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.     

Biological N2 Fixation in wetland rice fields: Estimation and contribution to nitrogen balance

This paper 1) reviews improvements and new approaches in methodologies for estimating biological N₂ fixation (BNF) in wetland soils, 2) summarizes earlier quantitative estimates and recent data, and 3) discusses the contribution of BNF to N balance in wetland-rice culture.Measuring acetylene reducing activity (ARA) is still the most popular method for assessing BNF in rice fields. Recent studies confirm that ARA measurements present a number of problems that may render quantitative extrapolations questionable. On the other hand, few comparative measures show ARA's potential as a quantitative estimate. Methods for measuring photodependent and associative ARA in field studies have been standardized, and major progress has been made in sampling procedures. Standardized ARA measurements have shown significant differences in associative N₂ fixation among rice varieties.The ¹⁵N dilution method is suitable for measuring the percentage of N derived from the atmosphere (% Ndfa) in legumes and rice. In particular, the ¹⁵N dilution technique, using available soil N as control, appears to be a promising method for screening rice varieties for ability to utilize biologically fixed N. Attempts to adapt the ¹⁵N dilution method to aquatic N₂ fixers (Azolla and blue-green algae [BGA]) encountered difficulties due to the rapid change in ¹⁵N enrichment of the water.Differences in natural ¹⁵N abundance have been used to show differences among plant organs and species or varieties in rice and Azolla, and to estimate Ndfa by Azolla, but the method appears to be semi-quantitative.Recent pot experiments using stabilized ¹⁵N-labelled soil or balances in pots covered with black cloth indicate a contribution of 10–30 kg N ha^-1 crop^-1 by heterotrophic BNF in flooded planted soil with no or little N fertilizer used.Associative BNF extrapolated from ARA and ¹⁵N incorporation range from 1 to 7 kg N ha^-1 crop^-1. Straw application increases heterotrophic and photodependent BNF. Pot experiments show N gains of 2–4 mg N g^-1 straw added at 10 tons ha^-1.N₂ fixation by BGA has been almost exclusively estimated by ARA and biomass measurements. Estimates by ARA range from a few to 80 kg N ha^-1 crop^-1 (average 27 kg). Recent extensive measurements show extrapolated values of about 20 kg N ha^-1 crop^-1 in no-N plots, 8 kg in plots with broadcast urea, and 12 kg in plots with deep-placed urea.Most information on N₂ fixed by Azolla and legume green manure comes from N accumulation measurements and determination of % Ndfa. Recent trials in an international network show standing crops of Azolla averaging 30–40 kg N ha^-1 and the accumulation of 50–90 kg N ha^-1 for two crops of Azolla grown before and after transplanting rice. Estimates of % Ndfa in Azolla by ¹⁵N dilution and delta ¹⁵N methods range from 51 to 99%. Assuming 50–80% Ndfa in legume green manures, one crop can provide 50–100 kg N ha^-1 in 50 days. Few balance studies in microplots or pots report extrapolated N gains of 150–250 kg N ha^-1 crop^-1.N balances in long-term fertility experiments range from 19 to 98 kg N ha^-1 crop^-1 (average 50 kg N) in fields with no N fertilizer applied. The problems encountered with ARA and ¹⁵N methods have revived interest in N balance studies in pots. Balances are usually highest in flooded planted pots exposed to light and receiving no N fertilizer; extrapolated values range from 16 to 70 kg N ha^-1 crop^-1 (average 38 kg N). A compilation of balance experiments with rice soil shows an average balance of about 30 kg N ha^-1 crop^-1 in soils where no inorganic fertilizer N was applied.Biological N₂ fixation by individual systems can be estimated more or less accurately, but total BNF in a rice field has not yet been estimated by measuring simultaneously the activities of the various components in situ. As a result, it is not clear if the activities of the different N₂-fixing systems are independent or related. A method to estimate in situ the contribution of N₂ fixed to rice nutrition is still not available. Dynamics of BNF during the crop cycle is known for indigenous agents but the pattern of fixed N availability to rice is known only for a few green manure crops.     

Contribution of N2 fixing cyanobacteria to rice production: availability of nitrogen from 15N-labelled cyanobacteria and ammonium sulphate to rice

This study investigate the potential contribution of nitrogen fixation by indigenous cyanobacteria to rice production in the rice fields of Valencia (Spain). N₂-fixing cyanobacteria abundance and N₂ fixation decreased with increasing amounts of fertilizers. Grain yield increased with increasing amounts of fertilizers up to 70 kg N ha^-1. No further increase was observed with 140 kg N ha^-1. Soil N was the main source of N for rice, only 8–14% of the total N incorporated by plants derived from ¹⁵N fertilizer. Recovery of applied ¹⁵N-ammonium sulphate by the soil–plant system was lower than 50%. Losses were attributed to ammonia volatilization, since only 0.3–1% of applied N was lost by denitrification. Recovery of ¹⁵N from labeled cyanobacteria by the soil–plant system was higher than that from chemical fertilizers. Cyanobacterial N was available to rice plant even at the tillering stage, 20 days after N application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Physiological changes in soybean (Glycine max) Wuyin9 in response to N and P nutrition

Phosphorus deficiency is a very common problem in the acid soil of central China. Previous research has shown that starter N and N topdressing at the flowering stage (Rl) increased soybean (Glycine max) yield and N₂ fixation (Gan et al, 1997, 2000). However, there is little information available concerning soybean response to P‐fertiliser in soybean production in central China (Gan, 1999). A field experiment was conducted to investigate the response to P (0 kg P ha^?1, 22 kg P ha^?1, 44 kg P ha^?1 before sowing) and N fertiliser application (N1: 0 kg N ha^?1, N2: 25 kg N ha^?1 before sowing, N3: N2 + 50 kg N ha^?1 at the V2 stage and N4: N2 + 50 kg N ha^?1 at the R1 stage) on growth, yield and N₂ fixation of soybean. Both N and P fertiliser increased growth and seed yield of soybean (P < 0.01). Application of basal P fertiliser at 22 kg P ha^?1 or 44 kg P ha^?1 increased total N accumulation by 11% and 10% (P < 0.01) and seed yield by 12% and 13% (P < 0.01), respectively, compared to the zero P treatment. Although application of starter N at 25 kg N ha^?1 had no positive effect on seed yield at any P level (P > 0.05), an application of a topdressing of 50 kg N ha^?1 at the V2 or R1 stage increased total N accumulation by 11% and 14% (P < 0.01) and seed yield by 16% and 21% (P < 0.01), respectively, compared to the zero N treatment. Soybean plants were grown on sterilised Perlite in the greenhouse experiment to study the physiological response to different concentrations of phosphate (P1: 0 mM; P2: 0.05 mM; P3: 0.5 mM; P4:1.0 mN) and nitrate (N1: 0 mM with inoculation, N2: 20 mM with inoculation). The result confirmed that N and P nutrients both had positive effects on growth, nodulation and yield (P < 0.01). The relative importance of growth parameters that contributed to the larger biomass with N and P fertilisation was in decreasing order: (i) total leaf area, (ii) individual leaf area, (iii) shoot/root ratio, (iv) leaf area ratio and (v) specific leaf area. The yield increase at N and P supply was mainly associated with more seeds and a larger pod number per plant, which confirmed the result from the field experiment.     

Effects of carbonized and dried chicken manures on the growth, yield, and N content of soybean

The present study was conducted to investigate the effects of nitrogen derived from dried or carbonized chicken manure on growth, nodulation, yield and N content of soybean. ¹⁵N labeled chicken manure used in this study was obtained from the droppings of chicken fed on hulled rice grown under field conditions and fertilized with ¹⁵N-labeled stable isotope ammonium sulphate and potassium nitrate fertilizers. Carbonized chicken manure was made by heat treatment in a muffle furnace in our laboratory. This study was conducted in pots filled with clay loam soil. Results from the study show that the application of carbonized chicken manure increased soybean seed yield by 23% and 43% for the 50 and 100 kg N ha⁻¹ rates respectively. Dried chicken manure application increased soybean seed yield by 7% and 30% for the 50 and 100 kg N ha⁻¹ rates respectively. There was no difference in the N manure yield of both manures when applied at the same rate. The percentage ¹⁵N recovery was 17.6% and 8.9% for carbonized chicken manure, 19.2% and 10.5% for dried chicken manure at 50 and 100 kg N ha⁻¹ rates respectively at peak flowering stage of soybean growth. We found high total nitrogen yields of soybean at the rate of 100 kg N ha⁻¹ for both manures. There was a positive relationship between number of nodules and seed yield of soybean. Total N content also showed positive relationship with number of nodules and seed yield of soybean. We supposed that the higher P content of carbonized chicken manure is responsible for the higher seed yield and nodule growth compared to dried chicken manure.     

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.     

Seasonal N2 fixation by cool-season pulses based on several15N methods

Summary Accurate estimates of N₂ fixation by legumes are requisite to determine their net contribution of fixed N₂ to the soil N pool. However, estimates of N₂ fixation derived with the traditional¹⁵N methods of isotope dilution and A_N value are costly.Field experiments utilizing¹⁵N-enriched (NH₄)₂SO₄ were conducted to evaluate a modified difference method for determining N₂ fixation by fababean, lentil, Alaska pea, Austrian winter pea, blue lupin and chickpea, and to quantify their net contribution of fixed N₂ to the soil N pool. Spring wheat and non-nodulated chickpea, each fertilized with two N rates, were utilized as non-fixing controls.Estimates of N₂ fixation based on the two control crops were similar. Increasing the N rate to the controls reduced A_N values 32, 18 and 43% respectively in 1981, 1982 and 1983 resulting in greater N₂ fixation estimates. Mean seasonal N₂ fixation by fababean, lentil and Austrian winter pea was near 80 kg N ha^–1, pea and blue lupin near 60 kg N ha^–1, and chickpea less than 10 kg N ha^–1. The net effects of the legume crops on the soil N pool ranged from a 70 kg N ha^–1 input by lentil in 1982, to a removal of 48 kg N ha^–1 by chickpea in 1983.Estimates of N₂ fixation obtained by the proposed modified difference method approximate those derived by the isotope dilution technique, are determined with less cost, and are more reliable than the total plant N procedure.Scientific paper No. 6605. College of Agriculture and Home Economics Research Center, Washington State University, Pullman, WA 99164, U.S.A.     

Management of biological N2 fixation in alley cropping systems: Estimation and contribution to N balance

Alley cropping is being widely tested in the tropics for its potential to sustain adequate food production with low agricultural inputs, while conserving the resource base. Fast growth and N yield of most trees used as hedgerows in alley cropping is due greatly to their ability to fix N₂ symbiotically with Rhizobium. Measurements of biological N₂ fixation (BNF) in alley cropping systems show that some tree species such as Leucaena leucocephala, Gliricidia sepium and Acacia mangium can derive between 100 and 300 kg N ha^-1 yr^–1 from atmospheric N₂, while species such as Faidherbia albida and Acacia senegal might fix less than 20 kg N ha^-1 yr^-1. Other tree species such as Senna siamea and S. spectabilis are also used in alley cropping, although they do not nodulate and therefore do not fix N₂. The long-term evaluation of the potential or actual amounts of N₂ fixed in trees however, poses problems that are associated with their perennial nature and massive size, the great difficulty in obtaining representative samples and applying reliable methodologies for measuring N₂ fixed. Strategies for obtaining representative samples (as against the whole tree or destructive plant sampling), the application of ¹⁵N procedures and the selection criteria for appropriate reference plants have been discussed.Little is known about the effect of environmental factors and management practices such as tree cutting or pruning and residue management on BNF and eventually their N contribution in alley cropping. Data using the ¹⁵N labelling techniques have indicated that up to 50% or more of the tree's N may be below ground after pruning. In this case, quantification of N₂ fixed that disregards roots, nodules and crowns would result in serious errors and the amount of N₂ fixed may be largely underestimated. Large quantities of N are harvested with hedgerow prunings (>300 kg N ha^-1 yr^-1) but N contribution to crops is commonly in the range of 40–70 kg N ha^-1 season. This represents about 30% of N applied as prunings; however, N recoveries as low as 5–10% have been reported. The low N recovery in maize (Zea mays) is partly caused by lack of synchronization between the hedgerow trees N release and the associated food crop N demand. The N not taken up by the associated crop can be immobilized in soil organic matter or assimilated by the hedgerow trees and thus remain in the system. This N can also be lost from the system through denitrification, volatilization or is leached beyond the rooting zone. Below ground contribution (from root turnover and nodule decay) to an associated food crop in alley cropping is estimated at about 25–102 kg N ha^-1 season^-1. Timing and severity of pruning may allow for some management of underground transfer of fixed N₂ to associated crops. However many aspects of root dynamics in alley cropping systems are poorly understood. Current research projects based on ¹⁵N labelling techniques or ¹⁵N natural abundance measurements are outlined. These would lead to estimates of N₂ fixation and N saving resulting from the management of N₂ fixation in alley cropping systems.     

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.     

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.     

Nitrogen gas (N2+N2O) flux from urea applied to lowland rice as affected by green manure

A field study was conducted on a clay soil (Andaqueptic Haplaquoll) in the Philippines to directly measure the evolution of (N₂+N₂O)−¹⁵N from 98 atom %¹⁵N-labeled urea broadcast at 29 kg N ha⁻¹ into 0.05-m-deep floodwater at 15 days after transplanting (DT) rice. The flux of (N₂+N₂O)−¹⁵N during the 19 days following urea application never exceeded 28 g N ha⁻¹ day⁻¹. The total recovery of (N₂+N₂O)−¹⁵N evolved from the field was only 0.51% of the applied N, whereas total gaseous¹⁵N loss estimated from unrecovered¹⁵N in the¹⁵N balance was 41% of the applied N. Floodwater (nitrate+nitrite)−N in the 5 days following urea application never exceeded 0.14 g N m⁻³ or 0.3% of the applied N. Prior cropping of cowpea [Vigna unguiculata (L.) Walp.] to flowering with subsequent incorporation of the green manure (dry matter=2.5 Mg ha⁻¹, C/N=15) at 15 days before rice transplanting had no effect on fate of urea applied to rice at 15 DT. The recovery of (N₂+N₂O)−¹⁵N and total¹⁵N loss during the 19 days following urea application were 0.46 and 40%, respectively. Direct recovery of evolved (N₂+N₂O)−¹⁵N and total¹⁵N loss from 27 kg applied nitrate-N ha⁻¹ were 20% and 53% during the same 19-day period. The failure of directly-recovered (N₂+N₂O)−¹⁵N to match total¹⁵N loss from added nitrate-¹⁵N might be due to entrapment of denitrification end products in soil or transport of gaseous end products to the atmosphere through rice plants. The rapid conversion of added nitrate-N to (N₂+N₂O)−N, the apparently sufficient water soluble soil organic C for denitrification (101 μg C g⁻¹ in the top 0.15-m soil layer), and the low floodwater nitrate following urea application suggested that denitrification loss from urea was controlled by supply of nitrate rather than by availability of organic C.     

Rate of accumulation and emission of N2, N2O and CH4 from a flooded rice soil

In a field experiment using microplots, a flooded Crowley silt loam (Typic Albaqualfs) rice soil was fertilized with ¹⁵N labelled (60–74 atom %) urea and KNO₃. Emission of N₂, N₂O and CH₄ and accumulation in soil were measured for 21 d after fertilizer application.Emission of ¹⁵N₂-N measured from the urea and KNO₃ treated plots ranged from <15 to 570 and from 330 to 3,420 g ha^–1 d^–1, respectively. Entrapped ¹⁵N₂-N in the urea treated microplots was significantly lower (<15 g to 2.1 kg ha^–1) on all sampling dates compared to the ¹⁵N₂-N gas accumulation in the KNO₃ treated plots (6.4 to 31.5 kg ha^–1). Emissions of N₂O-N were low and did not exceed 4 g ha^–1 d^–1. Fluxes of CH₄ from the fertilizer and control plots were low and never exceeded 33 g ha^–1 d^–1. Maximum accumulation of CH₄ in the flooded soil measured 460 and 195 g ha^–1 for the urea and KNO₃ treatments, respectively.     

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.     

Improving N management through intercropping alleviates the inhibitory effect of mineral N on nodulation in pea

Background and aims

Symbiotic N₂ fixation is essential in the development of sustainable agriculture, but the nodulation of legumes is usually inhibited by N fertilization. Here, the intercropping of maize and pea in strips under various N managements was used as a means to alleviate the inhibitory effect of mineral N on pea nodulation and N₂ fixation and to improve system performance.

Methods

N natural abundance (δ ¹⁵N) analysis was employed to quantify N₂ fixation in the 3 years (2012 to 2014) of field experiment in Hexi Corridor of Northwestern China. Four N management systems with N rate of 0 kg N ha^?1 (the control), 90?+?45 kg N ha^?1 (base N plus topdressing N), 90?+?90 kg N ha^?1, and 90?+?135 kg N ha^?1 were implemented in the maize/pea strip intercropping to form different ratios of base N to topdressing N.

Results

Intercropped pea improved nodule biomass per plant by 99 %, increased nitrogen derived from the atmosphere (Ndfa) by 35 %, and promoted aboveground plant tissue N accumulation by 35 % as compared with sole pea, averaged across the four N treatments. Compared to the highest N fertilizer treatment, a reduction of topdressing to 45 kg N ha^?1 increased the nodule biomass of intercropped pea by 116 %, Ndfa by 35 %, and grain yield by 6 %.

Conclusions

Adaptation of suitable N management in cereal/legume intercropping systems will allow an effective conversion of atmospheric N₂ into crop available N and thus maximizing the system productivity.

     

Studies on the availability of Azolla N and urea N for rice growth using 15N

Field experiments (20 m² plots) were conducted to compare Azolla and urea as N sources for rice (Oryza sativa L.) in both the wet and dry seasons. Parallel microplot (1 m²) experiments were conducted using ¹⁵N. A total of approximately 60 kg N ha^-1 was applied as urea, Azolla, or urea plus Azolla. Urea or Azolla applied with equal applications of 30 kg N ha^-1 at transplanting (T) and at maximum tillering (MT) were equally effective for increasing rice grain yields in both seasons. Urea at 30 kg N ha^-1 at T and Azolla 30 kg N ha^-1 at MT was also equally effective. Urea applied by the locally recommended best split (40 kg at T and 20 kg at MT) gave a higher yield in the wet season, but an equal yield in the dry season. The average yield increase was 23% in the wet season, and 95% in the dry season. The proportion of the N taken up by the rice plants which was derived from urea (%NdfU) or Azolla (%NdfAz) was essentially identical for the treatments receiving the same N split. Recovery of ¹⁵N in the grain plus straw was also very similar. Positive yield responses to residual N were observed in the succeeding rice crop following both the wet and dry seasons, but the increases were not always statistically significant. Recovery of residual ¹⁵N ranged from 5.5 to 8.9% for both crops in succeeding seasons. Residual recovery from the urea applications was significantly higher than from Azolla in the crop succeeding the dry season crop. Azolla was equally effective as urea as an N source for rice production on a per kg N basis.     

Growing vetches (Vicia villosa Roth) in irrigated cotton systems: inputs of fixed N, N fertiliser savings and cotton productivity

We compared symbiotic N₂ fixation by winter forage legumes (clovers, medics and vetches) using the ¹⁵N natural abundance technique in three experiments. Vetches (Vicia spp.) were the most productive legumes, and woollypod vetch fixed (shoot+root) up to 265 kg N ha^–1 (mean 227 kg N ha^–1) during a 4–5 months period over winter and early spring. Balansa and Berseem clovers, and Gama medic were highly productive in the first experiment, but fixed significantly less N than woollypod vetch in the second experiment. A 6-year study (1997–2003) compared cotton (Gossypium hirsutum L.) systems with and without vetch, or with faba beans (Vicia faba L.) to assess the effects of these crops on cotton production. Woollypod vetch was grown either between annual cotton crops, or between wheat (Triticum aestivumL.) and cotton crops. Vetch added 230 kg N ha^–1 (174 kg fixed N ha^–1) to the soil when incorporated as a green manure. Faba bean shoot residues and nodulated roots contributed 108 kg fixed N ha^–1 to the soil, following the removal of 80 kg N ha^–1 in the harvested seed (meaned over three crops). Lablab (Lablab purpureus L. – summer-growing and irrigated) added 277 kg N ha^–1 (244 kg fixed N ha^–1) before incorporation as a green manure in the first year of the experiment. The economic optimum N fertiliser rate for each cropping system was determined every second year when all systems were sown to cotton. Cotton following cotton required 105 kg fertiliser N ha^–1, but only 40 kg N ha^–1 when vetch was grown between each cotton crop. Cotton following wheat required 83 kg fertiliser N ha^–1 but no N fertiliser was needed when vetch was grown after wheat (the highest yielding system). Cotton following faba beans also required no N fertiliser. The vetch-based systems became more N fertile over the course of the experiment and produced greater lint yields than the comparative non-legume systems, and required less N fertiliser. While no cash flow was derived from growing vetch, economic benefits accrued from enhanced cotton yields, reduced N fertiliser requirements and improved soil fertility. These findings help explain the rotational benefits of vetches observed in other regions of the world.     

Selection of Rhizobium leguminosarum strains for lentil (Lens culinaris) under growth room and field conditions

Most of the production of lentil (Lens culinaris) on the Great Plains occurs on soils that are free of indigenous Rhizobium leguminosarum. Inoculation is required to increase yields through N₂ fixation. A screening program to evaluate the effectiveness of R. leguminosarum strains for lentil was initially carried out under controlled environments followed by an evaluation under field conditions. In two separate growth room experiments, the effectiveness of 185 and 24 different strains of R. leguminosarum were tested for Laird and Eston lentil. Significant differences between strains in number of nodules, shoot weight and nitrogenase activity (acetylene reduction activity, ARA) were found for lentil grown for 5 weeks. When lentil were grown for 7 weeks, significant differences between strains in number of nodules, total plant weight, total N, and % N were observed.Fourteen strains plus Nitragin C inoculant were selected for further field testing on Eston and Laird lentil at two locations in 1986 and one site in 1987. Inoculation increased yield up to 135%. Percent Ndfa and total N₂ fixed ranged from 0 to 76 and 0 to 105 kg ha^-1, respectively. N₂-fixing activity was site specific and higher spring soil NO₃-levels resulted in lower N₂-fixing activity. Depending on site and growing conditions, strains 99A1 and I-ICAR-SYR-Le20 appeared to be superior to the other strains tested. A good agreement was found between the estimates for N₂ fixation based upon the ¹⁵N-isotope dilution and the classical N difference methods. Number of nodules, dry weight of nodules and ARA of Eston and Laird lentil grown under growth room conditions failed to show positive correlations with total dry matter production, total N or total N₂ fixed of field grown lentil. However, total plant weight and total N of lentil grown under growth room conditions were highly correlated with field parameters, and were the most reliable screening parameters for the selection of superior rhizobial strains.     

The effect of a single application of cow urine on annual N2 fixation under varying simulated grazing intensity, as measured by four 15N isotope techniques

The effects of dairy cow urine and defoliation severity on biological nitrogen fixation and pasture production of a mixed ryegrass-white clover sward were investigated over 12 months using mowing for defoliation. A single application of urine (equivalent to 746 kg N ha^–1), was applied in late spring to plots immediately after light and moderately-severe defoliation (35 mm and 85 mm cutting heights, respectively) treatments were imposed. Estimates of percentage clover N derived from N₂ fixation (%Ndfa) were compared by labelling the soil with ¹⁵N either by applying a low rate of ¹⁵N-labelled ammonium sulphate, immobilising ¹⁵N in soil organic matter, adding ¹⁵N to applied urine, or by utilising the small differences in natural abundance of ¹⁵N in soil. Urine application increased annual grass production by 85%, but had little effect on annual clover production. However, urine caused a marked decline in %Ndfa (using an average of all ¹⁵N methods) from 84% to a low of 22% by 108 days, with recovery to control levels taking almost a year. As a result, total N fixed (in above ground clover herbage) was reduced from 232 to 145 kg N ha^–1 yr^–1. Moderately–severe defoliation had no immediate effect on N₂ fixation, but after 108 days the %Ndfa was consistently higher than light defoliation during summer and autumn, and increased by up to 18%, coinciding with an increase in growth of weeds and summer-grass species. Annual N₂ fixation was 218 kg N ha^–1 yr^–1 under moderately-severe defoliation compared to 160 kg N ha^–1 yr^–1 under light defoliation. Estimates of %Ndfa were generally similar when ¹⁵N-labelled or immobilised ¹⁵N were used to label soil regardless of urine and defoliation severity. The natural abundance technique gave highly variable estimates of %Ndfa (–56 to 24%) during the first 23 days after urine application but, thereafter, estimates of %Ndfa were similar to those using ¹⁵N-labelling methods. In contrast, in urine treated plots the use of ¹⁵N-labelled urine gave estimates of %Ndfa that were 20–30% below values calculated using conventional ¹⁵N-labelling during the first 161 days. These differences were probably due to differences in the rooting depth between ryegrass and white clover in conjunction with treatment differences in ¹⁵N distribution with depth. This study shows that urine has a prolonged effect on reducing N₂ fixation in pasture. In addition, defoliation severity is a potential pasture management tool for strategically enhancing N₂ fixation.     

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.     

Influence of urea on biological N2 fixation and N transfer from Azolla intercropped with rice

The N₂ fixed by Azolla before and after urea application during the rice cycle, the mineralisation of Azolla-N as well as its availability to rice was studied in two greenhouse experiments conducted in 1996 and 1997 and in June 1998 in Goettingen (Germany). Dry matter production of the various rice parts of experiment 1 showed a clear positive synergism between treatment with Azolla and urea with a resulting apparent N recovery by rice increasing from 40% (without Azolla) to 57% in the presence of Azolla. Part of this increase may be due to N fixed biologically by Azolla and transferred to the rice. The second experiment shed some light on the role of BNF. Using an iterative method of estimation, the daily rate of N fixation was estimated at 0.6 – 0.7 kg N ha^–1. The rate was not so much affected by the age of the Azolla crop. At this rate, the BNF would amount to up to 100 kg N ha^–1 over a 130-day season. Assuming that BNF may be inhibited for a period of 5 – 10 days following urea application due to high levels of N in the floodwater, this might reduce the BNF by between 6 and 14 kg N ha over the season. Using the mean-pool-abundance concept, it was estimated that around 75 – 80% of the Azolla-N mineralized during the growth period was actually absorbed by the rice plants. Of the N taken up by rice around 28% was derived from the biologically fixed Azolla N, the remainder was urea N cycled through the Azolla. Azolla also seems to help sustain the soil N supply by returning N to the soil in quantities roughly equal to those extracted from the soil by the rice plant.