Can the 15N dilution technique be used to study N2 fixation in tropical tree symbioses as affected by water deficit?

Three methods were used to study N2 fixation and effects of water deficit on N2 fixation: C2H2 reduction assay (ARA), 15N dilution technique and accumulated N content. In addition, 15N dilution was calculated both in a traditional way and in a modified way, which takes into consideration N and 15N content for the plants before the experiment started. The three methods were applied on the following Rhizobium-symbioses: Acacia albida Del (Faidherbia albida (Del) A. Chev.) and Leucaena leucocephala (Lam) de Wit., and the Frankia-symbiosis Casuarina equisetifolia L. The plants were about 4-months-old when they were harvested. Nitrogen derived from N2 fixation in control plants of Acacia albida was 54.2 mg as measured with ARA, while it was 28.5 mg as measured with the 15N dilution technique, compared to 30.7 mg calculated as accumulated N. In comparison, L. leucocephala fixed 41.6 mg N (ARA), 53.5 mg N (15N dilution technique) and 56.3 mg N (accumulated N). The Frankia-symbiosis had fixed 27.4 mg N as measured by ARA, 8.1 mg N as measured by 15N dilution technique and 12.3 mg N as accumulated N. There were no differences between the estimates based on traditional and modified ways of calculating 15N dilution. The immediate effect of water deficit treatment on N2 fixation was continuously measured in all species with ARA, which started to decrease approximately 10 d after the initiation of the treatment, and declined to less than 5% of the initial level after 21-28 d. The decrease in the amount of N derived from N2 fixation was studied in L. leucocephala during the period of treatment. There was a 26% decrease in amount of N derived from N2 fixation as result of water deficit (as measured with ARA), while the decrease was 23% when measured with both the N dilution method and as accumulated N. The three different methods for measuring N2 fixation and effects of water deficit on N2 fixation are discussed.