Current Microbiology

, Volume 26, Issue 2, pp 113–116 | Cite as

A membrane technique for producing protoplasts ofCryphonectria parasitica

  • Jyan-Chyun Jang
  • Susan D. McElreath
  • Frank H. Tainter


A method for the formation and regeneration of protoplasts of several strains of the chestnut blight fungus,Cryphonectria parasitica, is presented. The procedure utillizes cellophane membranes for growth and employs centrifugation for separation of protoplasts from hyphal fragments. Yields averaged 8.04×106 protoplasts per membrane. Regeneration frequencies were 40–50% with a soft-agar overlay. These protoplasts are suitable for use in experiments designed to determine the role of dsRNA in hypovirulence ofC. parasitica.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature Cited

  1. 1.
    Anagnostakis SL (1984) Nuclear gene mutations inEndothia (Cryphonectria) parasitica that affect morphology and virulence. Phytopathology 74:561–565Google Scholar
  2. 2.
    Barrett V, Lemke PA, Dixon RK (1989) Protoplast formation from selected species of ectomycorrhizal fungi. Appl Microbiol Biotechnol 30:381–387Google Scholar
  3. 3.
    Dales RBG, Croft JH (1977) Protoplast fusion and the isolation of heterokaryons and diploids from vegetatively incompatible strains ofAspergillus nidulans. FEMS Microbiol Lett 1:201–204.Google Scholar
  4. 4.
    Day PR, Dodds JA, Elliston JE, Jaynes RA, Anagnostakis SL (1977) Double-stranded RNA inEndothia parasitica. Phytopathology 67:1393–1396Google Scholar
  5. 5.
    de Vries OMH, Wessels JGH (1972) Release of protoplasts fromSchizophyllum commune by a lytic enzyme preparation fromTrichoderma viride. J Gen Microbiol 73:13–22Google Scholar
  6. 6.
    Dodds JA (1980) Revised estimates of the molecular weights of dsRNA segments in hypovirulent strains ofEndothia parasitica. Phytopathology 70:1217–1220Google Scholar
  7. 7.
    Elliston JE (1985) Characteristics of dsRNA-free and dsRNA-containing strains ofEndothia parasitica in relation to hypovirulence. Phytopathology 75:151–158Google Scholar
  8. 8.
    Fincham JRS (1989) Transformation in fungi. Microbiol Rev 53:148–170 53:148–170Google Scholar
  9. 9.
    Gaugy D, Fevre M (1985) Regeneration and reversion of protoplasts from different species ofPenicillium. Microbios 44:285–293Google Scholar
  10. 10.
    Grente J, Berthelay-Sauret S (1978) Biological control of chestnut blight in France. In: MacDonald W, Chech F, Luchok J, Smith C (eds) Proceedings of the American Chestnut Symposium. W. Va Univ and US Forest Service, pp 30–34Google Scholar
  11. 11.
    Hwang SC, Ko WH, Aragaki M (1975) A simplified method for sporangial production byPhytophthora cinnamomi. Mycologia 67:1233–1234Google Scholar
  12. 12.
    Jaynes RA, Elliston JE (1982) Hypovirulent isolates ofEndothia parasitica associated with large American chestnut trees. Plant Dis 66:769–772Google Scholar
  13. 13.
    Kitamoto Y, Mori N, Yamamoto M, Ohiwa T, Ichikawa Y (1988) A simple method for protoplast formation and improvement of protoplast regeneration from various fungi using an enzyme fromTrichoderma harzianum. Appl Microbiol Biotechnol 28:445–450Google Scholar
  14. 14.
    Kunkel W, Groth I, Jacob HE, Risch S, Harnisch M, May R, Berg H, Katenkamp U (1987) Electrofusion of protoplasts ofPenicillium chrysogenum. Stud Biophys 119:35–36Google Scholar
  15. 15.
    Layton AC, Kuhn DN (1988) Heterokaryon formation by protoplast fusion of drug-resistant mutants inPhytophthora megasperma f. sp.glycinea. Exp Mycol 12:180–194Google Scholar
  16. 16.
    Puhalla JE, Anagnostakis SL (1971) Genetics and nutritional requirements ofEndothia parasitica. Phytopathology 61:169–173Google Scholar
  17. 17.
    Sonnenberg ASM, Wessels JGH (1987) Heterokaryon formation in the basidiomyceteSchizophyllum commune by electrofusion of protoplasts. Theor Appl Genet 74:654–658Google Scholar
  18. 18.
    Van Alfen NK, Jaynes RA, Anagnostakis SL, Day PR (1975) Chestnut blight: biological control by transmissible hypovirulence inEndothia parasitica. Science 189:890–891Google Scholar
  19. 19.
    Yamada O, Magae Y, Kashiwagi Y, Kakimoto Y, Sasaki T (1983) Preparation and regeneration of mycelial protoplasts ofCollybia velutipes andPleurotus ostreatus. Eur J Appl Microbiol Biotechnol 17:298–300Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1993

Authors and Affiliations

  • Jyan-Chyun Jang
    • 1
  • Susan D. McElreath
    • 1
  • Frank H. Tainter
    • 1
  1. 1.Department of Forest ResourcesClemson UniversityClemsonUSA

Personalised recommendations