Aims:  The main focus of this study was to gain an overall view of Pseudomonas putida 06909 genes involved in the Pseudomonas–Phytophthora interaction as a biological control mechanism, and to understand the roles of these genes. Methods and Results:  Sixteen Ps. putida genes with increased expression on Phytophthora mycelial surfaces were identified using in vivo expression technology (IVET) screening. Sequence analysis of these Phytophthora mycelium‐induced (pmi) genes revealed that many of them display similarity to genes known or predicted to be involved in carbohydrate catabolism, energy metabolism, amino acid/nucleotide metabolism, and membrane transport processes. Disruption of three pmi genes encoding succinate semialdehyde dehydrogenase, a dicarboxylic acid transporter, and glyceraldehyde‐3‐phosphate dehydrogenase showed significant phenotypic differences involved in the colonization processes, including motility, biofilm formation on abiotic surfaces, colony morphology, and competitive colonization of fungal mycelia. All three of these pmi genes were induced by glycogen and other substances, such as organic acids and amino acids utilized by Ps. putida. Conclusions:  The IVET screening and mutant characterization can be used to identify bacterial genes that are induced on the mycelial surface and provide insight into the possible mechanisms of mycelial colonization by this bacterium. Significance and Impact of the Study:  The IVET screening through a bacterial genome library might be a huge task. However, because the genes involved in direct interaction with Phytophthora and in bacterial adaptation can be identified, the IVET system will be a valuable tool in studying biocontrol bacteria at the molecular and ecological levels.

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