Summary
The intracellular localization of the sterol-biosynthesis-inhibiting (SBI) fungicide (3H)triadimenol A is investigated in vitro in the fungus Ustilago avenae. For this purpose low temperature preparation techniques (shock freezing, freeze substitution, embedding in Lowicryl HM20) are combined with conventional electron microscopic (EM) autoradiography. In particular the suitability of Lowicryl HM20 embedded specimens for EM autoradiography with regard to the finestructure preservation is shown. For the localization of (3H)triadimenol the filamentous grain development as well as the application of the gold latensification method resulting in the appearance of spherical silver grains is tested. Fungicide sensitive wild type sporidia of U. avenae are compared with fungicide resistant cells of the mutant r8. A quantitative analysis of the autoradiographs of the wild type developed according to the gold latensification process shows a relatively homogeneous distribution of silver grains over the entire cell. On the other hand, the resistant mutant is characterized by an accumulation of silver deposits over the vacuoles as compared with the lower density of grains over the cell walls and cytoplasm. The data are discussed in the context of possible resistance mechanisms against SBI-fungicides.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Benveniste P (1986) Sterol biosynthesis. Annu Rev Plant Physiol 37:275–308
Carlemalm E, Kellenberger E (1982) The reproducible observation of unstained embedded cellular material in thin sections; visualization of an integral membrane protein by a new mode of imaging for STEM. EMBO J 1:63–67
Fuchs A, DeWaard MA (1984) Resistance to ergosterol biosynthesis inhibitors. I. Chemistry and phenomenological aspects. In: Dekker J, Georgopoulos SG (eds) Fungicide resistance in crop protection. Pudoc, Wageningen, pp 71–86
Hang C, Hoffmann H (1986) Chemistry of plant protection. 1. Sterol biosynthesis, inhibitors and antifeeding compounds. Springer, Berlin Heidelberg New York
Henry MJ, Sisler HD (1984) Effects of sterol-biosynthesis-inhibiting (SBI) fungicides on cytochrome P-450 oxygenations in fungi. Pestic Biochem Physiol 22:262–275
Hippe S (1984a) Electron microscopic investigations on the cytology of Ustilago avenae influenced by the systemic fungicides triadimefon, nuarimol, and imazalil-nitrate: a thin-section and freeze-fracture study. Pestic Biochem Physiol 21:170–183
Hippe S (1984b) Rapid cryofixation by a simple propane-doublejet device adapted to a modified specimen table of the BIOETCH 2005. Mikroskopie 41:289–301
Hippe S (1985a) Ultrastructure of haustoria of Erysiphe graminis f.sp. hordei. Protoplasma 129:52–61
Hippe S (1985b) Hexagonal clustering of intramembrane particles in the plasmalemma of Ustilago avenae after fungicide treatment. Pestic Biochem Physiol 24:161–173
Hippe S, Giesen U (1987) The effect of triadimenol on the cytology and growth of sensitive and resistant strains of Ustilago avenae. Ann Appl Biol 112 (in press)
Hippe S, Hermanns M (1986) Improved structural preservation in freeze-substituted sporidia of Ustilago avenae — a comparison with low temperature embedding. Protoplasma 135:19–30
Hippe S, Hermanns M (1987) Intracellular localization of the systemic fungicide triadimenol in phytopathogenic fungi. J Phytopathol (in press)
Hippe S, Köller W (1986) Ultrastructure and sterol composition of laboratory strains of Ustilago avenae resistant to triazole fungicides. Pestic Biochem Physiol 26:209–219
Hoch HC (1986) Freeze substitution of fungi. In: Aldrich HC, Todd WJ (eds) Ultrastructure techniques for microorganisms. Plenum Press, New York, pp 183–212
Howard RJ, Aist JR (1979) Hyphal tip cell ultrastructure of the fungus Fusarium: improved preservation by freeze-substitution. J Ultrastruct Res 66:224–234
Hunziker EB, Herrmann W, Schenk RK, Mueller M, Moor H (1984) Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. I. Chondrocyte ultrastructure—implications for the theories of mineralization and vascular invasion. J Cell Biol 98:267–276
Köller W (1987) Mechanism of fungal resistance to sterol biosynthesis inhibitors. I. Initial and long-term effect of triadimenol on the accumulation of lanosterol in a sensitive and resistant laboratory strain of Ustilago avenae. Pestic Science (in press)
Kopriwa BM (1975) A comparison of various procedures for fine grain development in electron microscopic radioautography. Histochemistry 44:201–224
Kopriwa BM, Levine GM, Nadler NJ (1984) Assessment of resolution by half distance values for tritium and radioiodine in electron microscopic radioautographs using Ilford L4 emulsion developed by “Solution physical” or D19b methods. Histochemistry 80:519–522
Lancelle SA, Callaham DA, Hepler PK (1986) A method for rapid freeze fixation of plant cells. Protoplasma 131:153–165
Roth J, Bendayan M, Carlemalm E, Villiger W, Garavito M (1981) Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue. J Histochem Cytochem 29:663–671
Salpeter MM (1982) High resolution autoradiography. In: Baker PF, Tichen DA, Linden RJ, Otis AB (eds) Techniques in the life sciences, Vol P1/I. County Clare, Ireland, Elsevier North-Holland, Amsterdam, pp 1–45
Salpeter MM, Bachmann L (1972) Autoradiography. In: Hayat MA (ed) Principles and techniques of electron microscopy. Van Nostrand Reinhold, New York, pp 220–278
Sisler HD, Ragsdale NN (1984) Biochemical and cellular aspects of the antifungal action of ergosterol biosynthesis inhibitors. In: Trinci APJ, Ryley JF (eds) Mode of action of antifungal agents. University Press, Cambridge, pp 247–282
Stumpf WE (1976) Techniques for the autoradiography of diffusible compounds. In: Prescott DM (ed) Methods in cell biology. Academic Press, New York, pp 171–192
Stumpf WE, Roth LJ (1966) High resolution autoradiography with dry mounted, freeze-dried, frozen sections. Comparative study of six methods using two diffusible compounds. 3H-mesobilirubinogen. J Histochem Cytochem 14:274–287
Vrensen GFJM (1970) Some new aspects of efficiency of electron microscopic autoradiography with tritium. J Histochem Cytochem 18:278–290
Weibull C, Christiansson A (1986) Extraction of proteins and membrane lipids during low temperature embedding of biological material for electron microscopy. J Microsc 142:79–86
Wieneke J, Führ F (1983) Microautoradiography as a tool to elucidate sites of pesticide and mineral penetration into plant tissue. In: Duncan WP, Susan AB (eds) Synthesis and applications of isotopically labeled compounds. Proceedings of an International Symposium, Kansas City, MO, USA, pp 373–374
Wroblewski R, Wroblewski J (1984) Freeze drying and freeze substitution combined with low temperature-embedding. Histochemistry 81:469–475
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hippe, S. Combined application of low temperature preparation and electron microscopic autoradiography for the localization of systemic fungicides. Histochemistry 87, 309–315 (1987). https://doi.org/10.1007/BF00492583
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00492583