Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts

Abstract

The sensitivities of various methods for the detection of Ralstonia solanacearum following dilution in healthy potato tuber tissue macerate were compared. Estimated pathogen populations in undiluted macerates, from samples of 200 heel‐end vascular cores each containing a single diseased and 199 healthy tubers, ranged from 1.2 × 106–7.4 × 107 colony‐forming units per ml. Following concentration by high‐speed centrifugation and resuspension in phosphate buffer, the pathogen was detected by all methods studied, including culture on semi‐selective media, enzyme‐linked immunosorbent assay (ELISA), indirect immunofluorescent‐antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFCS), detection of specific DNA sequences following amplification by the polymerase chain reaction (PCR) and bioassay in tomato seedlings. Both ELISA and PCR methods were improved by pre‐enrichment of samples in semi‐selective broth prior to testing. A nested PCR method was evaluated which could detect fewer than 10 cells per ml in the potato extracts. Of the other methods only dilution plating on semi‐selective medium and tomato bioassay could detect fewer than 104 cells per ml. In order to combine ease and speed of use with sensitive detection, it was recommended that a series of methods be used for routine screening of potato tuber stocks for infection by R. solanacearum.

Ralstonia solanacearum requires F-box-like domain-containing type III effectors to promote disease on several host plants

“…For each IGC time point, 3 plants were collected in the order they were placed in the tray and pooled; this procedure was repeated three times. The IGC was performed as described (52), except that 5 l of the diluted solutions were spotted on SMSA plates (53). The typical R. solanacearum phenotype of the bacteria counted on the different dilutions was checked on B rich medium.…”

Survival strategies and pathogenicity of Ralstonia solanacearum phylotype II subjected to prolonged starvation in environmental water microcosms

“…Stems from wilted plants were cut at different levels above the inoculation point and segments of 2-3 cm were analysed for presence of R. solanacearum. Extracts were plated onto a modified semiselective medium South Africa (SMSA) agar (Elphinstone et al, 1996) and colonies were PCRtested (OLI1 Y2 primers) as described in EU Directives (Anonymous, 1998(Anonymous, , 2006. Stems from inoculated non-wilted plants were processed in the same way after 6 weeks.…”

Epidemiological analysis of clones and cultivars of potato in soil naturally infested with Ralstonia solanacearum biovar 2

“…Seal et al (1993) reported that PCR can detect as few as one to ten R. solanacearum cells, although this level of sensitivity could not be reproduced by other workers (Elphinstone et al, 1996;Martins, 2000). New methods have produced an increase in the sensitivity of detection techniques, an example being the use of a pretreatment protocol involving semi-selective enrichment medium plus the addition of 0.05 M NaOH and heating at 100 °C for 6 minutes during the PCR (Elphinstone et al, 1996). Martins (2000) determined the sensitivity of the PCR technique for potato tuber samples as being 3.7 x 10 3 CFU mL -1…”

“…However, some tubers were positive for R. solanacearum by DAS-ELISA and PCR, indicating the latent nature of the infection in resistant genotypes as well as in susceptible ones (Table 4) Using the same DNA extraction method and primers (Oli 1 and Y2) as were used by us, Llop et al (1999) found that PCR was more efficient at detecting latent infections of potato tubers as compared to ELISA and immunofluorescence tests using polyclonal anti-sera or traditional microbiological methods, with PCR being able to detect as few as 100 colony forming units (CFU).mL aimed at minimizing dissemination of the pathogen through infected tubers (Elphinstone et al, 1996;Priou et al, 1999;Llop et al, 1999;Martins, 2000). Seal et al (1993) reported that PCR can detect as few as one to ten R. solanacearum cells, although this level of sensitivity could not be reproduced by other workers (Elphinstone et al, 1996;Martins, 2000). New methods have produced an increase in the sensitivity of detection techniques, an example being the use of a pretreatment protocol involving semi-selective enrichment medium plus the addition of 0.05 M NaOH and heating at 100 °C for 6 minutes during the PCR (Elphinstone et al, 1996).…”