Isaac Scientific Publishing

Annals of Advanced Agricultural Sciences

Development of Carrier Based in Vitro Produced Arbuscular Mycorrhizal (AM) Fungal Inocula for Organic Agriculture

Download PDF (291.4 KB) PP. 26 - 37 Pub. Date: August 14, 2017

DOI: 10.22606/as.2017.11004


  • Rodrigues Kim Maria*
    Department of Botany, Goa University, Taleigao Plateau, Goa 403 206 India
  • Rodrigues Bernard Felinov
    Department of Botany, Goa University, Taleigao Plateau, Goa 403 206 India


Studies on the advantageous effects of arbuscular mycorrhizal (AM) fungi are providing new possible ways to exploit them as biofertilizers in sustainable agriculture. Many studies have described the potential of root organ culture (ROC) system for production of AM fungal inocula. However there is a need for development of a suitable carrier formulation to support in vitro produced AM fungal inocula when mixed with substrate, so as to enable the delivery of inocula in the rhizosphere. The aim of this study was to assess the performance of the organic carrier formulation consisting of vermiculite as the main component along with cattle manure, wood powder and wood ash in different proportions; and its ability to retain inoculum potential of the in vitro produced AM fungal propagules of Rhizoglomus intraradices and Funneliformis mosseae. Treatment 5 comprising of carrier formulation (vermiculite: cow dung powder: wood powder: wood ash) in the ratio of 20:8:2:1 was observed to be as the best carrier treatment for both the in vitro produced AM species. The in vitro produced propagules of both AM species were viable and effectively colonized the roots of Eleusine coracana Gaertn. The method established shows the efficiency of the carrier formulation in sustaining the inoculum potential of in vitro produced AM propagules for mass multiplication and possibility in application.


In vitro, AM fungal propagules, carrier formulation, inoculum.


[1] D. Owen, A. P. Williams, G. W. Griffith, and P. J. A. Withers, “Use of commercial bio-inoculants to increase agricultural production through improved phosphorus acquisition,” Applied Soil Ecology, vol. 86, pp. 41–54, 2015.

[2] S. E. Smith and D. J. Read, Mycorrhizal Symbiosis. Academic Press, London, 2008.

[3] M. W. Schwartz, J. D. Hoeksema, C. A. Gehring, N. C. Johnson, J. N. Klironomos, L. K. Abbott, and A. Pringle, “The promise and the potential consequences of the global transport of mycorrhizal fungal inoculum,” Ecology Letters, vol. 9, pp. 501–515, 2006.

[4] D. D. Douds, V. Gadkar, and A. Adholeya, “Mass production of VAM fungus biofertilizer,” in Mycorrhizal Biology. Kluwer Academic Press, New York, 2000, pp. 197–215.

[5] S. Singh, “Mass production of AM fungi: Part 1,” Mycorrhiza News, vol. 14, pp. 2–9, 2002.

[6] S. Gianinazzi and M. Vosátka, “Inoculum of arbuscular mycorrhizal fungi for production systems: science meets business,” Canadian Journal of Botany, vol. 82, pp. 1264–1271, 2004.

[7] G. Bécard and J. A. Fortin, “Early events of vesicular-arbuscular mycorrhiza formation on Ri T-DNA transformed roots,” New Phytologist, vol. 108, pp. 211–218, 1988.

[8] D. D. Douds, G. Nagahashi, and P. R. Hepperly, “On-farm production of inoculum of indigenous arbuscular mycorrhizal fungi and assessment of diluents of compost for inoculum production,” Bioresource Technology, vol.101, pp. 2326–2330, 2010.

[9] C. Accinelli, S. M. Ludovica, H. K. Abbas, R. M. Zablotowicz, and J. R. Wilkinson, “Use of a granular bioplastic formulation for carrying conidia of a non-aflatoxigenic strain of Aspergillus flavus,” Bioresource Technology, vol.100, pp. 3997–4004, 2009.

[10] G. P. Brahmaprakash and P. K. Sahu, “Biofertilizers for Sustainability,” Journal of Indian Institute of Science, vol. 92, pp. 37–62, 2012.

[11] J. H. G. Stephens and H. M. Rask, “Inoculant production and formulation,” Field Crops Research, vol. 65, pp. 249–258, 2000.

[12] F. B. Rebah, R. D. Tyagi, and D. Prevost, “Wastewater sludge as a substrate for growth and carrier for rhizobia: the effect of storage conditions on survival of Sinorhizobium meliloti,” Bioresource Technology, vol. 83, no. 2, pp.145–151, 2002.

[13] M. C. Rivera-Cruz, A. T. Narcía, G. C. Ballona, J. Kohler, F. Caravaca, and A. Roldán, “Poultry manure and banana waste are effective biofertilizer carriers for promoting plant growth and soil sustainability in banana crops,” Soil Biology and Biochemistry, vol. 40, no. 12, pp. 3092–3095, 2008.

[14] B. C. Mallesha, D. J. Bagyaraj, and G. Pai, “Perlite-soilrite mix as a carrier for mycorrhiza and rhizobia to inoculate Leucaena leucocephala,” Leucaena Research Reports, vol. 13, pp. 32–33, 1992.

[15] D. Redecker, H. Thierfelder, and D. Werner, “A new cultivation system for arbuscular mycorrhizal fungi on glass beads,” Angewandte Botanik, vol. 69, pp. 189–191, 1995.

[16] Y. Bashan, “Inoculants of plant growth-promoting bacteria for use in agriculture,” Biotechnology Advances, vol. 16, no. 4, pp. 729–770, 1998.

[17] A. Gaur and A. Adholeya, “Effects of the particle of soil-less substrates upon AM fungus inoculum production,” Mycorrhiza, vol. 10, no. 1, pp. 43–48, 2000.

[18] D. F. Herridge, M. B. Peoples, and R. M. Boddey, “Global inputs of biological nitrogen fixation in agricultural systems,” Plant and Soil, vol. 311, no. 1, pp. 1–18, 2008.

[19] E. Malusá, L. Sas-Paszt, and J. Ciesielska, “Technologies for beneficial microorganisms inocula used as biofertilizers,” Scientific World Journal, pp. 2012:491206, 2012.

[20] L. Herrmann and D. Lesueur, “Challenges of formulation and quality of biofertilizers for successful inoculation,” Applied Microbiology and Biotechnology, vol. 97, no. 20, pp. 8859–8873, 2013.

[21] H. Wang, L. Shen, L. Zhai, J. Zhang, T. Ren, B. Fan, and H. Liu, “Preparation and utilization of phosphate biofertilizers using agricultural waste,” Journal of Integrative Agriculture, vol. 14, no. 1, pp. 158–167, 2015.

[22] R. P. John, R. D. Tyagi, S. K. Brar, R. Y. Surampalli, and D. Prevost, “Bio-encapsulation of microbial cells for targeted agricultural delivery,” Critical Reviews in Biotechnology, vol. 31, no. 3, pp. 211–226, 2011.

[23] J. W. Gerdemann and T. H. Nicolson, “Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting,” Transactions of the British Mycological Society, vol. 46, no. 2, pp. 235–244, 1963.

[24] B. Mosse, “The regular germination of resting spores and some observations on the growth requirements of an Endogone sp. causing vesicular-arbuscular mycorrhiza,” Transactions of the British Mycological Society, vol. 42, no. 3, pp. 273–286, 1959.

[25] S. Declerck, D. G. Strullu, and C. Plenchette, “Monoxenic culture of the intraradical forms of Glomus sp. isolated from a tropical ecosystem: a proposed methodology for germplasm collection,” Mycologia, vol. 90, no. 4, pp. 579–585, 1998.

[26] S. Cranenbrouck, L. Voets, C. Bivort, L. Renard, D. G. Strullu, and S. Declerck, “Methodologies for in vitro cultivation of arbuscular mycorrhizal fungi with root-organs,” in In vitro culture of mycorrhizas. Springer-Verlag, Heidelberg, 2005, pp. 341–375.

[27] A. Walkley and J. A. Black, “An examination of the Degtjareff method for determining soil organic matter and a proposed modification of the chromic titration method,” Soil Science, vol. 37, no. 1, pp. 29–38, 1934.

[28] R. H. Bray and L. T. Kurtz, “Determination of total, organic and available forms of phosphorus in soils,” Soil Science, vol. 59, no. 1, pp. 39–46, 1945.

[29] J. J. Hanway and H. Heidel, “Soil analysis method as used in Iowa State College Soil Testing Laboratory,” Iowa Agriculture, vol. 57, pp. 1–31, 1952.

[30] W. L. Lindsay and W. A. Norvell, “Development of DTPA soil test for zinc, iron, manganese and copper,” Soil Science Society of America Journal, vol. 42, no. 3, pp. 421–428, 1978.

[31] K. C. Berger and E. Truog, “Boron determination in soils and plants,” Industrial and Engineering Chemistry Analytical Edition, vol. 11, no. 10, pp. 540–545, 1939.

[32] D. R. Hoagland and D. I. Arnon, “The water-culture method for growing plants without soil. Berkeley, Calif. : University of California, College of Agriculture, Agricultural Experiment Station,” Circular 347, 1950.

[33] J. M. Phillips and D. S. Hayman, “Improved procedures for clearing roots and staining parasitic and vesiculararbuscular mycorrhizal fungi for rapid assessment of infection,” Transactions of the British Mycological Society, vol. 55, no. 1, pp. 158–161, 1970.

[34] Ministry of Agriculture, Government of India, “Biofertilizers and organic fertilizers covered in fertilizer (Control) Order, 1985 (as amended, March 2006 and November 2009),” Official Gazette 3 November, 2009.

[35] D. J. Read, H. K. Koucheki, and J. Hodgson, “Vesicular-arbuscular mycorrhiza in natural vegetation systems. I. The occurrence of infection,” New Phytologist, vol. 77, no. 3, pp. 641–653, 1976.

[36] A. Adholeya, “Commercial production of AMF through industrial mode and its large scale application.” Proceedings of the 4th international conference Mycorrhizae (ICOM4), Montreal, Canada, 2003.

[37] Y. Okon and H. Hadar, “Microbial inoculants as crop-yield enhancers,” Critical Reviews in Biotechnology, vol. 6, no. 1, pp. 61–85, 1987.

[38] M. St-Arnaud, C. Hamel, B. Vimard, M. Caron, and J. A. Fortin, “Enhanced hyphal growth and spore production of the arbuscular mycorrhizal fungus Glomus intraradices in an in vitro system in the absence of host roots,” Mycological Research, vol. 100, no. 3, pp. 328–332, 1996.

[39] Y. Dalpé and M. Monreal, “Arbuscular mycorrhiza inoculum to support sustainable cropping systems,” Online Crop Management 3, 2004.

[40] A. Adholeya, P. Tiwari, and R. Singh, “Large-scale inoculum production of arbuscular mycorrhizal fungi on root organs and inoculation strategies,” in In vitro culture of mycorrhizas. Springer, Heidelberg, 2005, pp. 315–338.

[41] A. Gaur and A. Adholeya, “Arbuscular-mycorrhizal inoculation of five tropical fodder crops and inoculum production in marginal soil amended with organic matter,” Biology and Fertility of Soils, vol. 35, no. 3, pp. 214– 218, 2002.

[42] M. Gryndler, H. Hrselová, R. Sudová, H. Gryndlerová, V. Rezácová, and V. Merhautová, “Hyphal growth and mycorrhiza formation by the arbuscular fungus Glomus claroideum BEG23 is stimulated by humic substances,” Mycorrhiza, vol. 15, no. 7, pp. 483–488, 2005.

[43] H. Perner, D. Schwarz, and E. George, “Effects of mycorrhizal inoculation and compost supply on growth and nutrient uptake of young leek plants grown in peat-based substrates,” HortScience, vol. 41, no. 3, pp. 628–632, 2006.

[44] K. Saranya and K. Kumutha, “Standardization of the substrate material for large scale production of arbuscular mycorrhizal inoculum,” International Journal of Agriculture Sciences, vol. 3, no. 1, pp. 71–77, 2011.

[45] A. Tanwar, A. Aggarwal, A. Yadav, and V. Parkash, “Screening and selection of efficient host and sugarcane bagasse as substrate for mass multiplication of Funneliformis mosseae,” Biological Agriculture and Horticulture, vol. 29, no. 2, pp. 107–117, 2013.

[46] A. Gaur and A. Adholeya, “Diverse response of five ornamental plant species to mixed indigenous and single isolate arbuscular mycorrhizal inocula in marginal soil amended with organic matter,” Journal of plant nutrition, vol. 28, no. 4, pp. 707–723, 2005.

[47] F. S. B. Silva, A. M. Yano-Melo, J. A. C. Brand?o, and L. C. Maia, “Sporulation of arbuscular mycorrhizal fungi using Tris-HCl buffer in addition to nutrient solutions,” Brazilian Journal of Microbiology, vol. 36, no. 4, pp. 327–332, 2005.

[48] D. D. Jr. Douds, G. Nagahashi, P. E. Pfeffer, C. Reider, and W. M. Kayser, “On-farm production of AM fungus inoculum in mixture of compost and vermiculite,” Bioresource Technology, vol. 97, no. 6, pp. 809–818, 2006.

[49] I. R. Coelho, M. V. L. Pedone-Bonfim, F. S. B. Silva, and L. C. Maia, “Optimization of the production of mycorrhizal inoculum on substrate with organic fertilizer,” Brazilian Journal of Microbiology, vol. 45, no. 4, pp. 1173–1178, 2014.

[50] N. S. Bolan and A. D. Robson, “Increasing phosphorus supply can increase the infection of plant roots by vesicular-arbuscular mycorrhizal fungi,” Soil Biology and Biochemistry, vol. 16, pp. 419–420, 1984.

[51] F. S. B. Silva, “Fase assimbiótica, produ??o, infectividade e efetividade de fungos micorrízicos em substratos com adubos organicos [PhD Thesis],” Centro de Ciências Biológicas, Recife; pp. 297, 2006.

[52] A. Gildon and P. B. Tinker, “A heavy metal-tolerant strain of a mycorrhizal fungus,” Transactions of the British Mycological Society, vol. 77, pp. 648–649, 1981.

[53] F. M. S. Moreira and J. O. Siqueira, “Micorrizas,” in Microbiologia e Bioquímica do Solo. UFLA, Lavras, 2002, pp. 473–539.

[54] M. Luis, I. Carvalho, M. Cacador, and A. Martins-Loucao, “Arbuscular mycorrhizal fungi enhance root cadmium and copper accumulation in the roots of the salt marsh plant Aster tripolium L.,” Plant and Soil, vol. 285, no. 1, pp. 161–169, 2006.

[55] I. Ortas and C. Akpinar, “Response of kidney bean to arbuscular mycorrhizal inoculation and mycorrhizal dependency in P and Zn deficient soils,” Acta Agriculturae Scandinavica, Section B – Plant and Soil Science, vol. 56, pp. 101–109, 2006.

[56] S. V. Motha, H. Amballa, and N. R. Bhumi, “Arbuscular mycorrhizal fungi associated with rhizosphere soils of brinjal cultivated in Andhra Pradesh, India,” International Journal of Current Microbiologyand Applied Sciences, vol. 3, no. 5, pp. 519–529, 2014.

[57] S. E. Smith and D. J. Read, Mycorrhizal symbiosis. Academic Press, San Diego, 1997.

[58] H. Vierheilig, B. Bago, C. Albrecht, M. J. Poulin, and Y. Piche, “Flavonoids and arbuscular mycorrhizal fungi,” in Flavonoids in the living system. Plenum, New York, 1998a, pp. 9–33.

[59] C. Sbrana and M. Giovanetti, “Chemotropism in the arbuscular mycorrhizal fungus Glomus mosseae,” Mycorrhiza, vol. 15, no. 7, pp. 539–545, 2005.

[60] J. M. Scervino, M. A. Ponce, R. Erra-Bassells, H. Vierheilig, J. A. Ocampo, and A. Godeas, “Flavonoids exhibit fungal species and genus specific effects on the presymbiotic growth of Gigaspora and Glomus,” Mycological Research, vol. 109, no. 7, pp. 789–794, 2005b.

[61] J. M. Scervino, M. A. Ponce, R. Erra-Bassells, J. Bompadre, H. Vierheilig, J. A. Ocampo, and A. Godeas, “The effect of flavones and flavonols on colonization of tomato plants by arbuscular mycorrhizal fungi of the genera Gigaspora and Glomus,” Canadian Journal of Microbiology, vol. 53, pp. 702–709, 2007.