LYSOGENIC INDUCTION OF PHYTOPATHOGENIC BACTERIA
Burlaka V, Ainietdinova H, Dreus A, Kurahina N, Sklyar T.
Oles Honchar Dnipro National University
Genomes of phytopathogenic bacteria contain a lot of prophage elements, which integrate into the host genome and replicate as its part. Upon induction, the cells of phytopathogenic bacteria are able to secrete bacteriocins of a prophage nature that can lyse closely related bacterial strains.
The aim of the study was to carry out the induction of phytopathogenic bacteria prophages and to identify the obtained lysates.
Materials and methods. Three strains of phytopathogenic bacteria have been used for phage induction (grown on LB medium and M9+glucose medium) and detection as indicator strains: Erwinia dissolvens and Pectobacterium carotovorum from museum’s strain collection of the Department of Microbiology, Virology and Biotechnology Oles Honchar Dnipro National University; Pantoea ananatis (isolated from onion); E. coli C600 has been used only as indicator strain. Microbiological laboratory methods for phage induction, isolation and detection have been used: induction by nalidixic acid (NAL) in concentrations 16 and 20 mg/mL; centrifugation of NAL-induced cultures with chloroform adding (40 µL chloroform per 1 mL supernatant); soft-agar overlay technique for detection of phages; plaque transfer method for distinguishing between phages and bacteriocins.
Results. As a result of NAL-induction after 19 hours of incubation at a temperature 28° C (for phytopathogens) and 37° C (for E. coli C600), lysis spots have been observed, depending on the NAL-concentration and cultivation medium. The detected lysis zones were large with a non-sharp border. Supernatant from P. ananatis grown on M9+glucose medium with NAL concentration 20 mg/mL gave plaques on all indicator strains. LB-cultivated P. ananatis gave plaques on E. сoli C600 and P. carotovorum by NAL-concentration 20 mg/mL; on E. dissolvens indicator strain plaques were observed by NAL concentration 16 mg/mL. Phage lysate from P. carotovorum and E. dissolvens (LB cultivated) gave plaques only in induction with NAL in concentration 16 mg/mL. Plaque-forming activity in case of NAL concentration 20 mg/mL and/or M9+glucose medium cultivated cultures has not been observed. Plaque transfer method has demonstrated the multiplication of plaques. Thus, lysis zones have been caused by phages, but not bacteriocins.
Conclusions. Lysogenic phages from phytopathogenic strains (P. ananatis in particular) can be seen as attractive agents in further studies of microbial ecology (host-phytopathogenic bacteria interactions and the ways of controlling them) as well as for pre-harvest applications (crop protection).