The acetylene assay was used to find how soon nitrogenase activity could be detected in young, nodulating alder plants. Samples taken at intervals initially of 2–3 days were assayed, starting as soon as external signs of nodulation were seen, and continuing for a further 33 days. Squash preparations made from the nodules of the assayed plants were examined microscopically to detect whether there was any relation between nitrogenase activity and the stage of differentiation of the nodule endophyte.
No nitrogenase activity was detected on the first two assay occasions, on which the endophyte was confined to the hyphal stage in all the nodules present. Activity was detected on the subsequent occasions, its onset coinciding with the production of vesicles by the endophyte. Activity rose as the vesicles became more abundant, and in agreement with the limited previous evidence it was concluded that the vesicles, but not the hyphae, are nitrogen-fixing. The third type of structure produced by the alder endophyte, namely the bacteroid stage, was not detected in the young nodules studied here.
The onset of nitrogenase activity in the nodules preceded by 16 days the increased greening of the leaves signifying the arrival in the shoot of products of nitrogen fixation. Ethylene production per plant increased in exponential manner over the period of the experiment, while ethylene production per unit weight of nodule tissue rose ten-fold