Exploiting generic platform technologies for the detection and identification of plant pathogens

  • Neil Boonham
  • Rachel Glover
  • Jenny Tomlinson
  • Rick Mumford


The detection and identification of plant pathogens currently relies upon a very diverse range of techniques and skills, from traditional culturing and taxonomic skills to modern molecular-based methods. The wide range of methods employed reflects the great diversity of plant pathogens and the hosts they infect. The well-documented decline in taxonomic expertise, along with the need to develop ever more rapid and sensitive diagnostic methods has provided an impetus to develop technologies that are both generic and able to complement traditional skills and techniques. Real-time polymerase chain reaction (PCR) is emerging as one such generic platform technology and one that is well suited to high-throughput detection of a limited number of known target pathogens. Real-time PCR is now exploited as a front line diagnostic screening tool in human health, animal health, homeland security, biosecurity as well as plant health. Progress with developing generic techniques for plant pathogen identification, particularly of unknown samples, has been less rapid. Diagnostic microarrays and direct nucleic acid sequencing (de novo sequencing) both have potential as generic methods for the identification of unknown plant pathogens but are unlikely to be suitable as high-throughput detection techniques. This paper will review the application of generic technologies in the routine laboratory as well as highlighting some new techniques and the trend towards multi-disciplinary studies.


Plant health Diagnostics Detection Real-time PCR DNA barcoding Microarrays Isothermal amplification LAMP Direct tuber testing Molecular diagnostics TaqMan De novo sequencing Pyrosequencing 



The authors would like to acknowledge funding from Plant Health Division and Chief Scientist Group of Defra (, and also the European Union for funding under the fifth framework programme ( and also the sixth framework programme ( project (SSPE-CT-2004-502348). In addition the authors would like to acknowledge the help and support provided through the COST project ‘Agricultural Biomarkers for Array Technology’ (


  1. Armstrong, K. F., & Ball, S. L. (2005). DNA barcodes for biosecurity: invasive species identification. Philosophical Transactions of the Royal Society B, 360, 1813–1823.CrossRefGoogle Scholar
  2. Ball, S. L., & Armstrong, K. F. (2006). DNA barcodes for insect pest identification: a test case with tussock moths (Lepidoptera: Lymantriidae). Canadian Journal of Forest Research, 36, 337–350.CrossRefGoogle Scholar
  3. Blaxter, M. (2003). Molecular systematics: counting angels with DNA. Nature, 421, 122–124.PubMedCrossRefGoogle Scholar
  4. Blaxter, M. (2004). The promise of a DNA taxonomy. Philosophical Transactions of the Royal Society B, 359, 669–679.CrossRefGoogle Scholar
  5. Boonham, N., Tomlinson, J., & Mumford, R. (2007). Microarrays for rapid identification of plant viruses. Annual review of Phytopathology, 45, 307–328.PubMedCrossRefGoogle Scholar
  6. Boonham, N., Walsh, K., Mumford, R. A., & Barker, I. (2000). The use of multiplex real-time PCR (TaqMan®) for the detection of potato viruses. EPPO Bulletin, 30, 427–430.CrossRefGoogle Scholar
  7. Brunner, P. C., Fleming, C., & Frey, J. E. (2002). A molecular identification key for economically important thrips species (Thysanoptera: Thripidae) using direct sequencing and a PCR-RFLP-based approach. Agricultural and Forest Entomology, 4, 127–136.CrossRefGoogle Scholar
  8. Clare, E. L., Lim, B. K., Engstron, M. D., Eger, J. L., & Hebert, P. D. N. (2007). DNA bar coding of neotropical bats: species identification and discovery within Guyana. Molecular Ecology Notes, 7, 184–190.CrossRefGoogle Scholar
  9. Clark, M. F., & Adams, A. N. (1977). Characteristics of the microplate method of Enzyme-Linked Immunosorbent Assay for the detection of plant viruses. Journal of General Virology, 34, 475–483.Google Scholar
  10. Coomans, A. (2002). Present status and future of nematode systematics. Nematology, 4, 573–582.CrossRefGoogle Scholar
  11. Cox-Foster, D. L., Conlan, S., Holmes, E. C., Palacios, G., Evans, J. D., Moran, N. A., et al. (2007). A metagenomic survey of microbes in honey bee colony collapse disorder. Science, 318(5848), 283–287, Oct 12, Epub 2007 Sep 6.PubMedCrossRefGoogle Scholar
  12. Gandelman, O., Church, V. L., Moore, C., Carne, C., Jalal, H., Murray, J. A. H., et al. (2006). Bioluminescent alternative to real-time PCR (BART). Luminescence, 21, 276–277.Google Scholar
  13. Hajibabaei, M., Singer, G. A. C., Clare, E. L., & Hebert, P. D. N. (2007). Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring. BMC Biology, 5, 24.PubMedCrossRefGoogle Scholar
  14. Hebert, P. D. N., Cywinska, A., Ball, S. L., & deWaard, J. R. (2003a). Biological identification through DNA barcodes. Proceedings of the Royal Society London B, 270, 313–321.CrossRefGoogle Scholar
  15. Hebert, P. D. N., Ratnasingham, S., & deWaard, J. R. (2003b). Bar coding animal life: Cytochrome C oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society London B, 270, S96–S99.CrossRefGoogle Scholar
  16. Hebert, P. D. N., Stoeckle, M. Y., Zemlak, T. S., & Francis, C. M. (2004). Identification of birds through DNA barcodes. PloS Biology, 2, e312.PubMedCrossRefGoogle Scholar
  17. Hill, S. A., & Jackson, E. A. (1984). An investigation of the reliability of ELISA as a practical test for detecting Potato leaf roll virus and Potato virus Y in tubers. Plant Pathology, 33, 21–26.CrossRefGoogle Scholar
  18. Hopkins, G. W., & Freckleton, R. P. (2002). Declines in the numbers of amateur and professional taxonomists: implications for conservation. Animal Conservation, 5, 245–249.CrossRefGoogle Scholar
  19. Hulcr, J., Miller, S. E., Setliff, G. P., Darrow, K., Mueller, N. D., Hebert, P. D. N., et al. (2007). DNA bar coding confirms polyphagy in a generalist moth, Homona mermerodes (Lepidoptera: Tortricidae). Molecular Ecology Notes, 7, 549–557.CrossRefGoogle Scholar
  20. Jaffrezic-Renault, N., Martelet, C., Chevolot, Y., & Cloarec, J. P. (2007). Biosensors and bio-bar code assays based on biofunctionalized magnetic microbeads. Sensors, 7, 589–614.CrossRefGoogle Scholar
  21. Kerr, K. C. R., Stoeckle, M. Y., Dove, C. J., Weight, L. A., Francis, C. M., & Hebert, P. D. N. (2007). Comprehensive DNA barcode coverage of North American Birds. Molecular Ecology Notes, 7, 535–543.CrossRefPubMedGoogle Scholar
  22. Klerks, M. M., Leone, G. O. M., Verbeek, M., van den Heuvel, J. F. J. M., & Schoen, C. D. (2001). Development of a multiplex AmpliDet RNA for the simultaneous detection of Potato leafroll virus and Potato virus Y in potato tubers. Journal of Virological Methods, 93, 115–125.PubMedCrossRefGoogle Scholar
  23. Leone, G., van Schijndel, H. B., van Gemen, B., & Schoen, C. D. (1997). Direct detection of Potato leafroll virus in potato tubers by immunocapture and the isothermal nucleic acid amplification method NASBA. Journal of Virological Methods, 66, 19–27.PubMedCrossRefGoogle Scholar
  24. Ledford, H. (2007). Rapid sequencer puts virus in the frame for deaths. Nature, 447, 12–13.Google Scholar
  25. Mumford, R. A., Barker, I., & Wood, K. R. (1994). The detection of Tomato Spotted Wilt Virus using the polymerase chain reaction. Journal of Virological Methods, 46(3), 303–311.PubMedCrossRefGoogle Scholar
  26. Mumford, R., Boonham, N., Tomlinson, J., & Barker, I. (2006). Advances in molecular phytodiagnostics - new solutions for old problems. European Journal of Plant Pathology, 116, 1–19.CrossRefGoogle Scholar
  27. Nelson, L. A., Wallman, J. F., & Dowton, M. (2007). Using COI barcodes to identify forensically and medically important blowflies. Medical and Veterinary Entomology, 21, 44–52.PubMedCrossRefGoogle Scholar
  28. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N., & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28, e63.Google Scholar
  29. O’Donnell, K. J., Canning, E., & Young, L. G. A. (1996). Detection of Potato virus Y using ligase chain reaction (LCR), in combination with a microtitre plate based method for product detection. In G. Marshall (Ed.) Diagnostics in Crop Production: BCPC Symposium Proceedings No. 65 (pp. 187–192). Farnham, UK: British Crop Protection Council.Google Scholar
  30. Pfunder, M., Holzgana, O., & Frey, J. E. (2004). Development of microarray-based diagnostics of voles and shrews for use in biodiversity monitoring studies, and evaluation of mitochondrial cytochrome oxidase I vs. cytochrome b as genetic markers. Molecular Ecology, 13, 1277–1286.PubMedCrossRefGoogle Scholar
  31. Puchta, H., & Sanger, H. L. (1989). Sequence analysis of minute amounts of viroid RNA using the polymerase chain reaction (PCR). Archives of Virology, 106, 335–340.PubMedCrossRefGoogle Scholar
  32. Ratnasingham, S., & Hebert, P. D. N. (2007). BOLD: The barcode of life data system. Molecular Ecology Notes, 7, 355–364. ( Scholar
  33. Schoen, C. D., Knorr, D., & Leone, G. (1996). Detection of Potato leafroll virus in dormant potato tubers by immunocapture and a fluorogenic 5ô nuclease RT-PCR assay. Phytopathology, 86, 993–999.CrossRefGoogle Scholar
  34. Seifert, K. A., Samson, R. A., deWaard, J. R., Houbraken, J., Levesque, C. A., Moncalvo, J. M., et al. (2007). Prospects for fungus identification using CO1 DNA barcodes, with Penicillium as a test case. Proceedings of the National Academy of Sciences USA, 104, 3901–3906.CrossRefGoogle Scholar
  35. Spiegel, S., & Martin, R. R. (1993). Improved detection of Potato leafroll virus in dormant potato tubers and microtubers by the polymerase chain reaction and ELISA. Annals of Applied Biology, 122, 493–500.CrossRefGoogle Scholar
  36. Stoeckle, M. (2003). Taxonomy, DNA and the barcode of life. BioScience, 53, 2, 3.CrossRefGoogle Scholar
  37. Tan, E., Wong, J., Nguyen, D., Zhang, Y., Erwin, B., Van Ness, L. K., et al. (2005). Isothermal DNA amplification coupled with DNA nanosphere-based colorimetric detection. Analytical Chemistry, 77, 7984–7992.PubMedCrossRefGoogle Scholar
  38. Tomlinson, J. A., Barker, I., & Boonham, N. (2007). Faster, simpler, more specific methods for improved molecular detection of Phytophthora ramorum in the field. Applied and Environmental Microbiology, 73, 4040–4047.PubMedCrossRefGoogle Scholar
  39. Vincent, M., Xu, Y., & Kong, H. M. (2004). Helicase-dependent isothermal DNA amplification. EMBO Reports, 5, 795–800.PubMedCrossRefGoogle Scholar
  40. Ward, R. D., Zemlak, T. S., Innes, B. H., Last, P. R., & Hebert, P. D. N. (2005). DNA bar coding Australia’s fish species. Philosophical Transactions of The Royal Society of London B, 360, 1847–1857.CrossRefGoogle Scholar
  41. Yi, J. Z., Zhang, W. D., & Zhang, D. Y. (2006). Molecular Zipper: A fluorescent probe for real-time isothermal DNA amplification. Nucleic Acids Research, 34, e81.PubMedCrossRefGoogle Scholar
  42. Zhang, D., Wu, J., Ye, F., Feng, T., Lee, I., & Yin, B. J. (2006). Amplification of circularizable probes for the detection of target nucleic acids and proteins. Clinica Chimica Acta, 363, 61–70.CrossRefGoogle Scholar

Copyright information

© KNPV 2008

Authors and Affiliations

  • Neil Boonham
    • 1
  • Rachel Glover
    • 1
  • Jenny Tomlinson
    • 1
  • Rick Mumford
    • 1
  1. 1.Central Science LaboratoryYorkUK

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