Application of N2/Ar ratio method for N2 fixation studies in submerged soil

Abstract

Recently there has been developments in the measurement of N₂ fixation due mainly to the C₂H₂ reduction method (1). This method, however, has several disadvantages, especially for submerged soil, and the estimated amount of fixed N₂ on the basis of the C₂H₂ reduction activity is not very reliable. The tracer ¹⁵N₂ technique which gives a reliable estimation of the fixed N₂ is too expensive for common use. Development of an alternative method suitable for submerged soil would therefore be desirable. The present authors expected that the measurement of the ratio N₂/Ar in the soil solution might provide advantages for the estimation of the fixed N₂ in submerged soil.     

Nitrogen fixation in paddy soils

Several important features of the N. fixation in paddy fields which were reported previously were confirmed and some new additional results regarding the evaluation of the N₂ fixation in the rhizosphere were obtained by reinvestigation in the fields. In addition, rice plants were cultivated in the submerged soil in pots and various parts of the soil were analyzed for the N₂-fixing activity as well as several other properties. The results of the pot experiments were found to be fairly similar to those observed in the field investigations, indicating the validity of the submerged soil in a pot as a rather simulated model for the actual paddy field. By using this model system, the following facts were ascertained: (1) Water-percolation had almost no effect on the N₂-fixing activities of both the rhizosphere and the non-rhizosphere soils. (2) Suppressing effect of washing the root of rice plant on the N₂-fixing activity was slight in the seedling stage and marked in the tillering and flowering stages. (3) The N₂-fixing activity of a single rice root varied from tip to base.     

Nitrogen fixation in paddy soils

The 4 long-term experimental plots (Umbric haplaquept) with different fertilizer treatment at Cent. Agric. Exp. Sta. in Konosu City, Saitama Prefecture, were used for the sites of investigation. The 4 plots were NF (applied with no fertilizer), IF (applied with inorganic fertilizers), GM (applied with green manure and CaCO₂), and OM (applied with manure and inorganic fertilizers). Flooded water, floating weed, upper (0-2cm) and lower (2-10cm) parts of Apg horizon and rhizosphere were collected from each plot before flooding, during flooding, and after drainage. These samples were analyzed for N₂-fixing activity by acetylene reduction method, pH, Eh, and contents of Fe²⁺, NH₄ ⁺, chlorophyll-type compounds, and water-soluble carbohydrates.

The N₂-fixing activity of all samples showed almost the same pattern of change with time: very low before flooding, rapidly increased after flooding, the maximum value at the maximum tillering stage of rice plant, declined afterwards and reached a very low value after drainage.

Rough estimation of the “N₂-fixing capacity” of each part of the paddy field revealed that the most important site of the N₂ fixation was the reduced Apg horizon, that the importance of flooded water and/or the oxidized layer in the N₂ fixation was rather low except in infertile soil, and that the role of rhizosphere in the N₂ fixation could not be neglected also in Japan.

Reduced condition and content of easily decomposable organic substances were judged to be main factors which control the N₂-fixing activity in the flooded soil on the basis of correlations between the Nt-fixing activity and several analytical data of the paddy soils.     

Influences of organic debris and rice root on the nitrogen fixation in the submerged soil

Abstract

A series of experiments has been conducted on the N2 fixation in the paddy soils by the authors (1–4). The amount of organic substrates for microorganisms and the degree of reduction of the soil are found to be two major factors affecting the N₂-fixing activity of the heterotrophic microorganisms in the submerged soil. Organic debris, rice root and their neighboring soils are identified to be the important micro-sites for the heterotrophic N₂ fixers. The organic debris and the rice root are considered to play dual roles by supplying the organic substances; (1) increase of the population of the heterotrophic N₂ fixers—the amount of nitrogenase, (2) preparation of the reduced conditions favorable for the nitrogenase activity.

However, it is not yet clearly known which of these two roles of the organic substrates is more essential to the N₂-fixing activity in the paddy soil. In addition, it is expected that there must be some differences between the organic debris and rice root in their contribution to the N₂ fixation in the paddy soil.

An experiment was carried out to clarify these problems. Moist soil sample was collected from the plough layer of the paddy field at Central Agric. Exp. Sta. in Konosu City, Saitama Pref., passed through a 5 mm sieve and placed in pots (3 kg moist soil/pot). Ammonium sulfate, calcium superphosphate, and potassium chloride at the rate of 0.4-0.4-0.4 (N-P₂O₅-K₂0) g/pot were incorporated into the soil 7 days before transplanting. Split application of ammonium sulfate at the rates of 0.2 and 0.4 g N/pot were also incorporated at 30 and 41 days after transplanting respectively. These pots were divided into three series; planted (P-series), non-planted (N-series), and non-planted and applied with organic manure (O-series). In case of O-series, 60 g of fairly rotted organic manure was applied to each pot. Each pot of P-series was planted with two 4O-day-old seedlings of rice plant at 7 days after submergence. The Nseries was regarded as a control. Each series was not replicated in this preliminary experiment.