Pseudomonas chlororaphis strain 30-84 is a plant-beneficial bacterium that is able to control take-all disease of wheat caused by the fungal pathogen Gaeumannomyces graminis var. tritici. The production of phenazines (PZs) by strain 30-84 is the primary mechanism of pathogen inhibition and contributes to the persistence of strain 30-84 in the rhizosphere. PZ production is regulated in part by the PhzR/PhzI quorum-sensing (QS) system. Previous flow cell analyses demonstrated that QS and PZs are involved in biofilm formation in P. chlororaphis (V. S. R. K. Maddula, Z. Zhang, E. A. Pierson, and L. S. Pierson III, Microb. Ecol. 52:289-301, 2006). P. chlororaphis produces mainly two PZs, phenazine-1-carboxylic acid (PCA) and 2-hydroxy-PCA (2-OH-PCA). In the present study, we examined the effect of altering the ratio of PZs produced by P. chlororaphis on biofilm formation and pathogen inhibition. As part of this study, we generated derivatives of strain 30-84 that produced only PCA or overproduced 2-OH-PCA. Using flow cell assays, we found that these PZ-altered derivatives of strain 30-84 differed from the wild type in initial attachment, mature biofilm architecture, and dispersal from biofilms. For example, increased 2-OH-PCA production promoted initial attachment and altered the three-dimensional structure of the mature biofilm relative to the wild type. Additionally, both alterations promoted thicker biofilm development and lowered dispersal rates compared to the wild type. The PZ-altered derivatives of strain 30-84 also differed in their ability to inhibit the fungal pathogen G. graminis var. tritici. Loss of 2-OH-PCA resulted in a significant reduction in the inhibition of G. graminis var. tritici. Our findings suggest that alterations in the ratios of antibiotic secondary metabolites synthesized by an organism may have complex and wide-ranging effects on its biology.Bacteria in nature exist within surface-attached communities, termed biofilms, and interact cooperatively and competitively with other members of the microbial community (reviewed in references 13, 14, 22, and 33). Biofilm communities are "microniches" that differ dramatically from surrounding conditions (33), confer resistance to deleterious agents such as antibiotics and detergents (15), and enable cells to coordinately carry out functions not typically undertaken outside of the biofilm (33). Bacteria associated with plants may form biofilms on or within plant tissues (reviewed in references 13, 33, and 41). The associations between plants and microorganisms encompass beneficial, neutral, detrimental, and pathogenic associations. The effects of deleterious or pathogenic microorganisms on plants can be ameliorated by the presence of bacteria that reduce the impact of the pathogen on the plant. Many of these beneficial bacteria (often termed biological control bacteria) produce secondary metabolites that interfere with the pathogen's disease potential by either direct inhibition of pathogen growth, induction of plant defenses, or enhancement of plant...