Analysis of rhizosphere bacterial communities in Arabidopsis: impact of plant defense signaling

In the rhizosphere, numerous microbial and plant-microbe interactions occur. Of special interest is the ability of specific rhizosphere bacteria to elicit induced systemic resistance (ISR), a state of enhanced defensive capacity of the plant that is effective against a wide range of pathogens. The goal to minimize the use of agrochemicals in crop protection stimulates the development of practical applications of ISR-eliciting bacteria as an environmentally friendly alternative. However, application of these bacteria on a large scale and at high densities may perturb the indigenous microflora. This thesis is focused on effects of plant defense signaling on the indigenous bacterial rhizosphere microflora. Population densities of the bacterial and Pseudomonas spp. microflora were determined by selective dilution plating, whereas bacterial community structures were studied by DGGE analysis of amplified 16S rDNA, obtained from DNA directly extracted from rhizosphere and bulk soil. As model plant we used Arabidopsis thaliana accession Col-0, since numerous defense signaling mutants and transgenic lines are available and rhizobacteria-mediated ISR has been characterized in detail in this species. To determine if the indigenous bacterial microflora in the rhizosphere is affected by plant defenses, Arabidopsis genotypes with altered defense signaling were used. Whereas no differences were observed on microbial community structures, in some defense signal-transduction mutants rhizosphere population densities of culturable bacteria or Pseudomonas spp. were different from those of the parent Col-0. These differences were observed only in one type of soil. Apparently, soil is a predominant factor shaping microbial communities. In a complementary approach, jasmonic acid (JA)- or salicylic acid (SA)-dependent defenses were chemically activated by application of these hormones. Neither the abundance, nor the community structure of the bacterial rhizosphere microflora was affected by activation of the JA- or SA-dependent responses. Whereas Pseudomonas putida WCS358r and Pseudomonas fluorescens WCS417r elicit ISR against the bacterial leaf pathogen Pseudomonas syringae pv. tomato (Pst) in Arabidopsis, P. fluorescens WCS374r does not. The root-colonizing capacity of these three bacterial strains was studied on wild-type Arabidopsis and on a non ISR-expressing mutant, myb72. Whereas WCS358r and WCS417r proliferated on the roots of the wild type, this was not the case for WCS374r. However, none of the strains proliferated on the roots of the myb72 mutant. Apparently, MYB72 is not only essential for the expression of ISR, but also influences root colonization by rhizobacteria. Metabolic profiling revealed that treatment of wild-type plants and the myb72 mutant with the Pseudomonas spp. strains significantly altered the amounts of sugars, organic acids and amino acids. Most annotated metabolite fragments could be linked to known plant-microbe or plant-pathogen interactions, but not to the expression of ISR. Finally, population densities of total culturable bacteria and Pseudomonas spp. in the phyllosphere were determined upon infection with Pst. Arabidopsis mutants differed in their sensitivity to Pst and the most sensitive mutants also had the highest bacterial and Pseudomonas spp. populations on their leaves. Collectively, these results suggest that control of plant diseases by elicitation of induced systemic resistance will not significantly affect the indigenous rhizosphere bacterial microflora.