Exploring the Biocontrol Potential of Insecticidal Fluorescent Pseudomonads Applied Alone and in Combination with Entomopathogenic Nematodes and Fungi

Abstract

This thesis focuses on the biocontrol of insect pests using insecticidal Pseudomonas bacteria. Fluorescent pseudomonads are well-known for their abilities to promote plant growth, suppress pathogens and induce systemic resistance. In the last decade, research focused on the mechanisms underlying oral insecticidal activity of P. chlororaphis and P. protegens subgroups as well as their ecological interactions with insects. We aimed at exploiting the insecticidal activity of pseudomonads for controlling below-ground insect pests alone and in combination with entomopathogenic nematodes and fungi. Furthermore, we explored the phyllosphere competence of fluorescent pseu- domonads, a prerequisite to control foliar pests and pathogens. In the first part of this thesis, we investigated the potential of P. chlororaphis and P. pro- tegens strains to control the cabbage maggot Delia radicum, an important pest of Brassicacean crops for which no satisfactory control exists. We then combined the most potent strain, P. chlororaphis PCLRT03, with the entomopathogenic nematode Stein- ernema feltiae RS5 and the entomopathogenic fungus Metarhizium brunneum Bip5 in screening, greenhouse, semi-field experiments and a field trial. The consortium of P. chlororaphis, S. feltiae and M. brunneum could successfully reduce D. radicum damage in the field trial and the individual members had no impact on the survival of each other on roots and in the soil. The biocontrol agents applied alone were also effective, yet the Pseudomonas strain was more efficient than the nematode and the fungus. Under screening and semi-field conditions, combinations of pseudomonads with either nematodes or fungi resulted in synergistic interactions. In a next step, the consortium was applied in laboratory assays against the leaf-feeding large cabbage white Pieris brassicae and the root-feeding banded cucumber beetle Diabrotica balteata and the interaction between the three biocontrol agents inside the larvae was investigated. The triple consortium was the most lethal and fastest killing treatment against both insects. A combination of plating and qPCR approaches allowed us to simultaneously monitor all biocontrol agents including the nematode-associated bacterium Xenorhabdus bovienii in the same insect. After simultaneous application, all three agents as well as the xenorhabds established inside the larvae in the early stages of the infection. P. chlororaphis seems to profit from the other BCA and reached the highest colonisation densities in co-infections in both insects. S. feltiae and M. brunneum, however, seemed to be mutually exclusive in double applications of nematodes and fungi. Interestingly, all four insecticidal organisms could be detected in several individual larvae. These results suggest that P. chlororaphis, S. feltiae and M. brunneum can indeed co-infect the same insect. In the last part of this thesis, fluorescent pseudomonads were isolated from radish leaves and screened for their abilities to kill insects, suppress pathogens and persist in the phyllosphere. Unfortunately, no P. chlororaphis and P. protegens strains were discovered, but strains from the subgroups P. fluorescens, P. koreensis and the group P. putida. Several strains showed insecticidal activity upon injection into Galleria mellonella larvae. Two leaf isolates of the P. fluorescens subgroup showed potent oral insecticidal activity against the diamondback moth Plutella xylostella, comparable to P. chlororaphis PCLRT03 and P. protegens CHA0. Furthermore, the new leaf isolates persisted better in the phyllosphere than the tested P. chlororaphis and P. protegens strains. The results obtained in this thesis suggest that insecticidal pseudomonads from the P. chlororaphis subgroup can be used to control below-ground insect pests. Furthermore, our findings show that insecticidal pseudomonads are compatible and co-operate with entomopathogenic nematodes and fungi. The consortium of P. chlororaphis, S. feltiae and M. brunneum can potentially be used to control a variety of below-ground insect pests. Applying the consortium might improve efficacy or stability of biocontrol. How- ever, further research on performance in the field and efficacy against different insect pests is needed. The approaches used in this thesis can be used to build and evaluate further consortia against other pests. Finally, the insights gained in this thesis are highly valuable for the development of biocontrol strategies based on insecticidal pseudomonads and consortia of biocontrol agents.