Induction, Resuscitation, And The Mechanism Of VBNC State In Clavibacter Michiganensis Subsp. Michiganensis

Clavibacter michiganensis subsp. michiganensis （Cmm）, the causal agent of bacterial canker of tomato, is one of the most destructive threats to tomato seed production and tomato field production worldwide. 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. According to this theory, experiements were conducted to study the effect of copper ion and low pH on the induction of VBNC state in Cmm and the resuscitation and mechanism of VBNC state in Cmm in this research. It will contribute to analyzing the survival strategy of Cmm in harsh enrivonments, and revealing the bacteria in VBNC state could be the primary inoculum of bacterial canker in the field. The main results are as followed.1) A flow cytometry （FCM） method using the nucleic acid dyes, SYTO 9 and propidium iodide （PI）, combined with agar plating was developed to detect and count VBNC cells. The optimal population range of Cmm for FCM detecting was 105-107 cell/mL. The final concentrations of SYTO 9 and PI were 50 μmol/L and 150μmol/L, respectively. The Cmm cells were incubated with SYTO 9 and PI separately for 30 min and 60 min, respectively. The settings of FCM were as follows:amplifier mode, Log; voltage of FSC, E00; voltage of SSC,440V; voltage of FL1 for SYTO 9,640V; voltage of FL2 for PI,625V; speed, medium.2) Cmm could be induced into the VBNC state when exporsure to low concentration of copper ion and mild acid in oligotrophic environments. In 0.85% NaCl solution supplemented with 0.5-50 μM CuSO4, all the viable cells of Cmm could enter the VBNC state 37 d-2 h after induction. In 0.85%NaCl solution with pH 3.5-5.5, all the viable Cmm cells entered the VBNC state 3-107 d after induction. In the neutral NaCl solution without Cu2+, all the viable Cmm cells lost culturability after ca.110 d. With the increase of copper ion concentration or decrease of pH value, Cmm could enter the VBNC state more quickly.3) The resuscitation of VBNC Cmm cells could be achieved by removal of inducer, adding nutrients and inoculating the hose plants. The VBNC cells, which induced by 50 μM Cu2+ for 2 h or by 5 μM Cu2+ for 8 h and 1 d, could recover the culturability by adding EDTA in the inducing microcosms at the final concentration of 75 μM and 7.5 μM, respectively. The VBNC cells, which induced by 50 μM and 5 μM Cu2+ for 1 d or by pH 3.5 for 11 d, could recover the culturability in 0.1 ×LB broth. The VBNC cells, which induced by 5 μM Cu2+ for 1 d and 5 d or by pH 3.5 for 11d, could recover the culturability in 5% tomato seedling homogenate broth. The cell-free supernatant of Cmm in LB broth could recover the VBNC cells induced by 50 μM Cu2+ for 1 d. Inoculation of tomato seedlings could help Cmm cells, induced with 50 μM Cu2+ for 2 h-3 d,5μM Cu2+ for 1 d and 0.5μM Cu2+ for 4 d, recover from VBNC state. The pathogenicity of resuscitated Cmm cells was recovered with the restoration of culturability.4) The mechanism of formation and maintanence of VBNC state in Cmm cells was studied by RNA-Seq techinique and evaluating the expression levels of 16 key genes with RT-qPCR assay. One hundred and fourteen genes were up-regulated and 38 genes were down-regulated in Cu2+-induced Cmm cells from 5 min to 10 d. A big regulatory network was involved in the VBNC mechanism. Sigma factor （sigH） and some metal-sensitive transcriptional regulator families, such as MarR family （CMM₀414）, played important roles in the network. The genes associated with copper resistance （CMM₁035, CMM₁037）, stress （CMM₀581, CMM₀737）, stringent response （CMM₁809, CMM₁465）, and so on, all contributed to this physiological state. Most of genes responsible for cell wall were up-or down-regulated significantly. The VBNC cells kept energy and carbohydrate metabolism, however, the cell division related genes （CMM₀842, CMM₀012）, were repressed and lead to the non-culturability. The function of sigH （CMM₁319） and katA （CMM₀737） were preliminarily studied. Compared with the parent strains, deletion of katA resulted in slower growth rate in Cmm but showed no significant difference to copper response, and the time of log phase of AsigH mutant was extended and the mutant was more sensitive to copper. It was deduced that the sigH in Cmm act as a homologous gene of rpoS in Escherichia coli and was predicted to be a key regulator in VBNC regulatory networks.The current study is the first report of the VBNC state in a Gram-positive plant pathogen. The results have improved our understanding of biology and physiology of phytopathogenic bacteria. Most importantly, since CuSO4 and low pH conditions are used for disease management in tomato production, induction of the VBNC state and subsequent resuscitation of Cmm cells on tomato seedlings may affect the pathogen detection based on culture-based assays, and as well as the disease development and management in the field.