The Use of Nitrogen-15-depleted Ammonium Sulfate for Estimating Nitrogen Fixation by Leguminous Trees

The feasibility of using N-depleted (NH4)2S04 for assessing biological N2 fixation of leguminous trees was investigated. A slowgrowing species, lebbeck [Albizia lcbbeck (L.) Benthl, and a fastgrowing species, leucaena [Leucaena leucocephala (Lam.) de Wit] were inoculated with appropriate Rkizobium strains and grown under four different levels of N. Each week, 0, 5, 12.5, or 25 mg of Ndepleted (NH4)2S04 was applied to the upper chamber of the Leonard assembly. After 10 and 18 weeks growth for leucaena and lebbeck respectively, plants were separated into shoots, roots, and nodules for analysis. The atom% N in shoots and roots was linearly correlated to the amount of applied 'N-depleted (NH4)ZS04. Significant differences in atom%N and percentage N derived from N2 fixation were measured in lebbeck and leucaena when, respectively, more than 8.3 and 2.9% of the total N was derived from (NH4)2SO4. Nodules derived a larger portion of their total N from N2 fixation than did the roots or shoots, indicating that most of the N present in the nodules was derived from N2 fixation. Nodules of both species at the no N treatment showed slightly higher atom% N values than the N-natural abundance level of atmospheric N2. Additional index words. Leucaena, Leucaena leucocephala (Lam.) de Wit, lebbeck, Albizia lebbeck (L.) Benth, Nitrogen-15 dilution, Rkizobium, Nitrogen-allocation. SINCE the heavy, stable isotope of nitrogen, N, has been available (Thade and Urey, 1939), there have been numerous reports of the use of this isotope for the estimation of biological N2 fixation (Hauck and Bystrom, 1970). In field studies, biological N2 fixation (BNF) has been estimated by applying N-enriched salts to the soil and measuring the level of N uptake into both N2-fixing plants and non-N2-fixing reference plants. The N enrichment level of the material applied varies, depending on factors such as the total N applied, the available N in the soil, and the expected total N uptake by the growing crop. Although current ratio mass spectrometers can precisely measure differences as small as 0.0005 atom% N, few field studies have been conducted where materials were applied with an enrichment level of < 1 atom% N. 2 AGRONOMY JOURNAL, VOL. 78, MAY-JUNE, 1986 1.1 for leucaena and 50.9 + 5.4, 75.5 + 8.6, and 19.2 + 3.1 for lebbeck, respectively. Levels of (NH4)2SO4 applied were probably too low to cause detectable responses in dry matter or plant N accumulation. Tables 1 and 2 show the atom% N and the calculated percent N derived from N2 fixation (%NdfA) for the shoots, roots, and nodules of the leucaena and lebbeck. The atom% N in shoots and roots was linearly correlated to the amount of applied N-depleted (NH4)2SO4. The corresponding correlation coefficients for those regression lines for shoots and roots were 0.94 and 0.93 for leucaena and 0.96 and 0.93 for lebbeck, respectively. The lowest level of applied N did not generally produce significant differences for atom% N as compared with the no N treatment. Differences occurred as the amount of N-depleted (NH4)2SO4 applied was increased to a level of 12.5 and 25 mg (NH4)2SO4 per week. Data indicate that when more than 3% of total N in leucaena and 8.5% in lebbeck is derived from the inorganic N source, significant differences in atom% N can be measured when compared with the seedlings receiving no (NH4)2SO4. Values for atom% N are lower for lebbeck than for leucaena at every level of applied N. This difference is due to the slower growth of lebbeck and the greater amount of isotope it received in comparison to leucaena. Lebbeck seedlings had received 0, 40, 100, or 200 mg more Ndepleted (NH4)2SO4 than the leucaena seedlings at the same treatment level at the time of harvest. The atom% N values of the nodules remained much closer to the value of atmospheric N2 than did the other plant parts. In the 0 mL N treatment the different plant parts of both species had an atom% N close to the natural N abundance level of 0.3663 (Junk and Svec, 1958). Nodules of both species at the no N treatment showed slightly higher atom% N values than the corresponding values for roots and shoots or the N-natural abundance level of atmospheric N2. Similar higher atom% 15N values have been reported Results and Discussion Total N of the different plant parts of leucaena and lebbeck were not significantly different, regardless of the N application. Total mg N + SE of shoots, roots, and nodules were 84.5 + 7.6, 55.2 + 4.1, and 12.0 + After seed germination, lebbeck seedlings were inoculated with turbid suspensions of Rhizobium strains TAL 1597 and leucaena with TAL 1145 (NifTAL Project, Box 0, Paia HI). Treatments were replicated five times in a completely randomized design. The leucaena and lebbeck seedlings were harvested at 10 and 18 weeks after planting, respectively. Shoots, roots, and nodules were separated, dried for 48 h at 70°C, weighed, and ground in a Cyclone mill. Plant samples were digested in a salicylic acid-sulfuric acid mixture, using a Se-KZSO4/CuSO4 catalyst (Bremner and Mulvaney, 1982) to include nitrate and nitrite. The digests were made alkaline with 13 M NaOH, steam distilled for 7 min, and the NH3 trapped in 10 mL of 0.01 M H2SO4Twenty milliliters of ethyl alcohol was distilled through the steam apparatus between all samples. The distillate was subsampled and total N determined by the indophenol blue method (Keeney and Nelson, 1982). The pH of the remaining distillate was adjusted to a value between 4 and 5 and evaporated to near dryness for mass spectrometer analysis. The N analyses were conducted by Isotope Service, Inc., Los Alamos, NM, and at the laboratory of Dr. R.H. Burris, University of Wisconsin, Madison, WI. The percentage N derived from N2 fixation was calculated using the following equations: 1. atom% N excess sample = atom% N sample atom% N of comparable part from plants receiving no mineral N. 2. atom%N-excess of mineral N applied =atom%N mineral N applied 0.3663. Due to the large seed size, particular for lebbeck, a correction was made for calculating the percent N derived from N2 of shoots and roots. Total N in seed of lebbeck and leucaena was 7.9 and 2.5 mg N, respectively. The partitioning of seed N between shoot and root was assumed to be equal and seed N transported to the initial nodule formation was considered to be insignificant. The difference in atom% N between atmospheric N2 and seed N was found to be insignificant. 3. Percent N derived from the N2 fixation and seed N (%NdfAS) =