Genotypic diversity and rhizosphere competence of antibiotic-producing Pseudomonas species

The phenolic antibiotic 2,4-diacetylphloroglucinol (DAPG) has been implicated in biological control of multiple plant pathogens by fluorescent Pseudomonas species. DAPG-producing Pseudomonas strains are effective biocontrol agents, however, their ecological performance is often highly variable resulting in inconsistent disease suppression. The ecological performance is complex and determined by many bacterial traits and environmental factors, including the host plant. In this thesis, several genotypic and phenotypic characteristics underlying the ecological performance of DAPG-producing Pseudomonas were investigated. To discriminate between genotypically different DAPG-producing Pseudomonas strains directly in rhizosphere samples without their prior isolation or enrichment on nutrient media, a simple and rapid method was developed based on polymorphisms in the polyketide synthase gene phlD. Denaturing Gradient Gel Electrophoresis (DGGE) analysis, sequencing and phylogenetic analyses of indigenous phlD+ isolates obtained from the rhizosphere of wheat, sugar beet and potato plants, resulted in the identification of seven phlD+ genotypes, designated A, B, C, D, E, F, and Z, five of which were not described previously (C, D, E, F and Z). The phlD-DGGE analysis allowed simultaneous detection of multiple phlD+ isolates in the rhizosphere and, compared to cultivation-based approaches, this technique does not have the bias toward detecting either the most dominant genotype or the genotype with higher growth rates or competitive abilities during cultivation. Subsequent studies with representative strains of each of the Pseudomonas genotypes showed that three genotypes (A, Z and G) were superior in long-term colonization of roots of wheat, sugar beet and potato plants. These results suggest that their rhizosphere competence is not linked to a specific plant species, but is due to yet unknown characteristics that enable these strains to be competitive in different rhizosphere environments. In contrast, the rhizosphere competence of Pseudomonas genotypes E, C and F was dependent on the plant species and, therefore, these strains are considered to be specialists instead of generalists. Results of this thesis further showed that the host plant species also have a significant effect on DAPG-production by indigenous phlD+ Pseudomonas: the wheat and potato rhizospheres supported significantly higher amounts of DAPG produced per cell basis than the rhizospheres of sugar beet and lily. In the same context, the eight Pseudomonas genotypes differed significantly in their ability to produce DAPG in the rhizosphere of sugar beet plants with in situ DAPG concentrations ranging from 1 to 144 ng per 105 cells. Based on these data, significant correlations were established between the rhizosphere competence of a genotype and in situ DAPG production levels. In general, these correlations suggest that Pseudomonas genotypes that produce high amounts of DAPG per cell basis in situ establish lower population densities in the sugar beet rhizosphere than genotypes that produce small amounts of DAPG. To our knowledge, this is the first study that shows an inverse correlation between rhizosphere competence of Pseudomonas strains and in situ antibiotic production. Biocontrol assays showed that P. ultimum was effectively controlled by all eight Pseudomonas strains and differential effects were observed in biocontrol activity against A. cochlioides. Pseudomonas genotype G was the most effective in biocontrol of Pythium and Aphanomyces damping-off, and its biocontrol activity was due, at least in part, to DAPG production as its DAPG-deficient mutant was significantly less effective. Comparative analysis of the eight DAPG-producing Pseudomonas genotypes revealed a highly significant correlation between their rhizosphere competence and efficacy to control Aphanomyces damping-off of sugar beet. These results indicate that the more rhizosphere competent DAPG-producing Pseudomonas strains are, the higher their efficacy is to control A. cochlioides in sugar beet. The promising results obtained with genotypes A, Z and G in the sugar beet bioassays provide a strong basis for their implementation in the current integrated disease management strategies in sugar beet. The results acquired in this thesis have shown that the identification of the genotypic diversity and rhizosphere competence of antibiotic-producing Pseudomonas species is of great value, because it may allow maximizing root colonization and disease suppression. Knowledge of genetic traits involved in host preference of these antagonistic bacteria will help to identify strains that are adequately adapted to specific host-pathogen systems. Similarly, looking into plant traits that promote the growth and activity of introduced biocontrol strains can be highly complementary and further contribute to sustainability in agriculture.