Abstract
The speed and sensitivity of PCR-based assays allow shorter turnaround times for the detection of pathogens for which culture and serological methods are difficult or unavailable. PCR was performed with primer sets Cms50 and Cms72, designed previously by Millset al. (1997) through subtractive hybridization to detectClavibacter michiganensis subspeciessepedonicus (Cms). In bacterial suspensions, fewer than three cells/10 ul reaction were detected after PCR amplicons were hybridized with specific DIG-labeled DNA probes in an enzyme-linked oligonucleosorbent assay (ELOSA). In naturally infected tuber samples representing three cultivars of potato, the diagnostic sensitivity of PCR/ ELOSA was 96%, while the specificity exceeded 99%. PCR/ELOSA detectedCms in infected tuber samples with equal sensitivity regardless of colony morphology, potato cultivar, or primer sets.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Literature Cited
Baer, D., and N.C. Gudmestad. 1993. Serological detection of nonmucoid strains ofClavibacter michiganensis subsp.sepedonicus in potato. Phytopathology 83:157–163.
Bertoni, G., and D. Mills. 1987. A simple method to monitor growth of bacterial populations in leaf tissue. Phytopathology 77:832–835.
Boucher, A. 1995. BRR protocol, Version 1. Canada Seed Potato Certification Program, Canadian Food Inspection Agency, Agriculture and Agri-Food Canada, 850 Lincoln Road P.O. Box 20280, Fredericton, NB. pp. 30–33.
Chandler, D.P. 1998. Redefining relativity: quantitative PCR at low template concentrations for industrial and environmental microbiology. J Industrial Microbiol Biotech 21:128–140.
De Boer, S.H., and M. McCann. 1989. Determination of population densities ofCorynebacterium sepedonicum in potato stems during the growing season. Phytopathology 79:946–951.
DeBoer, S.H., and A. Wieczorek. 1984. Production of monoclonal antibodies toCorynebacterium sepedonicum. Phytopathology 74:1431–1434.
Dinesen, I.G., and S.H. DeBoer. 1995. Extraction ofClavibacter michiganensis subsp.sepedonicus from composite samples of potato tubers. Am Potato J 72:133–142.
Drennan, J.L., A.A.G. Westra, S.A. Slack, L.M. Delserone, A. Collmer, N.C. Gudmestad, and A.E. Oleson. 1993. Comparison of a DNA hybridization probe and ELISA for the detection ofClavibacter michiganensis subsp.sepedonicus in field-grown potatoes. Plant Dis 77:1243–1247.
Henson, J.M., and R. French. 1993. The polymerase chain reaction and plant disease diagnosis. Annu Rev Phytopathol 31:81–109.
Li, X., and S.H. DeBoer. 1995. Selection of polymerase chain reaction primers from an RNA intergenic spacer region for specific detection ofClavibacter michiganensis subsp.sepedonicus. Phytopathology 85:837–842.
Mills, D., B.W. Russell, and J.W. Hanus. 1997. Specific detection ofClavibacter michiganensis subsp.sepedonicus by amplification of three unique DNA sequences isolated by subtraction hybridization. Phytopathology 87:853–861.
Mogen, B.D., A.E. Oleson, R.B. Sparks, N.C. Gudmestad, and G.A. Secor. 1988. Distribution and partial characterization of pCS1, a highly conserved plasmid present inClavibacter michiganense subsp.sepedonicum. Phytopathology 78:1381–1386.
Rademaker, J.L.W., and J.D. Janse. 1994. Detection and identification ofClavibacter michiganensis subsp.sepedonicus andClavibacter michiganensis subsp.michiganensis by nonradioactive hybridization, polymerase chain reaction, and restriction enzyme analysis. Can J Microbiol 40:1007–1018.
SAS (Statistical Analysis System). 1994. The SAS system for Microsoft Windows, ver. 6.10. SAS Institute, Cary, NC.
Schaad, N.W. 1988. Laboratory guide for identification of plant pathogenic bacteria, 2nd ed. American Phytopathological Society, Saint Paul, MN.
Schaad, N.W., Y. Berthier-Schaad, A. Sechler, and D. Knorr. 1999. Detection ofClavibacter michiganensis subsp.sepedonicus in potato tubers by BIO-PCR and an automated real-time fluorescence detection system. Plant Dis 83:1095–1100.
Schneider, J., J. Zhao, and C.S Orser. 1993. Detection ofClavibacter michiganensis subsp.sepedonicus by DNA amplification. FEMS Microbiol Lett 109:207–212.
Slack, S.A., J.L. Drennan, A.A.G. Westra, N.C. Gudmestad, and A.E. Oleson. 1996. Comparison of PCR, ELISA, and DNA hybridization for the detection ofClavibacter michiganensis subsp.sepedonicus. Plant Dis 80:519–524.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Baer, D., Mitzel, E., Pasche, J. et al. PCR detection ofclavibacter michiganensis subsp.sepedonicus- infected tuber samples in a plate capture assay. Amer J of Potato Res 78, 269–277 (2001). https://doi.org/10.1007/BF02875692
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02875692