FIELD APPLICATION OF THE N-15 ISOTOPE-DILUTION TECHNIQUE FOR THE RELIABLE QUANTIFICATION OF PLANT-ASSOCIATED BIOLOGICAL NITROGEN-FIXATION

To apply the isotope dilution (ID) technique, it is necessary to grow the ''N-2-fixing'' crop in a soil where the mineral N is labelled with N-15. Normally the ''N-2-fixing'' crop and a suitable non-N-2-fixing control crop are grown in the same labelled soil and the N-15 enrichme nt of the control crop is assumed to be equal to the N-15 enrichment o f the nitrogen (N) derived from the soil in the ''N-2-fixing'' crop. I n this case the proportion of unlabelled N being derived from the air via biological N-2 fixation (BNF) in the ''N-2-fixing'' crop will be p roportional to the dilution of the enrichment of the N derived from th e labelled soil. To label the soil, the technique most often used is t o add a single addition of N-15-labelled N fertilizer shortly before, at, or shortly after, the planting of the crops. Data in the literatur e clearly show that this technique results in a rapid fall in the N-15 enrichment of soil mineral N with time. Under these conditions, if th e control and the ''N-2-fixing'' crops have different patterns of N up take from the soil they will inevitably obtain different N-15 enrichme nts in the soil-derived N. In this case the isotope dilution technique cannot be applied, or if it is, there will be an error introduced int o, the estimate of the contribution of N derived from BNF. Several exp eriments are described which explore different strategies of applicati on of the ID technique to attempt to attenuate the errors involved. Th e results suggest that it is wise to use slow-release forms of labelle d N, or in some cases, multiple additions, to diminish temporal change s in the N-15 enrichment of soil mineral N. The use of several control crops produces a range of different estimates of the BNF contribution s to the ''N-2-fixing'' crops, and the extent of this range gives a me asure of the accuracy of the estimates. Likewise the use of more than one N-15 enrichment technique in the same experiment will also give a range of estimates which can be treated similarly. The potential of ot her techniques, such as sequential harvesting of both control and test crops, are also discussed.