Characterization of an antibiotic produced by a strain of Pseudomonas fluorescens inhibitory to Gaeumannomyces graminis var. tritici and Pythium spp

Abstract

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...

Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici

“…Samples of cultures were extracted twice with equal volumes of benzene. Absorbance of the pooled extracts was determined spectrophotometrically at 349.5 nm, and readings were converted to micrograms of phenazine-1-carboxylate from a standard curve prepared with the purified antibiotic (17). Antibiotic concentrations were expressed relative to the total protein of the producing cultures, assayed by the Bradford method (5).…”

“…An antibiotic isolated from strain 2-79, phenazine-1-carboxylate, is active in vitro against G. graminis var. tritici and several other wheat root pathogens at concentrations as low as 1 ,ug/ml (6,17). The purpose of this study was to determine the importance of the phenazine antibiotic in suppression of G. graminis var.…”

6-Hydroxymethyl-1-phenazine-carboxamide and 1,6-phenazinedimethanol from a marine bacterium, Brevibacterium sp. KMD 003, associated with marine purple vase sponge

“…1-Phenazinemethanol possesses antifungal activity against Gaeumannomyces graminis var. tritici and Pythium spp., 25 and PCN displayed antimicrobial activity especially against plant pathogenic Gram-positive bacteria and fungi. 23 PCN is synthesized in Pseudomonas aeruginosa by the conversion of PCA in the pyocyanin biosynthetic pathway, which consists of two core loci responsible for the synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine and PCN.…”

Structure and intramolecular hydrogen bonding of 1-phenazinecarboxylic acid

“…shown evidences in favor of both a dimeric (Gurusiddaiah, Weller, Sarkar & Cook, 1986) and a monomeric (Brisbane, Janik, Tate & Warren, 1987) structure. The mass spectrum of our microbially produced material displayed a dimer peak in the gas phase, but vapor-pressure osmometry showed the material to be monomeric in solution (CH2C12).…”