3.3.10
DETECTION OF RALSTONIA SOLANACEARUM RACE 3 IN POTATO TUBERS BY PCR AMPLIFICATION AND SANDWICH HYBRIDIZATION IN MICROTITRATION PLATES

A CHANDELIER1, S COGNET1 and P LEPOIVRE1

1Unité de Phytopathologie, FUSAGx, 2 Passage des Deportes, B-5030 Gembloux, Belgium

Background and objectives
Ralstonia solanacearum (syn. Pseudomonas solanacearum), the causal agent of potato brown rot, is one of the most important quarantine bacteria in Belgium. Strains of R. solanacearum constitute a complex taxonomic group including five races, of which races 1 and 3 can infect potatoes. However, in cool, temperate climates, only race 3 induces severe damage. As this pathogen has recently been introduced into Europe, notably in Belgium, France, the Netherlands, Portugal, Spain, Sweden and the UK [1], quarantine measures aimed at eradicating the disease have been established.

From a practical point of view, there is a need for a sensitive test, able to detect the bacteria in latently infected tubers. Moreover, considering the great number of samples to analyse, it is also important to develop a test which can be automated. In that respect, we aim at developing a diagnostic kit based on PCR amplification of ribosomal DNA and detection of the amplicons by sandwich hybridization in microtitration plates.

Materials and methods
Bacterial DNA was extracted from pure culture or from infected potato tubers using a silica-based protocol previously described [2]. Two primers located in the 23S RNA gene were designed for the PCR amplification. The amplicons were resolved by electrophoresis in agarose gel. Concerning the sandwich hybridization tests carried out in microtitration plates, the capture probe and the biotinylated detection probes constitute part of a detection kit in development (Lambdatech s.a., Belgium).

Results and conclusions
Chromosomal DNAs from different bacteria were submitted to a PCR amplification with the primers selected for the detection of R. solanacearum. The two primers were found to amplify a 257-pb fragment specific to R. solanacearum race 3. No amplification product was observed either for R. solanacearum race 1 or R. pickettii (which displays a high degree of DNA homology with R. solanacearum).

A sandwich hybridization assay in microtitration plate was then performed using the kit in development from Lambdatech s.a.. The amplified DNA was hybridized between a capture probe and three biotinylated detection probes. The hybrids were then detected with a streptavidin-peroxydase conjugate after the addition of the revelation mixture. In contrast to non-targeted bacteria, positive samples gave rise to high optical density values (ca 2.0), thus suggesting that the proposed assay can distinguish between targeted and non-targeted bacteria.

In order to characterize the sensitivity of our test, known concentrations of R. solanacearum race 3 were added to 1 ml of plant sap and total nucleic acids were extracted using the silica-based protocol. The detection limit after sandwich hybridization was about 10,000 bacteria/ml plant sap. As this detection limit is not sufficient, we aim in the future at improving the sample preparation step, either by modifying the silica-based protocol or by performing a pre-culture in a selective medium of the infected tubers (bioPCR).

References
1. Elphinstone JG, 1996. Potato Research 39, 403-410.
2. Boom R, Sol CJA, Salimans MMM et al., 1990. Journal of Clinical Microbiology 28, 495-503.