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European Journal of Plant Pathology

, Volume 136, Issue 2, pp 217–224 | Cite as

A loop-mediated isothermal amplification-based method for confirmation of Guignardia citricarpa in citrus black spot lesions

  • J. A. TomlinsonEmail author
  • S. Ostoja-Starzewska
  • K. Webb
  • J. Cole
  • A. Barnes
  • M. Dickinson
  • N. Boonham
Short Communications

Abstract

Guignardia citricarpa Kiely (anamorph Phyllosticta citricarpa Van der Aa), the causal agent of citrus black spot disease, is subject to phytosanitary restrictions in the EU and USA, such that consignments of citrus are rejected at import if citrus black spot is identified on inspection. Due to the variability of black spot symptoms, positive identification solely on the basis of visual inspection is difficult, especially when lesions lack pycnidia (fruiting bodies of the anamorph Phyllosticta citricarpa). As an aid to visual inspection of symptoms, we have developed a method for detection of G. citricarpa using loop-mediated isothermal amplification (LAMP) which can be used to confirm the presence of G. citricarpa in black spot lesions, including those lacking pycnidia. The LAMP assay can be used to test crude extracts prepared directly from lesions on fruit, and the entire test can be completed in less than 40 min, making it faster than previously described PCR-based methods for detection of G. citricarpa. The method is sufficiently simple to allow deployment of the test in the field, for example in the course of import inspections. Recent years have seen the description of a number of newly recognised species in the genus Phyllosticta that are associated with citrus. As new species emerge, and the taxonomy of the genus is resolved, it will be important to periodically re-evaluate the performance of DNA-based methods for detection of G. citricarpa, including the LAMP assay described here, such that the accuracy of diagnosis can be assured.

Keywords

Detection Citrus black spot Isothermal amplification Rapid testing 

Notes

Acknowledgements

Funding for this work was provided by Defra. The authors would like to thank Jim Beckley of Fera Plant Health and Seeds Inspectorate for providing helpful feedback during development of these methods.

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Copyright information

© UK Crown Copyright 2013

Authors and Affiliations

  • J. A. Tomlinson
    • 1
    Email author
  • S. Ostoja-Starzewska
    • 1
  • K. Webb
    • 1
  • J. Cole
    • 1
  • A. Barnes
    • 1
  • M. Dickinson
    • 2
  • N. Boonham
    • 1
  1. 1.The Food and Environment Research AgencyYorkUK
  2. 2.University of Nottingham School of BiosciencesLoughboroughUK

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