Methods for quantification of nitrogen fixation associated with gramineae

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Abstract
In recent years, the question of whether grasses or cereals are able to obtain significant quantities of nitrogen from plant‐associated heterotrophic nitrogen‐fixing bacteria has raised a great deal of controversy. This controversy is principally due to the difficulties involved in the reliable quantification of plant‐associated biological nitrogen fixation (BNF). The acetylene reduction assay has contributed immensely to the understanding of diazotroph/ gramineae associations, but in its use for definitive quantification of BNF, it suffers from many disadvantages, mainly due to the fact that it is a short‐term assay of enzyme activity and such activity is drastically reduced when plants are disturbed. Few nitrogen‐balance studies have been performed (due no doubt to their laborious nature), but recent data on rice and sugar cane have proved most valuable. Isotope techniques (15N) have been more popular. Incorporation of 15N‐labeled N2 provides definitive proof of incorporation of biologically fixed N, but the technique is not easily adaptable to field studies. The 15N‐dilution technique holds out the best possibility for the reliable quantification in the field, but problems of uniform labeling of the soil and the choice of suitable non‐N2‐fixing control plants must be addressed. These techniques are discussed and recommendations are made for their application to quantify N2 fixation and the selection of plant/diazotroph associations with agronomic potential.