The interaction of WLIP (white line-inducing principle), a member of the viscosin group of Pseudomonas lipopeptides, with tolaasin, a lipopeptide mycotoxin secreted by Pseudomonas tolaasii, enables identification of the mushroom pathogen relying on formation of a lipopeptide coprecipitate between confronted colonies of an indicator strain (designated Pseudomonas ‘reactans’) and P. tolaasii. The WLIP non-ribosomal lipopeptide synthesis system of the mushroom isolate P. ‘reactans’ LMG 5329 (Wip) was identified and shown to be most similar to the Pseudomonas fluorescens SBW25 viscosin system (Visc), but remarkably different from the WLIP-generating Wlp system previously identified in the rice rhizosphere isolate Pseudomonas putida RW10S2. The Wlp machinery is composed of modules most similar to those recruited for biosynthesis of the non-viscosin-type lipopeptides putisolvin and entolysin by strains from the P. putida clade. In line with the pronounced synteny between the wip and visc flanking regions, strain LMG 5329 was identified as an authentic P. fluorescens closely related to strain SBW25. In both P. putida and P. fluorescens, WLIP production confers similar phenotypes of microbial antagonism and surface colonization. Genotypes other than wlp or wip were not identified among WLIP producers isolated from mushroom, maize rhizosphere or water.