1887

Abstract

Scanning electron micrographs of the nematode-egg-parasitic fungus infecting eggs of the root-knot nematode spp. suggested the involvement of lytic enzymes. When grown on a liquid mineral salts medium, supplemented with different substrates as the sole N-and C-source, the fungus produced an extracellular protease. Colloidal chitin, vitellin and intact eggs of the root-knot nematode induced proteolytic activity that was repressed by glucose. The protease was partially purified from the culture filtrate by affinity chromatography. It has a molecular mass of 33·5 kDa, a pH optimum of 10·3, a temperature optimum of 60°C and an isoelectric point above pH 10·2. The enzyme was completely inhibited by PMSF. The amino acid sequence, as derived from the nucleotide sequence of a cDNA clone, had high homology with several subtilisin-like serine proteases. It was shown that the purified enzyme degrades vitellin. The protease quantitatively bound to nematode eggs, and eggs incubated with the purified protease eventually floated. Incubation of the purified protease with nematode eggs significantly influenced their development as demonstrated by time-lapse microscopy. Immature eggs were highly vulnerable to protease treatments, whereas those containing a juvenile were more resistant. In addition, hatched larvae were not visibly affected by the protease. It can be concluded that the serine protease might play a role in penetration of the fungus through the egg-shell of nematodes.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-141-4-775
1995-04-01
2021-07-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/141/4/mic-141-4-775.html?itemId=/content/journal/micro/10.1099/13500872-141-4-775&mimeType=html&fmt=ahah

References

  1. den Belder E., Boekestein A., van Esch J. W. J., Thiel F. 1993; Low-temperature scanning electron microscopy in fungusnematode interaction. Scanning 15:37–42
    [Google Scholar]
  2. Bidochka M. J., Khachatourians G. G. 1988; Regulation of extracellular protease in the entomopathogenic fungus Beauveria bassiana . Exp Mycol 12:161–168
    [Google Scholar]
  3. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
    [Google Scholar]
  4. Cabanillas E., Barker K. R. 1989; Impact of Paecilomyces lilacinus inoculum level and application time on control of Meloidogyne incognita on tomato. J Nematol 21:115–120
    [Google Scholar]
  5. Cabanillas E., Barker K. R., Nelson L. A. 1989; Growth of isolates of Paecilomyces lilacinus and their efficacy in biocontrol of Meloidogyne incognita on tomato. J Nematol 21:164–172
    [Google Scholar]
  6. Dackman C., Chet I., Nordbring-Hertz B. 1989; Fungal parasitism of the cyst nematode Heterodera schachtii: infection and enzymatic activity. Microb Ecol 62:201–208
    [Google Scholar]
  7. Davidow L. S., O’Donnell M. M., Kaczmarek F. S., Pereira D. A., Zeeuw de J. R., Franke A. E. 1987; Cloning and sequencing of the alkaline extracellular protease gene of Yarrowia lipolytica . J Bacteriol 169:4621–4629
    [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. 1984; A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395
    [Google Scholar]
  9. Dunn M. T., Sayre R. M., Carrell A., Wergin W. P. 1982; Colonization of nematode eggs by Paecilomyces lilacinus (Thom) Samson as observed with scanning electron microscope. Scanning Electron Microsc 3:1351–1357
    [Google Scholar]
  10. Fitters P. F. L., den Belder E. 1993; A time-lapse technique to study the effect of fungal products on embryogenesis of nematode eggs. Meded Fac Landbouwwet Univ Gent 58/2b:751–756
    [Google Scholar]
  11. Galper S., Cohn E., Spiegel Y., Chet I. 1990; Nematicidal effect of collagen-amended soil and the influence of protease and collagenase. Rev Nematol 13:67–71
    [Google Scholar]
  12. Gaspard J. T., Jaffee B. A., Ferris H. 1990; Meloidogyne incognita survival in soil infested with Paecilomyces lilacinus and Verticillium chlamydosporium . J Nematol 22:176–181
    [Google Scholar]
  13. Genstat 5 Committee 1987 Genstat 5 Reference Manual Oxford: Clarendon Press;
    [Google Scholar]
  14. Geremia R. A., Goldman G. H., Jacobs D., Ardiles W., Vila S. B., van Montagu M., Herrera-Estrella A. 1993; Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum . Mol Microbiol 8:603–613
    [Google Scholar]
  15. Goettel M. S., St Leger R. J., Rizzo N. W., Staples R. C., Roberts D.W. 1989; Ultrastructural localization of a cuticledegrading protease produced by the entomopathogenic fungus Metarhizium anisopliae during penetration of host (Manduca sexta) cuticle. J Gen Microbiol 135:2233–2239
    [Google Scholar]
  16. Isogai T., Fukawa M., Kojo H., Kohsaka M., Aoki H., Imanaka H. 1991; Cloning and nucleotide sequences of the complementary and genomic DNAs for the alkaline protease from Acremonium chrysogenum . Agric Biol Chem 55:471–477
    [Google Scholar]
  17. Jany, K.-D., Lederer G., Mayer B. 1986; Amino acid sequence of proteinase K from the mold Tritirachium album Limber. FEBS Lett 199:139–144
    [Google Scholar]
  18. Jatala P., Kaltenbach R., Bocangel M., Devaux J., Campos R. 1980; Field application of Paecilomyces lilacinus for controlling Meloidogyne incognita on potatoes. J Nematol 12:226
    [Google Scholar]
  19. Jaton-Ogay G., Suter M., Crameri R., Falchetto R., Fatih A., Monod M. 1992; Nucleotide sequence of a genomic and a cDNA clone encoding an extracellular alkaline protease of Aspergillus fumigatus . FEMS Microbiol Lett 92:163–168
    [Google Scholar]
  20. Jaworski E. G., Hemming B. C., McPherson S. A., Drahos D. J., Lawson E. C., Jonsson C. B., Fuchs R. L. 1986; Nematode control using soil bacteria. European Patent Application EP 171381
    [Google Scholar]
  21. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
    [Google Scholar]
  22. Lingappa Y., Lockwood J. L. 1962; Chitin media for selective isolation and culture of Actinomycetes. Phytopathol 52:317–323
    [Google Scholar]
  23. Lopez-Llorca L. V. 1990; Purification and properties of extracellular proteases produced by the nematophagous fungus Verticillium suchlasporium . Can J Microbiol 36:530–537
    [Google Scholar]
  24. Lopez-Llorca L. V., Robertson W. M. 1992; Immunocytochemical localization of a 32-kDa protease from the nematophagous fungus Verticillium suchlasporium in infected nematode eggs. Exp Mycol 16:261–267
    [Google Scholar]
  25. McCullagh P., Nelder J. A. 1989 Generalized Linear Models 2nd edn London: Chapman & Hall;
    [Google Scholar]
  26. Mercer C. F., Greenwood D. R., Grant J. L. 1992; Effect of plant and microbial chitinases on the eggs and juveniles of Meloidogyne hapla Chitwood (Nematoda: Tylenchida). Nematologica 38:227–236
    [Google Scholar]
  27. Moehle C. M., Tizard R., Lemmon S. K., Smart J., Jones E. W. 1987; Protease B of the lysosome-like vacuole of the yeast Saccharomyces cerevisiae is homologous to the subtilisin family of serine proteases. Mol Cell Biol 7:4390–4399
    [Google Scholar]
  28. Morgan-Jones G., Rodriguez-Kabana R. 1985; Phytonematode pathology: fungal modes of action. A perspective. Nematropica 15:107–114
    [Google Scholar]
  29. Morgan-Jones G., White J. F., Rodriguez-Kabana R. 1983; Phytonematode pathology: ultrastructural studies. I. Parasitism of Meloidogyne arenaria eggs by Verticillium chlamydosporium . Nematropica 13:245–260
    [Google Scholar]
  30. Morrissey J. H. 1981; Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117:307–310
    [Google Scholar]
  31. North M. J. 1982; Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiol Rev 46:308–340
    [Google Scholar]
  32. Rinderknecht H., Geokas M. C., Silverman P., Haverback B. J. 1968; A new ultrasensitive method for the determination of proteolytic activity. Clin Chim Act 21:197–203
    [Google Scholar]
  33. Samal B. B., Karan B., Boone T. C., Osslund T. D., Chen K. K., Stabinsky Y. 1990; Isolation and characterization of the gene encoding a novel, thermostable serine proteinase from the mould Tritirachium album Limber. Mol Microbiol 4:1789–1792
    [Google Scholar]
  34. 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]
  35. Stepanov V. M., Rudenskaya G. N. 1983; Proteinase affinity chromatography on bacitracin-Sepharose. J Appl Biochem 5:420–428
    [Google Scholar]
  36. Stirling G. R., Mankau R. 1979; Mode of parasitism of Meloidogyne and other nematode eggs by Dactylella oviparasitica . J Nematol 11:282–288
    [Google Scholar]
  37. Tahseem Q. M., Shamim J., Ahmad I. 1991; Observations on the immature and post-immature development of Diploscapter orientalis (Nematoda: Rhabditida). Rev Nématol 14:251–260
    [Google Scholar]
  38. Terada I., Kwon S.-T., Miyata Y., Matsuzawa H., Ohta T. 1990; Unique precursor structure of an extracellular protease, aqualysin I, with NH2-, and COOH-terminal pro-sequences and its processing in Escherichia coli . J Biol Chem 265:6576–6581
    [Google Scholar]
  39. Wells J. A., Ferrari E., Henner D. J., Estell D. A., Chen E. Y. 1983; Cloning, sequencing and secretion of Bacillus amyloliqugaciens subtilisin in Bacillus subtilis . Nucleic Acids Res 11:7911–7925
    [Google Scholar]
  40. Wharton D. 1980; Nematode egg-shells. Parasitology 81:447–463
    [Google Scholar]
  41. Zaki F. A., Bhatti D. S. 1990; In vivo parasitism of Meloidogyne javanica by an oviparasitic fungus, Paecilomyces lilacinus . Nematol Mediter 18:141–143
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-141-4-775
Loading
/content/journal/micro/10.1099/13500872-141-4-775
Loading

Data & Media loading...

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