Factors Affecting the Reduction of Acetylene by Rhizobium-Soybean Cell Associations in Vitro

Abstract

The acetylene reduction assay was used to measure presumed N-reducing activity in Rhizobium-soybean cell associations in vitro. No acetylene reduction was observed in liquid suspensions of these organisms, but cells plated onto an agar medium from a liquid suspension of Rhizobiumn and soybean cells exhibited acetylene-dependent production of ethylene after 7 to 14 days. Aggregates of soybean cells 0.5 to 2.0 mm in diameter were required for this activity. Decreasing oxygen from 0.20 atm to 0.10, 0.04, or 0.00 atm completely inhibited acetylene reduction. The presence of 2,4-dichlorophenoxyacetic acid or kinetin increased endogenous ethylene production and inhibited acetylene-dependent ethylene production. Acetylene reduction was observed with three out of four strains of R. japonicum tested, and three rhizobial strains, which produce root nodules on cowpeas but not soybeans, formed an association capable of acetylene-dependent ethylene production.

Nitrogenase Activity of Rhizobium japonicum Growing on Agar Surfaces in Relation to Slime Production, Growth and Survival

“…Previously, nitrogenase activity had been observed only in what appeared to be loose associations of rhizobia with tissue cultured plant cells (Holsten et a/., 1971;Child & LaRue, 1974;Philips, 1974;Child, 1975;. Studies of rhizobia grown in defined liquid media showed that a low concentration of dissolved oxygen is required for development of nitrogenase activity (Keister, 1975;Tjepkema & Evans, 1975).…”

Effect of Protein Additives on Acetylene Reduction (Nitrogen Fixation) by Rhizobium in the Presence and Absence of Soybean Cells

“…In all cases, aqueous protein solutions were analyzed for total Kjeldahl nitrogen before addition to the basal medium. The basal medium used was a low nitrogen medium designed for soybean cellRhizobium associations and previously designated as the SCN medium (10). BSA, egg albumin, and a-casein were purchased from Sigma Chemical Co.…”

“…In vitro associations between Acme soybean cells and Rhizobium were established using techniques previously reported from this laboratory (10,11). Seeds utilized in this study to provide leguminous meal fractions were Glycine max (L.) Merr.…”

“…In vitro techniques have been reported which permit one to examine interactions between soybean cells and Rhizobium under various environmental conditions (1,10). Recent reports indicate that cultures of Rhizobium can be induced to fix N2 in the absence of plant cells (5-7, 9, 14).…”

Inhibition of Soybean Cell Growth by the Adsorption of Rhizobium japonicum

“…Under certain conditions, when cultured soybean cells are mixed with Rhizobium root nodule bacteria nitrogenase activity as determined by an acetylene reduction assay (1,5,8) is observed. We also reported a similar phenomenon (7), but in our experiment the reproducibility was very poor.…”