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European Journal of Plant Pathology

, Volume 153, Issue 3, pp 879–890 | Cite as

Identification, intra- and inter-laboratory validation of a diagnostic protocol for ‘Candidatus Liberibacter solanacearum’ in carrot seeds

  • Vincenza IlardiEmail author
  • Valentina Lumia
  • Elisa Di Nicola
  • Mario Tavazza
Article
  • 79 Downloads

Abstract

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.

Keywords

Ring test Molecular detection Apiaceae Plant-pathogen EPPO standards 

Notes

Acknowledgments

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.

Supplementary material

10658_2018_1606_MOESM1_ESM.pdf (390 kb)
Online Resource 1 Details of the 72 samples used in the present study (PDF 390 kb)

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Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2018

Authors and Affiliations

  • Vincenza Ilardi
    • 1
    Email author
  • Valentina Lumia
    • 1
  • Elisa Di Nicola
    • 1
  • Mario Tavazza
    • 2
  1. 1.Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria-Centro di ricerca difesa e certificazione sede di Roma (CREA-DC-RM)RomeItaly
  2. 2.ENEA CR Casaccia, SSPT-BIOAGRomeItaly

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