André Trigalet

Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region, AFLP and 16S rRNA sequence analysis, and identification of an African subdivision.

The genetic diversity among strains in a worldwide collection of Ralstonia solanacearum, causal agent of bacterial wilt, was assessed by using three different molecular methods. PCR-RFLP analysis of the hrp gene region was extended from previous studies to include additional strains and showed that five amplicons were produced not only with all R. solanacearum strains but also with strains of the closely related bacteria Pseudomonas syzygii and the blood disease bacterium (BDB). However, the three bacterial taxa could be discriminated by specific restriction profiles. The PCR-RFLP clustering, which agreed with the biovar classification and the geographical origin of strains, was confirmed by AFLP. Moreover, AFLP permitted very fine discrimination between different isolates and was able to differentiate strains that were not distinguishable by PCR-RFLP. AFLP and PCR-RFLP analyses confirmed the results of previous investigations which split the species into two divisions, but revealed a further subdivision. This observation was further supported by 16S rRNA sequence data, which grouped biovar 1 strains originating from the southern part of Africa.

Transcriptional organization and expression of the large hrp gene cluster of Pseudomonas solanacearum.

Cloning and localized mutagenesis of the larger cluster of hrp genes of Pseudomonas solanacearum strain GMI1000 allowed the definition of the borders of this cluster, which now extends about 2 kb to the left of the insert of the previously described plasmid pVir2 (Boucher et al. 1987, J. Bacteriol. 169:5626-5632). The size of the cluster has also been expanded 3 kb to the right to include a region previously described as dsp; our present data demonstrate that insertions occurring in these 3 kb lead to leaky mutations affecting both pathogenicity on tomato and ability to induce the hypersensitive response (HR) on tobacco. Therefore, the size of the entire hrp gene cluster is estimated to be about 22 kb. The use of transposon Tn5-B20, which promotes transcriptional gene fusions, allowed us to demonstrate that the hrp gene cluster is organized in a minimum of six transcriptional units, which are transcribed when the culture is grown in minimal medium but are repressed during growth in rich medium or in the presence of peptone or Casamino Acids. The level of expression in minimal medium is modulated by the carbon source provided; pyruvate is the best inducer. Under these conditions the level of expression observed in vitro appears to be representative of the actual expression observed in planta.

Host plant-dependent phenotypic reversion of #Ralstonia solanacearum# from non-pathogenic to pathogenic forms via alterations in the phcA gene

Ralstonia solanacearum is a plant pathogenic bacterium that undergoes a spontaneous phenotypic conversion (PC) from a wild‐type pathogenic to a non‐pathogenic form. PC is often associated with mutations in phcA, which is a key virulence regulatory gene. Until now, reversion to the wild‐type pathogenic form has not been observed for PC variants and the biological significance of PC has been questioned. In this study, we characterized various alterations in phcA (eight IS element insertions, three tandem duplications, seven deletions and a base substitution) in 19 PC mutants from the model strain GMI1000. In five of these variants, reversion to the pathogenic form was observed in planta, while no reversion was ever noticed in vitro whatever culture media used. However, reversion was observed for a 64 bp tandem duplication in vitro in the presence of tomato root exudate. This is the first report showing a complete cycle of phenotypic conversion/reversion in a plant pathogenic bacterium.

Detection and visualization of the major acidic exopolysaccharide of Ralstonia solanacearum and its role in tomato root infection and vascular colonization

Exopolysaccharides play an important role in the pathogenicity of Ralstonia solanacearum. We compared in vitro and in planta exopolysaccharide production of the pathogenic strain AW1 with that of three related mutant strains impaired in both their exopolysaccharide production and aggressiveness on tomato. The distinction between the two hexosamine-rich exopolysaccharides, namely the N-acetyl-glucosaminorhamnan and the major N-acetyl-galactosamine-containing acidic polymer was emphasized. The major acidic polymer was identified specifically by electron microscopy using glutaraldehyde/ruthenium red/uranyl acetate staining, by immunofluorescence using specific monoclonal antibodies and correlated to an appropriate biochemical analysis. The two mutant strains AW1-1 and AW-19A were totally devoid of any production of the major exopolysaccharide in vitro or in planta whatever the technique used. Infection and vascular colonization of tomato roots by the pathogenic strain were also compared to those of the mutant strains by light microscopy. Pathogenicity on tomato was assessed by root infection without any artificial injury. Light microscopy showed that the two mutant strains AW1-1 and AW-19A were poorly infective and unable to invade xylem vessels, while they induced defence mechanisms in root tissues and appeared aggregated or degenerated within cortical infection pockets. These two mutant strains were non-pathogenic or weakly aggressive, respectively. In contrast, the pathogenic strain AW1 and the hypoaggressive AW1-41 strains, which produce large amounts of the major acidic exopolysaccharide in planta, were both infective and invasive, and tomato root tissues exhibited only limited defence reactions. Thus, the major acidic exopolysaccharide produced by Ralstonia solanacearum is involved in root infection and vascular colonization, though its precise role is still unknown.

Xylem Colonization by an HrcV¯ Mutant of Ralstonia solanacearum Is a Key Factor for the Efficient Biological Control of Tomato Bacterial Wilt

Microscopic studies of the colonization of the vascular tissues of tomato by an HrcV¯ (formerly HrpO¯) mutant strain of Ralstonia solanacearum were carried out after either root inoculation of the mutant strain alone or delayed challenge inoculation by a pathogenic strain. The use of two different marker genes, lacZ and uidA, introduced into either mutant or wild-type strains, respectively, permitted histological observation for the presence of both strains simultaneously. In roots, both strains could be found together in infected root tips and in lateral root emergence sites (lateral root cracks), but these bacterial strains subsequently invaded separate xylem vessels in the root system. At the hypocotyl level, a novel staining procedure, in conjunction with bacterial isolation and counting, showed three vascular colonization patterns: exclusive colonization by each of the competitors or simultaneous presence of each strain in separate xylem vessels. The relative frequencies of these patterns depended up...