(From the Summary of Improving Yield and Nitrogen Fixation
of Grain Legumes in the Tropics and Sub-Tropics of Asia
IAEA-TECDOC-1027, 1998)

The restorative effects of legumes on agricultural soil are documented in Greek and Roman literature, and have been exploited by farmers since antiquity. However, the swellings or nodules on the roots were not linked with those benefits and were thought to be storage organs or of pathological origin until the pioneering research of Drs. Hellreigel and Wilfarth in Germany a little over a century ago.

Hellreigel and Wilfarth's experiments compared oat, buckwheat and rape, with pea, serradella and lupin, grown in sand culture with nitrogen-free nutrient solution. A water extract of soil, containing an insignificant amount of nitrogen (N), was added to each pot. The non-legumes remained N-deficient, whereas the legumes after a time recovered from “N-hunger,” became dark green and grew luxuriantly. It was concluded that the legumes obtained the N from the atmosphere with the assistance of bacteria from the soil extract, in the root nodules. Within a few months of publication of these results, the great Russian microbiologist Beijerinck isolated nodule bacteria, which he designated Bacillus radicicola, applied them back to legumes and satisfied Koch’s postulates. A new arena of scientific endeavour was born.

Bacillus radicicola has been replaced by several genera and many species of soil-borne bacteria, the rhizobia, that infect, with varying degrees of specificity, certain groups of legume species. The rhizobia proliferate in the rhizosphere of the potential host plant then penetrate the root epidermis, either directly or via root hairs, and invade cortical cells. Host-cell division takes place, and highly differentiated nodules develop within which atmospheric N2 is converted to ammonia by the microsymbiont and released to the host legume. Thus, an effectively nodulated legume can thrive in a soil that is deficient in mineral N.

Prior to the mid-1970s, research interest in N2 fixation was restricted to a few groups of scientists in Europe, the USA and Australia. The energy crisis of 1973-74 caused sudden, rapid increases in the cost of nitrogenous fertilizers, and stimulated interest in legume N2 fixation and the possibility of replacing chemical fertilizers with organic alternatives. Many developing countries were particularly adversely affected by the increased costs of crop production, which stimulated research on tropical legumes and their role in tropical agriculture.

Concomitant with this new interest in the biological fixation of atmospheric N2 in the 1970s and the possibility of its exploitation for improved agricultural production in the developing world, the isotope-dilution methodology was developed at the Agency, as a way of precisely measuring fixation by field-grown legumes. A small quantity of 15N-enriched fertilizer is applied to the soil and the legume assimilates atmospheric N2 supplied by the root-nodule symbionts, in addition to the isotope-enriched N from the soil. Since non-fixing plants do not have access directly to atmospheric N2, the resulting 15N/14N ratio of tissue in any two species differs in proportion with the amount of N fixed. The isotope-dilution technique has been used extensively over the past 20 years in co-ordinated research projects sponsored by the IAEA.

A limitation of the isotope-dilution methodology is uncertainty in the choice of suitable non-N2-fixing reference species. The 15N/14N absorbed from the soil by the fixing plant changes significantly with time, therefore, for a non-fixing species to be a suitable reference, it must absorb 15N/14N with the same temporal pattern, and both should obtain their mineral N from similar soil-N pools. Occasionally, estimates of the amount of N fixed are negative as a result of the non-fixing check having a different pattern of usage of the 14N and 15N pools; on the other hand, cereal species have been shown to be acceptable non-fixing checks for some legumes.


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