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

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.