Calibrating the xylem-solute method for nitrogen fixation measurement of ureide-producing legumes: cowpea, mungbean, and black gram

Simple, accurate methods for measuring legume N2 fixation are needed for research on, and ultimate management of, legumes in agricultural and forestry systems. The xylem-solute method provides a low cost alternative to 15N methods, although currently it is restricted to ureide-producing species. Calibration experiments are reported in which the relative abundances of ureide-N in vacuum-extracted (VES) and root-bleeding xylem sap (RBS) of cowpea (Vigna unguiculata), mungbean (V. radiata) and black gram (V. mungo) were correlated with proportional dependence of the plants on N2 fixation (Pfix). Effectively nodulated plants were cultured in large pots in a glasshouse. Different levels of Pfix were generated by supplying the plants throughout growth with N-free nutrients or nutrients containing 15N-labeled nitrate at rates 1–8 mM N (cowpea and mungbean) or 1–10 mM N (black gram). The 4–5 samplings for xylem sap, plant N, and 15N indicated that the different levels of nitrate supply effectively varied nodulation and Pfix. Mean values for the latter ranged between 4–100% (cowpea), 3–100% (mungbean) and 0–100% (black gram). Relative ureide-N values for VES of cowpea (range 9–39%) were substantially less than those of mungbean (12–62%) and black gram (9–54%). Values for RBS, however, were similar for the three species, 9–83% (cowpea), 11–80% (mungbean) and 8–82% (black gram). Regression analyses of relative ureide-N (y) in VES and RBS of the three species against Pfix (x) provided the following functions: cowpea VES, y=7.2+0.31x (r2=0.89); mungbean, black gram VES, y=11.7+0.49x (r2=0.90); cowpea, mungbean, black gram RBS, y=8.6+0.75x (r2=0.94). Quantification of the relationships between relative ureide-N in xylem sap and Pfix for these species may now facilitate increased adoption of the method for assessing legume N2 fixation.