Quantitative assessment of symbiotic nitrogen fixation in diverse mutant lines of field bean (Vicia faba minor)

Summary Homozygous mutant lines of field bean selected for (a) improved yielding potential and (b) altered plant architecture and/or physiological response were tested for symbiotic nitrogen fixing ability under field conditions in comparison with their parent cultivar.¹⁵N-tracer techniques were applied to determine %N derived from atmosphere. Data were collected on assimilate and nitrogen accumulation and distribution among various plant parts during two stages of reproductive growth.Symbiotic nitrogen fixation was closely correlated with total plant top biomass and nitrogen yield. A similar close association was found between crop yield and nitrogen harvest index. Both harvest indices tended to be negatively correlated with stage of maturity and with the amount of N derived from air per unit of area. The generally high %N derived from symbiotic N₂ fixation and its comparatively small variability implies that this parameter may be difficult to improve inVicia faba under field conditions.It is concluded, that the main genetic potential for improving the amount of biological nitrogen fixation in this crop depends upon factors that promote high photosynthetic productivity and efficient N-use under appropriate agronomic conditions and with effective rhizobial associations. The establishment of rational ideotypes with a possitive impact on yield appears to be of practical significance for increasing the amount of symbiotically fixed nitrogen.     

Comparative HPLC analysis of seed albumins fromVicia faba andV. kalakhensis (Fabaceae)

Previously reported electrophoretic seed albumin data have shown an unexpected association ofVicia faba withV. kalakhensis. In the present work, seed albumins ofV. faba (subsp.paucijuga and subsp.faba) were compared with those ofV. kalakhensis using ionexchange (IE) and reversed-phase (RP) high-performance liquid chromatography (HPLC). Two subspecies ofV. faba displayed similar seed albumin profiles. On the other hand, seed albumin profiles ofV. faba andV. kalakhensis showed no major protein peak in common either in IE-HPLC or RP-HPLC chromatograms. The reported differences in seed albumin composition ofV. faba andV. kalakhensis are consistent with other taxonomical data showingV. faba to be genetically distant from the wild relatives.     

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.     

Measurement of biological dinitrogen fixation in grassland: Comparison of the enriched 15N dilution and the natural 15N abundance methods at different nitrogen application rates and defoliation frequencies

A plant mixture of white clover (Trifolium repens L.), red clover (Trifolium pratense L.), and ryegrass (Lolium perenne L.) was established in the spring of 1991 under a cover-crop of barley. Treatments were two levels of nitrogen (400 and 20 kg N ha^-1) and two cutting intensities (3 and 6 cuts per season). Fixation of atmospheric derived nitrogen was estimated by two ¹⁵N dilution methods, one based on application of ¹⁵N to the soil, the other utilising small differences in natural abundance of ¹⁵N.Both methods showed that application of 400 kg N ha^-1 significantly reduced dinitrogen fixation, while cutting frequency had no effect. Atmospheric derived nitrogen constituted between 50 and 64% of harvested clover nitrogen in the high-N treatment, while between 73% and 96% of the harvested clover nitrogen was derived from the atmosphere in the low-N treatment. The amounts of fixed dinitrogen varied between 31–72 kg N ha^-1 and 118–161 kg N ha^-1 in the high-N and low-N treatment, respectively. The highest values for biological dinitrogen fixation were estimated by the enriched ¹⁵N dilution method.Estimates of transfer of atmospheric derived nitrogen from clover to grass obtained by the natural ¹⁵N abundance method were consistently higher than those obtained by the enriched ¹⁵N dilution method. Neither mineral nitrogen application nor defoliation frequency affected transfer of atmospheric derived nitrogen from clover to grass.Isotopic fractionation of ¹⁴N and ¹⁵N (B value) was estimated by comparing results for nitrogen fixation obtained by the enriched ¹⁵N dilution and the natural ¹⁵N abundance method, respectively. B was on average +1.20, which was in agreement with a B value determined by growing white clover in a nitrogen free media.     

Nitrogen fixation,transfer and turnover in upland and lowland grass-clover swards,using15N isotope dilution

The stable isotope¹⁵N is particularly valuable in the field for measuring N fixation by isotope dilution. At the same time other soil-plant processes can be studied, including¹⁵N recovery, and nitrogen transfer between clover and grass. Three contrasting sites and soils were used in the present work: a lowland soil, an upland soil, and an upland peat. Nitrogen fixation varied from 12 gm^–2 on lowland soil to 2.7 gm^–2 on upland peat. Most N transfer occurred on upland soil (4.2 gm^–2) which, added to nitrogen fixed, made a total of 8.7 gm² input during summer 1985.¹⁵N recovery for the whole experiment was small, around 25%.Measurement of dead and dying leaves, stubble and roots, suggests that plant organ death is the first stage in N transfer from white clover to ryegrass, through the decomposer cycle. Decomposition was fastest on lowland soils, slowest on peat. On lowland soil this decomposer nitrogen is apparently subverted before transfer, probably by soil microbes.Variations in natural abundance of¹⁵N in plants were found in the two species on the different soils. These might be used to measure nitrogen fixation without adding isotope, but the need for many replicates and repeat samples would limit throughput.     

The role of root-associatedKlebsiella pneumoniae in the nitrogen nutrition ofPoa pratensis andTriticum aestivum as estimated by the method of15N isotope dilution

Summary The technique of¹⁵N isotope dilution was used to verify that nitrogen was fixed and transferred to the plant byKlebsiella pneumoniae strain Pp in association withPoa pratensis orTriticum aestivum. Surface sterilized, sprouting seeds were inoculated withK. pneumoniae and grown in sand in modified Leonard jars. Potassium nitrate enriched with¹⁵N was used to provide N concentrations ranging from 10–40 mg Nl^–1 nutrient solution. After 10–18 weeks the shoots and roots were analyzed separately for dry matter, N content, total N, and atom %¹⁵N excess. The acetylene reduction technique was used to test for the presence of N₂-fixing organisms on the roots. The data from¹⁵N isotope dilution demonstrated that up to 33.8% of N in the shoots ofP. pratensis and 15.9% in those ofT. aestivum were derived from associative N₂ fixation byK. pneumoniae. In most experiments the dry matter yield, N content, and total N yield of the shoots ofP. pratensis were increased byK. pneumoniae inoculation, whereas inoculation had no significant effect on the dry matter yield, N content or total N of the shoots ofT. aestivum.     

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.     

Root nodulation and nitrogen accumulation and partitioning in legume crops as affected by soil solarization

Soil solarization is a preplanting technique used in hot climates to control weeds and soilborne pathogens consisting of mulching the soil surface with polyethylene sheets. The increase in temperature associated with solarized soil could affect nitrogen availability for grain legume crops through effects on nitrogen fixing soil microorganisms or other mechanisms. To examine the effects of solarization on natural root nodulation and nitrogen accumulation and partitioning in the plant, two solarization field experiments were carried out over two planting seasons, involving genotypes of both faba bean (Vicia faba) and chickpea (Cicer arietinum). The effect of sowing date was also studied in the first season. Solarization increased the maximum soil temperature by 9–10 °C in the first, and by 13–15 °C in the second season. At 5 cm below the solarized soil surface, a temperature of over 46 °C prevailed for 146 and 280 h over the two respective seasons, while this temperature was not attained in unmulched soil. Solarization delayed the initiation of nodulation and consistently reduced the nodule number per host plant, but generated an approximate doubling of mean nodule weight. The total nodule mass per plant was not affected by the treatment in the first season, but was reduced in the second season. Solarization significantly increased the concentrations of NO₃^– -N, Na⁺, Zn²⁺, Ca²⁺ and K⁺ in the soil extract, and the total nitrogen accumulated in the whole plant. This latter increase was due to both higher plant growth and a greater plant nitrogen concentration. The increased nitrogen level in the plant was not uniform with respect to plant component, varying from 57% in the roots to 198% in the pods and seeds. The plants grown in non-solarized soil accumulated about 31% of their total N content in the shoots of the parasitic weed Orobanche crenata. Solarization dramatically improved grain yield by 300–900% in both seasons and in all genotypes studied, due to increased N availability in soil, N accumulation in plants, improved plant growth, and complete control of the parasite weed O. crenata. On the basis of these beneficial effects, soil solarization, which avoids site contamination and is suited to organic farming, should be a good opportunity in Mediterranean areas where the level and stability of grain yields are low, and the infestation of O. crenata is high.     

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.     

Genetic specificity of nitrogen nutrition in leguminous plants

Summary Mineral nitrogen did not increase grain yield and seed protein levels ofVicia faba L. andLupinus luteus L. in field trials and pot experiments. Fixed N₂ was substituted by mineral nitrogen in these cases because of inhibition of N₂ fixation by mineral nitrogen. Contrary to these results mineral nitrogen increased grain yields and seed protein amounts ofLupinus albus L.,Pisum sativum L., andGlycine max. (L.) Merr. The nitrogen effect was caused at an early stage by saving energy due to inhibition of N₂ fixation (measurement of gas exchange by means of IRGA). In case of the N application after flowering grain, yields and seed protein levels increased because the mineral N was an additional nitrogen source for plants. At this stage the plants had ceased fixing atmospheric nitrogen. The high sink activity of growing fruits induced a lack of assimilates in nodules (determined by means of¹⁴CO₂ application). The N effect was therefore the consequence of the lower assimilate pool for supplying root nodules in these plants in comparison withVicia faba L. andLupinus luteus L. Hence it follows that response to mineral nitrogen can be a criterion for discovering more effective Rhizobium-host combinations.     

Field evaluation of N2-fixation and N-utilization by phaseolus bean varieties determined by15N isotope dilution*

Summary Differences in N₂-fixation byPhaseolus vulgaris bean cultivars were successfully evaluated in the field using¹⁵N isotope dilution technique with a non-fixing test crop of a different species (wheat). The Phaseolus cultivars could have been similarly ranked for N₂-fixation capacity from either seed yield or total nitrogen yield, but the isotope method provided a direct measure of N₂-fixation and made it possible to estimate the proportion of fixed to total nitrogen in the crop and in plant parts. Amounts of nitrogen fixed varied between 24.59 kg N/ha for the 60-day cultivar Goiano precoce to 64.91 kg N/ha for the 90-day cultivar Carioca. The per cent of plant nitrogen due to fixation was 57–68% for the 90-day cultivars and 37% for Goiano precoce (60-day cultivar). Fertilizer utilization was 17–30% of a 20 kg N/ha fertilizer application. 100 kg N/ha fertilizer application decreased N₂-fixation without suppressing it totally. Differences in yield between the highest yielding (Carioca) and the lowest (Moruna) 90-day cultivars were also due apparently to varietal differences in efficiency of conversion of nitrogen to economic matteri.e. seed, as well as to differences in capacity of genotypes for N₂-fixation. The work described here was in part supported by IAEA Research Contract No. RC/2084 UNDP/IAEA Project BRA/78/006     

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.     

Utilisation du15N2 pour estimer la fixation d'azote et sa repartition chez les legumineuses

Resumé Les auteurs proposent une méthode d'utilisation du¹⁵N₂ pour l'étude de la fixation d'azote dans les associations complexes légumineuses-Rhizobium cultivées sur sol. La procédure consiste, à marquer l'atmosphère du sol à l'aide de¹⁵N₂ et à calculer la quantité d'azote total fixé pendant cette période. Les premiers résultats obtenus sur des haricots et des trèfles démontrent qu'après seulement 7 h d'incubation, des quantités significatives de¹⁵N sont mesurées dans les plantes permettant ainsi de déterminer précisément la valeur du rapport C₂H₄/N₂ qui a été établi entre 2,6 et 3,1 dans les conditions de l'expérience. Sur de longues périodes, ces mêmes quantités se sont avérées suffisantes pour suivre la dynamique de l'azote des nodules vers les organes reproducteurs de plants de haricots.

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Use of¹⁵N to estimate dinitrogen fixation and distribution in legumes

Summary A method for use of¹⁵N₂ in the study of dinitrogen fixation by complex legumes-Rhizobium associations grown on soil is proposed. The procedure consists in labelling the soil atmosphere with¹⁵N₂ during short periods of time, measuring¹⁵N enrichment in the plants and calculating the total nitrogen fixed during this period. The first results obtained with bean and clover plants demonstrate that after only 7 h of incubation, significative amounts of¹⁵N are measured in the plants to allow precise determination of C₂H₄/N₂ ratio which ranged between 2.6 and 3.1 in this experiment. Over longer periods of time, such amounts are meaningfull to follow the pattern of N dynamic from the nodules to the reproductive organs of bean plants.

     

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.     

Early growth and final yield of autumn sownVicia faba L cultivars given different forms of fertiliser N over winter

Summary Between 3 Nov. 1983 and 9 Apr. 1984, six applications of fertiliser N (ammonium, nitrate or urea) were given to four autumn sown (26 Oct. 1983)Vicia faba L cultivars, Banner Winter (BW) and Maris Beagle (MBg), cold tolerant cultivars normally sown in the autumn, and Herz Freya (HF) and Maris Bead (MBd), cold sensitive cultivars more commonly sown in the spring. The effects of additional N were determined by comparison with plants given zero-N (controls). Application of N, regardless of form, had no effect on % emergence at the first sampling (15 Dec. 1983); >90% for BW, MBg and HF, but only 40–60% for MBd. At this time the dry weight, carbon content and nitrogen content of all cultivars was approximately 20% less than that of the seed on planting. No more plants emerged after 15 Dec. 1983. Between 15 Dec. 1983 and 20 Feb. 1984, all cultivars, regardless of N treatment, showed little change in dry weight, carbon content and nitrogen content but the proportion of total plant dry weight, carbon content and nitrogen content in the cotyledons decreased while the proportions in root, stem and leaf tissue increased. On 20 Feb. 1984 there were no N effects. All cultivars but especially BW and MBg, showed progressive increases in dry weight, carbon content and nitrogen content during the period 20 Feb. 1984 to 8 May 1984. Pooled results for all four cultivars indicated that on 8 May 1984, plants given ammonium and urea had a greater dry weight, carbon content and nitrogen content than controls. At harvest (1–3 Sep. 1984), BW and MBg outyielded (g dw seed m⁻²) HF and MBd. Pooled results for all cultivars indicated that application of N regardless of form gave increased yield and an increased N concentration (mg N g⁻¹ dw) in the seed.     

Partitioning of biologically fixed nitrogen in cowpea during pod development

Two days after exposure of roots to¹⁵N labeled N₂, partitioning of biologically fixed N into leaves, stems, peduncles, pods, roots and nodules was measured in the early pod development stage of cowpea (Vigna unguiculata (L.). The experimental objective was to determine the quantity of biologically fixed N that is incorporated into vegetative tissue before being mobilized to pods. For the three varieties of cowpea included in the experiment a maximum of 50% of the N, biologically fixed two days earlier, was contained in the pods. The remaining N was distributed throughout the vegetative portion of the plant with at least 30% in stems and leaves which indicates that much of the newly fixed N must cycle through a N pool in these tissues before reaching the pods.     

Assessment of potential sources of error in nitrogen fixation measurements by the nitrogen-15 isotope dilution technique

Although the use of ¹⁵N fertilizers to measure nitrogen (N₂) fixed in crops has increased substantially in recent years, some methodological uncertainties still remain unresolved. The results obtained from a greenhouse study of soybean [Glycine max. (L.) Merrill] inoculated by six different methods have been examined for potential errors arising from incorporating ¹⁵N labelled fertilizer into soil to estimate N₂ fixed in pods or shoots or the whole plant at three growth stages (50% flowering, pod-initiation and physiological maturity) using as reference crops, an uninoculated soybean cultivar and a non-nodulating soybean isoline. At the first harvest when N₂ fixed was very low, the estimates of N₂ fixed by the two reference crops did not match. At this stage the uninoculated soybean estimated about four times as much N₂ fixed in the symbiotic soybean as that measured using the non-nodulating soybean. For the second and third harvests, there were substantial increases in N₂ fixed, and both the non-nodulating and uninoculated soybean were equally suitable as reference crops for assessing N₂ fixed in the symbiotic soybean. These results indicate how critical and difficult the choice of the reference crop could be at early harvests, or when N₂ fixed is low. Even though there were significant differences in ¹⁵N enrichments in different organs (generally nodules < pods < roots < shoots), the estimates of N₂ fixed in soybean plants obtained by excluding roots and nodules did not differ much from those based on the whole plant. Of the above-ground organs, % N₂ fixed in pods (containing seeds) was closest to that of the whole plant (similar at P<0.05 at physiological maturity). However, the total N₂ fixed in pods or shoots was substantially lower than that fixed by the whole plant (P<0.05), although that for the pods and enclosed seeds once again was closer to N₂ fixed in the whole plant than that in the shoots.     

Effects of plant breeding and selection on yields and nitrogen fixation in soybeans under two soil nitrogen regimes

Summary Soybeans (Glycine max (L.) Merr.) have a high N requirement which is fulfilled by soil N uptake and N₂-fixation. This study was concerned with the effects of past yield selection on N₂-fixation in soybeans.The soybean cultivars, Lincoln, Shelby, and Williams, which represent successive improvements in the Lincoln germplasm, and a non-nodulating control were planted in a soil containing¹⁵N labelled organic matter. Two replications occurred on soil previously cropped to alfalfa and two on soil previously cropped to soybeans. Plants were harvested at five growth stages and leaf area, plant weight, total N, and atom percent¹⁵N were determined. Mature grain was harvested and yield components were also determined, as well as the total N and¹⁵N content.Cultivar differences in total dry matter were only evident at physiological maturity, when Williams contained the greatest dry matter. Williams exhibited the longest period of seed formation and seed fill and also had the highest grain yield which resulted from a larger weight per seed.The N content of the cultivars did not vary until physiological maturity when Williams contained the highest percent N. The quantity of N fixed at physiological maturity was highest for Williams and lowest for Lincoln. Fixed N contained in the harvested grain was greater for Williams than for the other two cultivars. The fraction of the total plant N derived from fixation was not greatly affected by cultivar and all cultivars acquired an average of 50% of their total N through N₂-fixation.Previous cropping history greatly affected the quantity of N fixed and the fraction of the total plant N derived from fixation. Soybeans following soybeans were more dependent upon N₂-fixation than soybeans following alfalfa with the former deriving 65% of the total plant N from fixation and the latter only 32%. These soybean cultivars apparently utilized soil N first and then used N₂-fixation to satisfy their N requirement.The past selection for higher yield has resulted in soybean cultivars with improved capacities to fix atmospheric N₂ and an improved ability to take up available soil N.     

Influence of combined nitrogen level andEucalyptus competition on dinitrogen fixation in nodulatedCasuarina

Summary Experiments were conducted to determine the effect on biological dinitrogen fixation byCasuarina of available nitrogen (N) in the substrate and competition by interplantedEucalyptus. In these experiments, combined N was applied to the plants after nodules were developed and functioning. Both environmental factors, nitrate and competition, were observed to influence biological dinitrogen fixation byCasuarina, but not yield (total dry weight). In one experiment, the proportion of nitrogen derived byCasuarina from atmospheric fixation (pNdfa) was observed to be inhibited by potassium nitrate in a linear fashion. However, substrate N did not significantly affect the weight of root nodules. Thus nodule dry weight was not highly correlated with the proportion of nitrogen fixed. In a second experiment, the presence of a non-fixing interplanted species,Eucalyptus, increased dinitrogen fixation inCasuarina.Casuarina interplanted withEucalyptus obtained a greater proportion of its nitrogen (94.75%) from fixation than didCasuarina grown alone (86.68%) suggesting that competition for substrate N influences the proportion of nitrogen fixed by this actinorhizal plant.Dedicated to the memory of Professor John G. Torrey     

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.