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

Abstract

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.

Ecological patterns of seed microbiome diversity, transmission, and assembly

“…of the seed microbiota may be inactive or dormant [65,66], it seems unlikely that priority effects play a major role in a seed ultimate community structure, though this remains to be tested specifically. Still, we can hypothesize the general drivers that may be niche or neutral in seed microbiota assembly.…”

Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR

“…The challenge in detecting Cmm is that the bacterium enters a viable but nonculturable (VBNC) state under stress conditions [ 14 ]. VBNC is defined as metabolically active but without the ability to grow on conventional media [ 15 ].…”

“…The LIVE/DEAD Bac Light Bacterial Viability Kit has been widely used to distinguish live cells [ 16 , 20 ]. Jiang et al used this chemical treating bacteria prior to measuring the Cmm population with the flow cytometry method to count viable Cmm cells, combined with agar plating assay to enumerate VBNC cells [ 14 ]. However, this method cannot be used to detect the VBNC Cmm cells from a real sample, such as seed extract, because the flow cytometry method cannot differentiate between Cmm and other bacterial cells.…”

Extraction and detection of guanosine 5'-diphosphate-3'-diphosphate in Clavibacter michiganensis cells

“…The bacterial cells can be recovered or resuscitated from harsh stress by suitable environmental conditions, allowing the resuscitated cells become culturable. It has been reported that Cm could be induced into VBNC state under stress conditions of low pH and Cu 2+ , and then resuscitated by addition of nutrient or inoculation in planta (Jiang et al 2016). It is well known that ppGpp can accumulate and act as an alarmone molecule in the regulation of growth and survival in bacteria when adverse conditions are encountered.…”