Biological control of Pythium ultimum by Stenotrophomonas maltophilia W81 is mediated by an extracellular proteolytic activity

<i>Stenotrophomonas maltophilia strain W81</i>, isolated from the rhizosphere of field-grown sugar beet, produced the extracellular enzymes chitinase and protease and inhibited the growth of the phytopathogenic fungus <i>Pythium ultimum in vitro.</i> The role of these lytic enzymes in the interaction between W81 and <i>P. ultimum</i> was investigated using Tn<i>5</i> insertion mutants of W81 incapable of producing extracellular protease (W81M1), extracellular chitinase (W81M2) or the two enzymes (W81A1). Lytic enzyme activity was restored in W81A1 following introduction of a 15 kb cosmid-borne fragment of W81 genomic DNA. Incubation of <i>P. ultimum</i> in the presence of commercial purified protease or cell-free supernatants from cultures of wild-type W81, the chitinase-negative mutant W81M2 or the complemented derivative W81A1 (pCU800) resulted in hyphal lysis and loss of subsequent fungal growth ability once re-inoculated onto fresh plates. In contrast, commercial purified chitinase or cell-free supernatants from cultures of the protease-negative mutant WS1M1 or the chitinase- and protease-negative mutant W81A1 had no effect on integrity of the essentially chitin-free <i>Pythium mycelium,</i> and did not prevent subsequent growth of the fungus. In soil microcosms containing soil naturally infested by <i>Pythium</i> spp., strains W81, W81M2 and W81A1(pCU800) reduced the ability of <i>Pythium</i> spp. to colonize the seeds of sugar beet and improved plant emergence compared with the untreated control, whereas W81A1 and W21M1 failed to protect sugar beet from damping-off. Wild-type W81 and its mutant derivatives colonized the rhizosphere of sugar beet to similar extents, it was concluded that the ability of <i>S. maltophilia</i> W81 to protect sugar beet from Pythium -mediated damping-off was due to the production of an extracellular protease.