Cavity disease in white button mushrooms is caused by Burkholderia gladioli pv. agaricicola. We describe the isolation and characterization of six mutants of the strain BG164R that no longer cause this disease on mushrooms. The mutations were mapped to genes of the general secretory pathway (GSP). This is the first report of the association of the type II secretion pathway with a disease in mushrooms. Phenotypes of the six avirulent mutants were the following: an inability to degrade mushroom tissue, a highly reduced capacity to secrete chitinase and protease, and a reduced number of flagella. Using these mutants, we also made the novel observation that the factors causing mushroom tissue degradation, thereby leading to the expression of cavity disease, can be separated from mycelium inhibition because avirulent mutants continued to inhibit the growth of actively growing mushroom mycelia. The GSP locus of B. gladioli was subsequently cloned and mapped and compared to the same locus in closely related species, establishing that the genetic organization of the gsp operon of B. gladioli pv. agaricicola is consistent with that of other species of the genus. We also identify the most common indigenous bacterial population present in the mushroom fruit bodies from a New Zealand farm, one of which, Ewingella americana, was found to be an apparent antagonist of B. gladioli pv. agaricicola. While other investigators have reported enhanced disease symptoms due to interactions between endogenous and disease-causing bacteria in other mushroom diseases, to the best of our knowledge this is the first report of an antagonistic effect. Copyright © 2006, American Society for Microbiology. All Rights Reserved.