European Journal of Plant Pathology

, Volume 138, Issue 4, pp 695–709 | Cite as

Development and evaluation of Taqman assays for the differentiation of Dickeya (sub)species

  • J. M. van der WolfEmail author
  • B. H. de Haas
  • R. van Hoof
  • E. G. de Haan
  • G. W. van den Bovenkamp


TaqMan assays were developed for the detection of seven Dickeya species, namely D. dianthicola, D. dadantii, D. paradisiaca, D. chrysanthemi, D. zeae, D. dieffenbachiae and D. solani. Sequences of the gene coding for dnaX were used for the design of primers and probes. In studies with axenic cultures of bacteria, the assays were highly specific and only reacted with strains of the target species, and not with non-target bacteria, including those belonging to other Dickeya species and other genera. The detection thresholds for DNA extracted from pure cultures of target strains ranged from 10 to 100 fg. The TaqMan assays for D. dianthicola and D. solani were more extensively evaluated as part of a method validation procedure. The threshold level for target bacteria added to a potato peel extract diluted ten-times in a semi-selective broth, was strain dependent and ranged from 1,000 to 100,000 cfu/ml. The coefficients of variation for repeatability and reproducibility were low and results were largely independent of the type of substrate, i.e. potato tuber or carnation leaf extracts. However, during routine testing of seed potatoes, false-positive reactions were found with the assay for D. solani. The use of the TaqMan assays for inspection of plant propagation material, ecological studies and studies on the effect of control strategies in disease management strategies is discussed.


Blackleg dnaX Primer and probe design Potato Carnation 



This work has been financially supported by the Dutch Ministry of Economic Affairs, (project BO-06-004-2.1)and FES project Strengthening the infrastructure of plant health. Thanks are indebted to Dr M.C.M. Pérombelon (Dundee, UK) for his editorial work.


  1. Anonymous (2005). Accreditation programme building materials decree. Section: Soil composition. In Soil Management Board (Ed.), Incorporated in Foundation Infrastructure for Quality Assurance of Soil Management (SIKB) in Gouda, Netherlands, Gouda. AP04-SC, version 7, sikb/documents/05.7191, 70 pages.Google Scholar
  2. Anonymous (2010). Nationale richtlijn voor de validatie van detectie- en identificatiemethoden voor plantpathogenen en plaagorganismen. Nationaal Referentielaboratorium, Plantenziektenkundige Dienst, Wageningen, 25 pages.Google Scholar
  3. Anonymous. (2013). Scientific Opinion on the risk of Dickeya dianthicola for the EU territory with identification and evaluation of risk reduction options. EFSA Panel on Plant Health (PLH). EFSA Journal, 11, 3072. 115 pp. doi: 10.2903/j.efsa.2013.3072. Available online: Scholar
  4. Bogdanove, A. J., Koebnik, R., Lu, H., Furutani, A., Angiuoli, S. V., Patil, P. B., et al. (2011). Two new complete genome sequences offer insight into host and tissue specificity of plant pathogenic Xanthomonas spp. Journal of Bacteriology, 193, 5450–5464.PubMedCentralPubMedCrossRefGoogle Scholar
  5. Brady, C. L., Cleenwerck, I., Denman, S., Venter, S. N., Rodríguez-Palenzuela, P., Coutinho, T. A., & De Vos, P. (2012). Proposal to reclassify Brenneria quercina (Hildebrand and Schroth 1967) Hauben et al. 1999 into a new genus, Lonsdalea gen. nov., as Lonsdalea quercina comb. nov., descriptions of Lonsdalea quercina subsp. quercina comb. nov., Lonsdalea quercina subsp. iberica subsp. nov. and Lonsdalea quercina subsp. britannica subsp. nov., emendation of the description of the genus Brenneria, reclassification of Dickeya dieffenbachiae as Dickeya dadantii subsp. dieffenbachiae comb. nov., and emendation of the description of Dickeya dadantii. International Journal of Systematic and Evolutionary Microbiology, 62, 1592–1602.Google Scholar
  6. Brunet-Galmés, I., Busquets, A., Peña, A., Gomila, M., Nogales, B., García-Valdés, E., et al. (2012). Complete genome sequence of the naphthalene-degrading bacterium Pseudomonas stutzeri AN10 (CCUG 29243). Journal of Bacteriology, 194, 6642–6643.PubMedCentralPubMedCrossRefGoogle Scholar
  7. Czajkowski, R., de Boer, W. J., van der Zouwen, P. S., Kastelein, P., Jafra, S., de Haan, E. G., et al. (2012). Virulence of ‘Dickeya solani’ and Dickeya dianthicola biovar-1 and -7 strains on potato (Solanum tuberosum). Plant Pathology, 62, 597–610.CrossRefGoogle Scholar
  8. Del Cerro, A., Mendoza, M. C., & Guijarro, J. A. (2002). Usefulness of a TaqMan-based polymerase chain reaction assay for the detection of the fish pathogen Flavobacterium psychrophilum. Journal of Applied Microbiology, 93, 149–156.PubMedCrossRefGoogle Scholar
  9. Dickey, R. S. (1979). Erwinia chrysanthemi: a comparative study of phenotypic properties of strains from several hosts and other Erwinia species. Phytopathology, 69, 324–329.CrossRefGoogle Scholar
  10. Elphinstone, J. G., Hennessey, J., Wilson, J. K., & Stead, D. E. (1996). Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts. EPPO Bulletin, 26, 663–678.CrossRefGoogle Scholar
  11. Holland, P. M., Abramson, R. D., Watson, R., & Gelfand, D. H. (1991). Detection of specific polymerase chain reaction product by utilizing the 5′ 3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proceedings of the National Academy of Sciences of the United States of America, 88, 7276–7280.PubMedCentralPubMedCrossRefGoogle Scholar
  12. Janse, J. D., & Ruissen, M. A. (1988). Characterization and classification of Erwinia chrysanthemi strains from several hosts in The Netherlands. Phytopathology, 78, 800–808.CrossRefGoogle Scholar
  13. Koshkin, A. A., Nielsen, P., Meldgaard, M., Rajwanshi, V. K., Singh, S. K., & Wengel, J. (1998). LNA (locked nucleic acid): an RNA mimic forming exceedingly stable LNA: LNA duplexes. Journal of the American Chemical Society, 120, 13252–13253.CrossRefGoogle Scholar
  14. Laurila, J., Ahola, V., Lehtinen, A., Joutsjoki, T., Hannukkala, A., Rahkonen, A., et al. (2008). Characterization of Dickeya strains isolated from potato and river water samples in Finland. European Journal of Plant Pathology, 122, 213–225.CrossRefGoogle Scholar
  15. Najioullah, F., Thouvenot, D., & Lina, B. (2001). Development of a real-time PCR procedure including an internal control for the measurement of HCMV viral load. Journal of Virological Methods, 92, 55–64.PubMedCrossRefGoogle Scholar
  16. Nassar, A., Darrasse, A., Lemattre, M., Kotoujansky, A., Dervin, C., Vedel, R., et al. (1996). Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR- amplified fragments of pel genes. Applied and Environmental Microbiology, 62, 2228–2235.PubMedCentralPubMedGoogle Scholar
  17. Parkinson, N., Stead, D., Bew, J., Heeney, J., Tsror, L., & Elphinstone, J. (2009). Dickeya species relatedness and clade structure determined by comparison of recA sequences. International Journal of Systemic and Evolutionary Microbiology, 59, 2388–2393.CrossRefGoogle Scholar
  18. Pritchard, L., Humphris, S., Saddler, G. S., Parkinson, N. M., Bertrand, V., Elphinstone, J. G., et al. (2012). Detection of phytopathogens of the genus Dickeya using a PCR primer prediction pipeline for draft bacterial genome sequences. Plant Pathology, 62, 587–596.CrossRefGoogle Scholar
  19. Samson, R., Legendre, J. B., Christen, R., Fischer-Le Saux, M., Achouak, W., & Gardan, L. (2005). Transfer of Pectobacterium chrysanthemi (Burkholder et al. 1953) Brenner et al. 1973 and Brenneria paradisiaca to the genus Dickeya gen. nov. as Dickeya chrysanthemi comb. nov. and Dickeya paradisiaca comb. nov. and delineation of four novel species, Dickeya dadantii sp. nov., Dickeya dianthicola sp. nov., Dickeya dieffenbachiae sp. nov. and Dickeya zeae sp. nov. International Journal of Systemic and Evolutionary Microbiology, 55, 1415–1427.CrossRefGoogle Scholar
  20. Shin, S. H., Kim, S., Kim, J. Y., Lee, S., Um, Y., Oh, M.-K., et al. (2012). Complete genome sequence of Enterobacter aerogenes KCTC 2190. Journal of Bacteriology, 194, 2373–2374.PubMedCentralPubMedCrossRefGoogle Scholar
  21. Silby, M., Cerdeno-Tarraga, A., Vernikos, G., Giddens, S., Jackson, R., Preston, G., et al. (2009). Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens. Genome Biology, 10, R51.PubMedCentralPubMedCrossRefGoogle Scholar
  22. Sławiak, M., Łojkowska, E., & Van der Wolf, J. M. (2009a). First report of bacterial soft rot on potato caused by Dickeya sp. (syn. Erwinia chrysanthemi) in Poland. Plant Pathology, 58, 794.Google Scholar
  23. Sławiak, M., van Beckhoven, J. R. C. M., Speksnijder, A. G. C. L., Czajkowski, R., Grabe, G., & Van der Wolf, J. M. (2009b). Biochemical and genetical analysis reveal a new clade of biovar 3 Dickeya spp. strains isolated from potato in Europe. European Journal of Plant Pathology, 125, 245–261.CrossRefGoogle Scholar
  24. Stephan, R., Lehner, A., Tischler, P., & Rattei, T. (2011). Complete genome sequence of Cronobacter turicensis LMG 23827, a food-borne pathogen causing deaths in neonates. Journal of Bacteriology, 193, 309–310.PubMedCentralPubMedCrossRefGoogle Scholar
  25. Toth, I. K., van der Wolf, J. M., Saddler, G., Lojkowska, E., Helias, V., Pirhonen, M., et al. (2011). Dickeya species: an emerging problem for potato production in Europe. Plant Pathology, 60, 385–399.CrossRefGoogle Scholar
  26. Tsror, L., Erlich, O., Lebiush, S., Hazanovsky, M., Zig, U., Slawiak, M., et al. (2009). Assessment of recent outbreaks of Dickeya sp (syn. Erwinia chrysanthemi) slow wilt in potato crops in Israel. European Journal of Plant Pathology, 123, 311–320.CrossRefGoogle Scholar
  27. Tsror, L., Erlich, O., Hazanovsky, M., Ben Daniel, B., Zig, U., & Lebiush, S. (2012). Detection of Dickeya spp. latent infection in potato seed tubers using PCR or ELISA and correlation with disease incidence in commercial field crops under hot-climate conditions. Plant Pathology, 61, 161–168.CrossRefGoogle Scholar
  28. Van der Wolf, J. M., Nijhuis, E. H., Kowalewska, M. J., Saddler, G. S., Parkinson, N., Elphinstone, J. G., et al. (2013). Dickeya solani sp. nov., a pectinolytic plant pathogenic bacterium isolated from potato (Solanum tuberosum). International Journal of Systematic and Evolutionary Microbiology. doi: 10.1099/ijs.0.052944-0
  29. van Doorn, J., Vreeburg, P. J. M., van Leeuwen, P. J., & Dees, R. H. L. (2011). The presence and survival of soft rot (Erwinia) in flower bulb production systems. In J. E. v. d. Ende (ed.), Xth ISHS on flower bulbs and herbaceous perennials. Acta Horticultura, 886, 365–380Google Scholar
  30. Van Vaerenbergh, J., Baeyen, S., De Vos, P., & Maes, M. (2012). Sequence diversity in the Dickeya fliC gene: phylogeny of the DickeyaGenus and TaqMan® PCR for ‘D. solani’, New Biovar 3 Variant on Potato in Europe. PloS One, 7, e35738.PubMedCentralPubMedCrossRefGoogle Scholar
  31. Weiss, J., Wu, H., Farrenkopf, B., Schultz, T., Song, G., Shah, S., et al. (2004). Real time TaqMan PCR detection and quantification of HBV genotypes A-G with the use of an internal quantitation standard. Journal of Clinical Virology, 30, 86–93.PubMedCrossRefGoogle Scholar
  32. Zeigler, D. R. (2003). Gene sequences useful for predicting relatedness of whole genomes in bacteria. International Journal of Systemic and Evolutionary Microbiology, 53, 1893–1900.CrossRefGoogle Scholar

Copyright information

© KNPV 2013

Authors and Affiliations

  • J. M. van der Wolf
    • 1
    Email author
  • B. H. de Haas
    • 1
  • R. van Hoof
    • 1
    • 3
  • E. G. de Haan
    • 2
  • G. W. van den Bovenkamp
    • 2
  1. 1.Plant Research InternationalWageningenThe Netherlands
  2. 2.Dutch General Inspection Service for Agricultural Seed and Seed Potatoes (NAK)EmmeloordThe Netherlands
  3. 3.RIKILTWageningenThe Netherlands

Personalised recommendations