Submitted to: Applied and Environmental Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 3/20/2004
Publication Date: 7/20/2004
Citation: Huang, Z., Bonsall, R.F., Mavrodi, D.V., Weller, D.M., Thomashow, L.S. 2004. Transformation of pseudomonas fluorescens with genes for biosynthesis of phenazine-1-carboxylic acid improves biocontrol activity and in situ antibiotic production. FEMS Microbiol. Ecol. 49:243-251. Interpretive Summary: Root diseases caused by soilborne pathogens are major yield-limiting factors in the production of food, fiber and ornamental crops. As agriculture becomes more sustainable and less dependent on chemical fungicides, there is increasing need for ecologically sound methods to control these diseases. Biological control, which exploits the natural antifungal activity of certain root-colonizing bacteria, is one such approach. The most effective biocontrol bacteria against take-all, Rhizoctonia root rot and Pythium root rot, important root diseases of wheat grown in the Pacific Northwest, act by producing antifungal compounds, either phenazine-1-carboxylic acid (PCA) or 2,4-diacetylphloroglucinol (DAPG). In this study the PCA biosynthetic genes from one strain were cloned into another strain that produces DAPG in an attempt to broaden the range of diseases controlled. The resulting genetically modified strains produced increased quantities of both compounds, retained their ability to colonize the roots of wheat, and were more protective against root pathogens when applied to seed at lower doses than either of the parental strains. These results suggest that combining known biocontrol genes into a single organism can help to overcome barriers related to strain performance and inoculum preparation, formulation and cost that until now have limited more widespread use of these bacterial biocontrol agents.
Technical Abstract: Wheat growing worldwide can be damaged by one or more of the root diseases take-all, Pythium root rot, and Rhizoctonia root rot, which often occur in the Pacific Northwest as a complex in the same field. Strains of fluorescent Pseudomonas spp. can suppress these diseases, but most strains fail to perform consistently and no single strain is effective against all three diseases. The goal of this study was to extend the range of diseases controlled by P. fluorescens Q8r1-96, an aggressive root colonizer that produces 2,4-diacetylphloroglucinol (2,4-DAPG) and consistently suppresses take-all of wheat. A seven-gene operon for the synthesis of phenazine-1-carboxylic acid (PCA) was cloned distal to a constitutive tac promoter and introduced into Q8r1-96 via a mini-Tn5 vector that enabled stable insertion of the biosynthetic gene cluster at random sites in the genome. The recombinant strains produced more 2,4-DAPG than did Q8r1-96 and more PCA than strain 2-79, the source of the phenazine operon, both in vitro and in the rhizosphere of wheat. The recombinant strains grew more slowly than Q8r1-96 in vitro but maintained population sizes comparable to those of Q8r1-96 over a seven-week period in the rhizosphere of wheat. PCA-producing derivatives were no more suppressive of take-all and only moderately more effective against Pythium root rot than was Q8r1-96, but suppressed Rhizoctonia root rot at a dose of only 102 CFU per seed, one to two orders of magnitude lower than the dose of Q8r1-96 required for comparable disease control. Recombinant strains effective at low doses against multiple target root pathogens may aid in overcoming barriers related to inoculum preparation, formulation and cost that now limit more widespread use of Pseudomonas biocontrol agents.