Journal of Experimental Botany, Vol. 53, No. 371, pp. 1109-1118,
May 2002
© 2002 Oxford University Press
Original Papers |
Difference in 
15N signatures between nodulated roots and shoots of soybean is indicative of the contribution of symbiotic N2 fixation to plant N
1Institute of Ecology and Conservation Biology, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria
2Botany Department, The University of Western Australia, Nedlands, WA 6907, Australia
Received 30 June 2001; Accepted 7 December 2001
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Abstract |
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Symbiotic N2 fixation has a variable effect on the 15N abundance of different parts of legumes. Increases in fixation result in 15N enrichment of nodules, while decreases, in combination with an increased uptake of mineral N, result in 15N depletion of the root system. The difference between soybean shoot and below-ground
15N (
15N=15Nshoot-15Nbelowground) was assessed in hydroponic culture over a range of rates of supply of mineral N. The fractional contribution of N2 fixation to N uptake (%Ndfa) was determined using the natural abundance (NA) technique with ryegrass as a reference plant. 15N and %Ndfa were highly correlated, and the relationship was tested using the same soybean cultivar grown in pots in N-rich soil. Estimates of %Ndfa derived from the NA method and from the 15N approach yielded near-identical values. A literature survey showed similar relationships between %Ndfa and 15N with different growth stages of soybeans grown under glasshouse and field conditions, different cowpea (Vigna unguiculata) cultivars in the field, and tagasaste (Chamaecytisus proliferus) in hydroponic culture. Possible confounding and species-specific (either plant or Rhizobium spp.) influences are discussed. The difference in 15N signatures between nodulated roots and shoots is confirmed as a robust means of quantifying %Ndfa: it is independent of reference plants and offers the possibility of estimating %Ndfa in soils where the isotope composition of mineral N closely matches that of atmospheric N2.
Key words:
Glycine max, mineral nitrogen uptake, 15N, nitrogen fixation, soybean.
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Introduction |
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The natural abundance of the heavy nitrogen isotope, 15N, is commonly expressed as a
15N value, denoting the relative deviation (in 
) from the ratio 15N:14N in atmospheric N2: 15N=1000(Rsample-Rair)/Rair, where R is the ratio 15N:14N of sample and air, respectively. The relative abundances of these stable isotopes of N vary substantially among N pools in ecosystems. In particular, soil processes of mineralization, nitrification and denitrification (Koba et al., 1998) are frequently accompanied by strong fractionation that may leave product pools depleted in 15N and substrate pools 15N-enriched. Additions of atmospheric N2 or other gaseous forms of N further modify 15N signatures of mineral and organic N pools in soil (Heaton et al., 1997). Consequently, the 15N signatures of plant-available N forms range from <-15 (atmospheric ammonium) to >+10 (soil pools) (Yoneyama, 1996). Observed differences in 15N among plant species in the field are regularly hypothesized to reflect different N acquisition strategies (Hobbie et al., 2000; Michelsen et al., 1998). These include the role of mycorrhizal symbioses, the utilization of mineral or organic soil N forms (Handley and Scrimgeour, 1997; Michelsen et al., 1996) or the use of N sources from precipitation (Hietz et al., 1999). The 15N signatures of plants relying solely on N2 fixation by bacterial symbionts are similar to those of atmospheric N2 (i.e. close to 0) as opposed to the much wider range of signatures of plants entirely relying on soil N sources (Tjepkema et al., 2000; Sprent et al., 1996; Shearer and Kohl, 1986).
Modern techniques for estimating time-integrated rates of symbiotic N2 fixation, like the 15N natural abundance (NA; Shearer and Kohl, 1986; Unkovich et al., 1994) and 15N isotope dilution (ID) methods (Chalk and Ladha, 1999), rely on source signatures as outlined below. When the 15N abundance of plant-available soil N is significantly different from that of air N2, an estimate of the proportion of legume N derived from each source, air or soil, can be obtained with the NA method. The relative abundances of 15N and 14N in plant-available pools in the soil are usually estimated using a non-N2-fixing reference plant that is totally dependent on soil N for growth. The primary assumption is that reference plants exhibit similar mineral N acquisition characteristics, including preference for different forms of N, rooting zones, and growth behaviour, to the legume species studied. The percentage of plant N derived from atmospheric N2 (P or %Ndfa) is then calculated for the NA method as follows:
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15Nrefplant represents the respective reference plant 15N value grown under the same conditions as the legume, and B is the 15N value of the legume grown with N2 as the sole source of N. A 15N enrichment technique is sometimes used (ID method; McAuliffe et al., 1958) especially when the 15N natural abundance of soil N is close to that of atmospheric N2. This technique introduces extra 15N in the form of small amounts of fertilizer N, thereby increasing the difference in relative abundance of N isotopes in soil (mineral N) and the atmosphere (N2).
Nonetheless, reference plants are generally significant sources of error for both isotope methods, largely as a result of temporal and spatial variation in 15N signatures of soil N pools (Danso et al., 1993). Alternative approaches to the use of reference plants were recently reviewed (Chalk and Lahda, 1999), but their use remains restricted by methodological limitations to the measurement of temporal variability in the isotopic composition of plant-available soil N (Chalk and Ladha, 1999; Handley and Scrimgeour, 1997).
15N signatures may vary markedly between parts of a single plant. This variation is due variously to ammonium or nitrate acquisition (Evans et al., 1996; Evans, 2001), preferential nitrate reduction in roots or shoots (Pate et al., 1993; Unkovich et al., 2000), drought stress (Robinson et al., 2000) and N2 fixation (Shearer et al., 1980). By contrast to non-nodular tissues, nodules of a wide range of legume species are strikingly enriched in 15N compared to their source (Shearer et al., 1982). Earlier studies have shown that the extent of nodule 15N enrichment is affected by the metabolic activity of nodules and the ‘N2 fixation efficiency’ of rhizobial strains (Kohl et al., 1983; Steele et al., 1983; Ledgard, 1989; Bergersen et al., 1986). The term ‘N2 fixation efficiency’ is generally defined as the amount of N fixed relative to the standing N mass of nodules (Kohl et al., 1983). Metabolically active (N2-fixing) nodules of soybean were significantly enriched in 15N, whereas nodules from plants grown under N-rich conditions exhibited little 15N enrichment (Shearer et al., 1980). In parallel, shoots were slightly 15N-depleted under favourable conditions for N2 fixation whereas shoot 15N varied greatly when plants relied on mineral N. These changes in 15N among plant parts with decreasing N2 fixation are probably due to shifts in internal 15N partitioning resulting in the specific 15N distribution patterns between nodules, roots and shoots. While several studies have related patterns of nodule 15N enrichment to varying reliance on N2 fixation (e.g. N2 fixation efficiency or nodule mass per plant), no direct examination of the relationship to %Ndfa could be found.
Here the examination of whether shifts in within-plant 15N distribution are correlated with the proportion of legume N derived from atmospheric N2 is reported. Soybean and ryegrass plants were grown in hydroponic culture with a range of nitrate concentrations to (a) alter nodulation and N2 fixation, (b) identify changes in natural 15N distribution among soybean plant parts (nodulated roots and shoots) in relation to N2 fixation activity, and (c) calibrate the shifts in intra-plant 15N distribution using the NA technique. These factors were also investigated in soil-grown soybeans. The aim was to develop a technique for quantifying %Ndfa that is independent of reference plants. Advantages and restrictions of the proposed technique are discussed using further data obtained from published literature.
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Hydroponic culture
Soybean (Glycine max (L.) Merill) seeds (var. ‘Yellow SB’, country of origin: Brazil, supplier: Davert Mühle, Senden, Germany) were surface sterilized with 10% sodium hypochlorite for 5 min, extensively rinsed with deionized water and inoculated with the effective rhizobial strain VDDA16 (HUP-; kindly supplied by Dr Rebecca C Hood-Novotny, FAO/IAEA Agricultural and Biotechnology Laboratories, Seibersdorf, Austria) in water for 24 h. Two seeds were planted into pots containing 1 kg of acid-washed coarse quartz sand. Temperature and relative humidity were kept constant in a growth chamber at 28/23 °C and 60/80% (day/night). PPFD was 800 µmol m-2 s-1 at plant level. From day 5 onwards, five pots were each irrigated with half-strength N-free Hoagland solution which was supplemented with either 0.25, 2.5 or 25 mM KNO3. The daily irrigation regime was optimized to ensure full exchange of the nutrient solution which might otherwise develop changes in isotopic composition as well as in the relative abundance of forms of N, and to keep root systems moist. The nitrate source used had a mean
15N value of 24.1±1.8 versus atmospheric air. Seeds of reference plants, ryegrass (Lolium perenne L.), were surface-sterilized as described above, thinly sown into pots filled with quartz sand, and five replicate pots for each treatment were irrigated with the different nutrient solutions. Plants were harvested 72 d after planting, at the onset of flowering of soybean plants. Ryegrass plants from each pot were harvested together and separated into root and shoot biomass. Soybean plants were divided into nodules, roots, stems, and leaves. Nodules were counted and all plant fractions were dried and weighed.
Soil culture
After surface-sterilization of soybean and ryegrass seeds, soybean seeds were inoculated with the rhizobial strain VDDA16, and seeds of both species were sown into separate 8.0 l pots (12 per plant species) containing a mixture of peat:sand:soil (1:1:2; by vol.). Plants were cultivated under the same conditions as in hydroponic culture. No additional fertilizers were added. The pots were irrigated daily to maintain soil moisture at field capacity. After 65 d of growth, watering was withheld from four pots per species for 7 d (water deficit treatment). Total harvest of soybean and ryegrass was performed as given above, roots and nodules were carefully separated from adhering soil and washed.
Sample preparation and isotope analyses
Fresh plant material was shock-inactivated in a microwave oven (three times for 10 s at 1200 W; Popp et al., 1996) and further dried for 48 h at 60 °C in a drying oven. Dried samples were coarsely chopped and a subsample then ground to a fine powder in a ball mill (Retsch MM2, Vienna, Austria). A further subsample was then weighed into tin capsules for isotope ratio mass spectrometry (IRMS). The continuous-flow IRMS system consisted of an elemental analyser (EA 1110, CE Instruments, Milan, Italy) interfaced via ConFlo II (Finnigan MAT, Bremen, Germany) to the gas isotope ratio mass spectrometer (DeltaPLUS, Finnigan MAT). Reference gas (high purity N2 gas, AGA, Vienna, Austria) was calibrated to the atmospheric N2 standard using IAEA-NO-3, IAEA-N-1 and -2 reference material (International Atomic Energy Agency, Vienna, Austria). The standard deviation of repeated measurements of a laboratory standard was 0.15 for 15N.
15N values of above- and below-ground biomass of soybeans were calculated as the sum of the products of N yield and 15N signatures of individual organs, after dividing this by the N yield of shoots or nodulated roots. The proportion (%) of plant N derived from atmospheric N2 (P or %Ndfa) was estimated as given in the introduction. Only 15N values of whole plants were used in the calculations. The B-value (-0.8) was obtained in a preliminary experiment in N-free solution and closely resembled that of Peoples et al. (Peoples et al., 1989).
Statistical analysis
Statistical analysis was performed by analysis of variance (ANOVA) using Statgraphics Plus 4.0 software (Statistical Graphics Corp., Manugistics Inc., Maryland, USA). Data were analysed by one-way ANOVA employing Fishers' LSD or Scheffé post-hoc test. Regression analysis and curve fitting was done with SigmaPlot for Windows Version 4.00 (SPSS Inc., San Rafael, California, USA) and Statgraphics Plus 4.0.
Literature data
In order to test whether a shift in the internal 15N distribution in legume organs might be more widely applicable in estimating legume reliance on N2 fixation, literature data for 15N of plant parts, biomass yields and %Ndfa were compiled.
Soybean
(Kohl et al., 1980; Shearer et al., 1980): Data of soybean (Glycine max (L.) Merill) from three experiments, N-free hydroponics, low-N soil in the glasshouse and a field study were combined. %Ndfa was calculated by the NA method using a non-nodulating soybean isoline as reference plant (Kohl et al., 1980). Organ-specific 15N values were derived from total plant 15N values (Kohl et al., 1980) and the differences in 15N abundance between a plant part and whole plants (Shearer et al., 1980). Average shoot values were obtained from a table, those of nodulated roots had to be calculated from individual values of roots and nodules. Values for three different growth stages were included, namely 56-63 (R5), 77-80 (R7) and 94-98 d after planting (R8; Fehr et al., 1971).
Cowpea
(Ayisi et al., 2000): Isotope signatures of nodulated roots and shoots of different cowpea (Vigna unguiculata L. Walp.) varieties grown in a N-rich field soil were obtained (Ayisi et al., 2000). 15N values could thus be directly calculated from shoot and nodulated root data. Given the 15N values of reference plants (maize), total plant 15N values for six cultivars at two different harvests and the respective B-value of cowpea, %Ndfa was recalculated by the NA method.
Tagasaste
(Unkovich et al., 2000): The nitrogen isotope composition of tagasaste (Chamaecytisus proliferus Link) plant parts grown at four different nitrate concentrations (0, 1, 5, 10 mM) in sand culture in the glasshouse were derived from graphical data. 15N values of nodulated roots had to be calculated from values of roots and nodules which were weighted by their N yield. However, as no biomass data were available in the reference, the relative contribution of nodule N to below-ground N was assumed to linearily decline from 15% to 5% (line 1) or 11% to 3% (line 2, Fig. 6C
) with increasing nitrate concentration. Nitrogen fixation (%Ndfa) was calculated by the NA method, 15N values of mineral N were directly measured in the nitrate supplied via nutrient solution.
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Results |
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Effect of nitrate concentration on N yield,
15N values and N2 fixation of soybeansThe numbers and mass of nodules (Table 1
) declined significantly with increasing concentrations of nitrate in the solution culture. Symbiotic N2 fixation declined markedly from 84 to 3%Ndfa when the nitrate concentration in the nutrient solutions was increased from 0.25 to 25 mM (Table 2; P<0.05). The inhibition of N2 fixation was most pronounced between 2.5 and 25 mM. Furthermore, the relative nodule N content (as a percentage of total plant N) was highly correlated with %Ndfa calculated by the NA method (Fig. 1A). N yield increased significantly only in stems and roots as nitrate concentration increased (Table 1) whereas in nodules it dropped by a factor of 20. In whole soybean plants, 15N signatures approached those of reference plants as nitrate concentration increased (Table 2). 15N signatures were more variable in stems and leaves (range >18.5) than in roots and nodules (range <12.4) in relation to changes in %Ndfa (Table 1).
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Plotting %Ndfa against
15N signatures of above- and below-ground biomass yielded two divergent relationships (Fig. 2A) and a strongly linear correlation (r2>0.98). At high rates of N2-fixation, above-ground 15N signatures were less positive compared to nodulated root signatures. This relationship was reversed when nodulation and N2 fixation declined (Fig. 2A). Regressing the fractional contribution of N2 fixation to soybean N against the difference between above- and below-ground 15N signatures (15N=15Nshoot-15Nnodulated root) (Fig. 2B) produced a linear relationship:
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15N (Fig. 1B
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) in relation to fractional contribution of N2 fixation to plant N (%Ndfa), and (B) relationship between |
Application of the 15N approach to soil-grown soybean plants and literature data
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With the exception of a greater dry mass and total N in the root fraction, the mass of plant parts was unchanged in response to short-term water deficits (Table 3
). Both drought-stressed ryegrass and soybean became more depleted in 15N. However, this change was only significant in ryegrass (Table 4). Reference plant 15N signatures were obtained for each treatment and these were used to calculate %Ndfa of control and drought-stressed soybean. The %Ndfa values varied widely and were not significantly different among treatments (P<0.05; Table 4). Figure 3 shows a similar decrease in 15N signatures of above-ground biomass when N2 fixation increases as Fig. 2A. This relationship was weak in below-ground parts of soybean plants. Nonetheless, the regression lines for above- and below-ground soybean biomass diverged showing a shift in intra-plant 15N distribution when N2 fixation changed.
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The %Ndfa estimates obtained using the equation for
15N values given above were highly correlated with %Ndfa values from the NA method (r2=0.87; Fig. 4). The regression line had a slope of 1.006. A greater scattering of estimates could only be observed at low N2 fixation rates (<20%Ndfa). The intercept of the regression line of -7% (Ndfa) indicates that the 15N method may underestimate the fractional contribution of N2 fixation in soybeans. The correlation of relative N mass in nodules was higher with %Ndfa calculated from 15N than from the NA technique (r2=0.93 versus 0.78, Fig. 5A, B).
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Only three studies, with soybean (Kohl et al., 1980; Shearer et al., 1980), cowpea (Ayisi et al., 2000) and tagasaste (Unkovich et al., 2000), were found where
15N values and %Ndfa could be calculated and compared to this study. The regression analysis of 15N and %Ndfa gave a similar slope (-11.2 versus -8.9), but a lower intercept with the y-axis for soybean plants of different growth stages and cultivation conditions (hydroponics, soil; glasshouse, field) (Kohl et al., 1980; Shearer et al., 1980) than this study (5.8 versus 19.8, Fig. 6). This was due to more positive 15N values at very low %Ndfa in this investigation. The negative relationships between 15N and %Ndfa were strong and highly significant for all three plant species (r2>0.83; P<0.019 in tagaste, P<0.0001 in cowpea/soybean, Fig. 6). However, large variation was found in the slopes of regression lines, these varying between -6 (cowpea) and -94 (tagasaste). The close correlation between %Ndfa obtained by the NA and 15N techniques is exemplified for the glasshouse and field data of soybean (Fig. 7). The regression equation is %Ndfa (15N)=0.704xNdfa(NA)+20.5 (r=0.945, P<0.0001). The intercept of regression was higher than zero and the slope lower than one, thus, leading to an increasing over-estimation of %Ndfa in the lower N2 fixation range when using the 15N method.
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15N distribution within soybean plants at different hydroponic nitrate supplies
The rate of supply of nitrate via nutrient solution significantly modified symbiotic N2 fixation, growth and N allocation to different plant parts of soybean (Table 2
). In addition, the pattern of 15N distribution within soybean changed markedly (Fig. 2A; Table 1). Nodules, and to a lesser extent roots, were considerably enriched in 15N at high levels of %Ndfa (Table 1) whereas shoots were 15N depleted. 15N enrichment of nodules is a common phenomenon in a wide range of legumes like soybeans, lupins and Vigna species (Bergersen et al., 1988; Kohl et al., 1983; Shearer et al., 1980, 1982; Boddey et al., 2000). This enrichment has been related to nodule metabolism, and was shown to increase with nodule age and to vary with rhizobial strains (Bergersen et al., 1986; Kohl et al., 1983; Ledgard, 1989; Shearer et al., 1984; Steele et al., 1983). However, the underlying isotope effects and discrimination processes leading to this non-uniform 15N distribution within legumes remain poorly described, notwithstanding recently developed models of the 15N signatures for nitrate-fed plants (Robinson et al., 1998). None of the nitrogenase reaction, ammonia export from bacteroids, denitrifying activity of rhizobial strains, synthesis of ureides versus amides as export compounds or isotope effects at metabolic branchpoints in nodule metabolism, could explain the observations (for review see: Shearer and Kohl, 1986; Kohl and Shearer, 1995).
The changing pattern of 15N distribution at enhanced nitrate supply (Table 1; Fig. 2A) was partly attributable to decreased activity and biomass of nodules (Fig. 1) and partly to increased incorporation of N derived from the uptake of mineral N into nodules and roots (Fig. 2A; Shearer et al., 1984). At the greatest nitrate concentration, biomass and activity of nodules diminished and 15N partitioning between soybean plant parts was reversed resulting in 15N enrichment of leaves compared to root systems (Fig. 2A). This is presumably due to a switch in isotope discrimination from nodule metabolism to mineral N acquisition. Several putative factors and processes govern 15N distribution within plants. 15N discrimination during N acquisition depends on N form and N concentration, commonly results in 15N depletion of plants with respect to its source (Yoneyama and Kaneko, 1989; Yoneyama et al., 1991, 2001; Kohl and Shearer, 1980; Evans, 2001), but does not per se lead to intra-plant variation of 15N. However, as outlined earlier (Evans et al., 1996), nitrate utilization resulted in a gradient in the 15N distribution between tomato roots and shoots. This was attributed to partial assimilation of the nitrate pool in roots, retention of a fraction of reduced N in below-ground biomass, and export of 15N-enriched nitrate to the shoots. Thus, leaf 15N was greater than root 15N when tomato plants were grown on nitrate. The same isotope pattern was obvious with soybeans relying to >90% on mineral N uptake (Fig. 2A).
These changes in type and site of N isotope fractionation within soybean plants were linearily correlated with the change in the dependence on symbiotic N2 fixation (Fig. 2B). The difference between 15N of above-ground and 15N of below-ground soybean biomass (15N) was regressed against soybean %Ndfa (%Ndfa=19.8-8.9x15N). An advantage of 15N is that it is independent of the isotope composition of available mineral N, since it results from internal 15N partitioning processes. These processes will be governed by intrinsic isotope fractionations during nodule or root/shoot metabolic activity. From the available evidence changes in source 15N alone (i.e. without accompanying change in amounts of N) will shift 15N signatures of all plant material and 15N should not be changed.
Soil-grown soybean and comparison with literature data
The precision and accuracy of the 15N approach (using the 15N difference between nodulated roots and shoots to quantify %Ndfa) was assessed with soil-grown soybean plants in the glasshouse. Estimates of soybean reliance on N2 fixation obtained by the 15N approach were highly correlated with %Ndfa from the NA method (Fig. 4). The nearly 1:1 slope of the regression line suggested no systematic trend or bias in calculating %Ndfa via the NA method or the 15N approach. A greater scatter of estimates at low rates of N2 fixation (<20%Ndfa) might well have been predicted. The intercept of -7%Ndfa suggests that the 15N method slightly underestimates fractional contributions of N2 fixation to soybean N. However, the intercept value might also be due to small uncertainties in measuring the exact isotopic composition of available soil N using the reference plant approach. The latter is a known influence on %Ndfa estimates (NA method), particularly at the low end of the range (Unkovich et al., 1994). The correlation of relative N mass in nodules as one indicator of N2 fixation was higher with %Ndfa calculated from 15N as from NA (Fig. 5A, B).
Data obtained from the literature revealed that, similar to soybean plants in the present study and as previously described (Kohl et al., 1980; Shearer et al., 1980), cowpea (Ayisi et al., 2000) and tagasaste (Unkovich et al., 2000), exhibited similar negative relationships between 15N and %Ndfa (by NA method, Fig. 6A–C). Striking differences were found in the slopes of regression lines. The precision of the 15N approach to estimate %Ndfa improves when the calculated difference between 15N at 0 and 100%Ndfa is large. This difference was low in tagasaste (1.2, Fig. 6C), higher in soybeans (8.9, Fig. 6B; 11.3 in this study) and highest in cowpea (16.6, Fig. 6A). As the precision of 15N measurements by modern isotope ratio mass spectrometry is higher than 0.15 (SD), this potentially allows %Ndfa to be estimated by 15N with a precision in excess of 16.6% (tagasaste), 2.2% (soybean) and 1.2% (cowpea). Clearly, the 15N approach will be restricted to legume species which exhibit large shifts in their internal 15N distribution in relation to N nutrition.
Earlier studies demonstrated marked effects of rhizobial strain and legume cultivar on nodule and thus below-ground 15N enrichment (Bergersen et al., 1986; Kohl et al., 1983; Steele et al., 1983). Nonetheless, in these studies variations in 15N were commonly the result of differences in N2 fixation of the studied legume–rhizobia associations, as indicated by the correlation of nodule 15N enrichment with the amount of nodule N or with relative N2 fixation efficiency of rhizobial strains (Kohl et al., 1983; Bergersen et al., 1986). Furthermore, the cowpea study of Ayisi et al. included six different cultivars with widely differing extent of nodulation in a field situation and this did not affect the relationship between Ndfa and 15N (Ayisi et al., 2000).
As demonstrated above, a high similarity of regression lines was obtained in this investigation and a soybean study including a great variety of growth stages under glasshouse and field conditions (Kohl et al., 1980; Shearer et al., 1980). This suggests that (a) soybean nodulation by natural rhizobial populations in the field or specific rhizobial strains in glasshouse studies did not significantly alter the relationship between %Ndfa and 15N, (b) this technique showed a marked robustness of the 15N values over different growth conditions (Fig. 6A) and (c) its applicability is further indicated by the strong correlation between %Ndfa (NA) and %Ndfa (15N) (Fig. 7).
As reported previously (Evans et al., 1996), ammonium uptake leads to a slightly different pattern in the 15N distribution within plants compared with nitrate. This might require the calibration of 15N with %Ndfa not only at different nitrate but also ammonium levels to account for possible differences in internal isotope fractionations in field situations where ammonium is highly available.
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Conclusions |
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It has been shown that the intra-plant variation in
15N of a specific soybean cultivar depended on the fractional contribution of symbiotic N2 fixation to plant N. Based on this, a new method to investigate N2 fixation by legumes is proposed. After ‘calibration’ for a specific legume species, the 15N technique offers a way to quantify %Ndfa without the need of reference plants. It might also circumvent the problems of spatial and temporal changes in the isotopic composition of plant available soil N. As the approach does not depend on the isotopic composition of source N, it might be suitable in situations where 15N of available soil N is close to atmospheric N2 and, hence, the NA method is of only limited use. Further studies are necessary to (a) show how significant cultivars and rhizobial strains will affect 15N values in soybean, and (b) to extend it to other crop and forage legume species that show significant shifts in their 15N distribution between plant parts in relation to changes in %Ndfa.
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Acknowledgements |
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We are indebted to the following practical students in helping to raise the plants and in supporting us during harvest and sample preparation: Andreas Blöchl, Julia Hofmann, Edith Huber, Nicole Mandl, Jan Mayrhofer, Gabi Pietsch, and Katja Pörtl. We also thank Mark Adams and two anonymous reviewers for their careful review of this manuscript.
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Footnotes |
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3 To whom correspondence should be addressed. Fax: +43 1 4277 9542. E-mail: wolfgang.wanek{at}univie.ac.at
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References |
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