Development of liquid rhizobial inoculants and pre-inoculation of alfalfa seeds



rhizobia, pre-inoculation, alfalfa seeds, liquid inoculants, Sinorhizobium meliloti


Paper description:

  • Inoculation of legume seeds with symbiotic nitrogen-fixing bacteria, rhizobia, is an efficient mechanism for improving the nitrogen content and legume growth. The use of pre-inoculated seeds simplifies sowing in the field and reduces the cost of cultivation.
  • We used the following additives: agar, sodium-alginate, calcium chloride, glycerol and ferric chloride as supplements for alfalfa rhizobium (Sinorhizobium meliloti L3Si) survival in liquid inoculants as the final commercial products, and for pre-inoculation of seeds and one month storage.
  • The liquid rhizobial inoculants were successful for inoculation and pre-inoculation of alfalfa seeds and improved the yield and nitrogen content in alfalfa plants.

Abstract: Application of liquid microbial inoculants on legume seeds is a sustainable agricultural practice that can improve plant nutrient uptake and increase crop productivity. Inoculants should provide long-term survival of rhizobia in the final product and after application, to legume seeds. Ten different medium formulations of microbial inoculants were examined (yeast mannitol broth with the addition of agar, sodium-alginate, calcium chloride, glycerol or ferric chloride and combinations thereof) for the survival of the efficient nitrogen-fixing rhizobium, Sinorhizobium (Ensifer) meliloti L3Si strain. The most suitable liquid inoculant for survival of L3Si during a storage time of 150 days was the medium formulation containing glycerol in combination with agar or sodium-alginate. Alfalfa seeds were pre-inoculated with four formulations (yeast mannitol broth (YMB), YMB with agar (1 g L-1), YMB with 1 or 5 g L-1 sodium-alginate) for up to three months. Seeds pre-inoculated and stored for one month produced successful alfalfa plants. The nitrogen content in alfalfa obtained from pre-inoculated seeds one month before sowing was adequate and ranged from 3.72-4.19%. Using S. meliloti-based liquid inoculants for alfalfa and application of the pre-inoculation technique can increase the quality of alfalfa crops and reduce cultivation cost.

Received: October 8, 2018; Revised: December 3, 2018; Accepted: December 20, 2018; Published online: December 27, 2018

How to cite this article: Buntić AV, Stajković-Srbinović OS, Knežević MM, Kuzmanović ĐŽ, Rasulić NV, Delić DI. Development of liquid rhizobial inoculants and pre-inoculation of alfalfa seeds. Arch Biol Sci. 2019;71(2):379-87.


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Author Biography

Olivera S. Stajković-Srbinović, Institute of Soil Science, Department of Microbiology, University of Belgrade, Teodora Drajzera 7, 11000, Belgrade


Young CC, Rekha PD, Lai WA, Arun AB. Encapsulation of plant growth‐promoting bacteria in alginate beads enriched with humic acid. Biotechnol Bioeng. 2006;95(1):76-83.

Li Z, Zhang H. Application of microbial fertilizers in sustainable agriculture. J Crop Prot. 2001:3(1):337-47.

Dresler-Nurmi A, Fewer DP, Räsänen LA, Lindström K. The Diversity and Evolution of Rhizobia. In: Pawlowski K, editor. Prokaryotic Symbionts in Plants. Vol 8. Berlin, Heidelberg: Springer; 2007. p. 3-41.

Biswas B, Gresshoff PM. The role of symbiotic nitrogen fixation in sustainable production of biofuels. Int J Mol Sci. 2014:15(5):7380-97.

Galleguillos C, Aguirre C, Barea JM, Azcon R. Growth promoting effect of two Sinorhizobium meliloti strains (a wild type and its genetically modified derivative) on a non-legume plant species in specific interaction with two arbuscular mycorrhizal fungi. Plant Sci. 2000;159(1):57-63.

Singleton P, Keyser H, Sande E. Development and evaluation of liquid inoculants. In: Herridge D, editor. Proceedings of a workshop held in Hanoi; 2001 Feb 17-18; Vietnam: Canberra; 2002. p. 52-66.

Biswas PK, Bhowmick MK. Effect of liquid and carrier based Rhizobium inoculants on growth, nodulation and seed yield of urdbean. J Crop Weed. 2007;3(2):7-9.

Tlepov A, Dzhaparov R, Akhmetov E. Nitrogen accumulation in chickpea organs by isotope indication and influence of mineral fertilizers and biopreparates on its productivity in Ural region of Kazakhstan. Soil Plant. 2017;66(2):9-66.

Rebah FB, Tyagi RD, Prevost D. Wastewater sludge as a substrate for growth and carrier for rhizobia: the effect of storage conditions on survival of Sinorhizobium meliloti. Bioresource Technol. 2002;83(2):145-51.

Buntić A, Stajković-Srbinović O, Rasulić N, Kuzmanović Dj, Delić D, Dimitrijević-Branković S. Influence of spray drying technique on survival of Bradyrhizobium onto sodium alginate based carriers. Soil Plant. 2015;64(2):9-16.

Bashan Y, de-Bashan LE, Prabhu SR, Hernandez JP. Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013). Plant Soil. 2014:378(1-2):1-33.

Bashan Y. Inoculants of plant growth-promoting bacteria for use in agriculture. Biotechnol Adv. 1998:16(4):729-770.

Deaker R, Hartley E, Gemell G. Conditions affecting shelf-life of inoculated legume seed. Agriculture. 2012;2(1):38-51.

Zommere Ž, Nikolajeva V. Immobilization of bacterial association in alginate beads for bioremediation of oil-contaminated lands. Environ Exp Bot. 2017:15:105-11.

Catroux G, Hartmann A, Revellin C. Trends in rhizobial inoculant production and use. Plant Soil. 2001;230(1):21-30.

Hartley EJ, Gemell LG, Deaker R. Some factors that contribute to poor survival of rhizobia on preinoculated legume seed. Crop Pasture Sci. 2013;63(9):858-65.

Leo-Daniel AE, Vanketeswarlu B. Suseelendra D. Praveen-Kumar G. Mirhassanahmad SK, Meenakshi T. Effect of polymeric additives, adjuvants, surfactants on survival, stability and plant growth promoting ability of liquid bioinoculants. J Plant Physiol Pathol. 2013:1(2):1-5.

Lee SK, Lur HS, Lo KJ, Cheng KC, Chuang CC, Tang SJ, Yang ZW, Liu CT. Evaluation of the effects of different liquid inoculant formulations on the survival and plant-growth-promoting efficiency of Rhodopseudomonas palustris strain PS3. Appl Microbiol Biot. 2016:100(18):7977-87.

Temprano FJ, Albareda M, Camacho M, Daza A, Santamaría C. Rodríguez-Navarro N. Survival of several Rhizobium/Bradyrhizobium strains on different inoculant formulation and inoculated seed. Int Microbiol. 2002;5(2):81-6.

Namasivayam SKR, Saikia SL, Bharani ARS. Evaluation of persistence and plant growth promoting effect of bioencapsulated formulation of suitable bacterial bio-fertilizers. Biosci Biotech Res Asia. 2014;11(2):407-415.

Vance CP, Heichel GH, Phillips DA. Nodulation and symbiotic nitrogen fixation. In: Hanson AA, Barnes DK, Hill RR, editors. Alfalfa and Alfalfa Improvement, Agronomy 29. Madison, Wisconsin: ASA-CSSA SSSA; 1988. p.229-57.

Provorov NA, Tikhonovich IA. Genetic resources for improving nitrogen fixation in legume-rhizobia symbiosis. Genet Resour Crop Evol 2003:50:89-99.

Stajković-Srbinović O, Delić D, Nerandžić B, Andjelović S, Sikirić B, Kuzmanović Dj, Rasulić N. Alfalfa yield and nutrient uptake as influenced by co-inoculation with rhizobium and rhizobacteria. Rom Biotech Lett. 2017;22(4);12834-41.

Delić D, Stajković-Srbinović O, Radović J, Kuzmanović D, Rasulić N, Simić A, Knežević-Vukčević J. Differences in symbiotic N2 fixation of alfalfa, Medicago sativa L. cultivars and Sinorhizobium spp. strains in field conditions. Rom Biotech Lett. 2013;18(6):8743-50.

Vincent JM. A manual for the practical study of the root nodule bacteria. Oxford: Blackwell Scientific Publications, 1970. 164 p. (IBP handbook; 15).

Tittabutr P, Payakapong W, Teaumroong N, Singleton PW, Boonkerd N. Growth, survival and field performance of bradyrhizobial liquid inoculant formulations with polymeric additives. Sci Asia. 2007;33(1):69-77.

Trivedi P, Pandey A, Palni LMS. Carrier-based preparations of plant growth-promoting bacterial inoculants suitable for use in cooler regions. World J Microb Biot. 2005:21(6-7):941-5.

Howieson JG, Dilworth MJ. Working with rhizobia. Canberra: Australian Centre for International Agricultural Research; 2016.

Rivera D, Obando M, Barbosa H, Rojas Tapias D, Bonilla Buitrago R. Evaluation of polymers for the liquid rhizobial formulation and their influence in the Rhizobium-Cowpea interaction. Univ Sci. 2014:19(3):265-75.

Rinaudi L, Fujishige NA, Hirsch AM, Banchio E, Zorreguieta A, Giordano W. Effects of nutritional and environmental conditions on Sinorhizobium meliloti biofilm formation. Res Microbial. 2006,157(9):867-75.

Deaker R, Roughley RJ, Kennedy IR. Legume seed inoculation technology - a review. Soil Biol Biochem. 2004;36(8):1275-88.

Biederbeck VO, Geissler HJ. Effect of storage temperatures on Rhizobium meliloti survival in peat-and clay-based inoculants. Can J Plant Sci. 1993;73(1):101-10.

Somasegaran P. Inoculant production with diluted liquid cultures of Rhizobium spp. and autoclaved peat: evaluation of diluents, Rhizobium spp., peats, sterility requirements, storage, and plant effectiveness. Appl Environ Microb. 1985;50(2):398-405.

Cortes-Patino SA, Bonilla RR. Polymers selection for a liquid inoculant of Azospirillum brasilense based on the Arrhenius thermodynamic model. Afr J Biotechnol. 2005:14(33):2547-53.

Albareda M, Rodríguez-Navarro DN, Camacho M, Temprano FJ. Alternatives to peat as a carrier for rhizobia inoculants: solid and liquid formulations. Soil Biol Biochem. 2008;40(11):2771-9.

Chen, JH. The combined use of chemical and organic fertilizers and/or biofertilizer for crop growth and soil fertility. In: International workshop on sustained management of the soil-rhizosphere system for efficient crop production and fertilizer use; 2006 Oct 16-20. Bangkok, Thailand: Land Development Department; 2006. p. 1-11.

Sehrawat A, Suneja S, Yadav A, Anand RC. Influence of different additives on shelf life of rhizobial inoculants for mungbean (Vigna radiata L.). Int J Recent Scientific Res. 2015:6(5):4338-42.

Sehrawat A, Yadav A, Anand RC, Kukreja K, Suneja S. Enhancement of shelf life of liquid biofertilizer containing Rhizobium sp. infecting mungbean (Vigna radiata L.). Legume Res. 2017:40(4):684-90.

Delić D, Stajković-Srbinović O, Kuzmanović D, Mrvić V, Knežević-Vukčević J. Effect of bradyrhizobial inoculation on growth and seed yield of mungbean in Fluvisol and Humofluvisol. Afr J Microbiol Res. 2011;5(23):3946-57.

Pochon J. Manuel technique D’Analyse microbiologique du sol. Paris : Masson et Cie, 1954.123p.

Bergmann W, Nutritional disorders of plants - development, visual and analytical diagnosis. Jena: Fischer; 1992. 347 p.

Delić D, Stajković O, Radović J, Stanojković A, Kuzmanović Đ, Rasulić N, Miličić B. Genotypic differences in symbiotic N2 fixation of some alfalfa (Medicago sativa L.) Genotypes. In: Huyghe C, editor. Sustainable use of Genetic Diversity in Forage and Turf Breeding. Vol 16. Dordrecht: Springer; 2010. p.79-84.




How to Cite

Buntić AV, Stajković-Srbinović OS, Knežević MM, Kuzmanović Đorđe Ž., Rasulić NV, Delić DI. Development of liquid rhizobial inoculants and pre-inoculation of alfalfa seeds. Arch Biol Sci [Internet]. 2019Jun.4 [cited 2024Feb.26];71(2):379-87. Available from: