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 in lost dollars in lost yields. It can not be controlled consistently using chemicals or cultural practices. Isolates of naturally occurring rhizobacteria have shown potential as biocontrol agents for downy brome. Centrifuged supernatant from aerobic shake cultures of Pseudomonas fluorescens strain D7 stressed germination of seeds and reduced root and shoot growth of downy brome in agar diffusion assays. The present article 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 purification of microbial products. Two unusual techniques for isolation and partial purification of the active compounds from supernatant of strain D7 were developed. With one technique, supernatant was concentrated at 30 to 35 C under vacuum to 10% of the initial volume. Subsequently, the concentrate was chromatographed on sephadex gels to separate the active fraction. With the second technique cell-free supernatant was 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 precipitation and crystallization of the active fraction. The active fraction 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 Gaeumannomyces graminis var. tritici. Chemical analysis of the active fraction 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 of activity against downy brome.