Abstract
An in vivo whole-plant bi-dimensional experimental system has been devised and tested with different host plants, in order to obtain extraradical mycelium (ERM) produced by different arbuscular mycorrhizal fungi (AMF). In this system, a host plant germling is inoculated with AMF to establish mycorrhizal symbiosis, and, after colonization, newly formed extraradical hyphae and spores are removed. Then the mycorrhizal root system is wrapped in a nylon net and placed between membranes in a Petri dish, allowing ERM to grow on the membrane surface. Such extraradical hyphae may be used for in situ morphometric analyses or collected for molecular or biochemical assays: in the latter case, the plant with its root sandwich may be reassembled to renew mycelium production. In this experimental system, which was tested with diverse host plant species and lines, values of explored membrane surface areas and densities of ERM showed wide ranges of variation, and its length ranged from 9.7 ± 2.0 to 48.8 ± 9.9 m per plant, depending on host and AMF identity. Across the different plant-AMF combinations tested, the whole-plant system produced 2.0 ± 0.6 to 5.3 ± 0.3 mg of ERM fresh biomass per plant per harvest. This experimental system can be used for a wide range of AMF and host plants species, either establishing arbuscular mycorrhizas or other mycorrhizal interactions. ERM produced and collected in the whole-plant system is suitable for morphological, physiological, and molecular analyses, facilitating studies on the different aspects of mycorrhizal symbiotic interactions.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Philippot L, Raaijmakers JM, Lemanceau P, van der Putten WH (2013) Going back to the roots: the microbial ecology of the rhizosphere. Nat Rev Microbiol 11:789–799
Avio L, Turrini A, Giovannetti M, Sbrana C (2018) Designing the ideotype mycorrhizal symbionts for the production of healthy food. Front Plant Sc 9:1089
Smith SE, Read DJ (2008) Mycorrhizal symbiosis, 3rd edn. Academic, San Diego
Clark RB, Zeto SK (2000) Mineral acquisition by arbuscular mycorrhizal plants. J Plant Nutr 23:867–902
Hodge A, Campbell CD, Fitter AH (2001) An arbuscular mycorrhizal fungus accelerates decomposition and acquires nitrogen directly from organic material. Nature 413:297
Smith SE, Jakobsen I, Grønlund M, Smith FA (2011) Roles of arbuscular mycorrhizas in plant phosphorus nutrition: interactions between pathways of phosphorus uptake in arbuscular mycorrhizal roots have important implications for understanding and manipulating plant phosphorus acquisition. Plant Physiol 156:1050–1057
Lehmann A, Rillig MC (2015) Arbuscular mycorrhizal contribution to copper, manganese and iron nutrient concentrations in crops–a meta-analysis. Soil Biol Biochem 81:147–158
Battini F, Grønlund M, Agnolucci M, Giovannetti M, Jakobsen I (2017) Facilitation of phosphorus uptake in maize plants by mycorrhizosphere bacteria. Sci Rep 7:4686
Pepe A, Giovannetti M, Sbrana C (2018) Lifespan and functionality of mycorrhizal fungal mycelium are uncoupled from host plant lifespan. Sci Rep 8:10235
Giovannetti M, Avio L, Sbrana C (2015) Functional significance of anastomosis in arbuscular mycorrhizal networks. In: Horton TR (ed) Mycorrhizal networks. Springer, Dordrecht, pp 41–67
López-Pedrosa A, Gonzalez-Guerrero M, Valderas A et al (2006) GintAMT1 encodes a functional high-affinity ammonium transporter that is expressed in the extraradical mycelium of Glomus intraradices. Fungal Genet Biol 43:102–110
Pérez-Tienda J, Testillano PS, Balestrini R et al (2011) GintAMT2, a new member of the ammonium transporter family in the arbuscular mycorrhizal fungus Glomus intraradices. Fungal Genet Biol 48:1044–1055
Calabrese S, Pérez-Tienda J, Ellerbeck M et al (2016) GintAMT3-a low-affinity ammonium transporter of the arbuscular mycorrhizal Rhizophagus irregularis. Front Plant Sci 7:679
González-Guerrero M, Azcón-Aguilar C, Mooney M et al (2005) Characterization of a Glomus intraradices gene encoding a putative Zn transporter of the cation diffusion facilitator family. Fungal Genet Biol 42:130–140
Tamayo E, Gómez-Gallego T, Azcón-Aguilar C, Ferrol N (2014) Genome wide analysis of copper, iron and zinc transporters in the arbuscular mycorrhizal fungus Rhizophagus irregularis. Front Plant Sci 5:547
Ferrol N, Azcón-Aguilar C, Pérez-Tienda J (2018) Arbuscular mycorrhizas as key players in sustainable plant phosphorus acquisition: an overview on the mechanisms involved. Plant Sci 280:441–447
Bago B, Cano C (2005) Breaking myths on arbuscular mycorrhizas in vitro biology. In: Declerck S, Strullu DG, Fortin A (eds) Vitro culture of mycorrhizas. Soil biology series, vol 4. Springer, Berlin, Heidelberg, New York, pp 111–138
Pepe A, Sbrana C, Ferrol N, Giovannetti M (2017) An in vivo whole-plant experimental system for the analysis of gene expression in extraradical mycorrhizal mycelium. Mycorrhiza 27:659–668
Giovannetti M, Sbrana C, Avio L, Strani P (2004) Patterns of belowground plant interconnections established by means of arbuscular mycorrhizal networks. New Phytol 164:175–181
de la Providencia IE, de Souza FA, Fernandez F et al (2005) Arbuscular mycorrhizal fungi exhibit distinct pattern of anastomoses formation and hyphal healing mechanism between different phylogenic groups. New Phytol 165:261–271
Pepe A, Giovannetti M, Sbrana C (2016) Different levels of hyphal self-incompatibility modulate interconnectedness of mycorrhizal networks in three arbuscular mycorrhizal fungi within the Glomeraceae. Mycorrhiza 26:325–332
Cárdenas-Flores A, Cranenbrouck S, Draye X et al (2011) The sterol biosynthesis inhibitor fenhexamid impacts the vegetative compatibility of Glomus clarum. Mycorrhiza 21:443–449
Purin S, Morton JB (2013) Anastomosis behaviour differs between asymbiotic and symbiotic hyphae of Rhizophagus clarus. Mycologia 12:589–602
Barreto de Novais C, Pepe A, Siqueira JO, Giovannetti M, Sbrana C (2017) Compatibility and incompatibility in hyphal anastomosis of arbuscular mycorrhizal fungi. Sci Agric 74:411–416
de Souza FA, Declerck S (2017) Mycelium development and architecture, and spore production of Scutellospora reticulata in monoxenic culture with Ri T-DNA transformed carrot roots. Mycologia 95:1004–1012
Ezawa T, Saito M, Yoshida T (1995) Comparison of phosphatase localization in the intraradical hyphae of arbuscular mycorrhizal fungi, Glomus spp. and Gigaspora spp. Plant Soil 176:57–63
Besserer A, Puech-Pagès V, Kiefer P et al (2006) Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria. PLoS Biol 4:e226
Sbrana C, Fortuna P, Giovannetti M (2011) Plugging into the network: belowground connections between germlings and extraradical mycelium of arbuscular mycorrhizal fungi. Mycologia 103:307–316
Acknowledgments
The financial support of the University of Pisa (Fondi di Ateneo) and of CNR is gratefully acknowledged.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Sbrana, C., Pepe, A., Ferrol, N., Giovannetti, M. (2020). A Whole-Plant Culture Method to Study Structural and Functional Traits of Extraradical Mycelium. In: Ferrol, N., Lanfranco, L. (eds) Arbuscular Mycorrhizal Fungi. Methods in Molecular Biology, vol 2146. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0603-2_3
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0603-2_3
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0602-5
Online ISBN: 978-1-0716-0603-2
eBook Packages: Springer Protocols