Survival and change in physiological state of Bradyrhizobium japonicum in soybean (Glycine max L. Merril) liquid inoculants after long-term storage

Commercial liquid inoculants for soybean, stored at 20 degreesC for 1-8 yea rs in 400 ml bottles or in 5000 ml containers, were assessed for their effi cacy and changes in the physiological activity of Bradyrhizobium japonicum. A decrease in viable counts and in bacterial survival on seeds was observe d in inoculants stored for several years. The number of nodules produced pe r plant in a growth chamber decreased and was correlated to the number of b acteria surviving on the seeds. Changes in physiological properties were as sessed using biochemical, physiological and microscopic methods. The cell t otal sugars content decreased with increased storage of the inoculants. Hig h calculated ratios of suspended solid dry matter/carbon/nitrogen/proteins weight per c.f.u. strongly suggested the presence of dead or viable but non -culturable (VBNC) cells in the inoculants. This was confirmed in a study o f bacterial respiratory activity, using p-iodonitrotetrazolium reduction. T he time of colony appearance on plates increased in the old inoculants stor ed for a long time, especially on yeast-free culture medium. The heterogene ity in colony size also increased with storage length. Inoculants stored fo r more than 2 years could be differentiated from the others by using nalidi xic acid against cellular division. Nucleic acid staining of cells showed t hat the percentage of membrane-compromised bacteria in all the inoculants i ncreased with increased storage length, whatever the type of packaging used for the inoculants. These results demonstrated that the physiological acti vity of B. japonicum cells in commercial liquid inoculants changes after st orage. To complete c.f.u. determination, three methods were proposed to ass ess the fitness of stored bradyrhizobia, but they remain to be checked for reliability on a variety of commercial inoculants.