Interaction in the rhizosphere of Arabidopsis thaliana

The present study investigated the effect of a common soil amoeba, Acanthamoeba castellanii, on bacterial community composition in the rhizosphere of Arabidopsis thaliana. In the first experiment a fingerprinting method was established to detect grazing induced shifts on bacterial communities in a sand/litter system. The effect of different extraction protocols on fingerprinting was assessed. The use of a method including a phenol/chloroform purification step proved to be most efficient for monitoring grazing-induced shifts with denaturing gradient gel electrophoreses (DGGE). Bacterivorous protozoa alter the structure of bacterial communities in the rhizosphere of plants, and these changes likely contribute to plant growth-promoting effects of protozoa. In the second experiment effects of protozoan grazing on bacterial community composition was investigated with DGGE and fluorescence in situ hybridisation (FISH). DGGE fingerprinting pattern demonstrated rapidly induced changes in the composition of the bacterial community, some bands already disappeared two days past inoculation of amoebae. Using FISH with probes for major bacterial phyla a decrease in metabolically active bacteria in presence of amoeba could be demonstrated. However, simultaneously their proportion strongly increased. The decrease in numbers was most pronounced in Betaproteobacteria and Firmicutes. The quantity of Gammaproteobacteria was not affected by protozoan grazing but DGGE with specific primers for pseudomonads revealed functional shifts in the gacA gene. Protozoa are known to increase plant growth by mobilization of nutrients due to grazing on bacteria and by grazing induced shifts in bacterial community composition favouring plant growth promoting rhizobacteria. In a third experiment it was investigated if protozoan-mediated changes in plant performance are based on changes in the composition of the rhizosphere bacterial community or on nutrient based effects. Arabidopsis thaliana was grown in a sand/litter substrate inoculated with single strains of bacteria or a diverse bacterial soil filtrate and Acanthamoeba castellanii. Plant rosette diameter, as indicator for plant biomass and fitness, was generally increased in presence of amoebae, whereas root biomass was reduced in both single strain bacteria treatments. Further amoebae increased plant tissue nitrogen concentration resulting in lower C-to-N ratio. Amoeba-mediated changes in rosette diameter and plant tissue C-to-N ratio were not significantly different between the single strain and diverse bacterial communities suggesting that the effects were independent of bacterial community composition. The results of the present study suggest that nutrient based effects caused by grazing of the amoeba on bacterial biomass and thereby mobilizing nutrients locked in bacterial tissue are more important than grazing induced shifts in bacterial community composition