Assessment of Mycorrhizal Frequency in the Roots of Fruit Plants Using Different Dyes
ABSTRACT
The aim of the experimental work was to optimize the previously used Phillips and Hayman (1970) method of staining roots for microscopic assessment of mycorrhizal frequency in the root material. The materials used in the experiment were the roots of strawberry plants of the cultivar “Elsanta” obtained in a greenhouse experiment in May 2012. Morphological features of the roots were assessed using a root scanner, and then attempts were made to stain the roots in four types of dyes: 0.01% methylene blue, 0.01% acridine orange, 0.01% malachite green, and 0.01% carbol fuchsin. A comparative microscopic assessment was made of the effects of staining with the four types of dyes the structures of mycorrhizal fungi formed in strawberry roots. Mycorrhizal frequency (F%) in the stained root samples was also compared. Next, the usefulness of these dyes for staining the roots of other fruit plant species, such as apple, sweet cherry, blackcurrant and sour cherry, was evaluated. It was concluded that the staining of roots at a temperature of up to 65°C did not cause tissue breakdown, and that the clearing time used—20 to 30 minutes, and the use of carbol fuchsin were found to be the most effective combinations in achieving good quality microscopic images. During microscopic examinations, a satisfactory contrast was noted between root tissues and the structures of mycorrhizal fungi. The use of carbol fuchsin for staining roots also helped to expose a greater number of fungal structures and obtain higher values of mycorrhizal frequency in the roots of strawberries, blackcurrants, sour cherries, sweet cherries and apples, in comparison with other dyes tested. The newly developed method of staining roots with carbol fuchsin, compared with the method used previously (Phillips and Hayman, 1970), is both less time consuming and less labour intensive.
The aim of the experimental work was to optimize the previously used Phillips and Hayman (1970) method of staining roots for microscopic assessment of mycorrhizal frequency in the root material. The materials used in the experiment were the roots of strawberry plants of the cultivar “Elsanta” obtained in a greenhouse experiment in May 2012. Morphological features of the roots were assessed using a root scanner, and then attempts were made to stain the roots in four types of dyes: 0.01% methylene blue, 0.01% acridine orange, 0.01% malachite green, and 0.01% carbol fuchsin. A comparative microscopic assessment was made of the effects of staining with the four types of dyes the structures of mycorrhizal fungi formed in strawberry roots. Mycorrhizal frequency (F%) in the stained root samples was also compared. Next, the usefulness of these dyes for staining the roots of other fruit plant species, such as apple, sweet cherry, blackcurrant and sour cherry, was evaluated. It was concluded that the staining of roots at a temperature of up to 65°C did not cause tissue breakdown, and that the clearing time used—20 to 30 minutes, and the use of carbol fuchsin were found to be the most effective combinations in achieving good quality microscopic images. During microscopic examinations, a satisfactory contrast was noted between root tissues and the structures of mycorrhizal fungi. The use of carbol fuchsin for staining roots also helped to expose a greater number of fungal structures and obtain higher values of mycorrhizal frequency in the roots of strawberries, blackcurrants, sour cherries, sweet cherries and apples, in comparison with other dyes tested. The newly developed method of staining roots with carbol fuchsin, compared with the method used previously (Phillips and Hayman, 1970), is both less time consuming and less labour intensive.
Cite this paper
Derkowska, E. , Paszt, L. , Dyki, B. and Sumorok, B. (2015) Assessment of Mycorrhizal Frequency in the Roots of Fruit Plants Using Different Dyes. Advances in Microbiology, 5, 54-64. doi: 10.4236/aim.2015.51006.
Derkowska, E. , Paszt, L. , Dyki, B. and Sumorok, B. (2015) Assessment of Mycorrhizal Frequency in the Roots of Fruit Plants Using Different Dyes. Advances in Microbiology, 5, 54-64. doi: 10.4236/aim.2015.51006.
References
[1] Brundrett, M.C. (1991) Mycorrhizas in Natural Ecosystems. Advances in Ecological Research, 21, 171-313.
http://dx.doi.org/10.1016/S0065-2504(08)60099-9 [2] Allen, M.F. (1991) The Ecology of Mycorrhizae. Cambridge University Press, Cambridge. [3] Li, X.L. and Feng, G. (2001) Ecophysiology of Arbuscular Mycorrhizas. Huawen Press, Beijing. [4] Gianinazzi, S., Gollotte, A., Binet, M.N., van Tuinen, D., Redecker, D. and Wipf, D. (2010) Agroecology: The Key Role of Arbuscular Mycorrhizas in Ecosystem Services. Mycorrhiza, 20, 519-530.
http://dx.doi.org/10.1007/s00572-010-0333-3 [5] Oehl, F., Sieverding, E., Ineichen, K., Mader, P., Boller, T. and Wiemken, A. (2003) Impact of Land Use Intensity on the Species Diversity of Arbuscular Mycorrhizal Fungi in Agroecosystems of Central Europe. Applied and Environmental Microbiology, 69, 2816-2824.
http://dx.doi.org/10.1128/AEM.69.5.2816-2824.2003 [6] Cameron, D.D. (2010) Arbuscular Mycorrhizal Fungi as (Agro) Ecosystem Engineers. Plant and Soil, 333, 1-5.
http://dx.doi.org/10.1007/s11104-010-0361-y [7] Raviv, M. (2010) The Use of Mycorrhiza in Organically-Grown Crops under Semi arid Conditions: A Review of Benefits, Constraints and Future Challenges. Symbiosis, 52, 65-74.
http://dx.doi.org/10.1007/s13199-010-0089-8 [8] Smith, S.E. and Read, D.J. (2008) Mycorrhizal Symbiosis. 3rd Edition, Academic Press, London. [9] Gluszek, S., Sas Paszt, L., Sumorok, B. and Derkowska, E. (2008) Wplyw mikoryzy na wzrost i plonowanie roslin ogrodniczych. Postepy Nauk Rolniczych, Warszawa, 11-22. [10] Dalpé, Y. and Séguin, S.M. (2013) Microwave-Assisted Technology for the Clearing and Staining of Arbuscular Mycorrhizal Fungi in Roots. Mycorrhiza, 23, 333-340.
http://dx.doi.org/10.1007/s00572-012-0472-9 [11] Vierheilig, H., Schweiger, P. and Brundrett, M. (2005) An Overview of Methods for the Detection and Observation of Arbuscular Mycorrhizal Fungi in Roots. Physiologia Plantarum, 125, 393-404. [12] Brundrett, M.C. (2004) Diversity and Classification of Mycorrhizal Associations. Biological Reviews, 78, 473-495.
http://dx.doi.org/10.1017/S1464793103006316 [13] Phillips, J.M. and Hayman, D.A. (1970) Improved Procedures for Clearing Roots and Staining Parasitic and Vesicular-Arbuscular Mycorrhizal Fungi for Rapid Assessment of Infection. Transactions of the British Mycological Society, 55, 158-161.
http://dx.doi.org/10.1016/S0007-1536(70)80110-3 [14] Brundrett, M.C., Piché, Y. and Peterson, R.L. (1984) A New Method for Observing the Morphology of Vesicular-Arbuscular Mycorrhizae. Canadian Journal of Botany, 62, 2128-2134. [15] Schaffer, G.F. and Peterson, R.L. (1993) Modifications to Clearing Methods Used in Combination with Vital Staining of Roots Colonized with Vesicular-Arbuscular Mycorrhizal Fungi. Mycorrhiza, 4, 29-35.
http://dx.doi.org/10.1007/BF00203248 [16] Vierheilig, H., Coughlan, A.P., Wyss, U. and Piche, Y. (1998) Ink and Vinegar, a Simple Staining Technique for Arbuscular-Mycorrhizal Fungi. Applied and Environmental Microbiology, 64, 5004-5007. [17] Pitet, M., Camprubi, C., Calvet, C. and Estaun, V. (2009) A Modified Staining Technique for Arbuscular Mycorrhizal Compatible with Molecular Probes. Mycorrhiza, 19, 125-131.
http://dx.doi.org/10.1007/s00572-008-0206-1 [18] Arsenault, J.L., Poulcur, S., Messier, C. and Guay, R. (1995) WIN-RHIZO a Root-Measuring System with a Unique Overlap Correction Method. HortScience, 30, 906. [19] Turnau, K., Ryszka, P., Tuinen van, D. and Gianinazzi-Pearson, V. (2001) Identification of Arbuscular Mycorrhizal Fungi in Soils and Roots of Plants Colonizing Zinc Wastes in Southern Poland. Mycorrhiza, 10, 169-174.
http://dx.doi.org/10.1007/s005720000073 [20] Trouvelot, A., Kough, J.L. and Gianinazzi-Pearson, V. (1986) Mesure du taux de mycorhization VA d’un systeme radiculaire. Recherche de methods d’estimation ayant une signification fonctionnelle. In: Gianinazzi-Pearson V. and Gianinazzi, S., Eds, Physiological and Genetical Aspects of Mycorrhizae, INRA, Paris, 217-221. [21] Carmichael, J.W. (1955) Lacto-Fuchsin a New Medium for Mounting Fungi. Mycologia, 47, 611. [22] McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L. and Swan, J.A. (1990) A New Method Which Gives an Objective Measure of Colonization of Roots by Vesicular-Arbuscular Mycorrhizal Fungi. New Phytologist, 115, 495-501.
http://dx.doi.org/10.1111/j.1469-8137.1990.tb00476.x
[1] Brundrett, M.C. (1991) Mycorrhizas in Natural Ecosystems. Advances in Ecological Research, 21, 171-313.
http://dx.doi.org/10.1016/S0065-2504(08)60099-9 [2] Allen, M.F. (1991) The Ecology of Mycorrhizae. Cambridge University Press, Cambridge. [3] Li, X.L. and Feng, G. (2001) Ecophysiology of Arbuscular Mycorrhizas. Huawen Press, Beijing. [4] Gianinazzi, S., Gollotte, A., Binet, M.N., van Tuinen, D., Redecker, D. and Wipf, D. (2010) Agroecology: The Key Role of Arbuscular Mycorrhizas in Ecosystem Services. Mycorrhiza, 20, 519-530.
http://dx.doi.org/10.1007/s00572-010-0333-3 [5] Oehl, F., Sieverding, E., Ineichen, K., Mader, P., Boller, T. and Wiemken, A. (2003) Impact of Land Use Intensity on the Species Diversity of Arbuscular Mycorrhizal Fungi in Agroecosystems of Central Europe. Applied and Environmental Microbiology, 69, 2816-2824.
http://dx.doi.org/10.1128/AEM.69.5.2816-2824.2003 [6] Cameron, D.D. (2010) Arbuscular Mycorrhizal Fungi as (Agro) Ecosystem Engineers. Plant and Soil, 333, 1-5.
http://dx.doi.org/10.1007/s11104-010-0361-y [7] Raviv, M. (2010) The Use of Mycorrhiza in Organically-Grown Crops under Semi arid Conditions: A Review of Benefits, Constraints and Future Challenges. Symbiosis, 52, 65-74.
http://dx.doi.org/10.1007/s13199-010-0089-8 [8] Smith, S.E. and Read, D.J. (2008) Mycorrhizal Symbiosis. 3rd Edition, Academic Press, London. [9] Gluszek, S., Sas Paszt, L., Sumorok, B. and Derkowska, E. (2008) Wplyw mikoryzy na wzrost i plonowanie roslin ogrodniczych. Postepy Nauk Rolniczych, Warszawa, 11-22. [10] Dalpé, Y. and Séguin, S.M. (2013) Microwave-Assisted Technology for the Clearing and Staining of Arbuscular Mycorrhizal Fungi in Roots. Mycorrhiza, 23, 333-340.
http://dx.doi.org/10.1007/s00572-012-0472-9 [11] Vierheilig, H., Schweiger, P. and Brundrett, M. (2005) An Overview of Methods for the Detection and Observation of Arbuscular Mycorrhizal Fungi in Roots. Physiologia Plantarum, 125, 393-404. [12] Brundrett, M.C. (2004) Diversity and Classification of Mycorrhizal Associations. Biological Reviews, 78, 473-495.
http://dx.doi.org/10.1017/S1464793103006316 [13] Phillips, J.M. and Hayman, D.A. (1970) Improved Procedures for Clearing Roots and Staining Parasitic and Vesicular-Arbuscular Mycorrhizal Fungi for Rapid Assessment of Infection. Transactions of the British Mycological Society, 55, 158-161.
http://dx.doi.org/10.1016/S0007-1536(70)80110-3 [14] Brundrett, M.C., Piché, Y. and Peterson, R.L. (1984) A New Method for Observing the Morphology of Vesicular-Arbuscular Mycorrhizae. Canadian Journal of Botany, 62, 2128-2134. [15] Schaffer, G.F. and Peterson, R.L. (1993) Modifications to Clearing Methods Used in Combination with Vital Staining of Roots Colonized with Vesicular-Arbuscular Mycorrhizal Fungi. Mycorrhiza, 4, 29-35.
http://dx.doi.org/10.1007/BF00203248 [16] Vierheilig, H., Coughlan, A.P., Wyss, U. and Piche, Y. (1998) Ink and Vinegar, a Simple Staining Technique for Arbuscular-Mycorrhizal Fungi. Applied and Environmental Microbiology, 64, 5004-5007. [17] Pitet, M., Camprubi, C., Calvet, C. and Estaun, V. (2009) A Modified Staining Technique for Arbuscular Mycorrhizal Compatible with Molecular Probes. Mycorrhiza, 19, 125-131.
http://dx.doi.org/10.1007/s00572-008-0206-1 [18] Arsenault, J.L., Poulcur, S., Messier, C. and Guay, R. (1995) WIN-RHIZO a Root-Measuring System with a Unique Overlap Correction Method. HortScience, 30, 906. [19] Turnau, K., Ryszka, P., Tuinen van, D. and Gianinazzi-Pearson, V. (2001) Identification of Arbuscular Mycorrhizal Fungi in Soils and Roots of Plants Colonizing Zinc Wastes in Southern Poland. Mycorrhiza, 10, 169-174.
http://dx.doi.org/10.1007/s005720000073 [20] Trouvelot, A., Kough, J.L. and Gianinazzi-Pearson, V. (1986) Mesure du taux de mycorhization VA d’un systeme radiculaire. Recherche de methods d’estimation ayant une signification fonctionnelle. In: Gianinazzi-Pearson V. and Gianinazzi, S., Eds, Physiological and Genetical Aspects of Mycorrhizae, INRA, Paris, 217-221. [21] Carmichael, J.W. (1955) Lacto-Fuchsin a New Medium for Mounting Fungi. Mycologia, 47, 611. [22] McGonigle, T.P., Miller, M.H., Evans, D.G., Fairchild, G.L. and Swan, J.A. (1990) A New Method Which Gives an Objective Measure of Colonization of Roots by Vesicular-Arbuscular Mycorrhizal Fungi. New Phytologist, 115, 495-501.
http://dx.doi.org/10.1111/j.1469-8137.1990.tb00476.x