ISOLATION AND CHARACTERIZATION OF METABOLITES FROM PSEUDOMONAS-FLUORESCENS-D7 FOR CONTROL OF DOWNY BROME (BROMUS-TECTORUM)

Downy brome is a grassy weed that infests millions of acres of winter wheat in the Western United States and costs farmers millions of dolla rs in lost yields. It can not be controlled consistently using chemica ls or cultural practices. Isolates of naturally occurring rhizobacteri a have shown potential as biological control agents for downy brome. C entrifuged supernatant from aerobic shake cultures of Pseudomonas fluo rescens strain D7 suppressed germination of seeds and reduced root and shoot growth of downy brome in agar diffusion assays. The present art icle relates to the isolation and characterization of active compounds from strain D7. The active compounds were denatured and lost activity with commonly used techniques of extraction, concentration, and purif ication of microbial products. Two unusual techniques for isolation an d partial purification of the active compounds from supernatant of str ain D7 were developed. With one technique, supernatant was concentrate d at 30 to 35 C under vacuum to 10% of the initial volume. Subsequentl y, the concentrate was chromatographed on sephadex gels to separate th e active fraction. With the second technique, cell-free supernatant wa s frozen, with or without prior concentration, at -12 C for 12 to 24 h . The frozen supernatant was then thawed at 2 to 4 C, resulting in pre cipitation and crystallization of the active fraction. The active frac tion isolated with either method inhibited downy brome. Inhibition was complete at concentrations as low as 1 mg total dry matter L-1 agar. Also, the active fraction inhibited the plant pathogenic fungus Gaeuma nnomyces graminis var. tritici. Chemical analysis of the active fracti on complex revealed the presence of chromopeptides and other peptides, fatty acid esters, and a lipopolysaccharide matrix. Separation of any of the components from the complex resulted in nearly complete loss o f activity against downy brome.