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.
This is a preview of subscription content, log in via an institution to check access.
References
Baayen, R. P., Bonants, P. J. M., Verkley, G., Carroll, G. C., van der Aa, H. A., de Weerdt, M., van Brouwershaven, I. R., Schutte, G. C., Maccheroni, W., Jr., Glienke de Blanco, C., & Azevedo, J. L. (2002). Nonpathogenic isolates of the citrus black spot fungus, Guignardia citricarpa, identified as a common endophyte of woody plants, G. mangiferae (Phyllosticta capitalensis). Phytopathology, 92, 464–477.
Bonants, P. J. M., Carroll, G. C., de Weerdt, M., van Brouwershaven, I. R., & Baayen, R. P. (2003). Development and validation of a fast PCR-based detection method for pathogenic isolates of the citrus black spot fungus, Guignardia citricarpa. European Journal of Plant Pathology, 109, 503–513.
EPPO. (2009). PM 7/17(2): Guignardia citricarpa. EPPO Bulletin, 39, 318–327.
Glienke, C., Pereira, O. L., Stringari, D., Fabris, J., Kava-Cordeiro, V., Galli-Terasawa, L., Cunnington, J., Shivas, R. G., Groenewald, J. Z., & Crous, P. W. (2011). Endophytic and pathogenic Phyllosticta species, with reference to those associated with Citrus Black Spot. Persoonia, 26, 47–56.
Kaneko, H., Kawana, T., Fukushima, E., & Suzutani, T. (2007). Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. Journal of Biochemical and Biophysical Methods, 70, 499–501.
Meyer, L., Sanders, G. M., Jacobs, R., & Korsten, L. (2006). A one-day sensitive method to detect and distinguish between the citrus black spot pathogen Guignardia citricarpa and the endophyte Guignardia mangiferae. Plant Disease, 90, 97–101.
Nagamine, K., Hase, T., & Notomi, T. (2002). Accelerated reaction by loop-mediated isothermal amplification using loop primers. Molecular and Cellular Probes, 16, 223–229.
Nagamine, K., Watanabe, K., Ohtsuka, K., Hase, T., & Notomi, T. (2001). Loop-mediated isothermal amplification reaction using a nondenatured template. Clinical Chemistry, 47, 1742–1743.
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.
Peres, N. A., Harakava, R., Carroll, G. C., Adaskaveg, J. E., & Timmer, L. W. (2007). Comparison of molecular procedures for detection and identification of Guignardia citricarpa and G. mangiferae. Plant Disease, 91, 525–531.
Suarez, M. B., Walsh, K., Boonham, N., O’Neill, T., Pearson, S., & Barker, I. (2005). Development of real-time PCR (TaqMan) assays for the detection and quantification of Botrytis cinerea in planta. Plant Physiology and Biochemistry, 43, 890–899.
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.
Tomlinson, J. A., Dickinson, M. J., & Boonham, N. (2010). Rapid detection of Phytophthora ramorum and P. kernoviae by two-minute DNA extraction followed by isothermal amplification and amplicon detection by generic lateral flow device. Phytopathology, 100, 143–149.
van Gent-Pelzer, M. P. E., van Brouwershaven, I. R., Kox, L. F. F., & Bonants, P. J. M. (2007). A TaqMan PCR method for routine diagnosis of the quarantine fungus Guignardia citricarpa on citrus fruit. Journal of Phytopathology, 155, 357–363.
Wang, X., Chen, G., Huang, F., Zhang, J., Hyde, K. D., & Li, H. (2012). Phyllosticta species associated with citrus diseases in China. Fungal Diversity, 52, 209–224.
Weller, S. A., Elphinstone, J. G., Smith, N. C., Boonham, N., & Stead, D. E. (2000). Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real time, fluorogenic PCR (TaqMan) assay. Applied and Environmental Microbiology, 66, 2853–2858.
Wheeler, E. K., Hara, C. A., Frank, J., Deotte, J., Hall, S. B., Benett, W., Spadaccini, C., & Beer, N. R. (2011). Under-three minute PCR: probing the limits of fast amplification. Analyst, 136, 3707–3712.
Wikee, S., Udayanga, D., Crous, P. W., Chukeatirote, E., McKenzie, E. H. C., Bahkali, A. H., Dai, D., & Hyde, K. D. (2011). Phyllosticta—an overview of current status of species recognition. Fungal Diversity, 51, 43–61.
Wulandari, N. F., To-anun, C., Hyde, K. D., Duong, L. M., de Gruyter, J., Meffert, J. P., Groenewald, J. Z., & Crous, P. W. (2009). Phyllosticta citriasiana sp. nov., the cause of Citrus tan spot of Citrus maxima in Asia. Fungal Diversity, 34, 23–39.
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.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Tomlinson, J.A., Ostoja-Starzewska, S., Webb, K. et al. A loop-mediated isothermal amplification-based method for confirmation of Guignardia citricarpa in citrus black spot lesions. Eur J Plant Pathol 136, 217–224 (2013). https://doi.org/10.1007/s10658-013-0168-9
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10658-013-0168-9