The production, isolation, and characterization of an antibiotic substance from cultures of Pseudomonas fluorescens 2-79 (NRRL B-15132) is described. P. fluorescens 2-79 originally was isolated from the roots of wheat and is suppressive to the wheat root disease take-all caused by Gaeumannomyces graminis var. tritici. The antibiotic was isolated from potato glucose broth cultures of strain 2-79 by solvent extraction. It was purified by silica gel column chromatography and was a greenish yellow, needle-shaped crystal with a melting point of 242°C (decomposition). It was soluble in methylene chloride, chloroform, acetone, 2 N sodium hydroxide, and 2 N hydrochloric acid and was insoluble in water, methanol, ethyl acetate, tetrahydrofuran, diethyl ether, carbon tetrachloride, hexane, and petroleum ether. On the basis of UV, infrared, 'H-nuclear magnetic resonance, "3C-nuclear magnetic resonance, mass spectral analysis, and elemental analysis, the structure of the antibiotic is proposed to be a dimer of phenazine carboxylic acid. Lithium aluminum hydride reduction of the antibiotic yielded hydroxymethyl phenazine as a major product which retained most of the biological characteristics of the parent molecule. There were no toxic symptoms when mice received this antibiotic by oral doses up to 464 mg/kg. The antibiotic showed excellent activity against several species of fungi, including the wheat pathogens Gaeumannomyces graminis var. tritici, Rhizoctonia solani, and Pythium aristosporum; and it may have a role in suppression of take-all in vivo by strain 2-79.Pseudomonasfluorescens 2-79 (NRRL B-15132) (12) originally was isolated from the roots of wheat growing in soil from a field where the wheat root disease take-all caused by Gaeumannomyces graminis var. tritici had spontaneously declined (9). When cells of this bacterium are applied to wheat seed, the bacteria colonize the roots and suppress the pathogen on the roots (12). Strain 2-79 is highly inhibitory to G. graminis var. tritici in vitro on several media (12), and antibiotic and siderophore production probably are important mechanisms of suppression of take-all in vivo (D. M. Weller, W. J. Howie, and R. J. Cook, Phytopathology 75:1301, 1985. In this report we describe an antimicrobial compound produced by P. fluorescens 2-79 that may be involved in suppression of take-all in vivo. This description deals with the production, purification, and characterization of the antibiotic.
MATERIALS AND METHODSMaintenance of stock culture of P. fluorescens 2-79. Strain 2-79 can be grown on most common media including King medium B (7), potato glucose agar, nutrient agar (Difco Laboratories, Detroit, Mich.), and nutrient agar containing 0.5% yeast extract (Difco). The strain can be maintained in a lyophilized state in sealed ampoules at 5°C and in sterile soil in screw-cap tubes at 8°C for a prolonged period with no loss in antibiotic-producing ability.Cultural conditions for production of the antibiotic. For the production of the antibiotic, strain 2-79 was grown in potat...