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 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.