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.Ralstonia solanacearum (Smith) (30) is the agent of bacterial wilt, infecting over 450 plant species, including many economically important crops (12). This species has been subclassified into biovars based on biochemical tests and host-dependent races. Biovar 2A (equivalent to race 3) is adapted to temperate climates, has a narrow host range, and is responsible for recent outbreaks of potato brown rot disease in several countries of Western Europe and elsewhere worldwide (13,27). Although other biovars can also infect potatoes, biovar 2A is the most destructive phenotype in temperate areas.R. solanacearum is listed as a quarantine organism in the European Union (EU) (2), where new legislation has been introduced to control and eradicate the organism (3). Latent infections in seed potato tubers (6) have lead to the spread of the organism, both locally and internationally, and effective control of brown rot is dependent on the reliability of detection of the pathogen at this latent stage. For practical purposes, a detection assay is required which is rapid, specific, and sensitive to levels lower than those occurring in naturally infected potatoes and should be applicable to a crude sample of the specimen of interest (25). Serological techniques such as immunofluorescence (IF) microscopy, the enzyme-linked immunosorbent assay (ELISA) (10,15,21), and molecular techniques involving the PCR (9, 24) have been described for detection of R. solanacearum. An EU control directive (3) allows for a variety of detection methods to be employed. Briefly, a primary screening test (i.e., IF and/or selective isolation) is conducted with extracts from vascular tissue sampled from 200 tubers per 25-tonne lot. To confirm th...

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.

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