Repetitive sequence based-PCR fingerprints based on REP primers allow a classification of plant pathogenic Pseudomonas spp. that supports most of the recognised species. The classification also supports a pathovar-based system but indicates that some clusters of strains were isolated from unrelated hosts. Standardisation of methods is essential, and improved DNA extraction methods may be necessary before identification can be made based on comparisons with a database.

Two TaqMan PCR assays have been developed to test for Ralstonia solanacearum. One assay (RS) was genus specific, reacting with all strains of R. solanacearum but also the Banana blood disease bacterium and R. syzygii. Another assay (B2) was specific for Biovar 2 (Race 3) strains which cause brown rot of potatoes. The selective media SMSA and Willbrinks medium have been modified into broth form to detect the pathogen in complex substrates, such as sewage and river water, where population numbers are often low. Using these media in a pre-enrichment real-time bio PCR assay facilitates the robust, highly sensitive detection of the potato brown rot pathogen in natural samples including tubers and waste water.

TaqMan™ PCR exploits the 5‘ nuclease activity of Taq DNA polymerase (4) in conjunction with fluoroenic DNA probes (6). Each probe, designed to hybridise specifically to the target PCR product, is labelled with a fluorescent reporter dye and a quencher dye. During PCR amplification the probe is digested by Taq DNA polymerase, separating the dyes, resulting in an increase in reporter fluorescence. Repeated PCR cycles result in exponential amplification of the PCR product and corresponding increase in fluorescence intensity.

TaqMan polymerase chain reaction (PCR) exploits the 5′-nuclease activity of Taq DNA polymerase in conjunction with fluorogenic DNA probes. A positive PCR reaction results in a measurable emission of fluorescence within 2 h. TaqMan assays for several plant-pathogenic bacteria have been designed, including the potato brown rot pathogen Ralstonia solanacearum. Enrichment techniques designed to improve the detection of low pathogen populations in potato tissue are also suitable for use with TaqMan PCR. The quantitative aspect of TaqMan PCR enables relative population level differences between pre-enriched and enriched samples to be demonstrated and thus cell viability to be evaluated.

ABSTRACT A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum , and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5′ nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to ≥10 2 cells ml −1 was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum .