The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats

  • An, Ji-Hye (Department of Plant Medicine, Chungbuk National University) ;
  • Noh, Young-Hee (Department of Plant Medicine, Chungbuk National University) ;
  • Kim, Yong-Eon (Department of Plant Medicine, Chungbuk National University) ;
  • Lee, Hyok-In (Animal and Plant Quarantine Agency) ;
  • Cha, Jae-Soon (Department of Plant Medicine, Chungbuk National University)
  • Received : 2014.09.24
  • Accepted : 2015.02.07
  • Published : 2015.03.01
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Abstract

Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens.

Keywords

References

  1. Bella, P., Licciardello, G., Tessitori, M. and Catara, V. 2008. A real-time PCR quantitative detection assay for Pseudomonas savastanoi pv. nerii in Nerium oleander. Phytopathol. Mediterr. 47:204-213.
  2. Cho, J. H., Jeong, M. J., Song, M. J., Yim, K. O., Lee, H. I., Kim, J. H., Baeg, J. H. and Cha, J. S. 2010. Development of PCR Primers to Detect Pseudomonas savastanoi pv. phaseolicola from the Bean Seeds. Res. Plant Dis. 16:129-135. https://doi.org/10.5423/RPD.2010.16.2.129
  3. Collins, D. J. 2010. Oat Diseases in Alabama: Circular ANR-631. Alabama Cooperative Extension Service.
  4. Delcher, A. L., Bratke, K. A., Powers, E. C. and Salzberg, S. L. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673-679. https://doi.org/10.1093/bioinformatics/btm009
  5. Elliott, C. 1920. Halo-blight of oats. J. Agric. Res. 19:139-172.
  6. Finetti-Sialer, M. M. and Ciancio, A. 2005. Isolate-specific detection of Grapevine fan-leaf virus from Xiphinema index through DNA-based molecular probes. Phytopathology 95:262-268. https://doi.org/10.1094/PHYTO-95-0262
  7. Harder, D. E. and Haber, S. 1992. Oat diseases and pathologic techniques. In Oat Science and Technology 33:307-402.
  8. Hyatt, D., Chen, G. L., Locascio, P. F., Land, M. L., Larimer, F. W. and Hauser, L. J. 2010. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform. 11:119-129. https://doi.org/10.1186/1471-2105-11-119
  9. Lukashin, A. and Borodovsky, M. 1998. GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res. 26:1107-1115. https://doi.org/10.1093/nar/26.4.1107
  10. Maes, M., Garbeva, P. and Kamoen, O. 1996. Recognition and detection in seed of the Xanthomonas pathogens that cause cereal leaf streak using rDNA spacer sequences and polymerase chain reaction. Phytopathology 86:63-69. https://doi.org/10.1094/Phyto-86-63
  11. Margulies, M., Egholm, M., Altman, W. E., Attiya, S., Bader, J. S., Bemben, L. A., Berka, J., Braverman, M. S., Chen, Y. J., Chen, Z., Dewell, S. B., Du, L., Fierro, J. M., Gomes, X. V., Godwin, B. C., He, W., Helgesen, S., Ho, C. H., Ho, C. H., Irzyk, G. P., Jando, S. C., Alenquer, M. L., Jarvie, T. P., Jirage, K. B., Kim, J. B., Knight, J. R., Lanza, J. R., Leamon, J. H., Lefkowitz, S. M., Lei, M., Li, J., Lohman, K. L., Lu, H., Makhijani, V. B., McDade, K. E., McKenna, M. P., Myers, E. W., Nickerson, E., Nobile, J. R., Plant, R., Puc, B. P., Ronan, M. T., Roth, G. T., Sarkis, G. J., Simons, J. F., Simpson, J. W., Srinivasan, M., Tartaro, K. R., Tomasz, A., Vogt, K. A., Volkmer, G. A., Wang, S. H., Wang, Y., Weiner, M. P., Yu, P., Begley, R. F. and Rothberg, J. M. 2005. Genome sequencing in microfabricated highdensity picolitre reactors. Nature 437:376-380. https://doi.org/10.1038/nature03959
  12. Martens, J. W., Seaman, W. L. and Atkinson, T. G. 1984. Diseases of field crops in Canada. Canadian Phytopathological Society.
  13. Schena, L., Nigro, F., Ippolito, A. and Gallitelli, D. 2004. Real-time quantitative PCR: A new technology to detect and study phytopathogenic and antagonistic fungi. Eur. J. Plant Pathol. 110:893-908. https://doi.org/10.1007/s10658-004-4842-9
  14. Walcott, R. R. 2003. Detection of seedborne pathogens. Hort-Technology 13:40-47.
  15. Wallwork, H. 1992. Cereal leaf and stem diseases. In Grains Research and Development Corporation.
  16. Young, J. M., Dye, D. W. and Wilkie, J. P. 1978. Genus VII. Pseudomonas Migula 1894. New Zealand J. Agric. Res. 21:153-177. https://doi.org/10.1080/00288233.1978.10427397

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