Roelofarendsveen, The Netherlands
October 30, 2013
Xanthomonas campestris pv. campestris causes black rot in cabbage. Development of molecular assays can improve fast and reliable detection of this important pathogen. The R&D laboratory of Naktuinbouw is responsible for the development and implementation of new assays for pathogen detection. During a workshop in July new methods for the detection of Xanthomonas campestris pv. campestris were compared.
Workshop
A three-day proficiency workshop on molecular assays for detection of Xanthomonas campestris pv. campestris (Xcc) took place at the R&D laboratory of Naktuinbouw. In the workshop three new protocols based on molecular detection were compared to the current ISTA method, dilution plating on semi-selective media. In this classic test, the suspect bacteria have to be tested in a pathogenicity assay and the whole procedure can take up to a month.
Comparison of protocols
Two out of three new protocols were based on a direct assay. In other words, DNA was isolated directly from seed extracts without any enrichment step, followed by a quantitative real-time PCR. The third molecular protocol (BioPCR) did use an enrichment step; the seed extract was first plated on a semi-selective medium (mFS) and colonies were harvested after two days of growth for PCR. For this proficiency test Thomas Baldwin of Vilmorin, Michel Lattier of HM-Clause (both Group Limagrain, France) and Marco Kruijt of Monsanto Holland B.V. visited the Naktuinbouw laboratory. The researchers of Naktuinbouw, Monsanto Holland B.V. and Vilmorin each performed their own protocol within the Naktuinbouw laboratory.
Results
The results of this proficiency workshop provide insight in the reliability and sensitivity of the molecular assays compared to the dilution plating. Both direct PCRs detect both viable and non-viable cells and fast quantitative prescreening is possible with these methods; absence of Xcc can be confirmed within a few days (instead of one month). The BioPCR is more laborious, however, it does distinguish between viable and non-viable cells, as does dilution plating. The results of the molecular methods are very similar and in line with the classic dilution plating method. The results are used to further develop and implement these new methods.
Species
