Measurements of nitrogen fixation in fababean at different N fertilizer rates using the 15N isotope dilution and ‘A-value’ methods

The ¹⁵N isotope dilution and A-value methods were used to measure biological nitrogen (N₂) fixation in field grown fababean (Vicia faba L.), over a 2-year period. Four N rates, 20, 100, 200 and 400 kg N ha^–1 were examined. The two isotope methods gave similar values of % N derived from the atmosphere (%Ndfa). With 20 kg N ha^–1, %Ndfa in fababean was about 85% in both years. Increasing the N rate to 100 kg N ha^–1 decreased N₂ fixation slightly to 75%. Further reductions in N₂ fixed to 60 and 43% occurred where 200 and 400 kg N ha^–1 were applied, respectively. Thus even higher rates of N than normally applied in farming practice could not completely suppress N₂ fixation in fababean.We also devised one equation for both the isotope dilution and A-value approaches, thereby (i) avoiding the need for different calculations for the ¹⁵N isotope methods, and (ii) showing once again that the isotope dilution and A-value methods are mathematically and conceptually identical.     

Nitrogen fixation in Faidherbia albida,Acacia raddiana,Acacia senegal and Acacia seyal estimated using the 15N isotope dilution technique

A pot experiment was conducted in a greenhouse using the ¹⁵N isotope dilution method and two reference plants, Parkia biglobosa and Tamarindus indica to estimate nitrogen fixed in four Acacia species: A raddiana, A. senegal, A. seyal and Faidherbia albida (synonym Acacia albida). For the reference plants, the ¹⁵N enrichments in leaves, stems and roots were similar. With the fixing plants, leaves and stems had similar ¹⁵N enrichments; they were higher than the ¹⁵N enrichment of roots. The amounts of nitrogen fixed at 5 months after planting were similar using either reference plant. Estimates of the percentage of N derived from fixation (%Ndfa) for the above ground parts, in contrast to %Ndfa in roots, were similar to those for the whole plant. However, none of the individual plant parts estimated accurately total N fixed in the whole plant, and excluding the roots resulted in at least 30% underestimation of the amounts of N fixed. Between species, differences in N₂ fixation were observed, both for %Ndfa and total N fixed. For %Ndfa, the best were A. seyal (average, 63%) and A. raddiana (average, 62%), being at least twice the %Ndfa in A. senegal and F. albida. Because of its very high N content, A. seyal was clearly the best in total N fixed, fixing 1.62 g N plant^–1 compared to an average of 0.48 g N plant^–1 for the other Acacia species. Our results show the wide variability existing between Acacia species in terms of both %Ndfa and total N fixed: A. seyal was classified as having a high N₂ fixing potential (NFP) while the other Acacia species had a low NFP.     

Nitrogen fixation in breeding lines of Phaseolus vulgaris L.

Nitrogen fixation in nine common bean (Phaseolus vulgaris L.) lines was estimated using the ¹⁵N isotope dilution method at two locations in two seasons. In the first season at one location no N₂ fixation was detected while in the second season up to 51 kg N ha^–1 were estimated. There were significant differences between lines and correlations between trials were significant for the amounts of N₂ fixed, but not for total shoot nitrogen. The plants that fixed the most nitrogen nodulated rapidly after germination. Differences in maximum nodule mass, but not specific nodule activity, were detected also.     

Symbiotic N2 fixation by soybean in organic and conventional cropping systems estimated by 15N dilution and 15N natural abundance

Nitrogen (N) is often the most limiting nutrient in organic cropping systems. N₂ fixing crops present an important option to improve N supply and to maintain soil fertility. In a field experiment, we investigated whether the lower N fertilization level and higher soil microbial activity in organic than conventional systems affected symbiotic N₂ fixation by soybean (Glycine max, var. Maple Arrow) growing in 2004 in plots that were since 1978 under the following systems: bio-dynamic (DYN); bio-organic (ORG); conventional with organic and mineral fertilizers (CON); CON with exclusively mineral fertilizers (MIN); non-fertilized control (NON). We estimated the percentage of legume N derived from the atmosphere (%Ndfa) by the natural abundance (NA) method. For ORG and MIN we additionally applied the enriched ¹⁵N isotope dilution method (ID) based on residual mineral and organic ¹⁵N labeled fertilizers that were applied in 2003 in microplots installed in ORG and MIN plots. These different enrichment treatments resulted in equal %Ndfa values. The %Ndfa obtained by NA for ORG and MIN was confirmed by the ID method, with similar variation. However, as plant growth was restricted by the microplot frames the NA technique provided more accurate estimates of the quantities of symbiotically fixed N₂ (Nfix). At maturity of soybean the %Ndfa ranged from 24 to 54%. It decreased in the order ORG > CON > DYN > NON > MIN, with significantly lowest value for MIN. Corresponding Nfix in above ground plant material ranged from 15 to 26 g N m^-2, with a decreasing trend in the order DYN = ORG > CON > MIN > NON. For all treatments, the N withdrawal by harvested grains was greater than Nfix. This shows that at the low to medium %Ndfa, soybeans did not improve the N supply to any system but removed significant amounts of soil N. High-soil N mineralization and/or low-soil P availability may have limited symbiotic N₂ fixation.     

N2 fixation in Thai soybeans: Effect of tillage and inoculation on15N-determined N2 fixation in recommended cultivars and advanced breeding lines

The practice of seeding soybeans following paddy rice in Thailand has encountered difficulties in seedling germination, nodulation and crop establishment. This research project evaluated the choice of a non-fixing control to quantify N₂ fixation by¹⁵N isotope dilution, and the effect of tillage regime, soybean cultivar, strain ofBradyrhizobium japonicum and P fertilization on yield and N₂ fixation after paddy rice in northern and central Thailand.Japanese non-nodulating lines Tol-0 and A62-2 were the most appropriatecontrol plants for¹⁵N isotope dilution for Thai soybeans in these soils which contained indigenous rhizobia. Cereals such as maize, sorghum and barley were also appropriate controls at some sites. The choice of the appropriate non-fixing control plant for the¹⁵N isotope dilution technique remains a dilemma and no alternative exists other than to use several possible controls with each experiment. Acetylene reduction assay (ARA) proved of little value for screening varieties on their N₂ fixing capacity.The recommended Thai soybean cultivars (SJ1, 2, 4, 5) and an advanced line 16–4 differed little in their ability to support N₂ fixation or yield, possibly due to similar breeding ancestry. The ten AVRDC (ASET) lines showed considerable genotypic control in their ability to utilize their three available N sources (soil, fertilizer, atmosphere) and to translate them into yields. None of these lines were consistently superior to Thai cultivars SJ4 or SJ5 although ASET lines 129, 209 and 217 showed considerable promise.Neither recommended Thai or ASET cultivars were affected by tillage regime. Zero tillage resulted in superior N₂ fixation and yield at two sites but conventional tillage was superior at another site. Soybean cultivars grown in Thailand were well adapted to zero tillage. Levels of N₂ fixation were similar to world figures, averaging more than 100 kg N ha^–1 and supplying over 50% of the plant's N yield. However, seed yields seldom exceeded 2 t ha^–1, well below yields for temperately-grown soybeans. It is not clear why Thai soybeans support N₂ fixation, but do not translate this into higher seed yields.     

A field evaluation using the 15N isotope dilution method of lines of Phaseolus vulgaris L. bred for increased nitrogen fixation

N₂ fixation in lines of Phaseolus vulgaris was measured by ¹⁵N-isotope dilution to determine whether a programme of crossing and recurrent selection had resulted in enhanced nitrogen fixation. In field experiments on an isohyperthermic Aquic Hapludoll soil the amount of N₂ fixed by the different lines ranged from 18 to 36 kg ha^–1 (32 to 47% of plant N) in 56 days. The quantity of N₂ fixed and the proportion of plant N derived from fixation was not significantly greater in the lines selected for N₂ fixation (RIZ lines) than parental lines. Total shoot N ranged from 53 to 77 kg ha^–1 and partitioning of N to pods differed from 28 to 52% among the lines which all had similar growth habit and duration. Nodulation patterns were also distinct. Nodules formed early (10 to 15 plant^–1 at 13 days) in many lines, and smallest amounts of fixation were observed in those lines which nodulated slowly and did not form substantial nodule mass until after 40 days. The screening criteria used in the selection of the RIZ lines had been largely indirect with other factors such as disease resistance also being included. Progress for increasing N₂ fixation over good-fixing parental lines such as BAT76 was not significant and it is recommended that more attention be paid to early nodulation, to the use of soils with lower available N and to inter-crossing of lines having different good N₂ fixation traits in order to further enhance the potential for N₂ fixation in beans.     

Nitrogen fixation in legumes and actinorhizal plants in natural ecosystems: values obtained using 15N natural abundance

Background: Nitrogen fixation has been quantified for a range of crop legumes and actinorhizal plants under different agricultural/agroforestry conditions, but much less is known of legume and actinorhizal plant N₂ fixation in natural ecosystems.

Aims: To assess the proportion of total plant N derived from the atmosphere via the process of N₂ fixation (%Ndfa) by actinorhizal and legume plants in natural ecosystems and their N input into these ecosystems as indicated by their ¹⁵N natural abundance.

Methods: A comprehensive collation of published values of %Ndfa for legumes and actinorhizal plants in natural ecosystems and their N input into these ecosystems as estimated by their ¹⁵N natural abundance was carried out by searching the ISI Web of Science database using relevant key words.

Results: The %Ndfa was consistently large for actinorhizal plants but very variable for legumes in natural ecosystems, and the average value for %Ndfa was substantially greater for actinorhizal plants. High soil N, in particular, but also low soil P and water content were correlated with low legume N₂ fixation. N input into ecosystems from N₂ fixation was very variable for actinorhizal and legume plants and greatly dependent on their biomass within the system.

Conclusions: Measurement of ¹⁵N natural abundance has given greater understanding of where legume and actinorhizal plant N₂ fixation is important in natural ecosystems. Across studies, the average value for %Ndfa was substantially greater for actinorhizal plants than for legumes, and the relative abilities of the two groups of plants to utilise mineral N requires further study.     

Nitrogen accumulation in sole and mixed stands of sweet-blue lupin (Lupinus angustifolius L.), ryegrass and oats

Nitrogen fixation was measured in monocropped sweet-blue lupin (Lupinus angustifolius), lupin intercropped with two ryegrass (Lolium multiflorum) cultivars or with oats (Avena sativa) on an Andosol soil, using the ¹⁵N isotope dilution method. At 117 days after planting and at a mean temperature below 10°C, monocropped lupin derived an average of 92% or 195 kg N ha⁻¹ of its N from N₂ fixation. Intercropping lupin with cereals increased (p<0.05) the percentage of N derived from atmospheric N₂ (% Ndfa) to a mean of 96%. Compared to the monocropped, total N fixed per hectare in intercropped lupin declined approximately 50%, in line with the decrease in seeding rate and dry matter yield. With these high values of N₂ fixation, selection of the reference crop was not a problem; all the cereals, intercropped or grown singly produced similar estimates of N₂ fixed in lupin. It was deduced from the ¹⁵N data that significant N transfer occurred from lupin to intercropped Italian ryegrass but not to intercropped Westerwoldian ryegrass or to oats. Doubling the ¹⁵N fertilizer rate from 30 to 60 kg N ha⁻¹ decreased % Ndfa to 86% (p<0.05), but total N fixed was unaltered. These results indicate that lupin has a high potential for N₂ fixation at low temperatures, and can maintain higher rates of N₂ fixation in soils of high N than many other forage and pasture legumes.     

Short communication: Screening of native and foreign Bradyrhizobium japonicum strains for high N2 fixation in soybean

Four local rhizobia isolates selected after two screening experiments and five USDA Bradyrhizobium japonicum strains were estimated for N₂ fixation in soybean using the ¹⁵N isotope dilution technique. Strain USDA 110 was superior to the local isolates in nodulation and N₂ fixation when inoculated onto soybean cv TGX 1497-ID in a Nigerian soil and could therefore be used as an inoculant for enhanced N₂ fixation in soybean in Nigeria.     

Estimates of seasonal nitrogen fixation of annual subterranean clover-based pastures using the15N natural abundance technique

Annual pasture legumes play a key role in ley farming systems of southern Australia, providing biologically fixed nitrogen (N) to drive the production of the pastures as well as subsequent crops grown in rotation. Seasonal inputs of biologically fixed N in shoot biomass of the subterranean clover (Trifolium subterraneum) component of grazed annual pastures were assessed using the¹⁵N natural abundance technique and appropriately timed sampling of herbage dry matter (DM) for N accumulation. At three study sites spanning a gradient across the Western Australian wheatbelt from 300 to 600 mm annual rainfall the performance of the clover and non-legume herbs and grasses was examined as paired comparisons involving two management treatments expected to give contrasting effects on pasture productivity, botanical composition and N₂ fixation. The proportion of clover N derived from atmospheric N₂ fixation (%Ndfa) ranged from 65 to 95% across sites, treatments and sampling times. Amounts of fixed N accumulated in clover shoot biomass ranged from 50 to 125 kg ha⁻¹, and paralleled trends in clover production. Substantial increases in pasture production in high yielding treatments generally occurred without decrease in %Ndfa, suggesting that N₂ fixation was essentially non-limiting to performance of the clover component. Seasonal profiles for accumulation of fixed N were skewed towards the late winter and spring period, particularly in low plant density pastures following a cereal crop. There were seasonal, site and treatment-specific effects on the proportion of clover and non-legume pasture components and consequently clover yield and N₂ fixation were variably affected by competition from non-legume species.     

N2 fixation and nitrogen allocation to above and below ground plant parts in red clover-grasslands

Leys, used for grazing or production of forage to be conserved as silage or hay, are very important crops in northern areas. In order to measure the N₂ fixation in leys of varying ages and during different parts of the season, detailed measurements were taken of yield, N₂ fixation and the amounts of N remaining in the field after harvesting red clover (Trifolium pratense L.)-grass leys at a site in northern Sweden, where they are generally harvested twice per growing season. Entire plants, including stubble and roots, were sampled at the time of first and second harvest and, in addition, at the end of the growing season in three neighbouring fields, carrying a first, a second and a third year ley, respectively. N₂ fixation was measured by both ¹⁵N isotope dilution (ID) and ¹⁵N natural abundance (NA) methods. The proportion of clover dry matter (DM) in the stands increased from the first to the second harvest, but the grasses dominated throughout the entire season, especially below ground. The N concentrations, in both herbage and whole plants, were about twice as high in the clover as in the grasses. Seasonal variations in N concentrations were minor, and total N contents followed the same trends as DM. The clover acquired nearly all of its N from N₂ fixation: the proportion of N in clover herbage derived from N₂ fixation was often >0.8 throughout the season. The variations in the amounts of N₂ fixed during the course of the season corresponded well to the seasonal changes in clover biomass. Amounts of fixed N₂ allocated to clover herbage during the whole season were in the range 4 to 6 g N m⁻² in this unusually rainy year. Calculations of daily N allocation rates to herbage showed that N uptake rates were similar, and high, in grasses during May–June and July–August, while N₂ fixation rates in clover were about 10-fold as high in July–August as in May–June, reflecting the need for N in clover growth. The proportion of N remaining in clover stubble and roots after the first and second harvests was about 60 and 25%, respectively, while about 60% of the N in grasses remained in stubble and roots after both harvests. The considerable amounts of biomass and N that were left in field after harvesting red clover-grass leys are important for re-growth of the plants and provide substantial N fertilization for the next crop in the crop rotation.     

Estimation of symbiotic N2 fixation in an Amazon floodplain forest

Sustainable management for existing Amazonian forests requires an extensive knowledge about the limits of ecosystem nutrient cycles. Therefore, symbiotic nitrogen (N₂) fixation of legumes was investigated in a periodically flooded forest of the central Amazon floodplain (Várzea) over two hydrological cycles (20 months) using the ¹⁵N natural abundance method. No seasonal variation in ¹⁵N abundance (δ ¹⁵N values) in trees which would suggest differences in N₂ fixation rates between the terrestrial and the aquatic phase was found. Estimations of the percentage of N derived from atmosphere (%Ndfa) for the nodulated legumes with Neptunia oleracea on the one side and Teramnus volubilis on the other resulted in mean %Ndfa values between 9 and 66%, respectively. More than half of the nodulated legume species had %Ndfa values above 45%. These relatively high N gains are important for the nodulated legumes during the whole hydrological cycle. With a %Ndfa of 4–5% for the entire Várzea forest, N₂ fixation is important for the ecosystem and therefore, has to be taken into consideration for new sustainable land-use strategies in this area.     

Minimizing the effect of mineral nitrogen on biological nitrogen fixation in common bean by increasing nutrient levels

Although common bean (Phaseolus vulgaris L.) has good potential for N₂ fixation, some additional N provided through fertilizer usually is required for a maximum yield. In this study the suppressive effect of N on nodulation and N₂ fixation was evaluated in an unfertile soil under greenhouse conditions with different levels of soil fertility (low=no P, K and S additions; medium = 50, 63 and 10 mg kg^–1 soil and high = 200, 256 and 40 mg kg^–1 soil, respectively) and combined with 5, 15, 60 and 120 mg N kg^–1 soil of ¹⁵N-labelled urea. The overall average nodule number and weight increased under high fertility levels. At low N applications, nitrogen had a synergistic effect on N₂ fixation, by stimulating nodule formation, nitrogenase activity and plant growth. At high fertility and at the highest N rate (120 mg kg^–1 soil), the stimulatory effect of N fertilizer on N₂ fixation was still observed, increasing the amounts of N₂ fixed from 88 up to 375 mg N plant^–1. These results indicate that a suitable balance of soil nutrients is essential to obtain high N₂ fixation rates and yield in common beans.     

Biological nitrogen fixation in four Gliricidia sepium genotypes

A field experiment was conducted at the Coconut Research Institute in Sri Lanka to examine the biological nitrogen fixation potential of three Gliricidia sepium provenances (OFI 14/84, 17/84, 12/86) and a local landrace (designated LL), using the ¹⁵N isotope dilution method. There was marked variation in dry matter, total N, nodulation and ¹⁵N enrichment among the Gliricidia genotypes (=0.001), and the dry matter yield of Cassia siamea (syn. Senna siamea), the non-N₂ fixing reference plant was higher than for G. sepium. In all cases, highest biomass and total N were aboveground, with roots on average accounting for < 20 % of total dry matter or the total N in plants. Atom % ¹⁵N excess was highest in C. siamea, and lowest in OFI 14/84. Although atom % ¹⁵N excess was lower in Gliricidia leaves than in the other organs (all of which had similar ¹⁵N enrichments), values of % N derived from atmospheric N₂ fixation (% Ndfa) calculated for any individual organ or for the whole plant were similar. This was because the relative distribution of ¹⁵N in the different parts of the fixing plant followed the same trend as in the reference plant. There were significant differences (p=0.01) in N₂ fixation between the Gliricidia genotypes. The values ranged from 17.8 g N tree^-1 (equivalent to 86 kg N ha^-1 at 5000 trees ha^-1) in OFI 12/86 to 61.7g N tree^-1 (equivalent to 309 kg N ha^-1) in OFI 14/84. Although most of this variability was due to differences in both % Ndfa and total N in plant, amount of N fixed was more correlated with total N in plant (r=0.935) than with % Ndfa (r=0.707). On average, % Ndfa in all three G. sepium provenances and LL was about 55 % or 34.6 g N tree^-1 (equivalent to some 166 kg N ha^-1) in the 9 months within which N₂ fixation was measured. This represents a substantial contribution of N into the soil-plant system.     

A proposed role for copper ions in cell wall loosening

The measurement of N₂ fixation by legumes is necessary for gaining an understanding of their contributions to the N economies of agricultural and forestry systems and for their management in those systems. We report research to determine whether N₂ fixation of four of the commonly-grown ureide-producing legumes, soybean (Glycine max), cowpea (Vigna unguiculata), mungbean (V. radiata) and black gram (V. mungo), could be quantified from a single sampling and N-solute analysis of xylem sap. Data were derived from a previously-published experiment involving six genotypes of soybean at five field sites and from a second, irrigated experiment in which two genotypes of soybean, and one each of cowpea, mungbean and black gram were assessed in low- and high-nitrate soils for nodulation, yields of shoot and grain dry matter and N, and N₂ fixation using xylem solute (ureide) and ¹⁵N methods. Regression analysis of the published soybean data set indicated that the early pod-fill (R3.5 and R4) samplings for xylem sap gave estimates of percentage of plant N derived from N₂ fixation (%Ndfa) which agreed well with %Ndfa for the entire growing season obtained from ¹⁵N analysis of the shoots at R6-7. There was a marginal benefit in combining the R3.5 and R4 samplings and using the average of the two, with regression coefficients (r ²) increasing from 0.86 (R3.5 or R4 alone) to 0.92 (average of R3.5+R4). There was no additional benefit in combining R3, R3.5 and R4. In the second experiment, agreement between ¹⁵N-determined %Ndfa and either measured (R4 sampling) or calculated ureide-determined %Ndfa (R3.5 sampling) was also good (r ² of 0.73 (R4) and 0.79 (R3.5)). We conclude that seasonal %Ndfa can be accurately estimated using the xylem solute (ureide) method from a single sampling of xylem sap during early pod-fill (R3.5) and that this simplification of the protocol of the technique may encourage expanded use.     

Low concentrations of nitrate and ammonium stimulate nodulation and N2 fixation while inhibiting specific nodulation (nodule DW g−1 root dry weight) and specific N2 fixation (N2 fixed g−1 root dry weight) in soybean

Nitrate N is a major inhibitor of the soybean/Bradyrhizobium symbiosis in legumes and although this inhibition has been studied for many years, as yet no consensus has been reached on the specific and quantitative interactions between nitrate and ammonium supply and N₂ fixation. The effect of nitrate and ammonium supply on plant growth, nodulation and N₂ fixation capacity during the full growth cycle was investigated in both greenhouse and growth chamber experiments with three soybean genotypes. The results show that a high concentration of mineral N (10 mM), either as nitrate or ammonium or ammonium nitrate significantly suppressed nodule number, nodule dry weight and total N₂ fixed per plant of nodulated soybeans. However, lower mineral N concentrations, either 1 mM or 3.75 mM significantly enhanced nodule number, nodule dry weight and total N₂ fixed per plant, while specific nodulation (nodule dry weight g^–1 root DW, SNOD) and specific N₂ fixation (total N₂ fixed g^–1 root DW, SNF) were significantly reduced, particularly at the early vegetative growth stage V4, compared to the treatment with N₂ fixation as the only N source, in both growth chamber and greenhouse experiments. Therefore, we suggest that SNOD or SNF might be better indicators to express the suppressing effect of mineral N addition on nodule performance and N₂ fixed. Our studies also showed that ammonium alone was the more efficient N source than either ammonium nitrate or nitrate for soybean, as it resulted in higher biomass accumulation, nodule dry weight, total N accumulation and total N₂ fixed by 23, 20, 18 and 44%, respectively, compared to NO₃ ^– as the N source.     

Measurement of N2-fixation in field-grown pigeonpea [Cajanus cajan (L.) Millsp.] using15N-labelled fertilizer

Two experiments were carried out from 1981 to 1983 in Vertisol field at ICRISAT Center, Patancheru, India to measure N₂-fixation of pigeonpea [Cajanus cajan (L.) Millsp.] using the¹⁵N isotope dilution technique. One experiment examined the effect of control of a nodule-eating insect on fixation while another in vestigated the effect of intercroping with cereals on fixation and the residual effect of pigeonpea on a succeeding cereal crop. Although both experiments indicated that at least 88% of the N in pigeonpea was fixed from the atmosphere, one result is considered fortuitous in view of the differential rates of growth of the legume and the control, sorghum [Sorghum bicolor (L.) Moench]. The difference method of calculation in dieated negative fixation and the results emphasized the problem of finding a suitable nonfixing control. In a second experiment, when all plants were confined to a known volume of soil to which¹⁵N fertilizer was added in the field, these problems were overcome, and isotope dilution and difference methods gave similar results of N₂-fixation of about 90%. In intercropped pigeonpea 96% of the total N was derived from the atmosphere. This estimate might be an artifact. There was no evidence of benefit from N fixed by pigeonpea to intercropped sorghum plants. Plant tissue¹⁵N enrichments of cereal crops grown after pigeonpea indicated that the cereal derived some N fixed by the previous pigeonpea. Thus residual benefits to cereals are not only an effect of ‘sparing’ of soil N.     

The regulation of symbiotic N2 fixation: a conceptual model of N feedback from the ecosystem to the gene expression level

Symbiotic nitrogen (N₂) fixation in legumes may give the host plant a distinct competitive advantage; at the same time it is mainly responsible for introducing N into terrestrial ecosystems which may ultimately benefit all organisms. Depending on environmental conditions, symbiotic N₂ fixation may be tuned to the plant's N demand or specifically inhibited (a disadvantage for plants which depend mainly on symbiotic N₂ fixation), or even prevented. Thus, the ecological range for symbiotic N₂ fixation can be narrower than that of the host plants. A shortage of mineral N is the only case in which adverse environmental conditions clearly favour symbiotic N₂ fixation. Variations in number or mass of nodules or nodule morphology are persistent features, that may represent one kind of regulation of N₂ fixation. In addition, varying O₂ permeability of nodules functions as a rapid and reversible control of N₂ fixation which may compensate partially or fully for poor nodulation. The plant's demand for symbiotically fixed N is thought to play a central role in modulating both nodulation and N₂ fixation activity; an N feedback mechanism is assumed. The control of symbiotic N₂ fixation operates through a series of ecophysiological triggers which are also influenced by complex interactions between legume plants and other organisms in the ecosystem. The proportion of legume biomass and the performance of symbiotic N₂ fixation in each individual legume are the main parameters which determine the amount of symbiotically fixed N introduced into a terrestrial ecosystem. The various triggers and N feedback mechanisms from the whole ecosystem to the gene expression level which regulate symbiotic N₂ fixation in terrestrial ecosystems are reviewed and discussed in terms of a conceptual model. Although the presented model is based primarily on our knowledge about the physiology of a few leguminous crop species and of ecosystem processes in managed, perennial grassland in temperate climatic conditions, it may stimulate thinking about functional relationships between symbiotic N₂ fixation and terrestrial ecosystems at various system levels.     

Response to inoculation and N fertilization for increased yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is able to fix 20–60 kg N ha^–1 under tropical environments in Brazil, but these amounts are inadequate to meet the N requirement for economically attractive seed yields. When the plant is supplemented with N fertilizer, N₂ fixation by Rhizobium can be suppressed even at low rates of N. Using the ¹⁵N enriched method, two field experiments were conducted to compare the effect of foliar and soil applications of N-urea on N₂ fixation traits and seed yield. All treatments received a similar fertilization including 10 kg N ha^–1 at sowing. Increasing rates of N (10, 30 and 50 kg N ha^–1) were applied for both methods. Foliar application significantly enhanced nodulation, N₂ fixation (acetylene reduction activity) and yield at low N level (10 kg N ha^–1). Foliar nitrogen was less suppressive to nodulation, even at higher N levels, than soil N treatments. In the site where established Rhizobium was in low numbers, inoculation contributed substantially to increased N₂ fixation traits and yield. Both foliar and soil methods inhibited nodulation at high N rates and did not significantly increase bean yield, when comparing low (10 kg N ha^–1) and high (50 kg N ha^–1) rates applied after emergence. In both experiments, up to 30 kg N ha^–1 of biologically fixed N₂ were obtained when low rates of N were applied onto the leaves.     

Measurements of N2 fixation by common bean in Central Brazil as affected by different reference crops

An experiment was conducted at EMBRAPA/CNPAF, Goiânia, Goias, Brazil, on a typic haplustox soil to evaluate growth and N₂ fixation-related parameters of Phaseolus vulgaris L. Bean lines, which had been selected for N₂ fixation at CNPAF, including production cultivars, germplasm bank entries, and parents and progenies of a cross made to improve this characteristic. Wheat (Triticum aestivum L.) and dwarf sorghum (Sorghum bicolor (L.) Moench) were evaluated as non-N₂-fixing reference crops for difference method (DM) and ¹⁵N isotope dilution technique (IDT) estimates of N₂ fixation. IDT estimates ranges from 4 to 18 kg N₂ fixed ha^-1. High variability associated with low levels of N₂ fixation precluded definitive identification of the best N₂ fixing bean lines. Due to differences in growth cycle and in patterns and amounts of soil N uptake during the season, neither of the reference crops tested appears to be an adequate control for either DM or IDT estimates of N₂ fixation. However, ranking of lines for effectiveness in N₂ fixation could be performed without the use of any reference crops.