<jats:title>ABSTRACT</jats:title> <jats:p> <jats:italic>Paenibacillus polymyxa</jats:italic> (formerly <jats:italic>Bacillus polymyxa</jats:italic> ) PKB1 has been identified as a potential agent for biocontrol of blackleg disease of canola, caused by the pathogenic fungus <jats:italic>Leptosphaeria maculans</jats:italic> . The factors presumed to contribute to disease suppression by strain PKB1 include the production of fusaricidin-type antifungal metabolites that appear around the onset of bacterial sporulation. The fusaricidins are a family of lipopeptide antibiotics consisting of a β-hydroxy fatty acid linked to a cyclic hexapeptide. Using a reverse genetic approach based on conserved motifs of nonribosomal peptide synthetases, a DNA fragment that appears to encode the first two modules of the putative fusaricidin synthetase ( <jats:italic>fusA</jats:italic> ) was isolated from PKB1. To confirm the involvement of <jats:italic>fusA</jats:italic> in production of fusaricidins, a modified PCR targeting mutagenesis protocol was developed to create a <jats:italic>fusA</jats:italic> mutation in PKB1. A DNA fragment internal to <jats:italic>fusA</jats:italic> was replaced by a gene disruption cassette containing two antibiotic resistance genes for independent selection of apramycin resistance in <jats:italic>Escherichia coli</jats:italic> and chloramphenicol resistance in <jats:italic>P. polymyxa</jats:italic> . Inclusion of an <jats:italic>oriT</jats:italic> site in the disruption cassette allowed efficient transfer of the inactivated <jats:italic>fusA</jats:italic> allele to <jats:italic>P. polymyxa</jats:italic> by intergeneric conjugation. Targeted disruption of <jats:italic>fusA</jats:italic> led to the complete loss of antifungal activity against <jats:italic>L. maculans</jats:italic> , suggesting that <jats:italic>fusA</jats:italic> plays an essential role in the nonribosomal synthesis of fusaricidins.