Sensitive detection of Ralstonia solanacearum in soil: a comparison of different detection techniques

Abstract

The sensitivity and specificity of various methods were compared for routine detection of Ralstonia solanacearum in a sandy loam soil. Populations fewer than 102 CFU per g soil were detected by dilution plating on a modified semiselective medium (SMSA). In comparison, a tomato bioassay was shown consistently to detect populations at or greater than 7·5 × 105 CFU per g soil. An indirect enzyme‐linked immunosorbent assay (ELISA) was as sensitive as the tomato bioassay, but detected as few as 104 CFU per g soil when the suspension was first incubated in SMSA broth prior to testing. Detection using a nested polymerase chain reaction (PCR) was equally as sensitive as that using culture on SMSA agar, but only when the infested soil sample was first enriched overnight in SMSA broth prior to the nested PCR. Longer incubation periods in SMSA broth also increased the sensitivity of pathogen detection using a conventional PCR method, permitting detection of as few as 102 CFU per g soil after 60 h enrichment in SMSA broth. When evaluated using naturally infected field soils in Nepal, isolation of R. solanacearum on SMSA was reliable only when pathogen populations were higher than those of saprophytic soilborne bacteria. As few as 5 × 102 CFU of R. solanacearum per g were recovered from naturally infested soil, whereas the sensitivity of indirect ELISA was 106 CFU g−1.

Ralstonia solanacearum em viveiros clonais de eucalipto no Brasil

“…A bactéria em meio de cultivo apresenta, geralmente, colônia esbranquiçada e fluída, com 3-5 cm de diâmetro após 2-3 dias de incubação a 29 ºC. A sensibilidade desse método permite detectar até 100 ufc/g solo (Pradhanang et al, 2000). Todavia, esse método requer treinamento para distinguir colônias típicas do patógeno de bactérias saprófitas, o que limita o seu uso em larga escala.…”

Occurrence and Distribution of Potato Pests and Diseases in Kenya

“…When bacterial wilt-like symptoms were observed, at least one plant per field was uprooted for analysis in the laboratory by dilution plating the rhizosphere soil on a modified semi-selective medium (SMSA) as described by Pradhanang et al (2000) to enrich the sample for bacteria. Suspect colonies were then tested using DAS-ELISA (as described above) for Ralstonia solanacearum (reagents purchased from CIP, Lima, Peru).…”

Impact of soil amendments and weather factors on bacterial wilt and yield of two tomato cultivars in Abeokuta, Nigeria

“…The supernatant collected from the settled suspension was subjected to serial dilution. Aliquots (0.1 ml each) of supernatants from the dilution series were spread on replicated plates of TTC agar medium and incubated at 28 °C for 48 h in accordance with the method of Pradhanang et al (2000). Ralstonia solanacearum colonies were distinctively bold, fluidal and creamy-white on the medium, turning characteristic reddish in the centre and whitish at the periphery of the TTC agar after 48 h incubation.…”