Immunofluorescence colony-staining and selective enrichment in liquid medium for studying the population dynamics of Ralstonia solanacearum (race 3) in soil

Survival of Ralstonia solanacearum (race 3) in different soil types in The Netherlands is currently under investigation to determine its significance in the epidemiology of brown rot. For detection of the brown-rot pathogen, immunofluorescence colony staining (IFC) and selective enrichment in liquid media were optimized and evaluated. In IFC, pour-plating of extracts from a clay soil and a loamy sand in the semi-selective SMSA medium resulted in relatively low recoveries. Using non-selective trypticase soy agar (10% TSA) medium, colonies of target bacteria added to extracts, particularly the sandy loam, remained very small and were weakly stained. Increased colony size and improved staining quality were found after addition of sucrose and crystal violet to 10% TSA. IFC detected 103-104 cfug-1 of dry soil. Detection of R. solanacearum by dilution plating on selective SMSA agar was complicated by the growth of many saprophytic bacteria which was confused with that of R. solanacearum. For detection of populations lower than 103 cfu g-1 of dry soil, selective enrichment in SMSA broth containing the same nutrients as SMSA agar medium was evaluated. After enrichment, R. solanacearum was detected by serial dilution of the sample and immunofluorescence cell staining. EFC was less suitable here, due to the high densities of non-target bacteria in the samples. In clay soil, initial populations of 1–10 cfu g-1 of dry soil were enlarged within 48h to higher than 108 cfu g-1. In sandy soil, enrichment occurred only when initial populations exceeded 100 cfu g -1 of dry soil. IFC was used to study the survival of R. solanacearum in sandy soil from the north-east of The Netherlands (Drenthe). The pathogen survived at 15°C in soil for at least 92 days, but was not detectable at 4 or 20°C after 50–92 days. When the soil was frozen for 24 h at -8°C, the pathogen could not be detected by IFC. In soil from three different locations in The Netherlands from which brown rot-infected potatoes were harvested, the pathogen was not detected by IFC, nor by bioassay in tomato plants. Cross-reacting bacteria were detected in IFC preparations at a low frequency. They were weakly stained and could be distinguished from target bacteria in control preparations. Reisolated bacteria from these weakly stained colonies did not grow on SMSA and were negative in PCR amplifications, based on primers OLI-1 and Y-2.