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Validation of a detection scheme based on PCR and isolation for the detection of Xanthomonas axonopodis pv. allii in vegetative parts of Alliaceae and in onion seeds. poster 22

Chabirand A., Moreau A., Terville M.A., Robene I., Maillot V., Pruvost O., Hostachy B.. 2016. In : 12èmes Rencontres Plantes-Bactéries - Book of Abstracts. Paris : SFP, p. 106-106. Rencontres Plantes-Bactéries. 12, 2016-01-11/2016-01-15, Aussois (France).

Xanthomonas axonopodis pv. allii (Xaa) is the causal agent of bacterial blight of onion (BBO), an emerging disease threatening the world onion production, and causing damage to other Alliaceae, including garlic, welsh onion, shallot, chive and leek. Xaa is an EPPO quarantine organism (A1 list i.e. absent from the EPPO region). The international spread of Xaa can probably be explained by its seedborne status (Roumagnac et al., 2000). Consequently it is of crucial importance to validate efficient detection methods which allow the detection of seedborne pathogens for which the level of inoculum can be very low in very large seed lots. We evaluate a detection scheme based on molecular tests (a triplex quantitative real-time PCR assay (Robène et al., 2015) and a duplex nested end-point PCR assay (Robène-Soustrade et al., 2010)) and isolation. The method assessment was performed following the European standard EN ISO 16140 and the EPPO standard PM7/98 (2). An intra-laboratory study was first conducted, where we characterized the analytical specificity (inclusivity and exclusivity), the analytical sensitivity and the repeatability. We finally tested the detection scheme on naturally contaminated onion seed lots showing different contamination rates. In addition to the intralaboratory study, an inter-laboratory reproducibility trial (including five different laboratories) was performed. This detection scheme was shown to be very efficient, combining a maximal inclusivity (100%), a good exclusivity (83%), a high analytical sensitivity (approximately 103 CFU.mL-1), an excellent repeatability (100% for contamination rates higher than 103 CFU.mL-1) and reproducibility (100% for contamination rates higher than 103 CFU.mL-1). These results allowed to validate the detection scheme as official method of analysis for the French officially approved laboratories (MA 038), and to propose it as an EPPO diagnostic protocol for the detection of this emerging pathogen. (Texte integral)

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