A polyphasic approach for studying the interaction between Ralstonia solanacearum and potential control agents in the tomato phytosphere

Ralstonia solanacearum biovar 2, the causative agent of brown rot in potato, has been responsible for large crop losses in Northwest Europe during the last decade. Knowledge on the ecological behaviour of R. solanacearum and its antagonists is required to develop sound procedures for its control and eradication in infested fields. A polyphasic approach was used to study the invasion of plants by a selected R. solanacearum biovar 2 strain, denoted 1609, either or not in combination with the antagonistic strains Pseudomonas corrugata IDV1 and P. fluorescens UA5-40. Thus, this study combined plating (spread and drop plate methods), reporter gene technology ( gfp mutants) and serological (imunofluorescence colony staining [IFC]) and molecular techniques (fluorescent in situ hybridization [FISH], PCR with R. solanacearum specific primers and PCR–DGGE on plant DNA extracts). The behaviour of R. solanacearum 1609 and the two control strains was studied in bulk and (tomato) rhizosphere soil and the rhizoplane and stems of tomato plants. The results showed that an interaction between the pathogen and the control strains at the root surface was likely. In particular, R. solanacearum 1609 CFU numbers were significantly reduced on tomato roots treated with P. corrugata IDV1( chr∷ gfp1) cells as compared to those on untreated roots. Concomitant with the presence of P. corrugata IDV1( chr∷ gfp1), plant invasion by the pathogen was hampered, but not abolished. PCR–DGGE analyses of the tomato rhizoplane supported the evidence for antagonistic activity against the pathogen; as only weak R. solanacearum 1609 specific bands were detected in profiles derived from mixed systems versus strong bands in profiles from systems containing only the pathogen. Using FISH, a difference in root colonization was demonstrated between the pathogen and one of the two antagonists, i.e. P. corrugata IDV1( chr∷ gfp1); R. solanacearum strain 1609 was clearly detected in the vascular cylinder of tomato plants, whereas strain IDV1 was absent. R. solanacearum 1609 cells were also detected in stems of plants that had developed in soils treated with this strain, even in cases in which disease symptoms were absent, indicating the occurrence of symptomless infection. In contrast, strain 1609 cells were not found in stems of several plants treated with either one of the two antagonists. The polyphasic analysis is valuable for testing antagonistic strains for approval as biocontrol agents in agricultural