A Quantitative and Direct PCR Assay for the Subspecies-Specific Detection of Clavibacter michiganensis subsp. michiganensis Based on a Ferredoxin Reductase Gene
Min Seok Cho1, Jang Ha Lee2, Nam Han Her2, ChangKug Kim1, Young-Joo Seol1, Jang Ho Hahn1, Ji Hyoun Baeg3, Hong Gi Kim4, and Dong Suk Park1*
1National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Republic of Korea, 2Nongwoo Bio, Suwon 443-372, Republic of Korea, 3Animal, Plant and Fisheries Quarantine and Inspection Agency, Incheon International Airport Regional Office, Baggage Quarantine and Inspection Division, Incheon 400-718, Republic of Korea, 4Department of Applied Biology, Chungnam National University, Daejeon 305-764, Republic of Korea
Journal of Microbiology, 50(3),496-501, 2012,
Clavibacter michiganensis subsp. michiganensis, detection, quantitation, tomato, ferredoxin reductase
The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.