| Bacterial canker of tomato,a seedborne bacterial disease caused by Clavibacter michiganensis subsp.michiganensis(Cmm),is one of the most destructive threats to tomato seed production and tomato field production worldwide.It has been reported that Cmm could enter the viable but nonculturable(VBNC)state in nutrient starvation and fail to produce a visible colony on routine media,while it would recover the culturable and virulence in suitable environment.In this study,the detection method with qPCR for VBNC cells of Cmm in tomato seed was developed,and the morphological characteristics and peptidoglycan structure of VBNC Cmm was analyzed.It will contribute to clear the formation mechanism of Cmm in VBNC state.The main results are as followed.1.qPCR method using the nucleic acid dyes PMAxx,combined with agar plating was developed to detect and count VBNC cells.The optimal PMAxx concentration was 20 ?M for detecting membrane-intact Cmm cells after 8 min of light avoidance and 10 min of straight light.High specificity and sensitivity were obtained at Cmm concentrations ranging from 103 to 107 CFU ml-1.The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells induced by 50 ?M Cu2+ for 0,3,24,72,144,240,360.480 and 720 h.Furthermore,the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set.2.Morphologies of Cmm were measeured in different states under a scanning electron microscopy(SEM)and a transmission electron microscopy(TEM).It showed that the cells surface would heave and crude in VBNC state,and the width was bigger than three other state of cells,and the width was 492 ±31 nm.385 ± 19 nm,364 ± 26 nm and 412± 14 nm respectively for Cmm VBNC state,exponential phase,heat treatment and alcohol treatment cells by SEM(60000 times magnified).The blank area of Cmm cells observed by TEM(50000 times magnified)was enlargement with the induction time,it might indicate the nucleonic acid and protein was degradation in VBNC state Cmm.3.The mechanism of formation and maintanence of VBNC state in Cmm cells was studied and evaluating the expression levels of peptidoglycan synthstic genes with RT-qPCR assay.The function of mraY and murJ were preliminarily studied.For the growth rate and the speed of entering VBNC state,it didn't have the significant difference bwtween the parent strains and the mutant strains.4.Cmm cells in exponential,stationary and VBNC states were broken by sonicated in a melting ice bath with VCX 150 sonicator.Results showed that the time of sonication pulses,which was applied to reduce the original absorbance of the culture OD580 by 50%,was three times for VBNC state cells than exponential phase and stationary phase.VBNC Cmm cells would also have the resistance to heat treatment than exponential phase cells.A rapid extraction method for peptidoglycan of Cmm combined ultrasonic and lysozyme was established.According to the analysis of peptidoglycan extractive in 0,2,24,48,120 and 240 h by UPLC-MS method,the percentage of muropeptides cross-linked became bigger in VBNC state(from 38.0%to 52.6%).The muropeptides-tetramers were first shown at 24 h.The acetylated peptidoglycan were also riched in VBNC state(from 23%to 36.2%).The current study is the first report of the VBNC state in a Gram-positive plant pathogen for the research of the morphological characteristics and peptidoglycan structure.The results have improved our understanding of the mechanism of VBNC state and stress-resistant physiology of Cmm and other plant pathogenic bacteria,and as well as the disease development and management in the field. |
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