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

Abstract

Stenotrophomonas maltophilia strain W81, isolated from the rhizosphere of field-grown sugar beet, produced the extracellular enzymes chitinase and protease and inhibited the growth of the phytopathogenic fungus Pythium ultimum in witro. The role of these lytic enzymes in the interaction between W81 and P. ultimum was investigated using T n 5 insertion mutants of W81 incapable of producing extracellular protease (W81 MI), extracellular chitinase (W81M2) or the two enzymes (W81A1). Lytic enzyme activity was restored in W81Al following introduction of a 15 kb cosmid-borne fragment of W81 genomic DNA. Incubation of P. ultimum in the presence of commercial purified protease or cell-free supernatants from cultures of wild-type W81, the chitinase-negative mutant W81 M2 or the complemented derivative W81Al(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 W81M1 or the chitinase-and protease-negative mutant W81A1 had no effect on integrity of the essentially chitin-free pvthium mycelium, and did not prevent subsequent growth of the fungus. In soil microcosms containing soil naturally infested by Pythium spp., strains W81, W81M2 and W81Al(pCU800) reduced the ability of PyWum spp. to colonize the seeds of sugar beet and improved plant emergence compared with the untreated control, whereas W81A1 and W81M1 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 5. maltophilia W81 to protect sugar beet from Pythium-mediated damping-off was due to the production of an extracellular protease.Keywords : biological control, Pythium ultimum, sugar beet, Stenotrophomonas maltophilia, protease INTRODUCTIONThe interactions between pathogenic or deleterious soil micro-organisms and the plant root take place in the rhizosphere, a complex environment in which those rhizosphere. Beneficial micro-organisms of interest for biocontrol of soil-borne pathogens and pests have been identified among fungi, actinomycetes and bacteria and they represent a promising alternative to the use of chemical pesticides for crop protection (Becker & Schwinn, 1993; Cook, 1993;Dunne et al., , 1997Cronin et al., 1997; Keel & Defago, 1997). In addition to their beneficial properties, a key feature of effective biocontrol agents is their ability to persist in soil and aggressively colonize the rhizosphere. Therefore, it has been proposed that isolation of biocontrol microorganisms indigenous to an environment similar to that in which they will be required to function may ensure greater efficacy of plant protection (Cook, 1993).Antibiosis is perhaps the most-studied mechanism of biocontrol in bacteria. Several secondary metabolites with antifungal properties have been identified, for example phenazines, pyrrol derivatives, hydrogen cyanide and phloroglucino...

Isolation and characterization of mutants of Pseudomonas maltophila PM-4 altered in chitinolytic activity and antagonistic activity against root rot pathogens of clusterbean (Cyamopsis tetragonoloba)

“…Role of protease and antibiotic like compound, xanthobactin from Stenotrophomonas sp. in biocontrol of sugarbeet have been proved 35,36 .…”

The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms

“…Production of extracellular lytic enzymes is quite common among antagonistic microorganisms (Adesina et al 2007), but it does not contribute to antagonism in all cases (Sharifi-Tehrani et al 1998;Dunne et al 1997). Extracellular lytic enzymes act in different ways: many of them can affect the cell wall of pathogens, and this is documented for cellulases, chitinases and proteases produced by various bacteria.…”

Chitinases from the Plant Disease Biocontrol Agent, Stenotrophomonas maltophilia C3

“…Besides strain C3, a number of S. maltophilia strains inhibit fungal growth or infection when applied to plants (2,5,6,(10)(11)(12)21), but there is no mechanism of antagonism that is common among these strains. Biological control by these strains has been attributed to induced resistance (6), antibiotics (21), and protease (5).…”

“…Biological control by these strains has been attributed to induced resistance (6), antibiotics (21), and protease (5). Chitinase production was expressed in most strains of S. maltophilia evaluated in vitro by O'Brien and Davis (22), but only 1 of 11 Verticillium-inhibitory strains tested by Berg et al (2) was chitinolytic, indicating that involvement of chitinase in fungal inhibition by S. maltophilia is strain specific.…”