Enhanced soybean nodulation and nitrogen fixation via modifications of bradyrhizobial inoculant and culture technologies

Soybean (Glycine max L. Merr.) and Bradyrhizobium japonicum can form a nitrogen fixing symbiosis. This symbiosis is important for most sustainable agriculture systems. This thesis examines two ways to enhance nodulation and nitrogen fixation by this symbiosis: coinoculation of plant growth promoting bacteria (PGPB) with B. japonicum, and addition of RNA to a bradyrhizobial culture medium. The optimal coinoculation dose of Serratia proteamaculans 1–102 and S. liquefaciens 2–68 was determined as 108 cells per plant under both optimal and suboptimal root zone temperatures (RZTs). Nodulation dynamics studies indicated that coinoculation of these two PGPB caused earlier nodule initiation and a higher nodulation rate, contributing to the higher nodule number and nodule weight. The coinoculation also increased nitrogen fixation efficiency under both optimal and suboptimal RZTs. A novel inducible activator only produced by the bacteria after addition of flavonoids to the culture system was prepared and evaluated in greenhouse and field experiments. Fourteen non-bradyrhizobial endophytic bacteria (NEB) were isolated from the surface sterilized root nodules, and three of these, designated NEB4, NEW and NEB17, showed soybean plant growth promotion under both greenhouse (with controlled RZTs) and field conditions. Alone, they were neither nodule inducers nor nitrogen fixers. Biolog tests and partial 16S rRNA gene sequence analyses placed the three strains in genus Bacillus: NEB4 and NEB5 are B. subtilis and NEB17 B. thuringiensis. Bradyrhizobium species grow slowly, making the culture process long and the cost of inoculant production higher. Addition of commercial yeast RNA to the bacterial culture medium accelerated the bacterial growth rate, shortened the culture time and increased the lipo-chitooligosaccharide (LCO) yield in flask cultures. Inoculation experiments in the greenhouse also showed that bradyrhizobial inoculant produced in the presence of RNA had better symbiotic competency (indicated by greater nodule number, nodule weight and plant weight) than the control. Collectively, these research findings have the potential to improve the overall utility of the nitrogen fixing symbiosis in soybean production and contribute to the use of this symbiosis as a tool in sustainable agriculture systems.