed by periodic prunings.     

Efficiency of N2 fixation in Vicia faba L. in combination with different Rhizobium leguminosarum strains

The efficiency of symbiotic dinitrogen (N₂) fixation in Vicia faba L. in combination with 3 different Rhizobium leguminosarum strains was studied in a pot experiment during vegetative and reproductive growth. The objective of the experiments was to assess variability among Rhizobium strains inoculated on single legume species and determine possible reasons for observed variations. Dry matter formation, N₂ fixation and the carbon (C) costs of N₂ fixation were determined in comparison with nodule free plants grown with urea. Nodule number and the capacity of different respiratory chains in the nodules were also measured. The plants inoculated with the Rhizobium strain A 37 formed less dry matter and fixed less N compared to the other two Rhizobium strains (Vic 1 and A 150). This coincided with a lower number of nodules and higher C costs of N₂ fixation. The C costs for N₂ fixation were in all cases significantly lower during reproductive growth compared to vegetative growth. Neither the latter nor the differences in C expenditure for N₂ fixation between the Rhizobium strains could be explained in terms of differences or shifts in the capacity of different respiratory chains in the nodules.     

Biological nitrogen fixation in faba bean (Vicia faba L.) in the Ethiopian highlands as affected by P fertilization and inoculation

 N₂ fixation by leguminous crops is a relatively low-cost alternative to N fertilizer for small-holder farmers in developing countries. N₂ fixation in faba bean (Vicia faba L.) as affected by P fertilization (0 and 20 kg P ha^–1) and inoculation (uninoculated and inoculated) with Rhizobium leguminosarium biovar viciae (strain S-18) was studied using the ¹⁵N isotope dilution method in the southeastern Ethiopian highlands at three sites differing in soil conditions and length of growing period. Nodulation at the late flowering stage was significantly influenced by P and inoculation only at the location exhibiting the lowest soil P and pH levels. The percentage of N derived from the atmosphere ranged from 66 to 74%, 58 to 74% and 62 to 73% with a corresponding total amount of N₂ fixed ranging from 169 to 210 kg N ha^–1, 139 to 184 kg N ha^–1 and 147 to 174 kg N ha^–1 at Bekoji, Kulumsa and Asasa, respectively. The total N₂ fixed was not significantly affected by P fertilizer or inoculation across all locations, and there was no interaction between the factors. However, at all three locations, N₂ fixation was highly positively correlated with the dry matter production and total N yield of faba bean. Soil N balances after faba bean were positive (12–58 kg N ha^–1) relative to the highly negative N balances (–9–44 kg N ha^–1) following wheat (Triticum aestivum L.), highlighting the importance of rotation with faba bean in the cereal-based cropping systems of Ethiopia. Received: 13 January 2000     

Enrichment of natural 15N abundance in frankia-infected nodules

Abstract

The enrichment of ¹⁵N in the nodules of some N₂-fixing leguminous plants is an interesting finding (Shearer et al. 1982). The extent of ¹⁵N enrichment differed depending on the plant species (Shearer et al. 1982; Yoneyama 1987) and bacterial strains (Steele et al. 1983), and in soybeans it was apparently related to the nitrogen fixation efficiency (Shearer et al. 1984)     

The role of potassium fertilizer in nodulation and nitrogen fixation of faba bean (Vicia faba L.) plants under drought stress

Three-week-old nodulated faba bean plants were subjected to different levels of drought stress (onehalf, one-quarter, or one-eighth field capacity) for 5 weeks. Half the stressed plants were treated with KCl at 10 mg kg^-1 soil or 150 mg kg^-1 soil at the beginning of the drought stress. Nodulation and nitrogenase activity were significantly decreased by increasing drought stress. Leghaemoglobin and protein contents of nodule cytosol were also severely inhibited by drought sttess. This decline was attributed to the induction of protease activity. However, carbohydrate contents of the nodule cytosol increased significantly. This accumulation was attributed to a sharp decline in invertase activity and low use of sugar by the bacteroids We conclude that harmful effects of water deficits can be alleviated by increasing K⁺ supplementation.     

铝诱导蚕豆气孔保卫细胞凋亡研究

植物叶表面的气孔保卫细胞是研究信号转导的模式实验系统,对环境变化反应灵敏而准确,采用蚕豆叶面气孔保卫细胞,研究了铝（AlCl3）对细胞的毒性效应。结果表明,在1～10 mmol.L^-1范围内,AlCl3可使气孔保卫细胞活性降低,部分细胞死亡,且随着浓度的增高细胞死亡率增高;死细胞呈现核固缩、核降解、凋亡小体等典型凋亡特征。凋亡抑制剂Z-Asp-CH2-DCB或TLCK与AlCl3共同作用时,保卫细胞死亡率显著降低;一定浓度的抗坏血酸（AsA）或过氧化氢酶（CAT）以及Ca2＋螯合剂乙二醇四乙酸酯（EGTA）或Ca2＋通道抑制剂LaCl3与AlCl3共同作用时,细胞死亡率降低。研究结果表明,铝诱导的蚕豆保卫细胞死亡可能是一种细胞凋亡过程,由胁迫诱发的活性氧介导,通过激活质膜钙通道,引起胞内Ca2＋水平改变,进而介导细胞凋亡。     

Time-course of dinitrogen fixation of promiscuous soybean cultivars measured by the isotope dilution method

 Soybean cultivars capable of nodulating with indigenous Bradyrhizobium spp. have been developed by the International Institute of Tropical Agriculture (IITA) and national programs in Africa in order to avoid artificial inoculation by resource-poor farmers in Africa. The current selection procedure for enhanced N₂ fixation is based on an assessment of nodule formation which does not directly quantify the proportions of crop N derived from the atmosphere. We have monitored N accumulation patterns and N₂ fixation in nine promiscuous soybean cultivars with different maturity periods, using the ¹⁵N dilution technique. Nodule development generally peaked at the early podfill stage for all cultivars except Tgx 1519-1D and Tgx 1447-2D in which it continued to increase. The proportion of crop N derived from fixation (%NDFA) ranged between 51% and 67%, 77% and 84%, and 66% and 73% at full bloom, early podfill, and physiological maturity stages, respectively. Total N accumulation increased in all soybean genotypes with increasing plant age. Significant correlations (P<0.001) were established between nodule weight and %NDFA, even though this did not explain the relationship between nodule development and N₂ fixation in cultivars such as Tgx 1519-1D. Promiscuous soybean cultivars retained between 10% and 19% of total N accumulated at the final harvest, in belowground biomass. Our results indicated that these soybean cultivars can derive substantial proportions of plant N from N₂ fixation in soils where compatible indigenous bradyrhizobia populations are adequate and effective. Also, we have substantiated the claims that qualitative nodulation parameters currently used to select varieties with a high N₂ fixation capacity need to be validated with other measurements of N₂ fixation. Received: 5 November 1998     

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%.     

Natural nitrogen-15 abundance and carbohydrate content and composition of organic matter particle-size fractions from a sandy soil

Sandy soil samples collected from under a woody/grass savanna in the Lamto experimental area (6°13N, 5°20W; Côte dIvoire, West Africa), were fractionated according to particle size with the aim of measuring the natural abundance of ¹⁵N and determining the contents and composition of hydrolysable carbohydrates of soil organo-mineral particles for a better understanding of the contribution of each individual fraction to the soil function. The contributions of the fractions <20 m to the total pool of organic matter were 77% for C and 84% for N. Larger amounts of carbohydrates were found in the clay and silt fractions (3,784–6,043 g g^–1 soil). The carbohydrate composition indicated that microbe-derived carbohydrates [e.g. galactose (Gal) and mannose (Man)] accumulated preferentially in the fine fractions while plant-derived sugars [e.g. arabinose (Ara) and xylose (Xyl)] were dominant in coarse fractions. A negative relationship was observed between C:N ratio and ¹⁵N natural abundance on the one hand, and on the other hand between C:N and (Gal+Man):(Ara+Xyl), Man:(Ara+Xyl) and Man:Xyl ratios, clearly indicating that the chemistry of the organic materials of the particle-size fractions reflects a change from soil chemistry dominated by plant materials to that dominated by microbial biomass and metabolites. The contribution of a given fraction to soil microbial activity is controlled by the quality or quantity of associated soil organic matter, its microbial biomass and also by the accumulation of microbial-derived carbohydrates which can be resynthesized or recycled.     

蚕豆亚有限生长型新种质的发现与鉴定

蚕豆是重要的生物固氮和植物蛋白源作物,在改良土壤培肥地力、改善温带农业区持续蛋白生产方面尤为重要。然而,蚕豆品种的无限生长习性是导致机械化程度低的主因之一,极大地制约了蚕豆的规模化种植。为了探讨亚有限型在蚕豆育种中的应用及其对合理株型矮化育种研究的推动作用,通过查阅相关文献和多年的选择鉴定,对亚有限生长型新种质的发现过程和主要特征进行了总结归纳。对3个具有不同生长习性的种质与青蚕17号×RF25 F2分离群体主要农艺性状的表现差异进行了分析。结果表明,在F2群体中,亚有限型在开花层数、株高、结荚层数上与有限、无限型均存在显著差异,无限型与亚有限型的单株荚数、单株粒数、单株产量差异不显著,二者均显著高于有限型。说明亚有限生长习性蚕豆在品种改良后,能兼顾产量与机械化收获,在生产中应用优势更明显。     

Short-term nitrogen transformation rates in riparian wetland soil determined with nitrogen-15

N transformation rates in soil from a riparian wetland that receives runoff from adjacent pastoral land were investigated in a short-term (250 min), anaerobic laboratory incubation (20°C). A joint ¹⁵N tracing-isotope dilution technique was employed that used paired incubations of labelled (99 atom % ¹⁵N) NO₃^–-unlabelled NH₄⁺ and unlabelled NO₃^–-labelled (99 atom % ¹⁵N) NH₄⁺ at three N input levels (0.4, 4 and 24 g N g^–1 soil). At each N input level NO₃^– and NH₄⁺ were added in equal proportions (0.2, 2 and 12 g N g^–1 soil). Soil and gas samples were analysed after 10, 70 and 250 min, and the fate of ¹⁵N and N transformation rates were determined for each time period; 0–10 min (phase 1), 10–70 min (phase 2) and 70–250 min (phase 3). N transformation rates for all processes except gross NH₄⁺ mineralisation were very high during phase 1. Processes favoured by aerobic conditions, NO₃^– immobilisation (0–17% NO₃^– removal, 0–8.2 g N g^–1 soil h^–1), autotrophic nitrification (~2% NH₄⁺ removal, 0.58–0.88 g N g^–1 soil h^–1) and heterotrophic nitrification (11–35 g N g^–1 soil h^–1) increased with increased N input while the anaerobic dissimilatory NO₃^– reduction to NH₄⁺ process (1–6% NO₃^– removal, 0.48–0.62 g N g^–1 soil h^–1) decreased, presumably due to the oxidising effect of higher NO₃^– inputs. Denitrification (8–78% NO₃^– removal, 3.8–9.6 g N g^–1 soil h^–1) exhibited no clear trend related to N input levels. NH₄⁺ immobilisation (39–72% NH₄⁺ removal, 15–19 g N g^–1 soil h^–1) was higher than NO₃^– immobilisation. Gross NH₄⁺ mineralisation (0.27–0.80 g N g^–1 soil h^–1) was the only process not detected in phase 1 and one of few processes measurable in phases 2 or 3.     

Root turnover of faba beans (Vicia faba L.) and its interaction with P and K supply

An increased root turnover can be a mechanism of improved nutrient‐uptake efficiency. The objectives of this study were to investigate P and K efficiency of faba beans (Vicia faba L.), to determine their root growth and root turnover, and to assess the relevance of root turnover on P and K uptake at limited supply. Faba beans were grown as part of a long‐term fertilization experiment on fertilized plots (control) and plots that had not received any P or K fertilizer for 16 years (P0, K0). Although the unfertilized soils were low and very low in their P‐ and K‐supply level, respectively, no differences in shoot‐dry‐matter production occurred compared to the control. However, relative K concentration in dry matter of the K0 plants (control plants = 100) decreased during the experiment and was only 60% of the control at the final harvest. This indicated a high K‐utilization efficiency of faba bean. Relative phosphorus concentration increased in the P0 treatment and was not different from the control at the last harvest, indicating an improvement in P‐uptake efficiency with time. The size of the standing root system determined by sequential auger sampling (net development) was not influenced by P and K supply. Total root production as measured by the ingrowth‐core method was about 6 times higher than the average size of the standing root system and even increased under low‐K conditions. This indicated a fast root turnover. Modeling soil nutrient transport and uptake revealed that calculated uptake of the control was up to 48% higher when root turnover was taken into account, compared to calculations based on the net development of the root systems. This is due to a better soil exploitation. Under K shortage, root turnover resulted in a 117% higher calculated uptake, which was close to measured K uptake. Root turnover was also of benefit for P uptake, but calculated P uptake was significantly less than measured, indicating that root turnover was of little importance for P uptake of faba beans.