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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.

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 upon the root inoculation techniques used. The presence of one population always influenced the density of the other challenge-inoculated population. In plants inoculated with both wild-type and mutant strains, the population of the wild-type strain is lower than in plants inoculated with the wild type alone. In contrast, growth of the HrcV¯ mutant strain was significantly increased in the presence of the pathogenic strain. Two agriculturally acceptable techniques for plant inoculation were tested. Inoculation of plants by transplanting them into soil amended with clay micro-granules impregnated with the HrcV¯ mutant strain gave higher and more reproducible colonization of the plants than inoculation by watering a bacterial suspension on the roots. Significant percentages of exclusive colonization by the HrcV¯ mutant strain were only obtained after the clay microgranule inoculation technique. Competition for space in xylem vessels is one of the possible explanations for the protective ability of the HrcV¯ mutant strain against subsequent invasion by a pathogenic strain.

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.

There have been many attempts to control bacterial wilt with antagonistic bacteria or spontaneous nonpathogenic mutants of Pseudomonas solanacearum that lack the ability to colonize the host, but they have met with limited success. Since a large gene cluster ( hrp ) is involved in the pathogenicity of P. solanacearum , we developed a biological control strategy using genetically engineered Hrp - mutants of P. solanacearum. Three pathogenic strains collected in Guadeloupe (French West Indies) were rendered nonpathogenic by insertion of an ω-Km interposon within the hrp gene cluster of each strain. The resulting Hrp - mutants were tested for their ability to control bacterial wilt in challenge inoculation experiments conducted either under growth chamber conditions or under greenhouse conditions in Guadeloupe. Compared with the colonization by a pathogenic strain which spread throughout the tomato plant, colonization by the mutants was restricted to the roots and the lower part of the stems. The mutants did not reach the fruit. Moreover, the presence of the mutants did not affect fruit production. When the plants were challenge inoculated with a pathogenic strain, the presence of Hrp - mutants within the plants was correlated with a reduction in disease severity, although pathogenic bacteria colonized the stem tissue at a higher density than the nonpathogenic bacteria. Challenge inoculation experiments conducted under growth chamber conditions led, in some cases, to exclusion of the pathogenic strain from the aerial part of the plant, resulting in high protection rates. Furthermore, there was evidence that one of the pathogenic strains used for the challenge inoculations produced a bacteriocin that inhibited the in vitro growth of the nonpathogenic mutants.

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.

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.

The pathogenic interactions between tomato roots and Pseudomonas solanacearum were examined microscopically following inoculation with either a pathogenic strain or a nonpathogenic mutant, both harboring a transposon containing a promoterless Escherichia coli lacZ gene, which is highly expressed. Several stages were distinguished during tomato root infection by the pathogenic strain. After colonization of exudation sites such as root extremities and axils of secondary roots the bacteria intercellularly infect the inner cortex and the vascular parenchyma. These latter tissues are separated by the endodermis which is not yet fully differentiated at root extremities and is reoriented by the development of lateral roots. Following infection, the pathogenic strain invades protoxylem vessels degrading cell walls. The colonization of approximately 25% of xylem vessels in each vascular bundle of the hypocotyl just above the collar zone is sufficient to induce partial wilting of a tomato plant. In contrast, the nonpathogenic strain was able to infect intercellular spaces of the inner cortex of some secondary roots, but was never observed in the vascular cylinder. The application of this approach should enable the characterization of the process of tomato root infection by genetically defined mutant strains 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.

La technique ELISA a été appliquée à la détection de Pseudomonas phaseolicola dans des graines et des feuilles contaminées de haricot. Le test ELISA a été réalisé selon la méthode sandwich d’après CLARK & ADAMS (1977), modifiée par l’emploi de tampon substrat : Tris-HCI, NaCI avec la phosphatase alcaline d’Escherichia coli. Les immunoglobulines ont été purifiées par chromatographie d’affinité avec soit la protéine A, soit la bactérie entière Ps. phaseolicola comme ligand (fig. 1). Des études préalables ont montré que la technique ELISA donnait des résultats positifs avec les suspensions bactériennes et les filtrats correspondants après passage sur membrane de 0,22 μm. Dans nos conditions expérimentales, il ressort que : - la limite de sensibilité de cette technique correspond à une suspension bactérienne de 104 bactéries/ml en 4 h de réaction (tabl. 1). - Les métabolites bactériens contenus dans les filtrats donnent des réactions parallèles à celles obtenues avec les suspensions (tabl. 2 et 3). - La concentration de métabolites solubles détectés est d’environ 10 ng par ml par référence à la sérum albumine. - La sensibilité de la technique ELISA ne peut être exprimée en terme de concentration bactérienne mais plutôt en terme de concentration d’antigènes bactériens, solubles ou non. Il serait d’un grand intérêt de tenter d’établir une corrélation entre une concentration bactérienne et la production de métabolites correspondant dans des conditions naturelles d’environnement. Les résultats obtenus pour la détection de Ps. phaseolicola dans les graines et les feuilles de haricot contaminées par les techniques d’immunofluorescence ou ELISA sont identiques (tabl. 4 et 5). L’extraction et la purification de quelques métabolites bactériens liés au pouvoir pathogène doivent conduire à l’obtention d’antisérums spécifiques qui permettront des études qualitatives et quantitatives, par la technique ELISA de la production et de la distribution de ces métabolites pathogènes in vivo. La standardisation dans la purification des immunoglobulines et dans leur couplage avec une enzyme plus active est nécessaire pour permettre des comparaisons et pour améliorer la sensibilité de la technique ELISA., The ELISA technique has been applied to the detection of Pseudomonas phaseolicola in contaminated seed and leaves of bean. ELISA tests were performed according to the sandwich method as described by CLARK & ADAMS (1977) and slightly modified by using Tris-HCl saline buffer for the enzymatic phase with alkaline phosphatase from Escherichia coli. Purified immunoglobulins were obtained by affinity chromatography using either protein A, or whole bacterial Ps. phaseolicola as ligands (fig. 1). Preliminary studies revealed that the ELISA technique was effective when applied to bacterial suspensions or to corresponding filtrates through 0.22 μm membranes as well. Under our experimental conditions we found : i) that the lower limit of detection of the EusA sandwich method was about 104 bacteria per ml within four hours (table 1) ; ii) that the bacterial metabolites in the filtrates led to heavy, rapid positive responses (tables 2 and 3) ; iii) that the detectable amount of soluble protein is about 10 ng per ml by reference to serum albumin ; iv) that ELISA ensitivity could not be expressed in terms of bacterial concentration, but rather in terms of the concentration of bacterial antigens soluble or not. A question of major interest is to establish the possible correlation between a bacterial concentration and its related metabolite production under natural environmental conditions. A good correlation was demonstrated between the immunofluorescence indirect method and ELISA in detecting Ps. phaseolicola in contaminated seeds and leaves of bean (tables 4 and 5). The extraction and purification of some bacterial metabolites involved in pathogenicity, should lead to the production of specific antisera. These should be of great help in qualitative and quantitative studies by ELISA on the production and distribution of these pathogenic metabolites in vivo. Standardization in purifying immunoglobulins and in conjugating them with more active enzymes is needed to allow valid comparisons and improve the sensitivity of ELISA.