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 °C for 1–8 years in 400 ml bottles or in 5000 ml containers, were assessed for their efficacy and changes in the physiological activity of Bradyrhizobium japonicum. A decrease in viable counts and in bacterial survival on seeds was observed in inoculants stored for several years. The number of nodules produced per plant in a growth chamber decreased and was correlated to the number of bacteria surviving on the seeds. Changes in physiological properties were assessed using biochemical, physiological and microscopic methods. The cell total sugars content decreased with increased storage of the inoculants. High 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 of bacterial respiratory activity, using p-iodonitrotetrazolium reduction. The time of colony appearance on plates increased in the old inoculants stored for a long time, especially on yeast-free culture medium. The heterogeneity in colony size also increased with storage length. Inoculants stored for more than 2 years could be differentiated from the others by using nalidixic acid against cellular division. Nucleic acid staining of cells showed that the percentage of membrane-compromised bacteria in all the inoculants increased with increased storage length, whatever the type of packaging used for the inoculants. These results demonstrated that the physiological activity of B. japonicum cells in commercial liquid inoculants changes after storage. To complete c.f.u. determination, three methods were proposed to assess the fitness of stored bradyrhizobia, but they remain to be checked for reliability on a variety of commercial inoculants.