Stenotrophomonas maltophilia W81 can protect sugar beet against Pythium-mediated damping-off disease through the production of an extracellular protease, Here, the proteolytic enzyme of W81 was purified by anion-exchangle chromatography and characterized as a serine protease, The purified enzyme was fungicidal against Pythium ultimum in vitro, Its synthesis was inducible by casein in W81, and mutagenesis of this strain using the luciferase (luxAB) reporter transposon Tn5-764cd resulted in the isolation of two mutant derivatives (W81M3 and W81M4) capable of producing significantly increased levels of extracellular protease in the presence of casein, Strain W81M4 also exhibited increased chitinolytic activity. The luxAB fusions in strains W81M3 and W81M4 were highly expressed in the absence of casein but not in its presence, suggesting that the corresponding loci were involved in downregulating extracellular protease production. Extracellular protease production in the W81 wild-type strain and protease overproduction in mutants W81M3 and W81M4 were also induced in the presence of the autoclaved fungal mycelium, In soil microcosms naturally infested by Pythium spp., inoculation of sugar beet seeds with W81M3 or W81M4 resulted in improved biocontrol of Pythium-mediated damping-off disease compared with W81, and the level of protection achieved was equivalent to that conferred by chemical fungicides, The wild-type W81 and its mutant derivatives did not differ in rhizosphere colonization. Therefore, the improved biocontrol ability of W81M3 and W81M4 resulted from their capacity to overproduce extracellular serine protease.