Isolation and characterization of bacteria from colonies exuded from “germ-free” mulberry shoots cultured in vitro

Bacteria-like colonies have been periodically observed to exude from the bottom of mulberry shoots grown in vitro, although the culture origins (buds) were sterilized before planting on MS medium. From these colonies, five bacterial strains were isolated and characterized. All strains, designated as Y1-Y5, formed yellow colonies on LB medium, but none of them grew on MS medium. The present strains were identified as Xanthomonas campestris on the basis of principle bacteriological properties and the analyses of API-20NE identification kit and 16S r-RNA gene sequence. When strain Y1 (RSF1010) (RSF1010 plasmid was introduced as a marker into strain Y1) was inoculated into the mulberry shoots grown in vitro, similar types of colonies appeared at the bottom of the shoots after 16 days. Strain Yl (RSF1010) was also isolated from most of the leaves of the inoculated plants after as long as 45 days. These findings suggest that the strain survives within plant tissues for a long time. Thus, it was verified that colonies exuded from mulberry shoots originated from X, campestris within buds that escaped surface-sterilization.