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 tSN labelled fertilizer into soil to estimate N 2 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 N 2 fixed was very low, the estimates of N 2 fixed by the two reference crops did not match. At this stage the uninoculated soybean estimated about four times as much N 2 fixed in the symbiotic soybean as that measured using the non-nodulating soybean. For the second and third harvests, there were substantial increases in N 2 fixed, and both the non-nodulating and uninoculated soybean were equally suitable as reference crops for assessing N 2 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 N 2 fixed is low. Even though there were significant differences in 15N enrichments in different organs (generally nodules < pods < roots < shoots), the estimates of N 2 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, % N 2 fixed in pods (containing seeds) was closest to that of the whole plant (similar at P < 0.05 at physiological maturity). However, the total N z 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 N 2 fixed in the whole plant than that in the shoots.

Legumes native to longleaf pine savannas exhibit capacity for high N2‐fixation rates and negligible impacts due to timing of fire

“…15 N‐enrichment of plant tissue was determined using a Finnegan Ratio Mass Spectrometer at the University of Georgia's Institute of Ecology Chemical Analytical Laboratory. To improve the accuracy of N 2 fixation estimates, shoots, coarse roots, and fine roots were analyzed individually for 15 N concentration, and N 2 ‐fixation was calculated by the weighted atom percentage 15 N excess (WAE) for whole plants using the following equations described by Danso & Kumarasinghe (1990):…”

“…Although percentage N dfa is often used as a comparative measure of N 2 fixation (Danso & Kumarasinghe, 1990; Hendricks & Boring, 1999), it cannot account for potential differences in tissue N between legumes. Estimates of total N 2 fixed that was incorporated into tissue N overcomes this, but makes comparisons among plants that differ in size difficult.…”

“…In annual legumes, N 2 fixation is sensitive to the supply of current photosynthate, and canopy loss can lead to nodule loss (Hartwig et al ., 1987; Pate, 1996), yet the role of stored carbon in perennial legumes has not been ascertained. Flowering phenology and maximum N 2 fixation rates are also closely related in legumes (Trinick et al ., 1976; Danso & Kumarasinghe, 1990; Twary & Heichel, 1991; Hansen et al ., 1993). Because growing season fires delay flowering in many native legumes (Platt et al ., 1988; Hiers et al ., 2000), reproduction may be displaced later in the growing season when evapotranspiration and water stress are higher (Entrekin, 1997; Hendricks & Boring, 1999).…”

Quantification of symbiotic nitrogen fixation by Elaeagnus angustifolia L. on salt-affected irrigated croplands using two 15N isotopic methods

“…The selection and number of appropriate reference plants is critical for estimating BNF, since they affect the accuracy of the 15 N isotopic methods (Sanginga et al 1989;Boddey et al 1995;Chalk and Ladha 1999). In the absence of appropriate non-fixing isolines, the use of several uninoculated reference species has become a common procedure (e.g., Ndoye and Dreyfus 1988;Sanginga et al 1989;Danso and Kumarasinghe 1990). The non-nodulating legume G. triacanthos and the non-N-fixing U. pumila used in this study fulfilled the main requirement of reference species (Fried et al 1983), as both relied solely on soil N. The use of lysimeters avoided possible cross-contamination that may occur between N-fixer and non-fixer in open-field trials (Sanginga et al 1989).…”