BackgroundMicroorganisms that activate plant immune responses are useful for application as biocontrol agents in agriculture to minimize crop losses. The present study was conducted to identify and characterize plant immunity–activating microorganisms inBrassicaceaeplants.ResultsA total of 25 bacterial strains were isolated from the interior of aBrassicaceaeplant,Raphanus sativusvar.hortensis. Ten different genera of bacteria were identified:Pseudomonas,Leclercia,Enterobacter,Xanthomonas,Rhizobium,Agrobacterium,Pantoea,Rhodococcus,Microbacterium, andPlantibacter. The isolated strains were analyzed using a method to detect plant immunity–activating microorganisms that involves incubation of the microorganism with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses. In this method, cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells serves as a marker of immune activation. Among the 25 strains examined, 6 strains markedly enhanced cryptogein-induced ROS production in BY-2 cells. These 6 strains colonized the interior ofArabidopsisplants, andPseudomonassp. RS3R-1 andRhodococcussp. RS1R-6 selectively enhanced plant resistance to the bacterial pathogensPseudomonas syringaepv.tomatoDC3000 andPectobacterium carotovorumsubsp.carotovorumNBRC 14082, respectively. In addition,Pseudomonassp. RS1P-1 effectively enhanced resistance to both pathogens. We also comprehensively investigated the localization (i.e., cellular or extracellular) of the plant immunity–activating components produced by the bacteria derived fromR. sativusvar.hortensisand the components produced by previously isolated bacteria derived from anotherBrassicaceaeplant species,Brassica rapavar.perviridis. Most gram-negative strains enhanced cryptogein-induced ROS production in BY-2 cells via the presence of cells themselves rather than via extracellular components, whereas many gram-positive strains enhanced ROS production via extracellular components. Comparative genomic analyses supported the hypothesis that the structure of lipopolysaccharides in the outer cell envelope plays an important role in the ROS-enhancing activity of gram-negativePseudomonasstrains.ConclusionsThe assay method described here based on elicitor-induced ROS production in cultured plant cells enabled the discovery of novel plant immunity–activating bacteria fromR. sativusvar.hortensis. The results in this study also suggest that components involved in the ROS-enhancing activity of the bacteria may differ depending largely on genus and species.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12866-023-02920-y.