Identification, intra- and inter-laboratory validation of a diagnostic protocol for ‘Candidatus Liberibacter solanacearum’ in carrot seeds
- 79 Downloads
‘Candidatus Liberibacter solanacearum’ (CaLsol) is a phloem-limited, unculturable, Gram-negative bacterium associated with emerging diseases in crops of the Solanaceae and Apiaceae families. As it has been shown to be seed-transmitted in carrot, emergency measures for exportation require carrot seed to be heat-treated or tested by PCR and found CaLsol free. Therefore, the identification and harmonization of a protocol for CaLsol diagnosis in carrot seed are becoming of socio-economic priority. We initially set up an improved DNA extraction method for Apiaceae seeds and identified, among the widely used PCR tests to detect and identify CaLsol, the real-time PCR developed by Li et al. (Journal of Microbiological Methods, 78(1), 59–65, 2009) and the end-point PCR by Ravindran et al. (Plant Disease, 95(12), 1542–1546, 2011) to be the most sensitive ones. The two PCR methods were initially intra-laboratory validated followed by a “Test Performance Study” involving 11 Italian laboratories that received both the samples and the material necessary to carry out the experiments. The results indicated that the improved DNA extraction method was robust and that the real-time PCR showed the highest analytical sensitivity in the intra-laboratory validation tests. Similarly, the real-time PCR outperformed the end-point PCR in the inter-laboratory comparison assay showing a higher percentage of accuracy, accordance, and concordance. The overall obtained data could be used for the appropriate application of phytosanitary measures against CaLsol.
KeywordsRing test Molecular detection Apiaceae Plant-pathogen EPPO standards
This work was supported by the Ministero per le Politiche Agricole Alimentari e Forestali (project Azioni a supporto della protezione delle piante ASPROPI: Linea di ricerca “Sviluppo protocollo analitico per ‘Candidatus Liberibacter solanacearum’”).
Compliance with ethical standards
The manuscript complies with the rules of good scientific practice and ethical rules of European Journal of Plant Pathology, as reported in the “Ethical Responsibilities of Authors” of the “Instructions for Authors” section. There are no potential conflicts of interest, and the research does not involve human participants and/or animals. All authors have approved the manuscript and agreed with its submission to European Journal of Plant Pathology.
- Ben Othmen, S., Morán, F. E., Navarro, I., Barbé, S., Martínez, C., Marco-Noales, E., et al. (2018). ‘Candidatus Liberibacter solanacearum’haplotypes D and E in carrot plants and seeds in Tunisia. Journal of Plant Pathology, 1–11.Google Scholar
- Catara, V., Licciardello, G., Linguaglossa, M., Salonia, F., Rapisarda, C., La Rosa, R., Cocuzza Massimino, G.E. (2017). First report of ‘Candidatus Liberibacter solanacearum’ in carrot in Italy. 15th Congress of the Mediterranean Phytopathological Union, June 20–23, 2017, Córdoba, Spain. Google Scholar
- Chabirand, A., Loiseau, M., Renaudin, I., & Poliakoff, F. (2017). Data processing of qualitative results from an interlaboratory comparison for the detection of “Flavescence dorée” phytoplasma: How the use of statistics can improve the reliability of the method validation process in plant pathology. PLoS One, 12(4), e0175247.CrossRefGoogle Scholar
- EPPO (2016). https://www.eppo.int/QUARANTINE/listA1.htm International Organization for Standardization. ISO/IEC 17025:2005. General requirements for the competence of testing and calibration laboratories.
- EPPO (2017). EPPO reporting service 2017/20.Google Scholar
- EPPO PM7/122(1). (2014). Guidelines for the organization of interlaboratory comparisons by plant pest diagnostic laboratories. EPPO Bulletin/Bulletin OEPP, 44(3), 390±9.Google Scholar
- EPPO PM7/76(4). (2017). Use of EPPO diagnostic protocols. EPPO Bulletin/Bulletin OEPP, 47(1), 7–9.Google Scholar
- EPPO PM7/98(2). (2014). Specific requirements for laboratories preparing accreditation for a plant pest diagnostic activity. EPPO Bulletin/Bulletin OEPP, 44, 117±47.Google Scholar
- Euphresco (2017) PHYLIB meeting Bologna, 27–28 April.Google Scholar
- FAO, IPPC Emergency actions (2015). Notification of phytosanitary measures to reduce the risk of introduction of ‘Candidatus Liberibacter solanacearum’ through the importation of carrot (seed and seedling) and celery (seedling) - Emergency Actions https://www.ippc.int/en/countries/japan/eventreporting/2015/05/emergency-measures-to-reduce-the-risk-of-introduction-of-candidatus-liberibacter-solanacearum-through-the-importation-of-carrot-seed-and-seedling-and-celery-seedling/
- FAO/IPPC Emergency Action (2016). Proposed revision of List of the plants subject to Specific Phytosanitary Measures to be carried out in Exporting Countries (Annexed Table 2–2 of the Ordinance for Enforcement of the Plant Protection Act) http://www.maff.go.jp/j/syouan/keneki/kikaku/pdf/04_at2_2_specific_measures.pdf
- Haapalainen, M. L., Wang, J., Latvala, S., Lehtonen, M. T., Pirhonen, M., & Nissinen, A. I. (2018). Genetic variation of ‘Candidatus Liberibacter solanacearum’ haplotype C and identification of a novel haplotype from Trioza urticae and stinging nettle. Phytopathology, (ja).Google Scholar
- Hajri, A., Loiseau, M., Cousseau-Suhard, P., Renaudin, I., & Gentit, P. (2017). Genetic Characterization of ‘Candidatus Liberibacter solanacearum’ Haplotypes Associated with Apiaceous Crops in France. Plant Disease, PDIS-11.Google Scholar
- Hansen, A. K., Trumble, J. T., Stouthamer, R., & Paine, T. D. (2008). A new huanglongbing species, ‘Candidatus Liberibacter solanacearum’ found to infect tomato and potato, is vectored by the psyllid Bactericera cockerelli (Sulc). Applied and Environmental Microbiology, 74(18), 5862–5865.CrossRefGoogle Scholar
- Holeva, M. C., Glynos, P. E., & Karafla, C. D. (2017). First report of ‘Candidatus Liberibacter solanacearum’ on carrot in Greece. Plant Disease, (ja).Google Scholar
- Ilardi, V., Di Nicola, E., & Tavazza, M. (2016a). First report of ‘Candidatus Liberibacter solanacearum’ in commercial carrot seeds in Italy. Journal of Plant Pathology, 98, 2.Google Scholar
- Ilardi V., E. Di Nicola, V. Lumia, M. Tavazza (2016b) “Report of ‘Candidatus Liberibacter solanacearum’ in commercial Apiaceae seeds in Italy”. XXII Convegno Società Italiana Patologia Vegetale. Rome, Italy 19-22 September 2016. Journal of Plant Pathology, 98, S28.Google Scholar
- IPPC/ISPM 27annex 21, (2017) Diagnostic protocols for regulated pests DP 21: ‘Candidatus Liberibacter solanacearum’ Adopted 2017 – Taxonomic Information p.3 https://www.ippc.int/static/media/files/publication/en/2017/04/DP_21_2017_En_2017-03-31.pdf
- Janse, J. D. (2012). Bacterial diseases that may or do emerge, with (possible) economic damage for Europe and the Mediterranean basin: Notes on epidemiology, risks, prevention and management on first occurrence. Journal of Plant Pathology, 94(4sup), 4–5.Google Scholar
- Johansson M.K. (2006) Choosing reporter-quencher pairs for efficient quenching through formation of intramolecular dimers. In: Didenko V.V. (eds) Fluorescent energy transfer nucleic acid probes. Methods in molecular biology™, vol 335. Humana Press. https://doi.org/10.1385/1-59745.
- Li, W., Abad, J. A., French-Monar, R. D., Rascoe, J., Wen, A., Gudmestad, N. C., et al. (2009). Multiplex real-time PCR for detection, identification and quantification of ‘Candidatus Liberibacter solanacearum’ in potato plants with zebra chip. Journal of Microbiological Methods, 78(1), 59–65.CrossRefGoogle Scholar
- Lin, H., Lou, B., Glynn, J. M., Doddapaneni, H., Civerolo, E. L., Chen, C., ... & Vahling, C. M. (2011). The complete genome sequence of ‘Candidatus Liberibacter solanacearum’, the bacterium associated with potato zebra chip disease. PLoS One, 6(4), e19135.Google Scholar
- Loiseau, M., Garnier, S., Boirin, V., Merieau, M., Leguay, A., Renaudin, I., et al. (2014). First report of ‘Candidatus Liberibacter solanacearum’ in carrot in France. Plant Disease, 98(6), 839–839.Google Scholar
- Loiseau, M., Renaudin, I., Cousseau-Suhard, P., Lucas, P. M., Forveille, A., & Gentit, P. (2017). Lack of Evidence of Vertical Transmission of ‘Candidatus Liberibacter solanacearum’ by Carrot Seeds Suggests That Seed is not a Major Transmission Pathway. Plant Disease, PDIS-04.Google Scholar
- Monger, W. A., & Jeffries, C. J. (2017). A survey of ‘Candidatus Liberibacter solanacearum in historical seed from collections of carrot and related Apiaceae species. European Journal of Plant Pathology, 1–13.Google Scholar
- Munyaneza, J. E., Fisher, T. W., Sengoda, V. G., Garczynski, S. F., Nissinen, A., & Lemmetty, A. (2010b). Association of ‘Candidatus Liberibacter solanacearum’ with the psyllid, Trioza apicalis (Hemiptera: Triozidae) in Europe. Journal of Economic Entomology, 103(4), 1060–1070.CrossRefGoogle Scholar
- Nelson, W. R., Fisher, T. W., & Munyaneza, J. E. (2011). Haplotypes of “Candidatus Liberibacter solanacearum” suggest long-standing separation. European Journal of Plant Pathology, 130(1), 5–12.Google Scholar
- Nelson, W. R., Sengoda, V. G., Alfaro-Fernandez, A. O., Font, M. I., Crosslin, J. M., & Munyaneza, J. E. (2013). A new haplotype of “Candidatus Liberibacter solanacearum” identified in the Mediterranean region. European Journal of Plant Pathology, 135(4), 633–639.Google Scholar
- Olivier, T., Šveikauskas, V., Demonty, E., De Jonghe, K., Gentit, P., Viršček-Marn, M., et al. (2016). Inter-laboratory comparison of four RT-PCR based methods for the generic detection of pospiviroids in tomato leaves and seeds. European Journal of Plant Pathology, 144(3), 645–654.CrossRefGoogle Scholar
- Teresani, G. R., Bertolini, E., Alfaro-Fernández, A., Martínez, C., Tanaka, F. A. O., Kitajima, E. W., et al. (2014). Association of ‘Candidatus Liberibacter solanacearum’ with a vegetative disorder of celery in Spain and development of a real-time PCR method for its detection. Phytopathology, 104(8), 804–811.CrossRefGoogle Scholar
- Williams, M. M., Taylor Jr., T. H., Warshauer, D. M., Martin, M. D., Valley, A. M., & Tondella, M. L. (2015). Harmonization of Bordetella pertussis real-time PCR diagnostics in the United States in 2012. Journal of Clinical Microbiology, 53, 118–123. https://doi.org/10.1128/JCM.02368-14.CrossRefGoogle Scholar