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

Abstract

The effect of protein additives on acetylene reduction (N2 fixation) by Rhizobium associated with soybean cells (Glycine max [L.] Meff.) in vitro was studied. Acetylene reduction was promoted on the basal medium supplemented with 1.4 mg of N/mi supplied as aqueous extracts of hexane-extracted soybean, red kidney beans (Phaseolus vulgaris L.), or peas (Pisum sativum L.). Commercial samples of a-casein, or bovine serum albumin also promoted acetylene reduction at a concentration of 1.4 mg of N/ml of basal medium, but egg albumin supplying an equal amount of nitrogen to the basal medium completely suppressed acetylene reduction. Autodaving the aqueous extract of hexane-extracted soybean meal had no effect on its ability to promote acetylene reduction.The presence of 40 mm succinate decreased acetylene reduction with leguminous proteins supplying 1.4 mg of N/ml but promoted acetylene reduction by Rhizobium 32Hl-soybean cell associations on media containing a-casein, bovine serum albumin, or egg albumin suppling 1.4 mg of N/ml. Similar results were obtained with both cowpea Rhizobium 32H1 and Rhizobium japonicum 61A96. Pure cultures of Rhizobium 32H1 developed acetylene-reducing activity in the presence of soybean extract on basal agar medium and in vermiculite supplied with N-free mineral salts plus crude soybean meal. The results suggest that in certain situations, free living Rhizobium may reduce N2 under field conditions. Recent reports indicate that symbiotic N2 fixation is unaffected and seed yields of field-grown soybeans (Glycine max L.) are increased by fertilizing with crude soybean meal at a rate of 134 kg of N/acre (4, G. Ham, personal communication). Supplying other forms of fertilizer nitrogen at this level normally would be expected to suppress both the development and functioning of the Rhizobium-legume symbiosis. Although fertilizing with soybean meal is not an economically feasible method for increasing crop productivity, it may provide some insight into mechanisms which control symbiotic N2 reduction in the presence of combined nitrogen. In a typical soybean plant grown under field conditions, only 25% of the nitrogen is derived from N2 (3); therefore, increasing biological N2 reduction is a worthy goal from both ecological and economic points of view. Nitrogenase activity in a legume root nodule which is not decreased by the presence of soil nitrogen presumably would increase the combined nitrogen available to other plants in a rotation either by

Trials to create artificial nitrogen-fixing symbioses and associations using in vitro methods: An outlook

“…Some reports indicated that cultures of Rhizobium can be induced to fix N 2 in the absence of plant cells when grown on a modified medium supplemented with pentose sugars and a tricarboxylic acid cycle intermediate (Kurz and LaRue, 1975;McComb et al, 1975;Pagan et al, 1975). Soybean meal extract promoted nitrogenase activity directly in pure cultures of Rhizobium (Anderson and Philips, 1976). These early experiments did not aim for plant regeneration, their main goal was the better understanding of Rhizobium±plant symbioses.…”

Substances from cultured soybean cells which stimulate or inhibit acetylene reduction by free‐living Rhizobium japonicum

“…The discovery that Rhizobium can exhibit nitrogenase (Anderson and Phillips 1976) have a stimulatory effect. activity in pure culture (Pagan er al 1975, Kurz and Soybean cells produce diffusible factors which induce LaRue 1975, Keister 1975, McCottib etal.…”