Evaluation of biological nitrogen fixation associated with non-legumes

Abstract

The method to quantify biological nitrogen fixation (BNF) that was most frequently utilized in early studies of associations between N2-fixing bacteria and non-legume plants was the acetylene reduction (AR) assay, which subsequently was examined critically and found to be difficult to apply to produce reliable BNF estimates. However, the AR technique can produce meaningful results if in-situ assays are performed on undisturbed intact plant/ soil systems and precautions are taken to inhibit BNF activity of cyanobacteria and tests are made to check for endogenous ethylene evolution. With the techniques based on the quantification of total N, namely total N balance, the simultaneous use of several non-N2-fixing control plants greatly improves the chances of successful quantification of plant-associated BNF. For those with access to isotopically labelled nitrogen (15N), and the facilities to analyse it, either isotopically labelled N2 gas may be utilized or the plant growth medium may be labelled and the so-called isotope dilution technique can be applied. The use of 15N-labelled atmospheres is useful to confirm BNF inputs to plants but only very short-term assays are feasible unless very sophisticated sealed chambers are available. The isotope dilution technique does not require enclosure of the plants, but application of 15N-enriched fertilizers to the soil results in non-uniform labelling of the soil mineral N with depth and time. In this case as different plants (e.g. inoculated and non-inoculated) can vary considerably in their patterns of soil N uptake, they will not accumulate soil N with the same specific 15N label, which is the principal source of error in this technique. The most successful applications of this technique have been achieved using soils in containers (pots or tanks) with finite depth and uniformly filled with 15N labelled soil. In this review we discuss the problems associated with the use of these techniques for quantifying plant-associated BNF, and the strategies that can be applied to ameliorate them, using examples from studies performed at CNPAB and elsewhere.