The characterization of mycorrhizal status in hosts can be a good indicator of symbiotic associations in inoculation experiments or in ecological research. The most common microscopic-based observation methods, such as (i) the gridline intersect method, (ii) the magnified intersections method and (iii) the five-class system of Trouvelot were tested to find the most simple, easily executable, effective and objective ones and their appropriate parameters for characterization of mycorrhizal status. In a pot experiment, white clover (Trifolium repens L.) host plant was inoculated with 6 (BEG144; syn. Rhizophagus intradices) in pumice substrate to monitor the AMF colonization properties during host growth. Eleven (seven classical and four new) colonization parameters were estimated by three researchers in twelve sampling times during plant growth. Variations among methods, observers, parallels, or individual plants were determined and analysed to select the most appropriate parameters and sampling times for monitoring. The comparability of the parameters of the three methods was also tested. As a result of the experiment classical parameters were selected for hyphal colonization: colonization frequency in the first stage or colonization density in the later period, and arbuscular richness of roots. A new parameter was recommended to determine vesicule and spore content of colonized roots at later stages of symbiosis.
Brundrett, M.: Mycorrhizal associations and other means of nutrition of vascular plants: Understanding the global diversity of host plants by resolving conflicting information and developing reliable means of diagnosis. Plant Soil 320(1–2), 37–77 (2009).
Smith, S. E., Read, D.: Mycorrhizal Symbiosis, 2nd edn. Academic Press, London (1997).
Arora, G. K., Rai, B., Mukerji, K. G., Knudsen, G. R.: Handbook of Applied Mycology, Vol. 1: Soil and Plants. Marcel Dekker Inc, New York, pp. 720 (1991).
Turnau, K., Haselwandter, K.: Arbuscular mycorrhizal fungi, an essential component of soil microflora in ecosystem restoration. In Gianinazzi, H., Schüepp, H., Barea, J. M., Haselwandter, K. (eds): Micorrhizal Technology in Agriculture from Genes to Bioproducts. Springer Birkhäuser, Switzerland, pp. 137–149 (2002).
Marschner, H.: The soil-root interface (rhizosphere) in relation to mineral nutrition. In Marschner, H. (ed): Mineral Nutrition of Higher Plants. Academic Press, London, pp. 537–594 (1997).
Augé, R. M.: Water relations, drought, and vesicular-arbuscular mycorrhizal symbiosis. Mycorrhiza 11, 3–42 (2001).
Birhane, E., Sterck, F. J., Fetene, M., Bongers, F., Kuyper, T. W.: Arbuscular mycorrhizal fungi enhance photosynthesis, water use efficiency, and growth of frankincense seedlings under pulsed water availability conditions. Oecologia 169(4), 895–904 (2012).
Evelin, H., Kapoor, R., Giri, B.: Arbuscular mycorrhizal fungi in alleviation of salt stress: A review. Ann Bot 104(7), 1263–1280 (2009).
Füzy, A., Biró, B., Tóth, T., Hildebrandt, U., Bothe, H.: Drought, but not salinity, determines the apparent effectiveness of halophytes colonized by arbuscular mycorrhizal fungi. J Plant Physiol 165, 1181–1192 (2008).
Ruiz-Lozano, J. M., Aroca, R.: Host response to osmotic stresses: Stomatal behaviour and water use efficiency of arbuscular mycorrhizal plants. In Koltai, H., Kapulnik, Y. (eds): Arbuscular Mycorrhizas: Physiology and Function. Springer, Berlin, pp. 239–256 (2010).
Giovannetti, M., Sbrana, C.: Meeting a non-host: The behaviour of AM fungi. Mycorrhiza 8, 123–130 (1998).
Barea, J. M., Pozo, M. J., Azcón, R., Azcón-Aguilar, C.: Microbial co-operation in the rhizosphere. J Exp Bot 56(417), 1761–1778 (2005).
Gianinazzi, S., Vosatka, M.: Inoculum of arbuscular mycorrhizal fungi for production systems, science meets business. Can J Bot 82(8), 1264–1271 (2004).
Vierheilig, H., Schweiger, P., Brundrett, M.: An overview of methods for the detection and observation of arbuscular mycorrhizal fungi in roots. Physiol Plant 125, 393–404 (2005).
Cseresnyés, I., Takács, T., Végh, K. R., Anton, A., Rajkai, K.: Electrical impedance and capacitance method: A new approch for detection of functional aspects of arbuscular mycorhrizal colonization in maize. European Journal of Soil Biology 54, 25–31 (2013).
Takács, T., Füzy, A., Rajkai, K., Cseresnyés, I.: Investigation of arbuscular mycorrhizal status and functionality by electrical impedance and capacitance measurement: New method for description of AMF functionality. Acta Biologica Szegediensis 58(1), 55–59. (2014).
Phillips, J. M., Hayman, D.S.: Improved procedures for clearing roots and staining parasitic and VAM fungi for rapid assessment of infection. Trans Brit Mycol Soc 55, 158–161 (1970).
Byrd, D. W., Kirkpatrick, T., Barker, K. R.: An improved technique for clearing and staining plant tissues for the detection of nematodes. J Nematol 15, 142–143 (1983).
Kormanik, P. P., McGraw, A. C.: Quantification of vesicular-arbuscular mycorrhiza in plant roots. In Schenck, N. C. (ed): Methods and Principles in Mycorrhizal Research. Am Phytopathol Soc, St Paul, Minn., pp. 37–45 (1982).
Brundrett, M.: Estimation of root length and colonization by mycorrhizal fungi. In Brundrett, M., Melville, L., Peterson, L. (eds): Practical Methods in Mycorrhiza Research. Mycologue Publications, Guelph, pp. 51–59 (1994).
Grace, C., Stribley, D. P.: A safer procedure for routine staining of vesicular-arbuscular mycorrhizal fungi. Mycol Res 95, 1160–1162 (1991).
Ambler, J. M., Young, J. L.: Techniques for determining root length infected by vesiculararbuscular mycorrhizae. Soil Sci Soc Am J 41, 551–556 (1977).
Biermann, B., Linderman, R. G.: Quantifying vesicular arbuscular mycorrhizae: A proposed method towards standardization. New Phytol 87, 63–67 (1981).
Brundrett, M. C., Piché, Y., Peterson, R. L.: A new method for observing the morphology of vesicular-arbuscular mycorrhizae. Can J Bot 62, 2128–2134 (1984).
Giovannetti, M., Mosse, B.: An evaluation of techniques for measuring vesicular-arbuscular mycorrhizal infection in roots. New Phytol 84, 489–500 (1980).
Hayman, D. S., Azcón, R., Barea, J. M.: Vesicular-arbuscular mycorrhizae in Southern Spain: Its distribution in crops growing in soil of different fertility. Phytopathol Mediterr 15, 1–6 (1976).
Li, A. R., Guan, K. Y.: Mycorrhizal and dark septate endophytic fungi of Pedicularis species from northwest of Yunnan Province, China. Mycorrhiza 17, 103–109 (2007).
McGonigle, T. P., Miller, M. H., Evans, D. G., Fairchild, G. L., Swan, J. A.: A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytol 115, 495–501 (1990).
Morandi, D., Prado, E., Segan, M., Duc, G.: Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants. Mycorrhiza 15, 283–289 (2005).
Newman, E. I.: A method of estimating the total length of root in a sample. J Appl Ecol 3, 139–145 (1966).
Plenchette, C., Morel, C.: External phosphorus requirement of mycorrhizal and nonmycorrhizal barley and soybean plants. Biol Fertil Soils 21, 303–308 (1996).
Tennant, D.: A test of modified line intersect method of estimating root length. J Ecol 63, 995–1001 (1975).
Trouvelot, A., Kough, J. L., Gianinazzi-Pearson, V.: Mesure du taux de mycorhization VA d’un systeme radiculaire. Recherche de methodes d’estimation ayant une significantion fonctionnelle. In Gianinazzi-Pearson, V., Gianinazzi, S. (eds): Physiological and Genetic Aspects of Mycorrhizae. INRA, Paris, pp. 217–221 (1986).
Öpik, M., Moora, M., Liira, J., Zobel, M.: Composition of root-colonizing arbuscular mycorrhizal fungal communities in different ecosystems around the globe. J Ecol 94, 778–790 (2006).
Kliromonos, J. N., Rillig, M. C., Allen, M. F.: Designing belowground field experiments with the help of semi-variance and power analyses. Appl Soil Ecol 12, 227–238 (1999).
Johnson, J. M. F., Morgan, J.: Plant sampling guidelines. In Follett, R. F. (ed.): Sampling Protocols. USDA, pp. 1–10 (2010).
Taylor, H. M.: Methods of studying root systems in the field. Hort Sci 21, 952–956 (1986).
Estaún, V., Calvet, C., Camprubí, A.: Effect of differences among crop species and cultivars on the arbuscular mycorrhizal symbiosis. In Koltai, H., Kapulnik, Y. (eds): Arbuscular Mycorrhizas: Physiology and Function. Springer, Netherlands, pp. 279–295 (2010).
Smith, K. P. and Goodman, R. M.: Host variation for interactions with beneficial plantassociated microbes. Annu Rev Phytopathol 37, 473–491 (1999).
Reich, L., Barnard, J.: Sampling strategies for mycorrhizal research. New Phytol 98, 475–479 (1984).
Rajapakse, S., Miller, J. C. J.: Methods for studying vesicular-arbuscular mycorrhizal root colonization and related root physical properties. In Norris, J. R., Read, D. J., Varma, A. K., (eds): Methods in Microbiology. Volume 24: Techniques for the Study of Mycorrhiza. Academic Press, London, pp. 301–316 (1992).
Utobo, E. B., Ogbodo, E. N., Nwogbaga, A. C.: Techniques for extraction and quantification of arbuscular mycorrgizal fungi. Libyan Agric Res Cent J Int 2, 68–78 (2011).
Bonfante-Fasolo, P.: Anatomy and morphology of VA mycorrhizae. In Powell, C. L., Bagyaraj, D. J. (eds): VA Mycorrhiza. CRC Press, Boca Raton, pp. 5–33 (1984).
Cox, G., Tinker, P. B.: Translocation and transfer of nutrients in vesicular-arbuscular mycorrhizas. I. The arbuscule and phophosrus transfer: A quantitative ultratructural study. New Phytol 77, 371–378 (1976).
Escandari, A., Danesh, Y. R.: Study on life cycle of arbuscular mycorrhizal fungus Glomus intraradices using in vitro culturing technique. J Phytol 2, 69–75 (2010).
Giovannetti, M.: Spore germination and pre-symbiotic mycelia growth. In Kapulnik, Y., Douds, D. D. (eds): Arbuscular Mycorrhizas: Physiology and Function. Kluwer Academic Publishers, Dordrecht, pp. 47–68 (2000).
Dickson, S.: The Arum-Paris continuum of mycorrhizal symbioses. New Phytol 163, 187–200 (2004).
Kubota, M., McGonigle, T. P., Hyakumachi, M.: Co-occurrence of Arum-and Paris-type morphologies of arbuscular mycorrhizae in cucumber and tomato. Mycorrhiza 15, 73–77 (2005).