Genetic analysis of the antifungal activity of a soilborne Pseudomonas aureofaciens strain

Abstract

Pseudomonas aureofaciens Q2-87 produces the antibiotic 2,4-diacetophloroglucinol (Phl), which inhibits Gaeumannomyces graminis var. tritici and other fungi in vitro. Strain Q2-87 also provides biological control of take-all, a root disease of wheat caused by this fungus. To assess the role of Phl in the antifungal activity of strain Q2-87, a genetic analysis of antibiotic production was conducted. Two mutants of Q2-87 with altered antifungal activity were isolated by site-directed mutagenesis with Tn5. One mutant, Q2-87::Tn5-1, did not inhibit G. graminis var. tritici in vitro and did not produce Phl. Two cosmids were isolated from a genomic library of the wild-type strain by probing with the mutant genomic fragment. Antifungal activity and Phl production were coordinately restored in Q2-87::Tn5-1 by complementation with either cosmid. Mobilization of one of these cosmids into two heterologous Pseudomonas strains conferred the ability to synthesize Phl and increased their activity against G. graminis var. tritici, Pythium ultimum, and Rhizoctonia solani in vitro. Subcloning and deletion analysis of these cosmids identified a 4.8-kb region which was necessary for Phl synthesis and antifungal activity.