Abstract
Many studies have described the potential of root organ culture (ROC) to study the biology of arbuscular mycorrhizal (AM) fungi. The dual in vitro cultivation of AM fungi and ROC permits the establishment of the mycorrhizal symbiosis and the completion of fungal life cycle by producing new and infective propagules. However, some differences in AM fungal development are evident when growing in synthetic medium versus pot microcosms or in nature. We aimed to develop a new in vitro method that allows the study of AM symbiosis in a more natural environment, but under controlled conditions. This new culture method combines an artificial growth medium and differently sterilized soil samples for the cultivation of the AM fungal species Rhizophagus intraradices and Rhizophagus aggregatus. An extensive extraradical mycelia network and abundant viable spores, with similar morphological characteristics to those obtained under pot cultures developed under this in vitro soil-based cultivation system. Formation of sporocarp-like structures by R. aggregatus was also detected for the first time using this method.


References
Bago B, Cano C (2005) Breaking myths on arbuscular mycorrhizas in vitro biology. In: Declerck S, Strullu DG, Fortin A (eds) In vitro culture of mycorrhizas. Springer-Verlag Berlin Heidelberg, pp111–138
Bécard G, Fortin JA (1988) Early events of vesicular–arbuscular mycorrhiza formation on Ri T-DNA transformed roots. New Phytol 108:211–218
Bethlenfalvay GJ, Dakessian S, Pacovsky RS (1984) Mycorrhizae in a southern California desert: ecological implications. Canad J Bot 62:519–524
Bi Y, Li X, Wang H, Christie P (2004) Establishment of monoxenic culture between the arbuscular mycorrhizal fungus Glomus sinuosum and Ri T-DNA-transformed carrot roots. Plant Soil 261:239–244
Błaszkowski J, Czerniawska B, Zubek S, Turnau K (2008) Glomus intraradices and Pacispora robiginia, species of arbuscular mycorrhizal fungi (Glomeromycota) new for Poland. Acta Mycol 43(2):121–132
Buchan D, Moeskops B, Ameloot N, De Neve S, Sleutel S (2012) Selective sterilisation of undisturbed soil cores by gamma irradiation: effects on free-living nematodes, microbial community and nitrogen dynamics. Soil Biol Biochem 47:10–13
Colombo RP, Benavidez M, Fernandez Bidondo L, Silvani VA, Bompadre MJ, Statello M, Scorza MV, Martínez A, Scotti A, Godeas AM (2019) First molecular and morphological description of the arbuscular mycorrhizal fungal community growing in the extremely polluted soil of Riachuelo river basin. Revista Argentina de Microbiología Argentina https://doi.org/10.1016/j.ram.2019.05001
Dalpé Y, Declerck S (2002) Development of Acaulospora rehmii spore and hyphal swellings under root-organ culture. Mycologia 94:850–855
Debiane D, Calonne M, Fontaine J, Laruelle F, Grandmougin-Ferjani A, Lounes-HadjSahraoui A (2011) Lipid content disturbance in the arbuscular mycorrhizal, Glomus irregulare grown in monoxenic conditions under PAHs pollution. Fungal Biol 115:782–792
Declerck S, Strullu DG, Plenchette C (1998) Monoxenic culture of the intraradical forms of Glomus sp. isolated from a tropical ecosystem: a proposed methodology for germplasm collection. Mycologia 90:579–585
Declerck S, D’Or D, Bivort C, de Souza FA (2004) Development of extraradical mycelium of Scutellospora reticulata under root-organ culture: spore production and function of auxiliary cells. Mycol Res 108:84–92
Declerck S, Strullu DG, Fortin JA (2005) In vitro culture of mycorrhizas. Springer-Verlag, Berlin
Fortin JA, Becard G, Declerck S, Dalpe Y, St-Arnaud M, Coughlan AP, Piche Y (2002) Arbuscular mycorrhiza on root-organ cultures. Can J Bot 80:1–20
Hewitt EJ (1952) Sand and water culture methods in the study of plant nutrition,” Techn Commun 22, commonwealth agricultural bureau, Farnham Royal, UK
Koske RE (1985) Glomus aggregatum emended: a distinct taxon in the Glomus fasciculatum complex. Mycologia 77(4):619–630
Maldonado-Mendoza IE, Dewbre GR, Harrison MJ (2001) A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment. Mol Plant Microbe Interact 14:1140–1148
Marsh B (1971) Measurement of length in random arrangement of lines. J Appl Ecol 8:265
Mendoza RE, García IV, de Cabo L, Weigandt CF, Fabrizio de Lorio A (2015) The interaction of heavy metals and nutrients present in soil and native plants with arbuscular mycorrhizae on the riverside in the Matanza-Riachelo River basin (Argentina). J Sci Tot Env 505:555–564
Newman E (1966) A method of estimating the total length of roots in a sample. J Appl Ecol 2:139–145
Nielsen JS, Joner EJ, Declerck S, Olsson S, Jakobsen I (2002) Phospho-imaging as a tool for visualization and noninvasive measurement of P transport dynamics in arbuscular mycorrhizas. New Phytol 154:809–820
Pawlowska TE, Charvat I (2004) Heavy-metal stress and developmental patterns of arbuscular mycorrhizal Fungi. Appl Environ Microb 70(11):6643–6649
Pawlowska TE, Douds DD, Charvat I (1999) In vitro propagation and life cycle of the arbuscular mycorrhizal fungus Glomus etunicatum. Mycol Res 103(12):1549–1556
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
Raman N, Sahadevan C, Srinivaan V (2001) Growth of AM fungi on in vitro root organ culture of Sorghum vulgare and Saccharum officinarum. Indian J Exp Biol 39:1293–1298
Rillig MC, Steinberg PD (2002) Glomalin production by an arbuscular mycorrhizal fungus: a mechanism of habitat modification? Soil Biol Biochem 34:1371–1374
Rodrigues KM, Rodrigues BF (2015) Endomycorrhizal association of Funneliformis mosseae with transformed roots of Linum usitatissimum: germination, colonization, and sporulation studies. Mycology 6(1):42–49
Sharma AK, Singh C, Akhauri P (2000) Mass culture of arbuscular mycorrhizal Fungi and their role in biotechnology. Proc Indian Natl Sci Acad 66(4–5):223–238
Silvani VA, Fernández Bidondo L, Bompadre MJ, Pérgola M, Bompadre A, Fracchia S, Godeas AM (2014) Growth dynamics of geographically different arbuscular mycorrhizal fungal isolates belonging to the ‘Rhizophagus clade’ under monoxenic conditions. Mycologia 106(5):963–975
St-Arnaud M, Hamel C, Vimard B, Caron M, Fortin JA (1995) Altered growth of Fusarium oxysporum. Sp. chrysanthemi in an in vitro dual culture system with the vesicular arbuscular mycorrhizal fungus Glomus intraradices growing on Daucus carota transformed roots. Mycorrhiza 5:431–438
St-Arnaud M, Hamel C, Vimard B, Caron M, Fortin JA (1996) Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus Glomus intraradices in an in vitro system in the absence of host roots. Mycol Res 100:328–332
Zocco D, Fontaine J, Lozanova E, Renard L, Bivort C, Durand R, Grandmougin-Ferjani A, Declerck S (2008) Effects of two sterol biosynthesis inhibitor fungicides (fenpropimorph and fenhexamid) on the development of an arbuscular mycorrhizal fungus. Mycol Res 112:592–601
Acknowledgements
We acknowledge to Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Tecnológicas and Agencia Nacional de Promoción Científica y Tecnológica for the financial support. To Matías Benavidez and Fernández Bidondo Laura for providing soil samples from the riverside of Riachuelo River.
Author information
Authors and Affiliations
Corresponding author
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Silvani, V.A., Statello, M., Scorza, M.V. et al. A novel in vitro methodology to cultivate arbuscular mycorrhizal fungi combining soil and synthetic media. Symbiosis 79, 163–170 (2019). https://doi.org/10.1007/s13199-019-00637-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13199-019-00637-z