Microbial biocontrol agents (MBCA) are getting increased attention since the use of chemical pesticides needs to be reduced. Current screening methods of MBCA strongly focus on the production of secreted metabolites and may not pick up MBCAs with novel modes of actions. This project aims to study novel biocontrol mechanisms against soilborne pathogens in five taxonomically related plant-associated Pseudomonas strains obtained from disease suppressive soils or rice plants that differ in in vitro and in planta activity against phytopathogens. Three strains that produce similar antifungal cyclic lipopeptides (CLPs) show strong antagonism in vitro against the fungus Rhizoctonia solani, but only one of them is active in a bean/R. solani pathosystem. One strain (COR33) is completely inactive in vitro, but very performant in planta, while a fifth isolate (COR58) with strong in planta bicontrol is only active in vitro when in direct contact with the pathogen. Genome mining has revealed that COR33 encodes a type 3 secretion system (T3SS) that may interfere with plant immunity, while COR58 harbours two type 6 secretion systems (T6SSs) that may inject toxins directly inside the pathogen upon contact. In this project, we will use a multidisciplinary approach that includes genome mining, mutant construction, reporter strains, (bio)chemical analysis, microscopy and bio-assays to investigate the role of CLPs, T3SS and T6SS in in vitro and in planta biocontrol.