Effects of Long-Term Preservation of Frankia Strains on Infectivity, Effectivity, and In Vitro Nitrogenase Activity

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Abstract
Frankia strain HFP ArI3 which had been preserved for 27 months by being lyophilized, frozen in glycerol, or stored in complex medium was successfully used as an inoculum after being subcultured for inducing nodulation and nitrogen fixation of Alnus rubra. Glycerol-preserved HFP ArI3 produced significantly lower rates of nitrogenase activity than did lyophilized or complex-medium-preserved inocula. Bacteria that had been preserved by all three methods were successfully induced to fix atmospheric nitrogen by being cultured in nitrogen-free medium. Subculturing of these cells in nitrogen-free medium a second and third time yielded increasing rates of nitrogenase activity. Initial nitrogenase activity was detected on days 5, 4, and 3 during the first, second, and third subcultures after preservation, respectively. Maximum activity was observed on days 11, 10, and 8 during the first, second, and third subcultures, respectively. A description is given of standard culture techniques used in our laboratory for Frankia isolates, and methods used to distribute Frankia cultures by mail are described.