In an investigation of the ability of alfalfa to fix nitrogen under field conditions in Scandinavia, N2 ... the crops because of nitrogen deficiency in the non-N2-fixing.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1984, p. 702-707 0099-2240/84/100702-06$02.00/0 Copyright ©D 1984, American Society for Microbiology
Vol. 48, No. 4
Nitrogen Fixation in an Establishing Alfalfa (Medicago sativa L.) Ley in Sweden, Estimated by Three Different Methods ANNA M. MARTENSSON* AND HANS D. LJUNGGREN
Department of Microbiology, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden Received 25 July 1983/Accepted 12 July 1984
In an investigation of the ability of alfalfa to fix nitrogen under field conditions in Scandinavia, N2 fixation during the establishment year ranged between 7.85 and 10.37 g of N m-2, depending on the method used. The methods used were an in situ acetylene reduction method, a '5N isotope dilution method using two reference crops, and a total-N difference method. The dynamics of nitrogenase activity in relation to plant development was studied by using the acetylene reduction method. Also, the diurnal variation in N2 fixation at the field site was studied with the acetylene reduction method; no diurnal change was detected, which is explained by the fact that the nodules within the soil were protected against short-term fluctuations in temperature. The significant amount of nitrogen fixed by alfalfa during its first year even at northern latitudes suggests that this crop offers an alternative to conventional field management of heavily fertilized nonlegume leys.
In the present study the N2 fixation in an establishing alfalfa ley was estimated with three different methods in order to get a reliable value for N, fixation, information which so far seems to have been neglected. Since the early investigation of Arny and Thatcher (2), several attempts have been made to estimate N, fixation by field-grown legumes by using various available methods and techniques. Following the discovery of the acetylene reduction (AR) method (6, 11, 19), this approach aroused enormous interest, not only in laboratory experiments but also in its application to field situations (3). However, in recent years, several questions concerning the reliability of using the AR method for total N2 fixation estimations have been raised. Fluctuations of the conversion factor (10, 20) and the influence of the plant phenology at the time of incubation (Matrtensson and Ljunggren, Plant Soil, in press) have been discussed. Accordingly, to obtain reliable data, sampling must be performed at frequent intervals, thus integrating N2 fixation during the season, which is quite easily done by using the in situ method described in this paper. Various techniques using '5N are attractive alternative methods to the AR method. Without doubt, the most sensitive method is incubation of the N2-fixing system in a 15Ncontaining atmosphere, resulting in incorporation of 15N in the plant. The disadvantage of the AR method as a shortterm kinetic reaction also applies to 15N) incubations. Another available technique using 15N is the isotope dilution method (15), in which low levels of 15N-labeled fertilizer are applied to both a N2-fixing crop and a non-N2-fixing (reference) crop. Thus, the 15N content of the N2-fixing crop is less than that of the non-N2-fixing crop due to uptake of unlabeled atmospheric nitrogen via N2 fixation. This method results in an integrated value for seasonal nitrogen fixation. However, if the same amount of 15N-labeled fertilizer is applied to the test crop and reference criop, the nitrogen fertilizer applied to the N2-fixing crop may disturb the nitrogenase activity, or its amount may be too low for the non-N2-fixing crop to develop normally. To overcome this problem, Fried and Broeshart (7) used different levels of 15N-labeled fertilizer for the crops. The estimations of total N2 fixation were based on the A-value concept (8), which states that if a crop is offered different nitrogen sources, it
will take up nitrogen from them in proportion to the quantities available. A less sophisticated method for estimating N2 fixation is the total-N difference method (2). The main disadvantage with this method is the differences in root activity between the crops because of nitrogen deficiency in the non-N2-fixing crop. Because of the uncertainty in the methods mentioned above, we compared the methods in order to be able to decide which method is preferable. The lack of up-to-date information on the dynamics of N2 fixation, especially during the establishment year at a field site in northern Europe, made this investigation pertinent for discussions of improvement of field management by using biological N2 fixation. MATERIALS AND METHODS Plant material. Alfalfa (Medicago sativa L. cv. Vertus; Weibull AB, Landskrona, Sweden) and meadow fescue (Festuca pratensis L. cv. Boris; Svalof AB, Svalov, Sweden) were sown in the middle of May at the field site. Appropiate seed was inoculated with a mixture of Rhizobium meliloti strains obtained from the Leguminous Plant Laboratory, Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala. Uninoculated alfalfa did not form nodules during the season since the soil lacked indigenous R. meliloti. Field site. The Kjettslinge Experimental Field is situated at 60°10' N and 17°38' E in central Sweden. Each crop was represented by four plots (14 by 40 m), and the plots were arranged as a randomzied block. The soil at this site consists of a sandy silt loam to a depth of 40 cm (clay content, 28%; organic matter, 2.4%; pH 6.9); underlying this layer is a heavy glacial clay. Plant sampling. Following a plant growth period of 13 weeks, the above-ground material was collected in midAugust. The final harvest of the year, also of above-ground material, was carried out at a plant age of 25 weeks in early November. Following each harvest, all plant material was air dried and weighed. The dry material was ground in a high-speed sample grinding mill and carefully mixed. Kjeldahl digestion was carried out to determine the total nitrogen content. The i5N-labeled material was analyzed by mass spectrometry (5).
* Corresponding author. 702
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Jun
Jul
Aug
Sep
Oct
Nov
FIG. 1. N2 fixation by field-grown alfalfa during the establishing year, as estimated by an in situ AR technique. n 10. Bars indicate standard deviations. Symbols: 0, N2 fixation rate; accumulated N2 fixation. =
In situ AR assay. In each of the inoculated alfalfa plots, microplots (1 by 2 m) were used for distribution of three plastic cylinders, which consisted of commercially available polyvinylchloride drainage tubes (inside diameter, 185 mm; length, 350 mm). These cylinders were pushed into the soil, leaving approximately 30 mm above the soil surface and enclosing a mean of 30 young plants. A rubber tire was placed around the rim. The cuvettes remained in the microplots during the season. At harvest, the plant material from each cuvette was collected separately. During incubation for AR measurements, gas-tight plastic bags were attached around the rims of the cuvettes with hose clamps, thus enclosing the plant-soil system. Then 300 ml of commercial-grade acetylene (AGA, Stockholm, Sweden) and 0.50 ml of propane were injected through sampling ports in the plastic bags. Gas samples (1.00 ml) were withdrawn after 20 and 75 min of incubation, stored in sealed glass bottles during transport to the laboratory, and immediately analyzed by gas chromatography. Calculations of N2 fixation were carried out by using the method of Balandreau and Dommergues (3) and a conversion factor of 4.41, which was estimated by using 15N2 incubations (18). The occurrence of endogenous ethylene formation was investigated by incubating a cuvette with 0.1 atmosphere (10 kPa) of C2H2 and 0.02 atmosphere (2 kPa) of CO, as recommended by Nohrstedt (14). Diurnal variation was investigated during the field season by using 12 randomly distributed field cuvettes. Three cuvettes were incubated every sixth hour during 24 h. The experiment was carried out twice, both times during the autumn. During the incubations, the light intensity, soil temperature, and air temperature were recorded. A study of the diurnal rhythm was also made with 6-month-old inoculated alfalfa raised in pots filled with sand. One week before measurement, the plants were transferred into two growth chambers. Each chamber was given
703
12 h of light (300 W m-2) and 12 h of dark, but the two were kept at different day/night temperatures; one was kept at a constant temperature of 20°C, and the other was kept at 20°C during light conditions and at 15°C during the night. Every sixth hour during a day, five pots were incubated with acetylene by placing the pots in plastic bags which were sealed and incubated with 200 ml of acetylene and 0.2 ml of propane. Samples were withdrawn after 30 and 60 min and analyzed as described above. A-value method. Four small microplots (1.25 by 1.25 m) were used in each of the inoculated and uninoculated alfalfa replicates, as well as in the meadow fescue replicates. The microplots were supplied with '5N-labeled fertilizer as calcium nitrate in aqueous solution between the plant rows by using a sprinkler bottle, within a 1- by 1-m square; the outer parts of the microplots were supplied with the same amounts of unlabeled fertilizer. At a plant age of 3 weeks, 6.30 g of N m-2 (0.858 atom% 15N excess) was sprinkled onto the uninoculated alfalfa microplots; 4.80 g of N m-2 (0.812 atom% 15N excess) was applied to the meadow fescue, and 3.10 g of N m-2 (1.303 atom% 15N excess) was applied to the inoculated alfalfa. During harvest, the above-ground plant material was collected in an inner area (0.75 by 0.75 m) of the 15N-labeled square. At 1 week following harvest, 4.10 g of N m2 (1.187 atom% 15N excess) was sprinkled onto the plots of the two reference crops; the inoculated alfalfa was supplied with 2.60 g of N m-2 (1.183 atom% 15N excess). N2 fixation was estimated by the method of Fried and Broeshart (7), using the uninoculated alfalfa and meadow fescue as examples of two different reference crops. Total-N difference method. The above-ground biomass was harvested in each of the inoculated and uninoculated alfalfa microplots (1 by 1 m). Total N2 fixation was determined as described previously (2). RESULTS AND DISCUSSION Figure 1 shows the N2 fixation rate (in grams of N per square meter per day) and the accumulated N2 fixation (in grams of N per square meter) during the field season as determined by the AR method. In this figure, the dynamics of N2 fixation during the field season are clearly demonstrated. As the plants emerged, N2 fixation increased until beginning of flowering (i.e., just before harvest). The harvest was followed by a regrowth of the plants. N2 fixation decreased rapidly, but after 3 weeks it increased until a final decline occurred in late autumn. This decline was due to climatic factors as the plants did not develop buds or flowers. The above-ground total nitrogen production and the rate of nitrogen accumulation (both in grams of N per square meter) are shown in Fig. 2. Both of these parameters are very well correlated with the N2 fixation activity during the first part of the season. At harvest N2 fixation supplied the crop, including the root biomass, with 5 g of N m-2 according to AR measurements. The above-ground nitrogen production at the same time was 12.5 g of N m-2. Consequently, N2 fixation at this stage supplied the crop with less than 40% of the nitrogen needed. Therefore, the dynamics of nitrogen accumulation seem to be a consequence of plant phenology rather than N2 fixation activity. The latter is probably regulated by a surplus of energy available from plant photosynthesis. When buds are being formed, the accumulated nitrogen in the plant tissues is translocated, and the available energy is made use of at bud formation and nitrogen translocation. Most of the soil nitrogen in Swedish
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TABLE 1. Nitrogen contents and N excesses in different crops Harvest
First
content Nitrogen (g of N m-2)
'5N excess
Inoculated alfalfa
13.78 10.60 12.71 12.02
0.348 0.361 0.326 0.338
Uninoculated alfalfa
13.70 13.15 11.92 12.31
0.378 0.432 0.395 0.385
Meadow fescue
8.98 8.50 7.92 8.00
0.308 0.381 0.352 0.350
Inoculated alfalfa
8.67 8.14 8.62 8.13
0.116 0.133 0.129 0.103
Uninoculated alfalfa
5.23 5.59 5.62 5.78
0.397 0.412 0.407 0.423
12.55 14.07 11.85 12.32
0.332 0.344 0.265 0.302
Crop
(N
,
IE
z
E z
Cn 0
r-
10 X,
0
E D
E
u
Cu
u U
(atom%)
z
Second z
S
r 23 Jun
Harvest
14 27 10 24 JuL Aug
11 23 12 4 Oct Nov Sep FIG. 2. N accumulation by field-grown alfalfa during the establishing year, as estimated by N content in shoot material. Symbols: 0, N accumulation rate; *, accumulated N. (A. Sjolander and E. Steen, personal communication.)
soils is available as NO3-, and the reduction of N03- is generally energetically as expensive as N2 fixation (16). During the latter part of the season the N2 fixation rate and the rate of above-ground nitrogen accumulation were not very well correlated. From a practical point of view, the latest harvest of perennial ley legumes takes place early enough to allow winter buds to develop or later, when winter buds are formed but too late for the buds to develop before frost (the actual harvest in this experiment). The last harvest in this area of Sweden should take place from the end of August to mid-September. It is also known that overwintering is enhanced by a high stubble setting. Based on our data,
Meadow fescue
the ideal time for the latest harvest, acquired by experience, corresponds to the time of the maximum rate of accumulation of nitrogen (11 to 23 September). There was no indication that an overestimate occurred due to endogenous ethylene formation, because no ethylene was detected when incubation was done with 0.1 atmosphere (10 kPa) of C2HI2 and 0.02 atmosphere (2 kPa) of CO. The variation in N2 fixation obtained among the cylinders was found to be correlated with differences in biomass in the cuvettes, thus reflecting the natural biological variation. The diurnal variation in N2 fixation was negligible at the field site (Fig. 3 and 4). This is in contrast to the results of
TABLE 2. Calculation of amount of N fixed by alfalfa using A-values and non-nodulated lucerne and meadow fescue as reference crops Harvest
First
Second
Crop
Nodulated alfalfa Non-nodulated alfalfa Meadow fescue
Nodulated alfalfa Non-nodulated alfalfa Meadow fescue
Amt of N
fixed
Amt of N applied (g of N m2)
N excess in fertilizer
3.10
1.803
13.21 + 0.71'
6.30
0.812
6.60 ± 0.75
6.61 ± 1.46
4.98 ± 1.44
4.80
0.858
7.11 ± 1.07
6.10 ± 1.78
4.59 ± 1.98
2.60
1.183
23.24 ± 3.05
4.10
1.187
7.78 ± 0.31
A-value"
AA-value
(atom%)
Fraction of fertilizer used by N-fixing system (FU)b
calculated from (g of N m-)
0.753 ± 0.074
0.328 ± 0.040 15.46 ± 3.36
5.07 ± 0.56
4.10 1.187 11.72 ± 1.84 11.52 t 4.94 3.78 ± 0.60 a Single A-values were calculated from the values in Table 1. Mean values were then calculated from the A-values. bFixation units (FU) were calculated as follows: ('5N excess in inoculated alfalfa/'5N excess in applied N) x (N content in inoculated alfalfa/amount of N applied). Single values were calculated from the values in Table 1; mean values were then calculated from these values. c Mean ± standard deviation.
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hour FIG. 3. N2(C2H2) fixation rates (A) at the Kjettslinge Experimental Field, 20 to 21 August 1982. Bars indicate standard deviations. n = 3. Irradiation (0), air temperature (0), and soil temperature (0) were recorded at 2-h intervals.
Abdel Wahab (1), who investigated alfalfa in Egypt. However, this result was obtained with air temperature fluctuations of 200C. We think that the reason for not detecting any diurnal variation at the Kjettslinge field site was due to the fact that the alfalfa nodules with the active enzyme system were protected against short-term fluctuations in air temperature as only slight changes in soil temperature occurred during the day. The field observation (i.e., that no daily changes in N2 fixation occurred) was also confirmed by an experiment with alfalfa plants in growth chambers. A diurnal variation in AR activity was observed only when the plants
were exposed to different day and night temperatures in the root medium. Several authors have reported similar results in investigations of different leguminous systems. For instance, a good correlation between changes in soil temperature and N2(C2H2) fixation was obtained by Zary and Miller (22), who investigated southern pea. In white clover kept at a constant temperature but with varying light and dark periods, AR activity remain constant during the day (12). Daily changes in N2(C2H2) fixation appear to parallel variations in soil temperature (9). Concerning the applications of the A-value method, the
0600 hour FIG. 4. N2(C2H2) fixation rates (A) in alfalfa at the Kjettslinge Experimental Field, 11 to 12 October 1982. Bars indicate standard deviations. n = 3. Irradiation (0), air temperature (0), and soil temperature (O) were recorded at 2-h intervals.
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TABLE 3. Calculation of amount of N fixed by alfalfa using the total-N difference method Harvest
Nitrogen content of inoculated alfalfa (g of NM 2)
First Second
12.65 ± 0.57 9.57 ± 0.98
Nitrogen content of uninoculated alfalfa (g of NM2)
Nitrogen fixed (g of N M-2)
8.83 ± 0.69 5.54 ± 0.32
3.82 ± 1.20 4.03 ± 1.30
goN
derived nitrogen contents and the 15N excesses are shown in Table 1. The calculated A-values, the AA-values, and the calculated fractions of fertilizer used by the inoculated alfalfa are shown in Table 2. The calculated amounts of N2 fixation during the two growth periods are also shown in Table 2. There were no statistically significant differences between the values derived from the two different perennial crops. Our conclusion is that under the given field situation, either of the two reference crop gives a proper value for N2 fixation during the first year of the ley. This is contrary to results obtained by Rennie et al. (17), who showed that great differences in N2 fixation estimates could be obtained by using different reference crops. However, these data were obtained with annual crops. The applied total-N difference method offered a cheap and simple alternative to the other methods. The results obtained may be quite misleading since nitrogen deficiency may have occurred in the uninoculated reference system, resulting in a lower root activity (Table 3). It is interesting to note that we obtained the same relationship between the estimates made with the total-N and 15N methods as Witty (21) reported (i.e., the total-N value was about 75% of the '5N value). For the estimated values of the amounts of nitrogen fixed by an alfalfa ley in Sweden (Table 4), we found a range of 7.85 to 10.37 g of N m2. If we compare these figures with values from other investigations made under temperate conditions but using just one method for the estimations, the values obtained seem quite promising for extending alfalfa cultivation in Scandinavia. For instance, in the IBP field experiments carried out in the United Kingdom (4), the average nitrogen fixed during the first year was reported to be 14.2 g of N m-2. However, this figure was obtained with three cuts; recalculating this figure to be valid for just two cuts gives 9.4 g of N m2, which is in agreement with our results in Sweden. If we compare our figures with reports of N2 fixation under hot climate conditions (for instance, the IBP field experiments in Bulgaria, India, and Tunisia, with an average of 5.2 g of N m-2 fixed during the first year [15]), evidence is obtained supporting the observations that alfalfa TABLE 4. Estimated total nitrogen fixation derived by an in situ AR method, a 15N dilution method, and a total-N difference method Method
AR A-value
Total-N difference
Reference crop
Uninoculated alfalfa Meadow fescue Uninoculated alfalfa
(g of N mf-2) 10.37 10.05 8.37 7.85
+ 3.97b ± 2.00 ± 2.58 ± 2.56
P value'
NSc NS 0.05
a Each nitrogen fixation value was compared with the AR method value. Mean + standard deviation. c Each A-value was compared with the other; there were no significant differences between the values obtained.
fixes more nitrogen under temperate conditions and that it seems to endure low temperatures better than high ones. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Council for Planning and Coordination of Research, the Swedish Council for Forestry and Agricultural Research, the Swedish Natural Science Research Council, and the Swedish National Environment Protection Board. The studies were carried out as part of a project entitled "Ecology of arable land. The role of organisms in nitrogen cycling."
LITERATURE CITED 1. Abdel Wahab, A. M. 1980. Diurnal variations of N2(C2H2)-fixing activity in Medicago sativa under water stress. Nat. Monspel. Ser. Bot. 35:1-7. 2. Arny, A. C., and R. W. Thatcher. 1915. The effect of different methods of inoculation on the yield and protein content of alfalfa and sweet clover. J. Am. Soc. Agron. 7:172-185. 3. Balandreau, J., and Y. Dommergues. 1973. Assaying nitrogenase (C2H2) activity in the field. Bull. Ecol. Res. Commun. 17:247-254. 4. Bell, F., and P. S. Nutman. 1971. Experiments on nitrogen fixation by nodulated lucerne. Plant Soil (Special Volume):231264. 5. Bergersen, F. J. 1980. Measurement of nitrogen fixation by direct means, p. 65-110. In F. J. Bergersen (ed.), Methods for evaluating biological nitrogen fixation. John Wiley & Sons, Inc., Chichester, England. 6. Dilworth, M. J. 1966. Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum. Biochim. Biophys. Acta 127:285-294. 7. Fried, M., and H. Broeshart. 1975. An independent measurement of the amount of nitrogen fixed by a legume crop. Plant Soil 43:707-711. 8. Fried, M., and L. A. Dean. 1953. A concept concerning the measurement of available nutrients. Soil Sci. Soc. Am. J. 73:263-271. 9. Halliday, J., and J. S. Pate. 1976. The acetylene reduction assay as a means of studying nitrogen fixation in white clover under sward and laboratory conditions. J. Br. Grassl. Soc. 31:29-35. 10. Hardy, R. W. F., R. C. Burns, and R. D. Holsten. 1973. Applications of the acetylene-ethylene assay for measurement of nitrogen fixation. Soil Biol. Biochem. 5:47-81. 11. Hardy, R. W. F., R. D. Holsten, E. K. Jackson, and R. C. Burns. 1968. The acetylene-ethylene assay for N2 fixation: laboratory and field evaluation. Plant Physiol. 43:1185-1207. 12. Masterson, C. L., and P. M. Murphy. 1976. Application of the acetylene reduction technique to the study of nitrogen fixation by white clover in the field, p. 299-316. In P. S. Nutman (ed.), Symbiotic nitrogen fixation in plants. IBP 7. Cambridge University Press, Cambridge. 13. McAuliffe, C., D. S. Chamblee, H. Uribe-Arango, and W. W. Woodhouse, Jr. 1958. Influence of inorganic nitrogen in nitrogen fixation by legumes as revealed by '5N. Agric. J. India 50:334337. 14. Nohrstedt, H-O. 1983. Natural formation of ethylene in forest soils and methods to correct results given by the acetylenereduction assay. Soil Biol. Biochem. 15:281-286. 15. Nutman, P. S. 1976. Field experiments on nitrogen fixation by nodulated legumes, p. 211-237. In P. S. Nutman (ed.), Symbiotic nitrogen fixation in plants. IBP 7. Cambridge University Press, Cambridge. 16. Postgate, J. R. 1982. Chemistry and mechanism, p. 184-196. In J. R. Postgate (ed.), The fundamentals of nitrogen fixation. Cambridge University Press, Cambridge. 17. Rennie, R. J., D. A. Rennie, and M. Fried. 1978. Concepts of 15N usage in dinitrogen fixation studies, p. 103-133. In Isotopes in biological dinitrogen fixation. Proceedings of an Advisory Group Meeting Organized by the Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture. International Atomic Energy Agency, Vienna. 18. Rice, W. A. 1980. Seasonal patterns of nitrogen fixation and dry
VOL. 48, 1984 matter production by clovers grown in the Peace River region. Can. J. Plant Sci. 60:847-858. 19. Scholihorn, R., and R. H. Burris. 1967. Acetylene as a competitive inhibitor of N2 fixation. Proc. NatI. Acad. Sci. U.S.A. 58:213-216.
20. Schubert, K. R., and H. J. Evans. 1976. Hydrogen evolution: a major factor affecting the efficiency of nitrogen fixation in nodulated symbionts. Proc. Natl. Acad. Sci. U.S.A. 73:1207-
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21. Witty, J. F. 1983. Estimating N2-fixation in the field using '5Nlabelled fertilizer: some problems and solutions. Soil Biol. Biochem. 15:631-639. 22. Zary, K. W., and J. C. Miller, Jr. 1980. The influence of genotype on diurnal and seasonal pattern of nitrogen fixation in southern pea (Vigna unguiculata (L.) Walp.). J. Am. Soc. Hortic. Sci. 105:699-701.
