Nitrogenase activity of endophyte suspensions derived from root nodules of Alnus, Hippophaë, Shepherdia and Myrica spp.

Abstract

STUDIES of nitrogenase of root nodule homogenates have focused chiefly on the Rhizobium–legume system. Little is known about the nitrogenase of actinomycete-like endophytes of root nodules on woody, non-leguminous plants. Although nitrogen fixation has been reported in suspensions of the endophyte from Myrica cerifera^1,2, the results were either of very low significance or irreproducible. In both cases dithionite and oxygen were added, which seems to be contradictory. Subsequent attempts to detect nitrogen fixation in non-leguminous nodule homogenates have been unsuccessful and investigations stopped at an early stage. We have found³, however, that reasonable levels of acetylene reduction by nodule homogenates of Alnus glutinosa can be measured if 0.3 M sucrose and 0.1 M dithionite are present during anaerobic homogenisation. The latter was essential because the nodule material contains large amounts of phenolic compounds, which after oxidation inhibit nitrogenase activity. While determining optimal conditions for the nitrogenase assay it was found that the reaction was ATP-dependent but that addition of an ATP-generating system (creatine phosphate (Cr ∼ P)/creatine phosphokinase) decreased the activity during short-term experiments. This inhibitory effect was not found when the same ATP-generating system was added to bacteroid suspensions derived from leguminous nodules. We report here the seemingly aberrant behaviour of the added ATP-generating system, the applicability of our method to other actinomycete-woody plant symbioses, and the separation of actively acetylene-reducing endophyte material from nodule homogenates.