ASSESSMENT OF BIOLOGICAL NITROGEN-FIXATION

The four commonly used methods for measuring biological nitrogen fixat ion (BNF) in plants are: the total nitrogen difference (TND) method, a cetylene reduction assay (ARA) technique, xylem-solute (or ureide prod uction) method and the use of N-15 labelled compounds. The TND method relies on a control non-N-2-fixing plant to estimate the amount of N a bsorbed by the fixing plant from soil. It is one of the simplest and l east expensive methods, but works best under low soil N conditions. Th e ARA technique measures the rate of acetylene conversion to ethylene by the N-2-fixing enzyme, nitrogenase. The ethylene produced can then be converted into N-2 fixed, using a conversion ratio, originally reco mmended as 3. Although the method is inexpensive and highly sensitive, its major disadvantages are, the short-term nature of the assays, the doubtful validity of always using a conversion ratio of 3 and the aut o-inhibition of acetylene conversion to ethylene. The ARA technique is therefore not a method of choice for measuring BNF. The xylem-solute technique can be used to measure BNF for those species that produce si gnificant quantities of ureide as product of BNF Although simple and r elatively inexpensive, it is an instantaneous assay and also needs to be calibrated against a known method. The most serious limitation is, that only a small proportion of N-2-fixing plants examined are ureide exporters, and the method is therefore not widely applicable. The N-15 methods, classified into the isotope dilution and A-value methods, ap pear to be the most accurate, but also the most expensive. They involv e labelling soil with N-15 fertilizer and using a non-N-2-fixing refer ence plant to measure the N-15/N-14 ratio in the soil. The N-15 isotop e dilution approach is both operationally and mathematically simpler t han the A-value approach. To limit potential errors in the selection o f reference crops, it is recommended to use N-15 labelled compounds or soil labelling methods that result in the slow release of N-15 or the slow decline of N-15/N-14 ratio in the soil. Additionally, the use of several reference plants rather than a single one can improve the acc uracy of the results.