Induction of the viable but nonculturable state in Clavibacter michiganensis subsp. michiganensis and in planta resuscitation of the cells on tomato seedlings

Bacterial canker of tomato is an economically important seedborne disease caused by Clavibacter michiganensis subsp. michiganensis (Cmm). Copper-based bactericides and seed treatment with hydrochloric acid are commonly used for bacterial canker management. Recent studies have shown that some bacteria can enter a viable but nonculturable (VBNC) state, and fail to form colonies on microbiological agar media. Bacteria in the VBNC state can recover their culturability when returned to favourable conditions. This study reports the induction of the VBNC state in Cmm by CuSO4 and low pH, and resuscitation of VBNC cells on tomato seedlings. Flow cytometry using the nucleic acid dyes SYTO 9 and propidium iodide, combined with agar plating, was used to assess VBNC cell counts. It was demonstrated that CuSO4 and low pH induced the VBNC state in Cmm and the rate of induction increased with copper ion concentration and acidity. Pathogenicity tests showed that some of the VBNC cells induced by CuSO4 retained their ability to colonize tomato seedlings but failed to produce typical bacterial canker symptoms by 2 months post-inoculation. This was probably due to low levels of resuscitation of VBNC Cmm cells resulting in low levels of initial inoculum. This study has improved understanding of the VBNC state of Gram-positive phytopathogenic bacteria. Most importantly, because copper-based chemicals and low pH conditions are used for disease management, induction of the VBNC state and subsequent resuscitation of Cmm cells on tomato seedlings may limit pathogen detection by culture-based assays yet present a risk for disease development in the field.