Effects of Exogenous Hormones on Endophytic Rhizobial Proliferation and Growth Promotion in Alfalfa

Endophytic rhizobia have the functions of dissolving organic phosphorus, secreting auxin, fixing nitrogen, and promoting growth. The proliferation of endophytic rhizobia in alfalfa and their symbiotic nodulation with alfalfa seedlings are regulated by various plant hormones. In this study, the alfalfa seeds (Medicago sativa L.) containing CFP-labeled rhizobium R.gn5f (isolated from the seeds of Gannong No.5 alfalfa) were used as materials, and the concentrations of 3-indoleacetic acid (3-IAA), 6-benzylaminopurine (6-BA) and homobrassinolide (HBR) suitable for the growth of R.gn5f were used for seed soaking treatment, and distilled water was used as the control. The proliferation of endophytic rhizobium, plant nodulation, nitrogen fixation performance and plant growth ability of alfalfa at different growth stages were determined. The effects of hormone types and concentrations on the proliferation and nitrogen fixation of endophytic rhizobia were analyzed to provide a theoretical basis for accurately promoting the nodulation, nitrogen fixation and growth-promoting ability of endophytic rhizobia in seeds. The results showed that the optimal concentrations of 3-IAA, 6-BA and HBR were 12 mg·L−1, 16 mg·L−1 and 2.47 mg·L−1, respectively. The nitrogen fixation performance of endophytic rhizobium plants containing three hormones was higher at the branching stage and budding stage. The growth ability of the plant was better at the flowering stage. The hormone 2.47 mg·L−1 of HBR was beneficial to the proliferation, nodulation, nitrogen fixation and plant growth of endophytic rhizobia in alfalfa at the vegetative and reproductive growth stages, and the number of R.gn5 f in the seeds of HBR plants at the mature stage was the largest (281.25 CFU·g−1). Therefore, the hormone 2.47 mg·L−1 of HBR was better for the proliferation of endophytic rhizobia R.gn5 f and plant growth in alfalfa. These findings provide a theoretical basis for precisely leveraging the nodulation and nitrogen-fixing capabilities of seed-borne endophytic rhizobia, thereby laying a foundation for the symbiotic breeding of alfalfa and rhizobia.