Detection of Ralstonia solanacearum from potato tissue by post‐enrichment TaqMan PCR*

Abstract

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.

Assessment of latent infection frequency in progeny tubers of advanced potatoclones resistant to bacterial wilt: A new selection criterion

“…The sensitivity of post-enrichment PCR was reported to be lower, c. 103 cells ml 1 (Elphinstone et al, 1996;Weller et al, 2000), higher sensitivity being reached with nested PCR (Elphinstone et al, 1996). NCM-ELISA is faster, cheaper and easier-to-use than PCR-based methods, but with serological methods there is a risk of unspecific reactions.…”

Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR

“…As would be expected C T values generally decreased at each incubation period. However, C T values below a value of 20 were not detected, unlike similar experiments for detection of R. solanacearum in latently infected potato tissues (Weller et al 2000d), suggesting that 10 ml of Medium 1A would not sustain increasing populations of A. radiobacter beyond 48 h. Alternatively, the possibility exists that the Ri-plasmid was not retained by all Agrobacterium cells in 48-h-old cultures. In either case the need for a prolonged incubation period was not supported by the results from this experiment.…”

“…Detection of plant-associated bacteria by PCR direct from plant tissue has proved problematic due to low populations and PCR inhibitors present within plant tissues. The use of selective media as a pre-enrichment treatment in PCR protocols has been described (Elphinstone et al 1996;Schaad et al 1999;Penyalver et al 2000), including Taq-Man PCR (Weller et al 2000d). Selective media allow increase of pathogen numbers and reduce levels of inhibitors in the PCR reaction, facilitating detection without the need for complex DNA puri®cation steps.…”

Validation of four real‐time TaqMan PCRs for the detection of Ralstonia solanacearum and/or Ralstonia pseudosolanacearum and/or Clavibacter michiganensis subsp. sepedonicus in potato tubers using a statistical regression approach

“…sepedonicus has been advocated by various national plant protection organizations and several Euphresco projects have been established from 2009 to 2012 (van Vaerenbergh et al, 2017) to validate the TaqMan PCRs of Weller et al (2000) and Schaad et al (1999). sepedonicus has been advocated by various national plant protection organizations and several Euphresco projects have been established from 2009 to 2012 (van Vaerenbergh et al, 2017) to validate the TaqMan PCRs of Weller et al (2000) and Schaad et al (1999).…”

“…More TaqMan PCR tests for the detection of R. solanacearum, R. pseudosolanacearum or C. michiganensis subsp. Only the TaqMan PCR published by Weller et al (2000) can detect both R. solanacearum and R. pseudosolanacearum (Weller et al, 2000). Development of TaqMan PCR tests for potato brown rot has been focused on R. solanacearum (described in papers as R. solanacearum race 3, biovar 2 subtypes), while other types can be present in Europe (Cruz et al, 2008; former species name R. solanacearum biovar 1, species name with new classification unknown).…”