European Journal of Plant Pathology (2000) 106, 123-133

J.M. van der Wolf, S.G.C. Vriend, P. Kastelein, E.H. Nijhuis, P.J. van Bekkum and J.W.L. van Vuurde (2000)
Immunofluorescence colony-staining (IFC) for detection and quantification of Ralstonia (Pseudomonas) solanacearum biovar 2 (Race 3) in soil and verification of positive results by PCR and dilution plating
European Journal of Plant Pathology 106 (2), 123-133
Abstract: A procedure was developed for specific and sensitive quantitative detection of Ralstonia (Pseudomonas) solanacearum biovar 2 (race 3) in soil. It is based on immunofluorescence colony-staining (IFC) followed by confirmation of the identity of fluorescent colonies by PCR-amplification or dilution plating on a semi-selective medium, SMSA. Addition of sucrose and the antibiotics cycloheximide and crystal violet to the non-selective trypticase soy broth agar resulted in increased colony size and staining intensity of R. solanacearum in IFC. Verification of IFC-results by picking cells from IFC-positive colonies followed by dilution plating of the suspended cells on SMSA was highly efficient. The success rate was 92% and 96% with 'spiked' and naturally contaminated soils respectively. Several other bacterial species which cross-reacted with polyclonal antibodies in IFC also grew on SMSA and were difficult to distinguish from R. solanacearum, thereby necessitating confirmation of the results. Rapid verification of IFC-positive results directly by PCR-amplification with primers D2/B specific to division 2 of R. solanacearum had a success rate of 86% and 96% with 'spiked' and naturally contaminated soil samples, respectively. Primers D2/B reacted with all R. solanacearum division 2 strains, and strains of R. syzygii and the banana blood disease bacterium, but not with saprophytic bacteria cross-reacting in IFC with R. solanacearum antibodies. In comparative tests, IFC was able to detect consistently ca. 100 cfu g^-1 of soil, a detection level similar to that found with direct plating on SMSA, but less laboriously, whereas detection level with a bioassay on tomato plants was only 10⁴-10⁵ cfu g^-1 of soil.
(The abstract is excluded from the Creative Commons licence and has been copied with permission by the publisher.)
Link to article at publishers website
Database assignments for author(s): Jan M. van der Wolf, Pieter Kastelein

Research topic(s) for pests/diseases/weeds:
identification/taxonomy

---

Pest and/or beneficial records:

Beneficial	Pest/Disease/Weed	Crop/Product	Country	Quarant.
	Ralstonia solanacearum