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Official Journal of the Japan Wood Research Society

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Immunogold labeling of an extracellular substance producing hydroxyl radicals in wood degraded by brown-rot fungusTyromyces palustris


A fraction containing low-molecular-weight peptides that catalyzes redox reactions between electron donors and O2 to produce ·OH, was partially purified from wood-decaying cultures of the brown-rot fungusTyromyces palustris. Polyclonal antibodies raised to the fraction were used for immunogold labeling of transverse sections of sapwood of spruce in various stages of degradation byT. palustris to demonstrate the cellular localization of the ·OH-producing substance. Initially, the wood cell wall was attacked primarily by fungal hyphae growing in the cell lumen. During the early stages of degradation, the gold label was localized in the fungal cytoplasm and cell wall and in the extracellular slime sheath surrounding the fungal cell wall. The gold label also was found throughout the wood cell wall, although the cell wall remained almost intact so long as the fungal hyphae remained in the lumen. Thus, the ·OH-producing substance is secreted by the hyphae into the lumen, and it diffuses through the S3 layer into the S2 layer and the middle lamella. The role of this ·OH-producing system in wood degradation byT. palustris is discussed.


  1. 1.

    Cowling EB (1961) Comparative biochemistry of the decay of sweetgum sapwood by white-rot and brown-rot fungi. US Department of Agriculture Technical Bulletin 1258, pp 1–79

  2. 2.

    Kirk TK, Highley TL (1973) Quantitative changes in structural components of conifer woods during decay by white- and brownrot fungi. Phytopathology 63:1338–1342

    CAS  Article  Google Scholar 

  3. 3.

    Highley TL (1987) Changes in chemical components of hardwood and softwood by brown-rot fungi. Mater Org 22:39–45

    CAS  Google Scholar 

  4. 4.

    Enoki A, Tanaka H, Fuse G (1988) Degradation of lignin-related compounds, pure cellulose, and wood components by white-rot and brown-rot fungi. Holzforschung 42:85–93

    CAS  Article  Google Scholar 

  5. 5.

    Itakura S, Hirano T, Tanaka H, Enoki A (1994) Relationship between degradation of wood, cellulose or lignin-related compounds and production of hydroxyl radical or accumulation of oxalic acid in cultures of brown-rot fungi. International Research Group on Wood Preservation Document No. IRG/WP/94-10062

  6. 6.

    Enoki A, Tanaka H, Fuse G (1989) Relationship between degradation of wood and production of H2O2-producing or one-electron oxidases by brown-rot fungi. Wood Sci Technol 23:1–12

    CAS  Article  Google Scholar 

  7. 7.

    Liese W (1970) Ultrastructural aspects of woody tissue degradation. Annu Rev Phytopathol 8:231–258

    Article  Google Scholar 

  8. 8.

    Highley TL, Murmanis LL, Palmer JG (1985) Micromorphology of degradation in western hemlock and sweetgum by the brown-rot fungusPoria placenta. Holzforschung 39:73–78

    CAS  Article  Google Scholar 

  9. 9.

    Kuo M, Stokke DD, McNabb HS (1988) Microscopy of progressive decay of cottonwood by the brown-rot fungusGloeophyllum trabeum. Wood Fiber Sci 20:405–414

    Google Scholar 

  10. 10.

    Flournoy DS, Kirk TK, Highley TL (1991) Wood decay by brownrot fungi: changes in pore structure and cell wall volume. Holzforschung 45:383–388

    CAS  Article  Google Scholar 

  11. 11.

    Srebotnik E, Messner K (1991) Immunoelectron microscopical study of the porosity of brown-rot degraded pine wood. Holzforschung 45:95–101

    Article  Google Scholar 

  12. 12.

    Enoki A, Hirano T, Tanaka H (1992) Extracellular substance from the brown-rot basidiomyceteGloeophyllum trabeum that produces and reduces hydrogen peroxide. Mater Org 27:247–261

    CAS  Google Scholar 

  13. 13.

    Hirano T, Tanaka H, Enoki A (1995) Extracellular substance from the brown-rot basidiomyceteTyromyces palustris that reduces molecular oxygen to hydroxyl radicals and ferric iron to ferrous iron. Mokuzai Gakkaishi 41:334–341

    CAS  Google Scholar 

  14. 14.

    Hirano T, Tanaka H, Enoki A (1997) Relationship between production of hydroxyl radicals and degradation of wood by the brown-rot fungus,Tyromyces palustris. Holzforschung 51:389–395

    CAS  Article  Google Scholar 

  15. 15.

    Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 72–78

    Google Scholar 

  16. 16.

    Palfreyman JW, Glancy H, Button D, Vigrow A, Score A, King B (1988) Use of immunoblotting for the analysis of wood decay basidiomycetes. International Research Group on Wood Preservation Document No. IRG/WP/2307

  17. 17.

    Schgger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kDa. Anal Biochem 166:368–379

    Article  Google Scholar 

  18. 18.

    Ploug M, Jensen AL, Barkholt V (1989) Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3. Anal Biochem 181:33–39

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Kenna JG, Major GN, Williams RS (1985) Methods for reducing non-specific antibody binding in enzyme-linked immunosorbent assays. J Immunol Methods 85:409–419

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Highley TL (1973) Influence of carbon source on cellulase activity of white-rot fungi and brown-rot fungi.Wood Fiber 5:50–58

    CAS  Google Scholar 

  21. 21.

    Kirk TK, Farrell RL (1987) Enzymatic “combustion”: the microbial degradation of lignin. Annu Rev Microbiol 41:465–505

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Archibald F, Roy B (1992) Production of manganic chelates by laccase from the lignin-degrading fungusTrametes (Coriolus) versicolor. Appl Environ Microbiol 58:1496–1499

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Enoki A, Takahashi M, Tanaka H, Fuse G (1985) Degradation of lignin-related compounds and wood components by white-rot and brown-rot fungi. Mokuzai Gakkaishi 31:397–408

    CAS  Google Scholar 

  24. 24.

    Kirk TK, Ibach R, Mozuch MD, Conner AH, Highley TL (1991) Characteristics of cotton cellulose depolymerized by a brown-rot fungus, by acid, or by chemical oxidants. Holzforschung 45:239–244

    CAS  Article  Google Scholar 

  25. 25.

    Chirat C, Lachenal D (1997) Effect of hydroxyl radicals on cellulose and pulp and their occurrence during ozone bleaching. Holzforschung 51:147–154

    CAS  Article  Google Scholar 

  26. 26.

    Gierer J, Yang E, Reitberger T (1992) The reactions of hydroxyl radicals with aromatic rings in lignins, studied with cresol and 4-methylveratrol. Holzforschung 46:495–504

    CAS  Article  Google Scholar 

  27. 27.

    Highley TL, Murmanis LL (1985) Determination of hydrogen peroxide production inCoriolus versicolor andPoria placenta during wood degradation. Mater Org 20:241–251

    CAS  Google Scholar 

  28. 28.

    Rättö M, Ritschkoff A-C, Viikari L (1997) The effect of oxidative pretreatment on cellulose degradation byPoria Placenta andTrichoderma reesei cellulases. Appl Microbiol Biotechnol 48:53–57

    Article  Google Scholar 

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Correspondence to Taeko Hirano.

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Hirano, T., Enoki, A. & Tanaka, H. Immunogold labeling of an extracellular substance producing hydroxyl radicals in wood degraded by brown-rot fungusTyromyces palustris . J Wood Sci 46, 45–51 (2000).

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Key words

  • Brown-rot fungi
  • Immunogold labeling
  • Transmission electron mircoscopy
  • Wood decay
  • Hydroxyl radical