European Journal of Plant Pathology

, Volume 125, Issue 3, pp 413–423 | Cite as

Design and development of a DNA microarray for rapid identification of multiple European quarantine phytopathogenic bacteria

  • Cosima Pelludat
  • Brion DuffyEmail author
  • Jürg E. Frey


The European and Mediterranean Plant Protection Organization (EPPO) lists for quarantine status 26 phytopathogenic bacteria which pose serious economic threats to agricultural and natural ecosystems. A prototype diagnostic DNA microarray was developed for the rapid and simple identification of 22 of these quarantine bacteria. The microarray has 38 probes targeted to the 16S rDNA and the house-keeping genes rpoB, groEL and ftsZ. The 16S rDNA probes were selected according to a multiple-probe concept taking into account the hierarchical structure of phytobacterial systematics. Hybridisation with Cy3-labelled PCR products of corresponding genes enabled differentiation of the quarantine bacteria down to the species and subspecies level.


Plant inspection Bacterial diagnostics Quarantine pathogen Microarray 



The authors thank Jörg Samietz for assistance with statistical evaluation and Alicia Timm for critical reading of the manuscript. This work was funded by the Swiss Secretariat for Education and Research (SBF, COST project no. C04.0204), and was conducted within the European research networking frameworks of COST Action 853 (Agricultural Biomarkers for Array Technology) and COST Action 873 (Bacterial diseases of stone fruits and nuts).


  1. Arahal, D. R., Llop, P., Alonso, M. P., & Lόpez, M. M. (2004). In silico evaluation of molecular probes for detection and identification of Ralstonia solanacearum and Clavibacter michiganensis subsp. sepedonicus. Systematic and Applied Microbiology, 27, 581–591.PubMedCrossRefGoogle Scholar
  2. Bodrossy, L., & Sessitsch, A. (2004). Oligonucleotide microarrays in microbial diagnostic. Current Opinions in Microbiology, 7, 245–254.CrossRefGoogle Scholar
  3. Boonham, N., Walsh, K., Smith, P., Madagan, K., Graham, I., & Barker, I. (2003). Detection of potato viruses using microarray technology: towards a generic method for plant viral disease diagnosis. Journal of Virological Methods, 108, 181–187.PubMedCrossRefGoogle Scholar
  4. Borneman, J., Skroch, P. W., O’Sullivan, K. M., Palus, J. A., Rumjanek, N. K., Jansen, J. L., et al. (1996). Molecular microbial diversity of an agricultural soil in Wisconsin. Applied and Environmental Microbiology, 62, 1935–1943.PubMedGoogle Scholar
  5. Deyong, Z., Willingmann, P., Heinze, C., Adam, G., Pfunder, M., Frey, B., et al. (2005). Differentiation of Cucumber mosaic virus isolates by hybridisation to oligonucleotides in a microarray format. Journal of Virological Methods, 123, 101–108.PubMedCrossRefGoogle Scholar
  6. Fessehaie, A., De Boer, S. H., & Lévesque, C. A. (2003). An oligonucleotide array for the identification and differentiation of bacteria pathogenic on potato. Phytopathology, 93, 262–269.PubMedCrossRefGoogle Scholar
  7. Fletcher, J., Bender, C., Budowle, B., Cobb, W. T., Gold, S. E., Ishimaru, C. A., et al. (2006). Plant pathogen forensics: capabilities, needs, and recommendations. Microbiology and Molecular Biology Reviews, 70, 450–471.PubMedCrossRefGoogle Scholar
  8. Hirano, S. S., & Upper, C. D. (2000). Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae—a pathogen, ice nucleus, and epiphyte. Microbiology and Molecular Biology Reviews, 64, 624–653.PubMedCrossRefGoogle Scholar
  9. Janse, J. D. (2005). Phytobacteriology: Principles and Practice. Wallingford, Oxfordshire, UK: CABI Publishing.Google Scholar
  10. Kim, B. C., Park, J. H., & Gu, M. B. (2004). Development of a microarray chip for the identification of sludge bacteria using an unsequenced random genomic DNA hybridisation method. Environmental Science and Technology, 38, 6767–6774.PubMedCrossRefGoogle Scholar
  11. Koh, Y. J., & Nou, I. S. (2002). DNA markers for identification of Pseudomonas syringae pv. actinidiae. Molecules and Cells, 13, 309–314.PubMedGoogle Scholar
  12. Lievens, B., & Thomma, B. P. H. J. (2005). Recent developments in pathogen detection arrays: implications for fungal plant pathogens and use in practice. Phytopathology, 95, 1374–1380.PubMedCrossRefGoogle Scholar
  13. Lievens, B., Brouwer, M., Vanachter, A. C. R. C., Lévesque, C. A., Cammue, B. P. A., & Thomma, B. P. H. J. (2003). Design and development of a DNA array for rapid detection and identification of multiple tomato vascular wilt pathogens. FEMS Microbiology Letters, 223, 113–122.PubMedCrossRefGoogle Scholar
  14. Lievens, B., Brouwer, M., Vanachter, A. C. R. C., Lévesque, C. A., Cammue, B. P. A., & Thomma, B. P. H. J. (2005). Quantitative assessment of phytopathogenic fungi in various substrates using a DNA macroarray. Environmental Microbiology, 7, 1698–1710.PubMedCrossRefGoogle Scholar
  15. Ludwig, W., Strunk, O., Westram, R., Richter, L., Meier, H., Yadhukumar, et al. (2004). ARB: a software environment for sequence data. Nucleic Acids Research, 32, 1363–1371.PubMedCrossRefGoogle Scholar
  16. Maiden, M. C., Bygraves, J. A., Feil, E., Morelli, G., Russell, J. E., Urwin, R., et al. (1998). Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proceedings of the National Academy of Sciences U.S.A., 17, 3140–3145.CrossRefGoogle Scholar
  17. Nicolaisen, M., & Bertaccini, A. (2007). An oligonucleotide microarray-based assay for identification of phytoplasma 16S ribosomal groups. Plant Pathology, 56, 332–336.CrossRefGoogle Scholar
  18. Park, D. S., Hyun, J. W., Park, Y. J., Kim, J. S., Kang, H. W., Hahn, J. H., et al. (2005). Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar-specific primers based on hrpW gene sequences. Microbiology Research, 161, 145–149.Google Scholar
  19. Pfunder, M., & Frey, J. E. (2005). Dissociation analysis in polymerase chain reaction and 1X SSC buffer as a prerequisite for selection of 13mer microarray probe sets with uniform hybridisation behavior. Molecular Biotechnology, 29, 1–10.PubMedCrossRefGoogle Scholar
  20. Pozhitkov, A., Noble, P. A., Domazet-Lošo, T., Nolte, A. W., Sonnenberg, R., Staehler, P., et al. (2006). Tests of rRNA hybridisation to microarrays suggest that hybridisation characteristics of oligonucleotide probes for species discrimination cannot be predicted. Nucleic Acids Research, 17(66), 2–12.Google Scholar
  21. Scally, M., Schuenzel, E. L., Stouthamer, R., & Nunney, L. (2005). Multilocus sequence type system for the plant pathogen Xylella fastidiosa and relative contributions of recombination and point mutation to clonal diversity. Applied and Environmental Microbiology, 71, 8491–8499.PubMedCrossRefGoogle Scholar
  22. Sholberg, P., O’Gorman, D., Bedford, K., & Lévesque, C. A. (2005). Development of a DNA microarray for detection and montitoring of economically important apple diseases. Plant Disease, 89, 1143–1150.CrossRefGoogle Scholar
  23. Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A., Kampfer, P., Maiden, M. C., et al. (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. International Journal of Systematic and Evolutionary Microbiology, 52, 1043–1047.PubMedCrossRefGoogle Scholar
  24. Stoughton, R. B. (2005). Applications of DNA microarrays in biology. Annual Reviews of Biochemistry, 74, 53–82.CrossRefGoogle Scholar
  25. Tobler, N. E., Pfunder, M., Herzog, K., Frey, J. E., & Altwegg, M. (2006). Rapid detection and species identification of Mycobacterium spp. using real-time PCR and DNA-microarray. Journal of Microbiological Methods, 66, 116–124.PubMedCrossRefGoogle Scholar
  26. Vora, G. J., Meador, C. E., Bird, M. M., Bopp, C. A., Andreadis, J. D., & Stenger, D. A. (2005). Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but non-culturable state in human pathogenic Vibrio spp. Proceedings of the National Academy of Sciences U.S.A., 102, 19109–19114.CrossRefGoogle Scholar
  27. Wang, D., Coscoy, L., Zylberberg, M., Avila, P. C., Boushey, H. A., Ganem, D., et al. (2002). Microarray-based detection and genotyping of viral pathogens. Proceedings of the National Academy of Sciences U.S.A., 99, 15687–15692.CrossRefGoogle Scholar
  28. Ward, E., Foster, S. J., Fraaije, B. A., & McCartney, H. A. (2004). Plant pathogen diagnostics: immunological and nucleic acid-based approaches. Annals of Applied Biology, 145, 1–16.CrossRefGoogle Scholar
  29. Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173, 697–703.PubMedGoogle Scholar
  30. Zhou, J. (2003). Microarrays for bacterial detection and microbial community analysis. Current Opinions in Microbiology, 6, 288–294.CrossRefGoogle Scholar

Copyright information

© KNPV 2009

Authors and Affiliations

  1. 1.Agroscope Changins-Wädenswil Research Station ACWWädenswilSwitzerland

Personalised recommendations