Detection of Ralstonia pseudosolanacearum in drain water based on concentration, enrichment and the use of a duplex TaqMan PCR test

Abstract

The Ralstonia solanacearum species complex (RSSC) can cause bacterial wilt in a wide variety of plant species, including a number of ornamental glasshouse crops. Recently in Europe, ornamental rose plants for the production of cut flowers and propagation materials have been strongly affected by Ralstonia pseudosolanacearum, phylotype I, biovar 3. To test for the presence of the pathogen in the glasshouse, sampling of water from a drainage gutter or well may be an efficient strategy since it is known that RSSC can be released from infected root systems in the water. A protocol was developed to detect low densities of R. pseudosolanacearum in drain water collected from rose growers. Drain water was filtered through a bacterial filter, the filtrate was collected and target bacteria enriched for 48 h in Semi‐selective Medium South Africa (SMSA) broth supplemented with sterilized tomato plant extracts. DNA extracted from the enrichment broth was analysed using a TaqMan test in a duplex format, based on specific egl sequences of RSSC and the use of an extraction and amplification control. The optimized protocol had a detection level of ≤1–10 colony forming units of R. pseudosolanacearum in drain water.