Nitrogen fixation (15N dilution) with soybeans under Thai field conditions

Controlled environment and field studies were conducted to determine relationships between various measurements of N₂ fixation using soybeans and to use these measures to evaluate a number ofBradyrhizobium japonicum strains for effectiveness in N₂ fixation in Thai soils.¹⁵N dilution measurements of N₂ fixation showed levels of fixation ranging from 32 to 161 kg N ha⁻¹ depending on bacterial strain, host cultivar and location. Midseason measures of N₂ fixation were correlated with each other, but not related measures taken at maturity. Ranking ofB. japonicum strains based on performance under controlled conditions in N-free media were highly correlated with rankings based on soybean seed yields and N₂ fixation under field conditions. This study showed that inoculation of soybeans with effectiveB. japonicum strains can result in significant increases in yield and uptake of N through fixation. The most effective strains tested for use in Thai conditions were those isolated from Thai soils; however, effective strains from other locations were also of benefit.     

15N-determined dinitrogen fixation potential of genetically diverse bean lines (Phaseolus vulgaris L.)

Improvement of dinitrogen fixation in beans (Phaseolus vulgaris L.) will depend on the selection of superior plant genotypes and the presence of efficient rhizobial strains. This study was conducted to evaluate diverse bean lines for N₂ fixation potential using the¹⁵N-depleted dilution technique under field conditions in Wisconsin, USA. Plants of 21 bean lines and three non-nodulating isolines of soybean received appliin Wisconsin, USA. Plants of 21 bean lines and three non-nodulating isolines of soybean received applications of¹⁵N-depleted ammonium sulphate. Shoots harvested at the V₆, R₃ and R₇ stages and dry seeds were analyzed for total N using the Kjeldahl procedure, and the ratio of¹⁵N to¹⁴N was determind on a MAT 250 mass spectrometer. Nodule occupancy of the applied strain ofR. leguminosarum biovarphaseoli, CIAT 899, was determined in five of the bean lines. Total shoot N content showed a pattern of accumulation similar to shoot dry weight and fixed N₂ in the shoot. Based on shoot total N, N₂ fixed in the shoot and shoot dry weight Riz 30 and Preto Cariri were identified as being as good fixers as Puebla 152 and Cargamanto appear to begin N₂ fixation early. Furthermore, some bean lines that fixed considerable N₂ did not translocate a large amount of N to the grains. Preto Cariri accumulated 21.2 kg N ha⁻¹ in the seeds compared to Puebla 152 which accumulated 43.8 kg N ha⁻¹ of the fixed N₂ into the grains. At the early sampling, Puebla 152 and 22–27 had a considerable higher percentage of their crown nodules formed by the inoculant strain CIAT 899, than did Rio Tibagi which has been considered a poor N₂ fixer.     

15N-Determined effect of inoculation with N2 fixing bacteria on nitrogen assimilation in Western Canadian wheats

Summary A two-year field study was undertaken using¹⁵N isotope techniques to differentiate between stimulation of N uptake and N₂ fixation in Western Canadian cultivars of spring wheat (Triticum aestivum L. emend Thell) and durum (T. turgidum L. emend Bowden) in response to inoculation with N₂-fixing bacteria. Bacterial inoculation either had no effect or lowered the % N derived from the fertilizer and the fertilizer use efficiency. Despite the depression of fertilizer uptake, inoculants did not alter the relative uptake from soil and fertilizer-N pools indicating that bacterial inoculation did not alter rooting patterns. Nitrogen-15 isotope dilution indicated that N₂ fixation did occur. In 1984, % plant N derived from the atmosphere (% Ndfa) due to inoculation with Bacillus C-11-25 averaged 23.9% while that withAzospirillum brasilense ATCC 29729 (Cd) averaged 15.5%. In 1985, higher soil N levels reduced these values by approximately one-half. Cultivar x inoculant interactions, while significant, were not consistent across years. However, these interactions did not affect cultivars ‘Cadet’ and ‘Rescue’. In agreement with previous results, ‘Cadet’ performed well with all inoculants in both years while ‘Rescue’ performed poorly. Among 1984 treatments, the N increament in inoculated plants was positively correlated with % Ndfa but no such correlation existed in 1985. N₂ fixation averaged over all cultivars and strains was 17.9 and 6.7 kg N fixed ha⁻¹ in 1984 and 1985, respectively. Highest rates of N₂ fixation were estimated at 52.4 kg N ha⁻¹ for ‘Cadet’ in 1984 and 31.3 kg N ha⁻¹ for ‘Owens’ in 1985, both inoculated with Bacillus C-11-25, an isolate from southern Alberta soils. Inoculation with either ofAzospirillum brasilense strain Cd (ATCC29729) or 245 did not result in as consistent or as high N₂ fixation, suggesting that these wheats had not evolved genetic compatability with this exogenous microorganism. These agronomically significant amounts of N₂ fixation occurred under optimally controlled experimental conditions in the field. It is yet to be determined if N₂ fixation would occur in response to bacterial inoculation under dryland conditions commonly occurring in Western Canada. Contribution from Agriculture Canada Research Station, Lethbridge, Alberta, Canada.     

Seasonal patterns of growth and nitrogen fixation in field-grown pea

The seasonal patterns of growth and symbiotic N₂ fixation under field conditions were studied by growth analysis and use of¹⁵N-labelled fertilizer in a determinate pea cultivar (Pisum sativum L.) grown for harvest at the dry seed stage. The patterns of fertilizer N-uptake were almost identical in pea and barley (the non-fixing reference crop), but more fertilizer-N was recovered in barley than in pea. The estimated rate of N₂ fixation in pea gradually increased during the pre-flowering and flowering growth stages and reached a maximum of 10 kg N fixed per ha per day nine to ten weeks after seedling emergence. This was the time of early pod-development (flat pod growth stage) and also the time for maximum crop growth rate and maximum green leaf area index. A steep drop in N₂ fixation rate occurred during the following week. This drop was simultaneous with lodging of the crop, pod-filling (round pod growth stage) and the initiation of mobilization of nitrogen from vegetative organs. The application of fertilizer-N inhibited the rate of N₂ fixation only during that period of growth, when the main part of fertilizer-N was taken up and shortly after. Total accumulation of fixed nitrogen was estimated to be 244, 238 and 213 kg N ha⁻¹ in pea supplied with nil, 25 or 50 kg NO ₃ ⁻ −N ha⁻¹, respectively. About one-fourth of total N₂ fixation was carried out during preflowering, one fourth during the two weeks of flowering and the remainder during post-flowering. About 55% of the amount of N present in pods at maturity was estimated to be derived from mobilization of N from vegetative organs. “Starter” N (25 or 50 kg NO ₃ ⁻ −N ha⁻¹) did not significantly influence either dry matter and nitrogen accumulation or the development of leaf area. Neither root length and root biomass determined 8 weeks after seedling emergence nor the yield of seed dry matter and nitrogen at maturity were influenced by fertilizer application.     

Measurement of N2 fixation in maize (Zea mays L.)—ricebean (Vigna umbellata [Thunb.] Ohwi and Ohashi) intercrops

The yield of N in maize (Zea mays L.) and ricebean (Vigna umbellata [Thumb.] Ohwi and Ohashi) were compared on a Tropoqualf soil in North Thailand in 1984 and 1985. Both species were grown in field plots in monoculture or as intercrops at a constant planting density equivalent to 8 maize or 16 ricebean plants per m². The contribution of symbiotic N₂ fixation to ricebean growth was estimated from measurements of the natural abundance of¹⁵N (δ¹⁵N) in shoot nitrogen and from analysis of ureides in xylem sap vacuumextracted from detached stems. The natural abundance of¹⁵N in the intercropped ricebean was found to be considerably less than that in monoculture in both growing seasons. Using maize and a weed (Ageratum conyzoides L.) as non-fixing¹⁵N reference plants the proportions (P ¹⁵N) of ricebean shoot N derived from N₂ fixation ranged from 0.27 to 0.36 in monoculture ricebean up to 0.86 when grown in a 75% maize: 25% ricebean intercrop. When glasshouse-derived calibration curves were used to calculate plant proportional N₂ fixation (Pur) from the relative ureide contents of field collected xylem exudates, the contribution of N₂ fixation to ricebean N yields throughout the 1985 growing season were greater in intercrop than in monocrop even at the lowest maize:legume ratio (25∶75). Seasonal patterns of sap ureide abundance indicated that N₂ fixation was greatest at the time of ricebean podset. The averagePur andP ¹⁵N in ricebean during the first 90 days of growth showed identical rankings of monocrop and intercrop treatments in terms of N₂ fixation, although the two sets ofP values were different. Nonetheless, seasonal estimates of N₂ fixation during the entire 147 days of legume growth determined from ureide analyses indicated that equivalent amounts of N could be fixed by ricebean in a 75∶25 intercrop and in monoculture despite the former being planted at one-quarter the density.     

Estimation of symbiotically fixed nitrogen in field grown soybeans: An application of natural15N/14N abundance and a low level15N-tracer technique

Summary Plants from agricultural and natural upland ecosystem were investigated for¹⁵N content to evaluate the role of symbiotic N₂-fixation in the nitrogen nutrition of soybean. Increased yields and lower δ¹⁵N values of nodulating soybeansvs, non-nodulating isolines gave semi-quantitative estimates of N₂ fixation. A fairly large discrepancy was found between estimations by δ¹⁵N and by N yield at 0 kg N/ha of fertilizer. More precise estimates were made by following changes in plant δ¹⁵N when fertilizer δ¹⁵N was varied near¹⁵N natural abundance level. Clearcut linear relationships between δ¹⁵N values of whole plants and of fertilizer were obtained at 30 kg N/ha of fertilizer for three kinds of soils. In experimental field plots, nodulating soybeans obtained 13±1% of their nitrogen from fertilizer, 66±8% from N₂ fixation and 21±10% from soil nitrogen in Andosol brown soil; 30%, 16% and 54% in Andosol black soil; 7%, 77% and 16% in Alluvial soil, respectively. These values for N₂ fixation coincided with each corresponding estimation by N yield method. Other results include: 1)¹⁵N content in upland soils and plants was variable, and may reflect differences in the mode of mineralization of soil organics, and 2) nitrogen isotopic discrimination during fertilizer uptake (δ¹⁵N of plant minus fertilizer) ranged from −2.2 to +4.9‰ at 0–30 kg N/ha of fertilizer, depending on soil type and plant species. The proposed method can accurately and relatively simply establish the importance of symbiotic nitrogen fixation for soybeans growing in agricultural settings.     

Dinitrogen fixation in white clover grown in pure stand and mixture with ryegrass estimated by the immobilized 15N isotope dilution method

Dinitrogen fixation in white clover (Trifolium repens L.) grown in pure stand and mixture with perennial ryegrass (Lolium perenne L.) was determined in the field using ¹⁵N isotope dilution and harvest of the shoots. The apparent transfer of clover N to perennial ryegrass was simultaneously assessed. The soil was labelled either by immobilizing ¹⁵N in organic matter prior to establishment of the sward or by using the conventional labelling procedure in which ¹⁵N fertilizer is added after sward establishment. Immobilization of ¹⁵N in the soil organic matter has not previously been used in studies of N₂ fixation in grass/clover pastures. However, this approach was a successful means of labelling, since the ¹⁵N enrichment only declined at a very slow rate during the experiment. After the second production year only 10–16% of the applied ¹⁵N was recovered in the harvested herbage. The two labelling methods gave, nonetheless, a similar estimate of the percentage of clover N derived from N₂ fixation. In pure stand clover, 75–94% of the N was derived from N₂ fixation and in the mixture 85–97%. The dry matter yield of the clover in mixture as percentage of total dry matter yield was relatively high and increased from 59% in the first to 65% in the second production year. The average daily N₂ fixation rate in the mixture-grown clover varied from less than 0.5 kg N ha⁻¹ day⁻¹ in autumn to more than 2.6 kg N ha⁻¹ day⁻¹ in June. For clover in pure stand the average N₂ fixation rate was greater and varied between 0.5 and 3.3 kg N ha⁻¹ day⁻¹, but with the same seasonal pattern as for clover in mixture. The amount of N fixed in the mixture was 23, 187 and 177 kg N ha⁻¹ in the seeding, first and second production year, respectively, whereas pure stand clover fixed 28, 262 and 211 kg N ha⁻¹ in the three years. The apparent transfer of clover N to grass was negligible in the seeding year, but clover N deposited in the rhizosphere or released by turnover of stolons, roots and nodules, contributed 19 and 28 kg N ha⁻¹ to the grass in the first and second production year, respectively. This revised version was published online in June 2006 with corrections to the Cover Date.     

The15N methodology in estimating N2 fixation by vetch and pea grown in pure stand or in mixtures with oat

Cereal-legume mixtures are frequently the best management decision for forage production instead of growing crops in pure stands. Nitrogen fertilization of cereal-legume mixtures is questionable since combined nitrogen could depress N₂ fixation by legumes. The objectives of this study were (1) to examine the effect of N fertilization on N₂ fixation by vetch and field peas in pure and in mixed stands with oats, and (2) to examine if there is any transfer of N from legumes to associated cereals. The field experiment was conducted for two growing seasons. The treatments were pure stands of vetch, pea and oats, and the mixtures of the two legumes with oats at the seeding ratios 90:10 and 75:25, fertilized with labelled¹⁵N at the rates of 15 and 90 kg N ha⁻¹. Nitrogen fertilization of 90 kg N ha⁻¹ suppressed N₂ fixation in both legumes grown in pure and in mixed stands. Crops grown in mixtures in many instances had lower atom %¹⁵N excess. Whether this was due to high N₂ fixation in the case of legume and transfer in the case of oat or the differences were due to practical problems of the¹⁵N technique is not clearly shown by the results, so based on the literature the aspect is discussed as well as the precautions which should be considered in using the¹⁵N technique in such studies.     

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.     

Evaluation of N2-fixation and nitrogen economy of a maize/cowpea intercrop system using15N dilution methods

Yields of above ground biomass and total N were determined in summer-grown maize and cowpea as sole crops or intercrops, with or without supplementary N fertilizer (25 kg N ha⁻¹, urea) at an irrigated site in Waroona, Western Australia over the period 1982–1985. Good agreement was obtained between estimates of N₂ fixation of sole or intercrop cowpea (1984/85 season) based on the¹⁵N natural abundance and¹⁵N fertilizer dilution techniques, both in the field and in a glasshouse pot study. Field-grown cowpea was estimated to have received 53–69% of its N supply from N₂-fixation, with N₂-fixation onlyslightly affected by intercropping or N fertilizer application. Proportional reliance on N₂-fixation of cowpea in glasshouse culture was lower (36–66%) than in the field study and more affected by applied N. Budgets for N were drawn up for the field intercrops, based on above-ground seed yields, return of crop residues, inputs of fixed N and fertilizer N. No account was taken of possible losses of N through volatilization, denitrification and leaching or gains of N in the soil from root biomass. N₂-fixation was estimated tobe 59 kg N ha⁻¹ in the plots receiving no fertilizer N, and 73 kg N ha⁻¹ in plots receiving 25 kg N ha⁻¹ as urea. Comparable fixation by sole cowpea was higher (87 and 82 kg N ha⁻¹ respectively) but this advantage was outweighed by greater land use efficiency by the intercrop than sole crops.     

Adaptation of methods for evaluating N2 fixation in food legumes and legume cover crops

Methods for partitioning the nitrogen assimilated by nodulated legumes, between nitrogen derived from soil sources and from N₂ fixation, are described as applied in peninsular Malaysia. The analysis of nitrogenous components translocated from the roots to the shoots of nodulated plants in the xylem sap is outlined, with some precautions to be observed for applications in the tropics. Some examples of the use of the technique in surverying apparent N₂ fixation by tropical legumes, in studying interrow cropping in plantation systems and in assessing effects of experimental treatments on N₂ fixation by food legumes, are described. Techniques for assesing N₂ fixation by means of¹⁵N abundance have been used to show that applications of nitrogenous fertilizers commonly used in Malaysia for soybeans depress N₂ fixation, that similar results are obtained with natural abundance and¹⁵N-enrichment methods and that, in at least two locations in Malaysia, differences between the natural abundance of¹⁵N in plant-available soil nitrogen and in atmospheric N₂ are great enough to permit application to measurement of N₂ fixation by leguminous crops.     

Field evaluation of symbiotic nitrogen fixation by rhizobial strains using15N methodology

Summary Small differences in N₂ fixation by nodulated soybeans (Glycine max. (L.) Merr.), inoculated with various strains ofRhizobium japonicum, were assessed in field experiments using¹⁵N methodology, and compared with yields of plant dry matter and total N. Percentage of plant-N derived from atmospheric N₂ and from fertilizer, and values of %¹⁵N atom excess had lower coefficients of variation than did total N and dry matter yield. Nevertheless the precision of estimates of kg N/ha fixed were sufficient to differentiate only the extremes of the range of strains tested, and there were discrepancies between ranking of strains based on % N derived from fertilizer and on total N yield.     

Inhibition of N2 fixation by white clover (Trifolium repens L.) at low concentrations of NO inf3 sup− in flowing solution culture

The impact of sustained low external concentrations of NO ₃ ^– (0, 10, 100 and 1000 mmol m^–3) on plant growth and the relative acquisition of N through N₂ fixation and NO ₃ ^– uptake by established, nodulated white clover (Trifolium repens L. cv. Blanca) was studied over 28 days in flowing solution culture. Nitrogen fixation was measured by N difference and ¹⁵N dilution methods. Plants supplied with NO ₃ ^– achieved higher relative growth rates (% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGabmiEayaara% aaaa!3702!\[\bar x\]=0.091 d^–1) compared with control plants dependent on N₂ fixation (0.073 d^–1). Nitrate plants showed progressive increases in shoot: root d.w. ratios from 4 to 6.5–7.6 between days 0–28, compared with 5.1 on day 28 for control plants. Increases in both nodule d.w. and numbers per plant were inhibited after day seven at all concentrations of NO ₃ ^– . The severity of inhibition of N₂ fixation increased with increasing NO ₃ ^– concentration and with time. The total amounts of N₂ fixed per plant between days 0–7 after supplying 10, 100 and 1000 mmol m^–3 NO ₃ ^– , respectively, were 37–39, 28–30 and 0–13%, of the total N acquired. Between days 7–28 the proportional contributions of N₂ fixation to total N acquisition declined to 3, 0.5 and 0%, respectively, in these treatments. The corresponding mean specific rates of N₂ fixation between days 0–7 were, respectively, 5.4, 3.2, and 2.0 mmol N d^–1 g^–1 nodule d.w., compared with 7.9 mmol N d^–1 g^–1 nodule d.w. for zero NO ₃ ^– plants. There was no evidence of a transitory increase in N₂ fixation following the addition of NO ₃ ^– , even at the lowest supply concentration.     

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.     

Interactive effects of exogenous combined nitrogen and phosphorus on growth and nitrogen fixation by azolla

Effects of N source and media-N and P levels were examined on growth, N uptake, and N₂ fixation ofAzolla pinnata withAnabaena azollae association (azolla) at two inoculum-P concentrations. Each expeiment was conducted for 7 days in a growth chamber using azolla at a predetermined inoculum-P concentration and the growth media containing a combination of four levels of P (0, 15, 75, and 200 M) and three levels (0, 1, and 5 mM) of either¹⁵N-enriched NH ₄ ⁺ as ammonium sulfate or¹⁵N-enriched NO ₃ ^– as potassium nitrate. Nitrogen uptake and N₂ fixation were measured by¹⁵N isotopic dilution method. Tissue P and N, N uptake, and N₂-fixation increased with increasing P concentration in the media regardless of the inoculum-P level of azolla. Increasing P concentration in the media increased growth of azolla at low inoculum P, but the effect on high inoculum-P azolla was either small or absen. High inoculum-P concentration resulted in increased growth, tissue-N and P concentrations, N uptake, and N₂ fixation by azolla. Ammonium in the growth media caused larger increase in tissue-N and greater repression of N₂ fixation than equimolar concentration of NO ₃ ^– . In the presence of NH ₄ ⁺ or NO ₃ ^– , in the growth media, N uptake by azolla exceeded the corresponding decrease in N₂ fixation, resulting in an overall increase in tissue-N concentration. Phosphorus in the media tended to negate the inhibitory effect of NH ₄ ⁺ or NO ₃ ^– on N₂ fixation. A multiple regression model showed that the effect of tissue-N on N₂ fixation was negative while that of tissue-P was positive. Therefore, a relative change in tissue-N and P appeared to regulate N₂ fixation. Tissue-N and P had similar effects on relative growth rate of azolla also. Inoculum-P level of azolla was important in determining the response to media-P.This research was supported by a grant from USAID under Indo-US Science and Technology Initiative.     

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 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.     

Variability in nitrogen fixation of common bean (Phaseolus vulgaris L.) intercropped with maize

Thirty one selected bean lines were evaluated in the field for ability to support N₂ fixation when intercropped with maize which received 0, 30 and 60 kg N ha^–1 as ammonium sulphate. The amount of fixed N₂ was estimated using the natural variation of ¹⁵N and wheat as the standard non-fixing crop. Nitrogen as low as 15 kg N ha^–1 at sowing suppressed nodule weight and activity (acetylene reduction activity) but not nodule number, suggesting that the main effect of mineral N was on nodule development and function. ¹⁵N data revealed a high potential of the bean genotypes to fix N₂, with the most promising ones averaging between 50–60% of seed N coming from fixation. Bean lines CNF-480, Puebla-152, Mexico-309, Negro Argel, CNF-178, Venezuela-350 and WBR22-3, WBR22-50 and WBR22-55 were ranked as good fixers.     

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.     

Seasonal accumulation and partitioning of nitrogen by lentil

Although wheat (Triticum aestivum L.) is the dominant crop of the semi-arid plains of Canada and the western United States, lentil (Lens culinaris Medik.) has become an important alternative crop. Sources and seasonal accumulation of N must be understood in order to identify parameters that can lead to increased N₂-fixing activity and yield. Inoculated lentil was grown in a sandy-loam soil at an irrigated site in Saskatchewan, Canada. Wheat was used as the reference crop to estimate N₂ fixation by the A-value approach. Lentil and wheat received 10 and 100 kg N ha⁻¹ of ammonium nitrate, respectively. Crops were harvested six times during the growing season and plant components analyzed. During the first 71 days after planting the wheat had a higher daily dry matter and N accumulation compared to lentil. However, during the latter part of the growing season, daily dry matter and N accumulation were greater for lentil. The maximum total N accumulation for lentil at maturity was 149 kg ha⁻¹. In contrast, wheat had a maximum N accumulation of 98 kg ha⁻¹ in the Feekes 11.1 stage, or 86 days after planting. The maximum daily rates of N accumulation were 3.82 kg N ha⁻¹ day⁻¹ for lentil and 2.21 kg N ha⁻¹ day⁻¹ for wheat. The percentage of N derived from N₂ fixation (% Ndfa) ranged from 0 at the first harvest to 92 % at final harvest. Generative plant components had higher values for % Ndfa than the vegetative components which indicates that N in the reproductive plant parts was derived largely from current N₂ fixation and lentil continued to fix N until the end of the pod fill stage. At final harvest, lentil had derived 129 kg N ha⁻¹ from N₂ fixation with maximum N₂-fixing activity (4.4 kg N ha⁻¹ day⁻¹) occurring during the early stages of pod fill. Higher maximum rates of N₂-fixing activity than net N accumulation (3.82 kg N ha⁻¹ day⁻¹) may have been caused by N losses like volatilization. In addition, lentil provided a net N contribution to the soil of 59 kg ha⁻¹ following the removal of the grain.