Nitrogen Fixation by Intact Annual Rangeland Species in Soil

The acetylene reduction technique is widely used as an assay of N₂ fixation, but its application to intact N₂-fixing systems in soil has been limited. The purpose of this research was to develop methods suitable for short-duration assays of entire nitrogenase systems and to utilize these methods in studies of N₂ fixation by annual rangeland species growing intact in potted soil. Experiments conducted with nodules excised from subclover plants (Trifolium subterraneum L.) indicated large diurnal variation in acetylene reduction rates; the greatest activity being observed between 1200 and 1600 hours and thee minimum at 0400 hours. All standard assays of intact cultures were conducted between 1200 and 1600 hours to standardize light conditions. The pots were sealed, cultures exposed to an air-acetylene atmosphere, and ethylene production was measured. Ethylene production rates of intact subclover plants were linear over a 75-rain period if the gases were mixed during an incubation by pumping a syringe inserted through a rubber serum stopper fitted in the bottom of the pot. Successive biweekly assays of 30 or 60 rain duration were conducted on seven species of annual clovers during complete growth cycles. All species exhibited major exponential phases in ethylene production activity which coincided with flowering and seed development. There was a significant linear relationship between ethylene production and both herbage dry matter production and N content. An average of 3.2 moles of ethylene was produced per mole of total plant N contained in four subclover cultures. Ethylene production during a 4-month period by soil organisms in the rhizosphere of an annual grass, soft chess (Bromus mollis L.), was significantly and inversely related to the amount of fertilizer N applied. Ethylene production was significantly higher in the grass rhizosphere than in comparable fallow soils, except where 160 mg N (100 ppm) was applied to the grass cultures. The results indicate that these short-duration, nondestructive assays are of value in making quantitative estimates of N₂ fixation. They are particularly useful for repeated samplings of cultures in situ during entire growing seasons.