Quantification of viable cells of Clavibacter michiganensis subsp. michiganensis using a DNA binding dye and a real-time PCR assay

Viable cells of Clavibacter michiganensis subsp. michiganensis (CMM), the causal agent of bacterial canker of tomato, were discriminated from the dead cells by quantitative real-time polymerase chain reaction (PCR), after the bacterial solution was treated with the DNA binding dye ethidium monoazide (EMA). The primers and TaqMan probe, based on the 16S-23S rDNA spacer sequences, were highly specific for CMM at the subspecies level. The detection limit of the direct real-time PCR was 103 colony forming units per mL (cfu mL−1) in samples and with an apparent sensitivity of 2 cfu of target cells in PCR reaction solution. Application of this method allows for selective quantification of viable cells of CMM and facilitates monitoring the pathogen in tomato seeds.

[1]  R. Levin,et al.  Discrimination of viable Vibrio vulnificus cells from dead cells in real-time PCR. , 2006, Journal of microbiological methods.

[2]  R. Frederick,et al.  Advances in molecular-based diagnostics in meeting crop biosecurity and phytosanitary issues. , 2003, Annual review of phytopathology.

[3]  Knut Rudi,et al.  Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR. , 2003, BioTechniques.

[4]  M. Schloter,et al.  A TaqMan-PCR protocol for quantification and differentiation of the phytopathogenic Clavibacter michiganensis subspecies. , 2003, Journal of microbiological methods.

[5]  N. Schaad,et al.  Survival of Clavibacter michiganensis ssp. michiganensis in infected tomato stems under natural field conditions in California, Ohio and Morocco , 2002 .

[6]  M. Gleason,et al.  Refinement of a nondestructive tomato seed assay for Clavibacter michiganensis subsp. michiganensis using seed fiber , 2000 .

[7]  Y. Berthier-Schaad,et al.  Detection of Clavibacter michiganensis subsp. sepedonicus in Potato Tubers by BIO-PCR and an Automated Real-Time Fluorescence Detection System. , 1999, Plant disease.

[8]  H. Jahr,et al.  Interactions between Clavibacter michiganensis and its host plants. , 1999, Environmental microbiology.

[9]  Jan LW Rademaker,et al.  THE THREE DS OF PCR-BASED GENOMIC ANALYSIS OF PHYTOBACTERIA: Diversity, Detection, and Disease Diagnosis. , 1999, Annual review of phytopathology.

[10]  A Bastiaansen,et al.  Evaluation of the NASBA nucleic acid amplification system for assessment of the viability of Campylobacter jejuni. , 1997, International journal of food microbiology.

[11]  Xiang Li.  Selection of polymerase chain reaction primers from an RNA intergenic spacer region for specific detection of Clavibacter michiganensis subsp. sepedonicus. , 1995 .

[12]  S. D. De Boer,et al.  Comparison of 16S ribosomal RNA genes in Clavibacter michiganensis subspecies with other coryneform bacteria. , 1995, Canadian journal of microbiology.

[13]  W. Bolton,et al.  Comparison of cell viability probes compatible with fixation and permeabilization for combined surface and intracellular staining in flow cytometry. , 1995, Cytometry.

[14]  R. Eichenlaub,et al.  Southern hybridization and PCR for specific detection of phytopathogenic Clavibacter michiganensis subsp. michiganensis , 1995 .

[15]  M. Ricker,et al.  Recent progress in understanding and controlling bacterial canker of tomato in eastern North America , 1993 .

[16]  J. Pataky,et al.  Dissemination of Clavibacter michiganensis subsp. michiganensis by practices used to produce tomato transplants. , 1991 .

[17]  M. Gleason,et al.  Survival and dissemination of Clavibacter michiganensis subsp. michiganensis in tomatoes , 1991 .

[18]  C. Stewart,et al.  Use of a photolabeling technique to identify nonviable cells in fixed homologous or heterologous cell populations. , 1991, Cytometry.

[19]  P. Bolton,et al.  Spectroscopic properties of ethidium monoazide: a fluorescent photoaffinity label for nucleic acids. , 1978, Nucleic acids research.

[20]  W. E. White,et al.  Selective covalent binding of an ethidium analog to mitochondrial DNA with production of petite mutants in yeast by photoaffinity labelling. , 1975, Journal of molecular biology.