Determination of specificity and test performance study of current and novel molecular methods for Ralstonia solanacearum (brown rot of potatoes) and identification of methods allowing for detection of non-European strains of brown rot

The project allowed to: 

(1) Study the occurrence of intraspecific diversity of Rsol populations on local scale in the agro-ecological environment. Fundamental knowledge on diversity of Rsol was gained by characterizing Rsol populations in plant and water ecosystems in various geographic locations in Spain. Populations were characterized using several methods, namely: biochemical and metabolic profiling, serological methods and molecular methods. The genetic diversity showed to reflect the geographical origin within the country.

(2) Develop  a new test method that covers a wide range of Rsol variants. The Rsol species taxon, known to possess a considerable variation, was proven to be a species complex, and is hence referred to as the “Ralstonia solanacearum species complex” (Rssc). In order to develop a method that covers 17 major groups of interest in the Rssc, in a single multiplex reaction, microarray technology (ArrayTube) was used. Validation data of the developed protocol showed strong intra-repeatability, inter-repeatability, and reproducibility as well as good specificity. A hierarchical analysis of the probe groups is suitable for an accurate characterization. Compared with single marker detection tests, the developed method efficiently combines several tests by testing large numbers of phylogenetic markers in a single reaction assay. This custom microarray (RsscAT) represents a significant improvement in the epidemiological monitoring of Rssc strains worldwide, and it has the potential to provide insights for phylogenetic incongruence of Rssc strains based on the host of isolation and may be used to detect emergent strains.

(3) Evaluate test options for the EU test protocols (as laid down in the EU Council Directive 2006/63/EC) for Rsol and Clavibacter michiganensis subsp. sepedonicus (Cms), the performance of real-time PCRs was compared with the performance of IF (Immuno-fluorescense). There was also a multiplex real-time PCR for detection of both Rsol and Cms included in this study. Performance data of the real-time PCR tests from this study together with the results from the previously performed EUPHRESCO inter-laboratory comparisons (Van Vaerenbergh et al., 2017) form a profound basis to include real-time PCR as option in the EU Council Directives 2006/63/EC and 2006/56/EC for latent detection of Rsol and Cms. This test methodology has shown to be fast, sensitive and specific. The need for an update of the EU council directives for detection of Rsol and Cms was pointed out to the EU standing committee. The addition of real-time PCR tests in both control directives will have great benefits for diagnostic laboratories, NPPOs and trade in terms of reliable and fast throughput in inspections and surveys.