Measurement of Nitrogenase Activity of Intact Legume Symbionts In Situ Using the Acetylene Reduction Assay

The acetylene reduction assay has been used extensively to detect and measure nitrogenase activity of free-living microorganisms, excised legume and non-legume nodules, and root systems, soil cores, and entire nodulated legume plants removed from their soil medium. This investigation was undertaken to determine whether or not the acetylene reduction assay can be used to measure nitrogenase activity of cultures of nodulated soybean (Glycine max.) plants in situ and to compare the nitrogenase activity of these cultures with the nitrogenase activity of detached nodules and excised root systems from comparable cultures. Entire cultures grown in Perlite or in soll were placed in polyethylene containers, covered with Plexiglas lids, exposed to acetylene, and assayed for ethylene production. Time course experiments showed that rates of acetylene reduction of plants growing in Perlite and in soil were linear for at least 90 min following an initial lag period of 15 min. Saturation of nitrogenase in intact cultures was obtained at a pC₂H₂ of 0.1 atm. The apparent Km for nitrogenase was 0.05 atm. For convemence, assays in the large containers were conducted at a pC₂H₂ of 0.025 atm. The Michaelis-Menten equation was used to calculate rates of acetylene reduction at saturating levels of acetylene. The correlation coefficient between rates of acetylene reduction and nodule fresh weight was 0.99 for plants of the same age and 0.79 for plants of different ages. Acetylene reduction rates of either intact nodulated plants in Perlite or nodulated root systems removed from Perlite were significantly greater than acetylene reduction rates of detached nodules from comparable cultures. Relatively little diurnal variation was observed in nitrogenase activity of potted plants grown under controlled environmental conditions. The method is useful for the assessment of nitrogenase activity of legume cultures in a porous medium under standardized conditions, but its application to legumes in soil proved to be complicated by the water content of the soil which influences rates of gas diffusion within the culture medium.