Interlaboratory test performance studies for identification of Ralstonia solanacearum and molecular confirmation of its virulence

The EU directive 2006/63/EC describes a detailed protocol for the official testing of Ralstonia solanacearum, that is internationally recognized and has been implemented in many diagnostic laboratories across Europe and beyond. In this protocol, the confirmation of the identity of the bacterium is performed by a laborious, time-consuming and expensive pathogenicity test. Additionally, the described protocols involved in the official testing of Ralstonia solanacearum (for detection and/or identification) require a lot of time, a high level of quarantine measures, and a high degree of expertise. The aim of this project was to develop, and evaluate molecular diagnostic methods for the detection and identification of Ralstonia solanacearum and verification of its virulence that would be faster, more specific and robust. Based on the current taxonomic insights, the high degree of heterogenity present inside the Ralstonia solanacearum species complex (RSSC) has resulted in the clear distinction of three new species inside this complex; the requirement defined in this Euphresco project was that the new molecular tests could be optimally implemented for the detection and identification of Ralstonia solanacearum and Ralstonia pseudosolanacearum, but not of Ralstonia syzygii.

The two main objectives of the project were:

1) To identify Ralstonia solanacearum virulence genes, and subsequently develop a PCR test on those virulence genes. The development of such a test could substitute the pathogenicity test required by EU directive 2006/63/EC for complete diagnosis of Ralstonia solanacearum.

2) To verify other molecular methods to detect or identify Ralstonia solanacearum strains. These methods include the real-time Loop-mediated isothermal amplication test (LAMP) and the recombinase-polymerase amplification assay (RPA).