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Modifications to clearing methods used in combination with vital staining of roots colonized with vesicular-arbuscular mycorrhizal fungi

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Leek, maize, and pigmented soybean roots colonized by vesicular-arbuscular mycorrhizal (VAM) fungi were assessed for succinate dehydrogenase (SDH) activity using the nitro blue tetrazolium chloride (NBT)-succinate method. NBT-succinate-reacted roots, cleared in a 55° C drying oven in 5% (w/v) KOH for 24 h or longer and observed as whole mounts, revealed signs of intraradical VAM fungus colonization more clearly than roots cleared by the standard 20% (w/v) boiling chloral hydrate method. Combined clearing of NBT-succinate-reacted roots using boiling chloral hydrate followed by clearing in 5% KOH at 55° C for prolonged periods also improved the visualization of intraradical fungal structures. Bleaching of NBT-succinate-reacted roots using the standard NH3-H2O2 method removed pigmentation from roots and did not alter the viability indicator, formazan. Pigmented, field-collected soybean roots were successfully cleared and bleached to reveal signs of viable and nonviable intraradical fungal structures. Counterstaining of NBT-succinate-reacted roots with acid fuchsin clearly revealed both viable and nonviable intraradical fungal structures. The NBT-succinate solution infiltrated all intraradical fungal structures after 24 h; formazan products were observed at similar concentrations in viable structures after 24, 36, and 48 h.

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  1. An Z-Q, Hendrix JW (1988) Determining viability of endogonaceous spores with a vital stain. Mycologia 80:259–361

  2. Brundrett MC, Piché Y, Peterson RL (1984) A new method for observing the morphology of vesicular-arbuscular mycorrhizae. Can J Bot 62:2128–2134

  3. Hamel C, Fyles H, Smith DL (1990) Measurement of development of endomycorrhizal mycelium using three different vital stains. New Phytol 115:297–302

  4. Kormanik PP, McGraw AC (1982) Quantification of vesicular-arbuscular mycorrhizae in plant roots. In: Schenck NC (ed) Methods and principles of mycorrhizal research. The American Phytopathological Society, St Paul, Minn

  5. Kough JL, Gianinazzi-Pearson V (1986) Physiological aspects of VA mycorrhizal hyphae in root tissue and soil. In: Gianinazzi-Pearson V, Gianinazzi S (eds) Physiological and genetical aspects of mycorrhizae. Proceedings of the 1st European Symposium on Mycorrhizae, Dijon, France, pp 223–236

  6. Kough JL, Gianinazzi-Pearson V, Gianinazzi S (1987) Depressed metabolic activity of vesicular-arbuscular mycorrhizal fungi after fungicide applications. New Phytol 106:707–715

  7. MacDonald RM, Lewis M (1978) The occurrences of some acid phosphatases and dehydrogenases in the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. New Phytol 80:135–141

  8. McGee PA, Smith SE (1990) Activity of succinate dehydrogenase in vesicular-arbuscular mycorrhizal fungi after enzymatic digestion from roots of Allium porum Mycol Res 94:305–308

  9. McGonigle T, Miller M, Evans D, Fairchild G, Swan J (1990) A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol 115:495–501

  10. Pearse AE (1972) Histochemistry theoretical and applied, vol 2, 3rd edn. Churchill Livingstone, Edinburgh

  11. Pearson JN, Smith SE, Smith FA (1991) Effect of photon irradiance on the development and activity of VA mycorrhizal infection in Allium porrum L. Mycol Res 95:741–746

  12. Phillips JM, Hayman DS (1970) Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc 55:158–160

  13. Schubert A, Marzachi C, Mazzitelli M, Gravero MC, Bonfante-Fasolo P (1987) Development of total and viable extraradical mycelium in the vesicular-arbuscular mycorrhizal fungus Glomus darum Nicol. & Schenck. New Phytol 107:183–190

  14. Smith SE, Dickson S (1991) Quantification of active vesicular-arbuscular mycorrhizal infection using image analysis and other techniques. Aust J Plant Physiol 18:637–648

  15. Smith SE, Gianinazzi-Pearson V (1990) Phosphate uptake and arbuscular activity in mycorrhizal Allium cepa L.: effects of photon irradiance and phosphate nutrition. Aust J Plant Physiol 17:177–188

  16. Sylvia DM (1988) Activity of external hyphae of vesicular-arbuscular mycorrhizal fungi. Soil Biol Biochem 20:39–43

  17. Troyer H (1980) Principles and techniques of histochemistry. Little, Brown & Co, Boston

  18. Vierheilig H, Ocampo JA (1991) Receptivity of various wheat cultivars to infection by VA-mycorrhizal fungi as influenced by inoculum potential and the relation of VAM-effectiveness to succinic dehydrogenase activity of the mycelium in the roots. Plant Soil 133:291–296

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Schaffer, G.F., Peterson, R.L. Modifications to clearing methods used in combination with vital staining of roots colonized with vesicular-arbuscular mycorrhizal fungi. Mycorrhiza 4, 29–35 (1993). https://doi.org/10.1007/BF00203248

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Key words

  • Vesicular-arbuscular mycorrhizae
  • Viability
  • Nitro blue tetrazolium chloride
  • Succinate dehydrogenase
  • Formazan