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Development of a real-time PCR assay for detection of Phytophthora kernoviae and comparison of this method with a conventional culturing technique

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Abstract

Phytophthora kernoviae is a recently described pathogen causing leaf blight, aerial dieback and bleeding cankers on trees and shrubs in parts of Great Britain and Ireland and recently reported in New Zealand. This paper describes the development of a TaqMan real-time PCR assay based on internal transcribed spacer (ITS) sequence to aid diagnosis of this pathogen in culture and in plant material. The assay showed no cross reaction with 29 other Phytophthora species, including the closely related species P. boehmeriae, and detected at least 1.2 pg of P. kernoviae DNA per reaction. A rapid and simple method can be used to extract DNA prior to testing by real-time PCR, and a plant internal control assay can be used to aid interpretation of negative results. A comparison of real-time PCR and plating for 526 plant samples collected in the UK indicated that this assay is suitable for use in routine screening for P. kernoviae.

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References

  • Anonymous (2004). Statutory instrument No. 3367. The Plant Health (Phytophthora kernovii Management Zone) (England) order 2004. HMSO. ISBN 0110513878

  • Anonymous. (2006). EPPO diagnostic protocol for regulated pests: Phytophthora ramorum. EPPO Bulletin, 36, 145–155.

    Article  Google Scholar 

  • Beales, P. A., Lane, C. R., Barton, V. C., & Giltrap, P. M. (2006). Phytophthora kernoviae on ornamentals in the UK. EPPO Bulletin, 36, 377–379.

    Article  Google Scholar 

  • Beales, P. A., Giltrap, P. M., Payne, A., & Ingram, N. (2009). A new threat to UK heathland from Phytophthora kernoviae on Vaccinium myrtillus in the wild. Plant Pathology, 58, 393.

    Article  Google Scholar 

  • Bilodeau, G. J., Lévesque, C. A., de Cock, A. W. A. M., Duchaine, C., Brière, S., Uribe, P., et al. (2007). Molecular detection of Phytophthora ramorum by real-time polymerase chain reaction using TaqMan, SYBR Green and molecular beacons. Phytopathology, 97, 632–642.

    Article  PubMed  CAS  Google Scholar 

  • Blair, J. E., Coffey, M. D., Park, S. Y., Geiser, D. M., & Kang, S. (2008). A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences. Fungal Genetics and Biology, 45, 266–277.

    Article  PubMed  CAS  Google Scholar 

  • Brasier, C. M., Beales, P. A., Kirk, S. A., Denman, S. & Rose, J. (2005). Phytophthora kernoviae sp. Nov., an invasive pathogen causing bleeding stem lesions on forest trees and foliar necrosis of ornamentals in the UK. Mycological Research, 109, 853–859

    Google Scholar 

  • Erwin, D. C., & Ribeiro, O. K. (1996). Phytophthora diseases worldwide. St. Paul, MN, USA: American Phytopathological Society.

    Google Scholar 

  • Hayden, K. J., Rizzo, D., Tse, J., & Garbelotto, M. (2004). Detection and quantification of Phytophthora ramorum from Californian forests using a real-time polymerase chain reaction assay. Phytopathology, 94, 1075–1083.

    Article  PubMed  CAS  Google Scholar 

  • Hayden, K., Ivors, K., Wilkinson, C., & Garbelotto, M. (2006). TaqMan chemistry for Phytophthora ramorum detection and quantification, with a comparison of diagnostic methods. Phytopathology, 96, 846–854.

    Article  PubMed  CAS  Google Scholar 

  • Hughes, K. J. D., Fulton, C. E., McReynolds, D., & Lane, C. R. (2000). Development of new PCR primers for identification of Monilinia species. EPPO Bulletin, 30, 507–511.

    Article  Google Scholar 

  • Hughes, K. J. D., Giltrap, P. M., Barton, V. C., Hobden, E., Tomlinson, J. A., & Barber, P. (2006). On-site real time PCR detection of Phytophthora ramorum causing dieback of Parrotia persica in the UK. Plant Pathology, 55, 813.

    Article  Google Scholar 

  • Hughes, K. J. D., Tomlinson, J. A., Griffin, R. L., Boonham, N., Inman, A. J., & Lane, C. R. (2006). Development of a One-Step Real-Time PCR Assay for Diagnosis of Phytophthora ramorum. Phytopathology, 96, 975–981.

    Article  PubMed  CAS  Google Scholar 

  • Jeffers, S. N., & Martin, S. B. (1986). Comparison of two media selective for Phytophthora and Pythium species. Plant Disease, 70, 1038–1043.

    Article  Google Scholar 

  • Kroon, L. P. N. M., Bakker, F. T., van den Bosch, G. B. M., Bonants, P. J. M., & Flier, W. G. (2004). Phylogenetic analysis of Phytophthora species based on mitochondrial and nuclear DNA sequences. Fungal Genetics and Biology, 41, 766–782.

    Article  PubMed  CAS  Google Scholar 

  • Lane, C. R., Hobden, E., Walker, L., Barton, V. C., Inman, A. J., Hughes, K. J. D., Swan, H., Colyer, A. & Barker, I. (2007). Evaluation of a rapid diagnostic field test kit for identification of Phytophthora species, including P. ramorum and P. kernoviae at the point of inspection. Plant Pathology, 56, 828–835

  • Martin, F. N., Tooley, P. W., & Blomquist, C. (2004). Molecular detection of Phytophthora ramorum, the causal agent of sudden oak death in California, and two additional species commonly recovered from diseased plant material. Phytopathology, 94, 621–631.

    Article  PubMed  CAS  Google Scholar 

  • 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.

    Article  CAS  Google Scholar 

  • Ramsfield, T. D., Dick, M. A., Beever, R. E. & Horner, I. J. (2007). Phytophthora kernoviae – of Southern Hemisphere origin? Abstracts of the Fourth IUFRO working party meeting 7.02.09. Phytophthoras in Forests and Natural Ecosystems, Monterey, CA, USA August 26–31 2007.

  • Schena, L., Hughes, K. J. D. & Cooke, D. E. L. (2006). Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola, and P. quercina in symptomatic leaves by multiplex PCR. Molecular Plant Pathology, 7, 365–379

  • Tomlinson, J. A., Barker, I., & Boonham, N. (2007). Faster, simpler, more-specific methods for improved detection of Phytophthora ramorum in the field. Applied and Environmental Microbiology, 73, 4040–4047.

    Article  PubMed  CAS  Google Scholar 

  • Tomlinson, J. A., Boonham, N., Hughes, K. J. D., Griffin, R. L., & Barker, I. (2005). On-site DNA extraction and real-time PCR for detection of Phytophthora ramorum in the field. Applied and Environmental Microbiology, 71, 6702–6710.

    Article  PubMed  CAS  Google Scholar 

  • Tomlinson, J. A., Dickinson, M., Hobden, E., Robinson, S., Giltrap, P. M. & Boonham, N. (2010). A five-minute DNA extraction method for expedited detection of Phytophthora ramorum following prescreening using Phytophthora spp. lateral flow devices. Journal of Microbiological Methods, 81, 116–120

  • Vettraino, A. M., Sukno, S., Vannini, A., & Garbelotto, M. (2010). Diagnostic sensitivity and specificity of different methods used by two laboratories for the detection of Phytophthora ramorum on multiple natural hosts. Plant Pathology, 59, 289–300.

    Article  CAS  Google Scholar 

  • Widmer, T. L. (2010). Phytophthora kernoviae oospore maturity, germination, and infection. Fungal Biology, 114, 661–668.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

Funding for this work was provided by Plant Health Division of Defra and work was performed under license number PHL 251A/4736 (03/20004). The authors would like to thank Dr Paul Beales and Dr Rebecca Weekes for their invaluable support and guidance.

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Authors and Affiliations

  1. The Food and Environment Research Agency, Sand Hutton, York, North Yorkshire, YO41 1LZ, UK

    Kelvin J. D. Hughes, Jenny A. Tomlinson, Patricia M. Giltrap, Victoria Barton, Ellie Hobden, Neil Boonham & Charles R. Lane

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  1. Kelvin J. D. Hughes
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  2. Jenny A. Tomlinson
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  3. Patricia M. Giltrap
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  4. Victoria Barton
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  5. Ellie Hobden
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  6. Neil Boonham
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  7. Charles R. Lane
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Correspondence to Jenny A. Tomlinson.

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Hughes, K.J.D., Tomlinson, J.A., Giltrap, P.M. et al. Development of a real-time PCR assay for detection of Phytophthora kernoviae and comparison of this method with a conventional culturing technique. Eur J Plant Pathol 131, 695–703 (2011). https://doi.org/10.1007/s10658-011-9843-x

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  • DOI: https://doi.org/10.1007/s10658-011-9843-x

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