1887

Microbiology Society logo

Volume 146, Issue 8

Overproduction of an inducible extracellular serine protease improves biological control of by strain W81 Free

Abstract

W81 can protect sugar beet against -mediated damping-off disease through the production of an extracellular protease. Here, the proteolytic enzyme of W81 was purified by anion-exchange chromatography and characterized as a serine protease. The purified enzyme was fungicidal against . Its synthesis was inducible by casein in W81, and mutagenesis of this strain using the luciferase () reporter transposon Tn-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 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 down-regulating 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 spp., inoculation of sugar beet seeds with W81M3 or W81M4 resulted in improved biocontrol of -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.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): AEBSF, 4-(2-aminoethyl)benzenesulphonyl fluoride , bacterial inoculants , Cm, chloramphenicol , Km, kanamycin , overproduction , phytopathogenic fungi , plant protection , Rif, rifampicin , serine protease , Sm, streptomycin , Tc, tetracycline and TNBS, 2,4,6-trinitrobenzenesulphonic acid
© Microbiology Society
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-8-2069
2000-08-01
2024-04-18
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/8/1462069a.html?itemId=/content/journal/micro/10.1099/00221287-146-8-2069&mimeType=html&fmt=ahah

References

  1. Agrios G. N. 1997 Plant Pathology, 4th edn. San Diego, CA: Academic Press;
     [Google Scholar]
  2. Bangera M. G., Thomashow L. S. 1999; Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2-87. J Bacteriol 181:3155–3163
     [Google Scholar]
  3. Becker J. O., Cook R. J. 1988; Role of siderophores in suppression of Pythium species and production of increased growth response of wheat by fluorescent pseudomonads. Phytopathology 78:778–782 [CrossRef]
     [Google Scholar]
  4. Becker J. O., Schwinn F. J. 1993; Control of soil-borne pathogens with living bacteria and fungi: status and outlook. Pestic Sci 37:355–363 [CrossRef]
     [Google Scholar]
  5. Berg G., Marten P., Ballin G. 1996; in the rhizosphere of oilseed rape: occurrence, characterisation and interaction with phytopathogenic fungi. Microbiol Res 151:19–27 [CrossRef]
     [Google Scholar]
  6. Berg G., Marten P., Bahl H. 1998; Population dynamics of bacteria including antifungal species in the rhizosphere of oilseed rape during its life cycle. Arch Phytopathol Plant Protect 31:215–224 [CrossRef]
     [Google Scholar]
  7. Boller T., Mauch F. 1988; Colorimetric assay for chitinase. Methods Enzymol 161:430–435
     [Google Scholar]
  8. Bollet C., Davin R. A., De Micco P. 1995; A simple method for selective isolation of Stenotrophomonas maltophilia from environmental samples. Appl Environ Microbiol 61:1653–1654
     [Google Scholar]
  9. Boonchan S., Britz M. L., Stanley G. A. 1998; Surfactant-enhanced biodegradation of high molecular weight polycyclic aromatic hydrocarbons by Stenotrophomonas maltophilia. Biotechnol Bioeng 59:482–494 [CrossRef]
     [Google Scholar]
  10. Boyer H. W., Roulland-Dussoix D. 1969; A complementation analysis of restriction and modification in Escherichia coli. J Mol Biol 41:459–472 [CrossRef]
     [Google Scholar]
  11. Carroll H., Moënne-Loccoz Y., Dowling D. N., O’Gara F. 1995; Mutational disruption of the biosynthesis genes coding for the antifungal metabolite 2,4-diacetylphloroglucinol does not influence the ecological fitness of Pseudomonas fluorescens F113 in the rhizosphere of sugarbeets. Appl Environ Microbiol 61:3002–3007
     [Google Scholar]
  12. Cook R. J. 1993; Making greater use of introduced microorganisms for biological control of plant pathogens. Annu Rev Phytopathology 31:53–80 [CrossRef]
     [Google Scholar]
  13. Cook R. J., Thomashow L. S., Weller D. M., Fujimoto D., Mazzola M., Bangera G., Kim D. S. 1995; Molecular mechanisms of defence by rhizobacteria against root disease. Proc Natl Acad Sci U S A 92:4197–4201 [CrossRef]
     [Google Scholar]
  14. Cronin D., Moënne-Loccoz Y., Dunne C., O’Gara F. 1997; Inhibition of egg hatch of the potato cyst nematode Globodera rostochiensis by chitinase-producing bacteria. Eur J Plant Pathol 103:433–440 [CrossRef]
     [Google Scholar]
  15. Delany I., Sheehan M. M., Fenton A., Bardin S., Aarons S., O’Gara F. 2000; Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of phlF as a transcriptional repressor. Microbiology 146:537–543
     [Google Scholar]
  16. Dietrich S. M. C. 1973; Carbohydrates from the hyphal walls of some oomycetes. Biochim Biophys Acta 313:95–98 [CrossRef]
     [Google Scholar]
  17. Ditta G., Stanfield S., Corbin D., Helinski D. R. 1980; Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A 77:7347–7351 [CrossRef]
     [Google Scholar]
  18. Dowling D. N., O’Gara F. 1994; Metabolites of Pseudomonas involved in the biocontrol of plant disease. Trends Biotechnol 12:133–141 [CrossRef]
     [Google Scholar]
  19. Dowling D. N., Sexton R., Fenton A., Delany I., Fedi S., McHugh B., Callanan M., Moënne-Loccoz Y., O’Gara F. 1996; Iron regulation in plant-associated Pseudomonas fluorescens M114: implications for biological control. In Molecular Biology of Pseudomonads pp. 502–511Edited by Nakazawa T., Furukawa K., Haas D., Silver S. Washington, DC: American Society for Microbiology;
     [Google Scholar]
  20. Dunne C., Delany I., Fenton A., Lohrke S., Moënne-Loccoz Y., O’Gara F. 1996; The biotechnology and application of Pseudomonas inoculants for the biocontrol of phytopathogens. In Biology of Plant–Microbe Interactions pp. 441–448Edited by Stacey G., Mullin B., Gresshoff P. M. St Paul, MI: International Society for Molecular Plant–Microbe Interactions;
     [Google Scholar]
  21. Dunne C., Delany I., Fenton A., O’Gara F. 1997a; Mechanisms involved in biocontrol by microbial inoculants. Agronomie 16:721–729
     [Google Scholar]
  22. Dunne C., Crowley J. J., Moënne-Loccoz Y., Dowling D. N., de Bruijn F. J., O’Gara F. 1997b; Biological control of Pythium ultimum by Stenotrophomonas maltophilia W81 is mediated by an extracellular proteolytic activity. Microbiology 143:3921–3931 [CrossRef]
     [Google Scholar]
  23. Dunne C., Moënne-Loccoz Y., McCarthy J., Higgins P., Powell J., Dowling D. N., O’Gara F. 1998; Combining proteolytic and phloroglucinol-producing bacteria for improved biocontrol of Pythium-mediated damping-off of sugar beet. Plant Pathol 47:299–307 [CrossRef]
     [Google Scholar]
  24. Fedi S., Tola E., Moënne-Loccoz Y., Dowling D. N., Smith L. M., O’Gara F. 1997; Evidence for signalling between the phytopathogenic fungus Pythium ultimum and Pseudomonas fluorescens F113: P. ultimum represses the expression of genes in P. fluorescens F113, resulting in altered ecological fitness. Appl Environ Microbiol 63:4261–4266
     [Google Scholar]
  25. Fenton A. M., Stephens P. M., Crowley J., O’Callaghan M., O’Gara F. 1992; Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain. Appl Environ Microbiol 58:3873–3878
     [Google Scholar]
  26. Flores A., Chet I., Herrera-Estrella A. 1997; Improved biocontrol activity of Trichoderma harzianum by overexpression of the proteinase-encoding gene prb1. Curr Genet 31:30–37 [CrossRef]
     [Google Scholar]
  27. Geremia R. A., Goldman G. H., Jacobs D., Ardiles W., Vila S. B., Van Montagu M., Herrera-Estrella A. 1993; Molecular characterisation of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum. Mol Microbiol 8:603–613 [CrossRef]
     [Google Scholar]
  28. Giesler L. J., Yuen G. Y. 1998; Evaluation of Stenotrophomonas maltophilia strain C3 for biocontrol of brown patch disease. Crop Protect 17:509–513 [CrossRef]
     [Google Scholar]
  29. Handelsman J., Stabb E. V. 1996; Biocontrol of soilborne plant pathogens. Plant Cell 8:1855–1869 [CrossRef]
     [Google Scholar]
  30. Haran S., Schickler H., Chet I. 1996; Molecular mechanisms of lytic enzymes involved in the biocontrol activity of Trichoderma harzianum. Microbiology 142:2321–2331 [CrossRef]
     [Google Scholar]
  31. Jacobi M., Winkelmann G., Kaiser D., Kempter C., Jung G., Berg G., Bahl H. 1996; Maltophilin: a new antifungal compound produced by Stenotrophomonas maltophilia R3089. J Antibiot 11:1101–1104
     [Google Scholar]
  32. Jeffers S. N., Martin S. B. 1986; Comparison of two media selective for Phytophthora and Pythium spp. Plant Dis 70:1038–1043 [CrossRef]
     [Google Scholar]
  33. Keel C., Défago G. 1997; Interactions between beneficial soil bacteria and root pathogens: mechanisms and ecological impact. In Multitrophic Interactions in Terrestrial Systems pp. 27–46Edited by Gange A. C., Brown V. K. London: Blackwell Scientific Publications;
     [Google Scholar]
  34. Kragelund L., Christoffersen B., Nybroe O., de Bruijn F. J. 1995; Isolation of lux reporter gene fusions in Pseudomonas fluorescens DF57 inducible by nitrogen or phosphorus starvation. FEMS Microbiol Ecol 17:95–106 [CrossRef]
     [Google Scholar]
  35. Lorito M., Peterbauer C., Hayes C. K., Harman G. E. 1994; Synergistic interaction between fungal cell wall degrading enzymes and different antifungal compounds enhances inhibition of spore germination. Microbiology 140:623–629 [CrossRef]
     [Google Scholar]
  36. Lugtenberg B. J. J., Dekkers L. C. 1999; What makes Pseudomonas bacteria rhizosphere competent?. Environ Microbiol 1:9–15 [CrossRef]
     [Google Scholar]
  37. McKellar R. C. 1981; Development of off-flavour in ultra-high temperature and pasteurized milk is a function of proteolysis. J Dairy Sci 64:2138–2145 [CrossRef]
     [Google Scholar]
  38. Milcamps A., de Bruijn F. J. 1999; Identification of a novel nutrient-deprivation-induced Sinorhizobium meliloti gene (hmgA) involved in the degradation of tyrosine. Microbiology 145:935–947 [CrossRef]
     [Google Scholar]
  39. Milcamps A., Ragatz D. M., Lim P. O., Berger K. A., de Bruijn F. J. 1998; Isolation of carbon- and nitrogen-deprivation-induced loci of Sinorhizobium meliloti 1021 by Tn5–luxAB mutagenesis. Microbiology 144:3205–3218 [CrossRef]
     [Google Scholar]
  40. Mitchell R., Hurwitz E. 1965; Suppression of Pythium debaryanum by lytic rhizosphere bacteria. Phytopathology 55:156–158
     [Google Scholar]
  41. Moënne-Loccoz Y., Powell J., Higgins P., Britton J., O’Gara F. 1998; Effect of the biocontrol agent Pseudomonas fluorescens F113 released as sugarbeet inoculant on the nutrient contents of soil and foliage of a red clover rotation crop. Biol Fertil Soils 27:380–385 [CrossRef]
     [Google Scholar]
  42. Moënne-Loccoz Y., Naughton M., Higgins P., Powell J., O’Connor B., O’Gara F. 1999; Effect of inoculum preparation and formulation on survival and biocontrol efficacy of Pseudomonas fluorescens F113. J Appl Microbiol 86:108–116 [CrossRef]
     [Google Scholar]
  43. Nelson E. B., Harman G. E., Nash G. T. 1988; Enhancement of Trichoderma induced biological control of Pythium seed rot and pre-emergence damping-off of peas. Soil Biol Biochem 20:145–150 [CrossRef]
     [Google Scholar]
  44. Nielsen M. N., Sorensen J., Fels J., Pedersen H. C. 1998; Secondary metabolite- and endochitinase-dependent antagonism towards plant-pathogenic microfungi of Pseudomonas fluorescens isolates from sugar beet rhizosphere. Appl Environ Microbiol 64:3563–3569
     [Google Scholar]
  45. O’Sullivan D. J., O’Gara F. 1992; Traits of fluorescent Pseudomonas spp. involved in suppression of plant root pathogens. Microbiol Rev 56:662–676
     [Google Scholar]
  46. Palleroni N. J., Bradbury J. F. 1993; a new bacterial genus for Xanthomonas maltophilia (Hugh 1980) Swings et al., 1983. Int J Syst Bacteriol 43:606–609 [CrossRef]
     [Google Scholar]
  47. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  48. Scher F. M., Baker R. 1982; Effect of Pseudomonas putida and a synthetic iron chelator on induction of soil suppressiveness to Fusarium wilt pathogens. Phytopathology 72:1567–1573 [CrossRef]
     [Google Scholar]
  49. Schnider U., Keel C., Voisard C., Défago G., Haas D. 1995; Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin. Appl Environ Microbiol 61:3856–3864
     [Google Scholar]
  50. Stasz T. E., Harman G. E., Marx G. A. 1980; Time and site of infection of resistant and susceptible germinating pea seeds by Pythium ultimum. Phytopathology 70:730–733 [CrossRef]
     [Google Scholar]
  51. Thrane C., Tronsmo A., Funck Jensen D. 1997; Endo-1,3-β-glucanase and cellulase from Trichoderma harzianum: purification and partial characterization, induction of and biological activity against plant pathogenic Pythium spp. Eur J Plant Pathol 103:331–344 [CrossRef]
     [Google Scholar]
  52. Twinning S. 1984; Fluorescein isothiocyanate-labelled casein assay for proteolytic enzymes. Anal Biochem 143:30–34 [CrossRef]
     [Google Scholar]
  53. Van Wees S. C. M., Pieterse C. M. J., Trijssenaar A., Van ‘t Westende Y. A. M., Hartog F., Van Loon L. C. 1997; Differential induction of systemic resistance in Arabidopsis by biocontrol bacteria. Mol Plant–Microbe Interact 10:716–724 [CrossRef]
     [Google Scholar]
  54. Wolk C. P., Cai Y., Panoff J. M. 1991; Use of a transposon with luciferase as a reporter to identify environmentally responsive genes in a cyanobacterium. Proc Natl Acad Sci U S A 88:5355–5359 [CrossRef]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-8-2069
Loading
Overproduction of an inducible extracellular serine protease improves biological control of Pythium ultimum by Stenotrophomonas maltophilia strain W81
Microbiology 146, 2069 (2000); https://doi.org/10.1099/00221287-146-8-2069
/content/journal/micro/10.1099/00221287-146-8-2069
/content/journal/micro/10.1099/00221287-146-8-2069
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error