Overproduction of an inducible extracellular serine protease improves biological control of Pythium ultimum by Stenotrophomonas maltophilia strain W81

Stenotrophomonas maltophilia W81 can protect sugar beet against Pythium-med iated damping-off disease through the production of an extracellular protea se, Here, the proteolytic enzyme of W81 was purified by anion-exchangle chr omatography and characterized as a serine protease, The purified enzyme was fungicidal against Pythium ultimum in vitro, Its synthesis was inducible b y casein in W81, and mutagenesis of this strain using the luciferase (luxAB ) reporter transposon Tn5-764cd resulted in the isolation of two mutant der ivatives (W81M3 and W81M4) capable of producing significantly increased lev els of extracellular protease in the presence of casein, Strain W81M4 also exhibited increased chitinolytic activity. The luxAB fusions in strains W81 M3 and W81M4 were highly expressed in the absence of casein but not in its presence, suggesting that the corresponding loci were involved in downregul ating extracellular protease production. Extracellular protease production in the W81 wild-type strain and protease overproduction in mutants W81M3 an d W81M4 were also induced in the presence of the autoclaved fungal mycelium , In soil microcosms naturally infested by Pythium spp., inoculation of sug ar beet seeds with W81M3 or W81M4 resulted in improved biocontrol of Pythiu m-mediated damping-off disease compared with W81, and the level of protecti on achieved was equivalent to that conferred by chemical fungicides, The wi ld-type W81 and its mutant derivatives did not differ in rhizosphere coloni zation. Therefore, the improved biocontrol ability of W81M3 and W81M4 resul ted from their capacity to overproduce extracellular serine protease.