Importance of biotic interactions for the fitness and activity of rhizosphere biocontrol pseudomonads

This work investigates the ecology of biocontrol bacteria in the rhizosphere of crop plants. It focuses on biotic interactions influencing the fitness and the activity of these bacteria, and on defence mechanisms increasing their competitiveness against other bacteria. A number of soil bacterial present antagonistic activity against soil borne plant pathogens by producing antibiotics and enzymes. Thereby they bear potential for developing environmentally friendly management of crop diseases, as an alternative to conventional fungicides or fumigants. The application of such biocontrol bacteria, however, is still limited by the lack of consistency in their survival and antagonistic activity. Introduced bacteria often fail to establish in soil or remain in an inactive state. Biotic interactions are central for the fitness of introduced strains. Bacteria in soil compete with indigenous microorganisms present in high density and diversity. Further, they are exposed to a complex community of predators, in particular protozoa and nematodes. In order to successfully use bacterial inoculants under field conditions there is a need to better understand which interactions are the most relevant for the survival of introduced strains, and which defence mechanisms help bacteria to establish stable and persisting populations. Especially toxins play an important role. Antibiotics responsible for phytopathogen inhibition are often inhibitors of bacterial growth, and are highly toxic against protozoan predators. We used as model organism the biocontrol bacterium Pseudomonas fluorescens CHA0, an efficient coloniser of crop plants with a strong antagonistic activity against fungal pathogens and root knot nematodes. We tested if bacterial toxicity enhances competitiveness against other rhizosphere bacteria and improve resistance against predation pressure, and if bacteria alter the production of toxins in response to predator chemical cues or to signal molecules involved in plant - bacteria communication The first two experiments investigated the impact of bacterial toxins and microfaunal predation on intra- and interspecific competition among bacteria in the rhizosphere. We used gnotobiotic or semi-natural simplified microcosms with and without predators. Predation favoured toxic phenotypes and increased their competitiveness against other rhizobacteria such as non-toxic spontaneous mutants. This suggests that toxins of biocontrol bacteria primarily function as antipredator defence, and that microfaunal predators promote toxic bacteria thereby enhancing soil suppressiveness. The third and fourth experiments investigated the chemical ecology of biocontrol bacteria. By using green fluorescent protein (gfp) reporter fusions reflecting the expression of the main biocontrol genes, we followed changes in toxin production in response to chemical cues from predators and the host plant. The results demonstrated that bacteria sense chemical cues from free living amoebae, and respond by increased toxin production. Bacterial toxicity was also influenced by the host plant, which modulated the expression of antifungal genes upon infection with a root pathogen. The results suggest that bacteria adjust the production of toxins in response to a wide range of environmental parameters in order to optimise the costs and benefits of defence mechanisms. The fifth experiment explored the integration of introduced biocontrol bacteria in soil food webs by RNA Stable Isotope Probing (SIP). In this experiment wildtype and gacS- strains of P. fluorescens CHA0 were labelled with 13C and introduced in an agricultural soil. Microfaunal predators consuming both strains were resolved by T-RFLP and RT-qPCR of the 18S rRNA. The results indicate that carbon is transferred rapidly to higher trophic levels, and that toxic bacteria were consumed by a distinct and more restricted eukaryote community than bacteria without defence mechanisms. In conclusion, the production of extracellular toxins by biocontrol bacteria appear thus to be crucial for their competitiveness in the soil. This overlapping of antipredator and crop protecting traits opens promising possibilities of improvement of the efficiency of microbial biocontrol agents by manipulating the predation regime