, Volume 37, Issue 2, pp 187–200 | Cite as

Characterization of the mucilage sheaths ofLemonniera aquatica by lectin-gold labelling

  • Doris Wai Ting Au
  • Steve Thomas Moss
  • Evan Benjamin Gareth Jones
  • Ivor John Hodgkiss
Original Papers


A pre-embedding lectin-gold labelling method was used to characterize the carbohydrate components in the mucilage ofLemonniera aquatica. A specific tissue processing protocol was developed, namely: a) primary fixation in 2% paraformaldehyde and 0.2% glutaraldehyde in PIPES buffer (pH 7.2) for 30 min; b) secondary fixation in 2% glutaraldehyde in the same buffer system for 1 h; c) post-fixation in 1% aqueous OsO4 for 1h; d) embedment in Möllenhaur's resin. The three gold conjugated lectins used were: concanavalin A, wheat germ agglutinin andLimax flavus agglutinin, allowing detection of their complementary saccharides, namely α-d-mannose/α-d-glucose,N-acetyl-d-glucosamine (GluNAc), andN-acetylneuraminic acid (NANA), respectively.N-Acetyl-d-glucosamine and NANA residues were the major components of germ tube mucilage with only a small amount of α-d-manose/α-d-glucose. However, NANA was restricted to the mucilage in the region of germ tube emergence from the conidial arm. The abundance of GluNAc and NANA residues on hyphae and appressoria was less than that on the germ tube. Conversely, α-d-mannose/α-d-glucose was more abundant in the appressorial mucilage. Variability of mucilage composition was found to exist between different structures of the germinated conidium and also between different regions of the same structure. Further, the conidial cell wall ofL. aquatica is not chitinous, and lacks NANA and α-d-mannose/α-d-gluocse.

Key Words

appressorium aquatic Hyphomycetes germ tube hyphal sheath mucilage composition 


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Literature cited

  1. Aldrich, H. C. and Möllenhauer, H. H. 1986. Secrets of successful embedding; sectioning and imaging. In: Ultrastructure techniques for microorganisms, (ed. by Aldrich, H. C. and Todd, W. J.), pp 103–130. Plenum Press, New York.Google Scholar
  2. Armbruster, B. L., Garavito, R. M. and Kellenberger, E. 1983. Dehydration and embedding temperatures affect the antigenic specificity of tubulin and immunolabeling by the protein A-colloidal gold technique. J. Histochem. Cytochem.31: 1380–1384.PubMedGoogle Scholar
  3. Au, W. T. 1993. Enzymatic studies of conidial attachment and lectin-gold histochemical investigation of the extracellular mucilage ofLemonniera aquatica de Wild. andMycocentrospora filiformis (Petersen) Iqbal. Hong Kong: The University of Hong Kong, PhD Thesis.Google Scholar
  4. Au, D. W. T., Jones, E. B. G. and Moss, S. T. 1996. Spore attachment and extracellular mucilage on aquatic Hyphomycetes. In: Proceedings of the IX International Congress on Biofouling, (ed. by Callow, M.). Harwood Academic Publ., Switzerland. (In press.)Google Scholar
  5. Barkai-Golan, R., Mirelman, D. and Sharon, N. 1978. Studies on growth inhibition by lectins of Penicillia and Aspergilli. Arch. Microbiol.116: 119–124.PubMedCrossRefGoogle Scholar
  6. Bendayan, M., Nanci, A. and Kan, F. W. K. 1987. Effect of tissue processing on colloidal gold cytochemistry. J. Histochem. Cytochem.35: 938–996.Google Scholar
  7. Benhamou, N. 1988. Ultrastructural localization of carbohydrates in the cell walls of two pathogenic fungi: A comparative study. Mycologia80: 324–337.Google Scholar
  8. Benhamou, N. and Charest, P. M. 1986. Mise en evidence ultrastructurale d'acide sialique et de fucose chez deux ascomycetes phytopathogenes par l'intermediaire de lectines complexes l'or colloidal. Phytoprotection67: 21–33.Google Scholar
  9. Benhamou, N. and Ouellette, G. B. 1986. Ultrastructural localization of glycoconjugates in the fungiAscocalyx abietina, the sclerodermis canker agent of conifers, using lectin-gold complexes. J. Histochem. Cytochem.34: 855–867.PubMedGoogle Scholar
  10. Benhamou, N., Chamberland, H., Ouellette, G. B. and Pauze, F. J. 1988. Detection of galactose in two fungi causing wilt diseases and in their plant host tissues by means of gold-complexedRicinus communis agglutinin I. Physiol. Mol. Plant Pathol.32: 249–266.Google Scholar
  11. Bonfante-Fasolo, P., Perotto, S., Testa, B. and Faccio, A. 1987. Ultrastructural localization of cell surface sugar residues in ericoid mycorrhizal fungi by gold-labelled lectins. Protoplasma139: 25–35.CrossRefGoogle Scholar
  12. Burr, A. W. 1991. Comparative diplanetic developmental processes of salmonid-pathogenic and saprophytic isolates of theSaprolegnia parasitica-diclina complex. University of Newcastle, UK, PhD Thesis.Google Scholar
  13. Chamberland, H., Charest, P. M., Ouellette, G. B. and Pauze, F. J. 1985. Chitinase-gold complex used to localize chitin ultrastructurally in tomato root cells infected byFusarium oxysporum f. sp.radicis-lycopersici, compared with a chitin specific gold-conjugated lectin. Histochem. J.17: 313–321.PubMedCrossRefGoogle Scholar
  14. Erdos, G. W. 1986. Localization of carbohydrate-containing molecules. In: Ultrastructure techniques for microorganisms, (ed. by Aldrich, H. C. and Todd, W. J.), pp. 339–420. Plenum Press, New York.Google Scholar
  15. Hamer, J. E., Howard, R. J., Chumley, F. G. and Valent, B. 1988. A mechanism for surface attachment in spores of a plant pathogenic fungus. Scienc239: 288–290.CrossRefGoogle Scholar
  16. Harder, D. E., Chong, J., Rohringer, R. and Kim, W. K. 1986. Structure and cytochemistry of the walls of urediospores, germ tubes and appressoria ofPuccinia graminis tritici. Can. J. Bot.64: 476–485.Google Scholar
  17. Hardham, A. R. 1986. Studies on the cell surface of zoospores and cysts of the fungusPhytophthora cinnamomi: The influence of fixation on patterns of lectin binding. J. Histochem. Cytochem.33: 110–118.Google Scholar
  18. Hardham, A. R. 1989. Lectin and antibody labelling of surface components of spores ofPhytophthora cinnamomi. Aust. J. Plant Physiol.16: 19–32.CrossRefGoogle Scholar
  19. Hayat, M. A. 1970. Principles and techniques of electron microscopy, vol. 1. Biological application. Van Nostrand Reinhold, New York.Google Scholar
  20. Hyde, K. D. 1985. Spore settlement and attachment in marine fungi. University of Portsmouth, UK, PhD Thesis.Google Scholar
  21. Hyde, K. D., Moss, S. T. and Jones, E. B. G. 1994. Ascospore ultrastructure ofHalosphaeria appendiculata (Halosphaeriaceae). Bot. Mar.37: 51–56.CrossRefGoogle Scholar
  22. Kellenberger, E., Durrenberger, M., Villiger, W., Carlemalm, E. and Wurtz, M. 1987. The efficiency of immunolabel on Lowicryl sections compared to theoretical predictions. J. Histochem. Cytochem.35: 959–969.PubMedGoogle Scholar
  23. Lei, J., Wong, K. K. Y. and Piche, Y. 1991. Extracellular concanavalin A-binding sites during early interactions betweenPinus banksiana and two closely related genotypes of the ectomycorrhizal basidiomyceteLaccaria bicolor. Mycol. Res.95: 357–363.CrossRefGoogle Scholar
  24. Lehninger, A. L., Nelson, D. L. and Cox, M. M. 1993. Carbohydrates. In: Principles of biochemistry, pp. 312–315. Worth Publ., New York.Google Scholar
  25. Manimohan, P., Jones, E. B. G. and Moss, S. T. 1993. Ultrastructural studies of ascospores of someRemispora species. Can. J. Bot.71: 385–392.Google Scholar
  26. Manocha, M. S. 1985. Specificity of mycoparasite attachment to host cell surface. Can. J. Bot.63: 772–778.Google Scholar
  27. Manocha, M. S., Chen, Y. and Rao, N. 1990. Involvement of cell surface sugars in recognition attachment and appressorium formation by a mycoparasite. Can. J. Microbiol.36: 771–778.CrossRefGoogle Scholar
  28. Mirelman, D., Galun, E., Sharon, N. and Lotan, R. 1975. Inhibition of fungal growth by wheat germ agglutinin. Nature256: 414–416.PubMedCrossRefGoogle Scholar
  29. Möllenhauer, H. H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol.39: 111–114.PubMedGoogle Scholar
  30. Newman, G. R., Jasani, B. and Williams, E. D. 1983. A simple post-embedding system for the rapid demonstration of tissue antigens under the electron microscope. Histochem. J.15: 543–555.PubMedCrossRefGoogle Scholar
  31. Read, S. J. 1990. Spore attachment in fungi with special reference to freshwater Hyphomycetes. University of Portsmouth, UK, PhD Thesis.Google Scholar
  32. Bread, S. J., Moss, S. T. and Jones, E. B. G. 1991. Attachment studies of aquatic Hyphomycetes. Phil. Trans. R. Soc. Lond. 334: 449–457.Google Scholar
  33. Read, S. J., Moss, S. T. and Jones, E. B. G. 1992a. Attachment and germination of conidia. In: The ecology of aquatic Hyphomycetes, (ed by Bärlocher, F.), pp. 134–151. Springer-Verlag, Berlin.Google Scholar
  34. Read, S. J., Moss, S. T. and Jones, E. B. G. 1992b. Germination and development of attachment structures by conidia of aquatic Hyphomycetes: light microscopic studies. Can. J. Bot.70: 831–837.Google Scholar
  35. Read, S. J., Moss, S. T. and Jones, E. B. G. 1992c. Germination and development of attachment structures by conidia of aquatic Hyphomycetes: a scanning electron microscopic study. Can. J. Bot.70: 838–845.Google Scholar
  36. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol.17: 208–212.PubMedCrossRefGoogle Scholar
  37. Roth, J. 1983. Application of lectin-gold complexes for electron microscopic localization of glycoconjugates on thin sections. J. Histochem. Cytochem.31: 987–999.PubMedGoogle Scholar
  38. Soares, M. A., Alviano, C. S., Angluster, J. and Travassos, L. R. 1993. Identification of sialic acids on the cell surface of hyphae and yeast forms of the human pathogenParacoccidioides brasilliensis. FEMS Microbiol. Lett.108, 31–34.PubMedCrossRefGoogle Scholar
  39. Spurr, A. R. 1969. A lowe-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res.26: 31–43.PubMedCrossRefGoogle Scholar
  40. Stanley, S. J. 1991. The autecology and ultrastructural interaction betweenMycosphaerella ascophylli Cotton,Lautitia danica (Berlese) Schatz,Mycaureola dilseae Marie et Chemin and their respective marine algal hosts. University of Portsmouth, UK, PhD Thesis.Google Scholar
  41. Tronchin, G., Poulain, D. and Vernes, A. 1984. Cytochemical and ultrastructural studies ofCandida albicans III. Evidence for modifications of the cell wall coat during adherence to human buccal epithelial cells. Arch. Microbiol.139: 221–224.PubMedCrossRefGoogle Scholar
  42. Van Wyk, P. W. J., Wingfield, M. J. and Van Wyk, P. S. 1991. Ascospore development inCeratocystis moniliformis. Mycol. Res.95: 96–103.Google Scholar
  43. Van Wyk, P. W. J. and Wingfield, M. J. 1994. Ultrastructure of ascus arrangement and ascospore development inOphiostoma seticolle. Mycologia86: 607–614.Google Scholar
  44. Yusoff, M., Read, S. J., Jones, E. B. G. and Moss, S. T. 1994. Ultrastructure ofAntennospora salina comb. nov. Mycol. Res.98: 997–1004.Google Scholar

Copyright information

© The Mycological Society of Japan 1996

Authors and Affiliations

  • Doris Wai Ting Au
    • 1
  • Steve Thomas Moss
    • 2
  • Evan Benjamin Gareth Jones
    • 2
  • Ivor John Hodgkiss
    • 3
  1. 1.Department of Biology and ChemistryCity University of Hong KongKowloonHong Kong
  2. 2.School of Biological SciencesUniversity of PortsmouthPortsmouthUK
  3. 3.Department of Ecology and BiodiversityThe University of Hong KongHong Kong

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