Assessment of potential sources of error in nitrogen fixation measurements by the nitrogen-15 isotope dilution technique

Abstract

Although the use of 15N fertilizers to measure nitrogen (N2) fixed in crops has increased substantially in recent years, some methodological uncertainties still remain unresolved. The results obtained from a greenhouse study of soybean [Glycine max. (L.) Merrill] inoculated by six different methods have been examined for potential errors arising from incorporating 15N labelled fertilizer into soil to estimate N2 fixed in pods or shoots or the whole plant at three growth stages (50% flowering, pod-initiation and physiological maturity) using as reference crops, an uninoculated soybean cultivar and a non-nodulating soybean isoline. At the first harvest when N2 fixed was very low, the estimates of N2 fixed by the two reference crops did not match. At this stage the uninoculated soybean estimated about four times as much N2 fixed in the symbiotic soybean as that measured using the non-nodulating soybean. For the second and third harvests, there were substantial increases in N2 fixed, and both the non-nodulating and uninoculated soybean were equally suitable as reference crops for assessing N2 fixed in the symbiotic soybean. These results indicate how critical and difficult the choice of the reference crop could be at early harvests, or when N2 fixed is low. Even though there were significant differences in 15N enrichments in different organs (generally nodules < pods < roots < shoots), the estimates of N2 fixed in soybean plants obtained by excluding roots and nodules did not differ much from those based on the whole plant. Of the above-ground organs, % N2 fixed in pods (containing seeds) was closest to that of the whole plant (similar at P<0.05 at physiological maturity). However, the total N2 fixed in pods or shoots was substantially lower than that fixed by the whole plant (P<0.05), although that for the pods and enclosed seeds once again was closer to N2 fixed in the whole plant than that in the shoots.