Azospirillum brasilense and organomineral fertilizer co-inoculated with Bradyrhizobium japonicumon oxidative stress in soybean

Document Type : Original Article


Federal University of Uberlândia (ICIAG - UFU), Minas Gerais state, Brazil


Purpose Nitrogen-fixing bacteria (NFB) are essential for biologically fixing nitrogen in natural and soybean crop systems. The use of organomineral fertilizers is a promising crop management tool made from the mixing of an organic source and mineral fertilizers. This study evaluated the efficacy of an organomineral fertilizer (sewage sludge-based) as a carrier for the Azospirillum brasilense inoculation and the influence of A. brasilense plus Bradyrhizobium japonicum on the soybean antioxidant metabolism.
Method The experiment was performed under greenhouse conditions. The soybean cultivar (AS 3680 IPRO) was evaluated in a 4×2(+1) factorial scheme, corresponding to four doses of A. brasilense [0, 100, 200, 300 mL ha-1 (2x108 viable cells mL-1) per 50 kg-1 of soybean seeds], with or without B. japonicum [100 mL ha-1 (5x109 viable cells mL-1) per 50 kg-1 of soybean seeds], and a control treatment (without inoculants or fertilizers). Soybean antioxidant metabolism (enzymes, oxidative processes, proline amino acid) was evaluated in different plant stages.
Results The seed inoculation with A. brasilense via organomineral fertilizer enhanced the soybean plant protection factors. Plant protection to stresses happened mainly by reductions in the superoxide dismutase activity, lipid peroxidation, and hydrogen peroxide concentration. The co-inoculation of A. brasilense with B. japonicum enhanced the proline activity at the V3 and R5 soybean phenological stage compared to A. brasilense inoculated alone.
Conclusion The studied organomineral fertilizer is an efficient inoculant carrier for A. brasilense in soybean plants via seed treatment. The protective results observed for A. brasilense were improved when co-inoculated with B. japonicum.


  • Nitrogen-fixing bacteria biologically fixes atmospheric nitrogen in natural and cropped systems.
  • Organomineral fertilizers are improving crop performance, resistance and productivity.
  • Azospirillum brasilense and Bradyrhizobium japonicum enhanced soybean proline activity.
  • Soybean protection was improved when brasilense and B. japonicum were co-inoculated.
  • Organomineral fertilizer is an efficient inoculant carrier for brasilense in soybean plants.


Ahmad P, Jaleel CA, Sharma S (2010) Antioxidant defense system, lipid peroxidation, proline-metabolizing enzymes, and biochemical activities in two Morus alba genotypes subjected to NaCl stress. Russ J Plant Physl 57(4):509-517.
Alam R, Das DK, Islam MR, Murata Y, Hoque MA (2016) Exogenous proline enhances nutrient uptake and confers tolerance to salt stress in maize (Zea mays L.). Progr Agric 27:409-417.
Alexieva V, Sergiev I, Mapelli S, Karanov E (2001) The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant Cell Environ 24:1337-1344.
Alfenas AC (1998) Electrophoresis of isoenzymes and related proteins: Fundamentals and applications in plants and microorganisms. Viçosa: Federal University of Viçosa
Alves Filho A, Camargo R, Lana RMQ, Moraes MRB, Maldonado ACD, Atarasi RT (2016) Treatment of sewage sludge with the use of solarization and sanitizing products for agricultural purposes. Afr J Agric Res 11(3):184-191.
Apel K, Hirt H (2004) Reactive oxygen species: Metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55(123):373-399.
Bárbaro IM, Brancalião SR, Ticelli M, Miguel FB, Silva JAA (2008) Técnica alternativa: co-inoculação de soja com Azospirillum e Bradyrhizobium visando incremento de produtividade. In: Infobibos - Informações Tecnológicas. Infobibos. Campinas: Infobibos
Bárbaro IM, Bárbaro Junior LS, Ticelli M, Machado PC, Miguel FB (2011) Resultados preliminares da co-inoculação de Azospirillum juntamente com Bradyrhizobium em soja. Pesquisa Tecnol 8(2):1-6
Bartoli CG, Casalongue CA, Simontacchi M, Marquez-Garcia B, Foyer CH (2012) Interactions between hormone and redox signaling pathways in the control of growth and cross-tolerance to stress. Environ Exp Bot 93:73-88.
Basal O, Szabó A (2020) Physiomorphology of soybean as affected by drought stress and nitrogen application. Scientifica. Article ID 6093836.
Bashan Y, de-Bashan LE, Prabhu SR, Hernandez JB (2014) Advances in plant growth-promoting bacterial inoculant technology: Formulations and practical perspectives (1998- 2013). Plant Soil 378:1-33.
Bates L, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205-207.
Beauchamp C, Fridovich I (1971) Superoxide dismutase: Improved assays and applicable to acrylamide gels. Anal Biochem 44(1):276-287.
Bela K, Horváth E, Gallé Á, Szabados L, Tari I, Csiszár J (2015) Plant glutathione peroxidases: emerging role of the antioxidant enzymes in plant development and stress responses. J Plant Physiol 176:192-201.
Benidire L, Madline A, Pereira SIA, Castro PML, Boularbah A (2021) Synergistic effect of organo-mineral amendments and plant growth-promoting rhizobacteria (PGPR) on the establishment of vegetation cover and amelioration of mine tailings. Chemosphere 262:127803.
Benites VM, Moutta RO, Coutinho HLC, Balieiro FC (2010) Análise discriminante de solos sob diferentes usos em área de mata atlântica a partir de atributos da matéria orgânica. Rev Árvore 34(4):685-690.
Berg J, Lässig M, Wagner A (2004) Structure and evolution of protein interaction networks: a statistical model for link dynamics and gene duplications. BMC Evol Biol 4:51.
Bieker S, Potschin M, Zentgraf U (2018) Study of hydrogen peroxide as a senescence-inducing signal. Plant Senes 1744:173-193.
Bor M, Özdemir F, Türkan I (2003) The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. and wild beet Beta maritima L. Plant Sci 164:77-84.
Bouhia Y, Hafidi M, Ouhdouch Y, Boukhari MM, Zeroual Y, Lyamlouli K (2021) Effect of the co-application of olive waste-based compost and biochar on soil fertility and Zea mays agrophysiological traits. Int J Recycl Org Waste Agric 10(2):111-127.
Brasil. Ministério da Agricultura, Pecuária e Abastecimento (2005) Instrução Normativa n.23, de 31 de agosto de 2005. Aprova as definições e normas sobre as especificações e as garantias, as tolerâncias, o registro, a embalagem e a rotulagem dos fertilizantes orgânicos simples, mistos, compostos, organominerais e biofertilizantes destinados à agricultura. Brasília, DF: MAPA
Bulegon LG, Guimarães VF, Laureth JCU (2016) Azospirillum brasilense affects the antioxidant activity and leaf pigment content of Urochloa ruziziensis under water stress. Pesqui Agropec Trop 46(3):343-349.
Camargo R, Lana RMQ, Franco MHR Magela MLM (2020) Fertilizantes organominerais: do surgimento até as novas possibilidades. Revista Campo & Negócios 1:26-29
Carvalho K, Campos MKF, Domingues DS, Pereira LFP, Vieira LGE (2013) The accumulation of endogenous proline induces changes in gene expression of several antioxidante enzymes in leaves of transgenic Swingle citrumelo. Mol Biol Rep 40:3269-3279.
CFSEMG: Comissão de Fertilidade de Solos do Estado de Minas Gerais (1999) Recomendações para o Uso de Corretivos e Fertilizantes em Minas Gerais: 5ª aproximação. Viçosa, MG. 289-302
Chibeba AM, Guimarães MF, Brito OR, Nogueira MA, Araujo RS, Hungria M (2015) Co-Inoculation of soybean with Bradyrhizobium and Azospirillum promotes early nodulation. Am J Plant Sci 6(10):1641-1649.
CONAMA: Conselho Nacional do Meio Ambiente (2006) Resolution no 375 – August 29, 2006. Ministério do Meio Ambiente, Brazil
Dan LGM, Dan HA, Piccinin GG, Ricci TT, Ortiz AHT (2011) Tratamento de sementes com inseticida e a qualidade fisiológica de sementes de soja. Rev Caatinga 25(1):45-51
Elhady A, Hallmann J, Heue H (2020) Symbiosis of soybean with nitrogen-fixing bacteria affected by root-lesion nematodes in a density-dependent manner. Sci Rep 10:1619.
El Moukhtari A, Cabassa-Hourton C, Farissi M, Savouré A (2020) How does proline treatment promote salt stress tolerance during crop plant development? Front Plant Sci 11:1127.
El Sabagh AE, Sorour S, Ragab A, Saneoka H, Islam M (2017) The effect of exogenous application of proline and glycine betaine on the nodule activity of soybean under saline condition. J Agri Biotec 2:1-5.
EMBRAPA: Empresa Brasileira de Pesquisa Agropecuária (2009) Manual de análises químicas de solos, Plantas e Fertilizantes. 2 ed. Brasília: Embrapa Informação Tecnológica
Ernst WHO, Nelissen HJM, Bookum WMT (2000) Combination toxicology of metal-enriched soils: Physiological responses of a Zn- and Cd-resistant ecotype of Silene vulgaris on polymetallic soils. Environ and Exp Bot 43(1):55-71.
Escobar N, Arenas NE, Marques SM (2020) Characterization of microbial populations associated with different organic fertilizers. Int J Recycl Org Waste Agric 9(2):171-182.
Fagan EB, Ono EO, Rodrigues JD, Soares LH, Durval Neto D (2016) Fisiologia Vegetal: Metabolismo e nutrição mineral. São Paulo
Foyer CH, Noctor G (2005) Oxidant and antioxidant signaling in plants: A reevaluation of the concept of oxidative stress in a physiological context. Plant Cell Environ 28:1056-71.
Gadjev I, Stone JM, Gechev TS (2008) Programmed cell death in plants: new insights into redox regulation and the role of hydrogen peroxide. Int Rev Cell Mol Bio 270:87-144.
García AC, Santos LA, Izquierdo FG, Sperandio MVL, Castro RN, Berbara RLL (2012) Vermicompost humic acids as an ecological pathway to protect rice plant against oxidative stress. Ecol Eng 47:203-208.
Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909-930.
Hasanuzzaman M, Bhuyan MHMB, Zulfiqar F, Raza A, Mohsin SM, Mahmud JA, Fujita M, Fotopoulos V (2020) Reactive oxygen species and antioxidant defense in plants under abiotic stress: revisiting the crucial role of a universal defense regulator. Antioxidants 9:681.
Havir EA, Mchale NA (1987) Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves. Plant Physiol 84(2):450-455.
Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A (2012) Role of proline under changing environments: A review. Plant Signal Behav 7(11):1456-1466.
Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125(1):189-198.
Hernandez JA, Ferrer MA, Jimenez A, Barcelo AR, Sevilla F (2001) Antioxidant systems and O2/H2O2 production in the apoplast of pea leaves. Its relation with salt-induced necrotic lesions in minor veins. Plant Physiol 127(3):827-831.
Hungria M, Nogueira MA, Araujo RS (2013) Co-inoculation of soybeans and common beans with rhizobia and azospirilla: Strategies to improve sustainability. Biol Fert Soils 49(7):791-801.
Hungria M, Nogueira MA (2014) Tecnologia de coinoculação: Rizobium e Azospirillum em soja e feijoeiro. Emprapa Soja Folders
Inzé D, Van Montagu M (1995) Oxidative stress in plants. Curr Opin Biotech 6:153-158.
Kadian N, Yadav K, Jangra E, Aggarwal A (2020) Influence of host plant and rice straw as substrate on mass multiplication of arbuscular mycorrhizal fungi for large‑scale agricultural application. Int J Recycl Org Waste Agric 8(Suppl 1):S21-S26.
Kar M, Mishra D (1976) Catalase, peroxidase, and polyphenoloxidase activities during rice leaf senescence. Plant Physiol 57:315-319.
Karuppanapandian T, Moon J, Kim C, Manoharan K, Kim W (2011) Reactive oxygen species in plants: Their generation, signal transduction, and scavenging mechanisms. Aust J Crop Sci 5(6):709-725
Kaur G, Asthir B (2015) Proline: A key player in plant abiotic stress tolerance. Biol Plantarum 59(4):609-619.
Khan T, Yusuf M, Fariduddin Q (2018) Hydrogen peroxide in regulation of plant metabolism: Signalling and its effect under abiotic stress. Photosynthetica 56:1237-1248.
Libório PHS, Bárbaro-Torneli IM, Sobrinho RM, Paula GFG, Silva CHA, Anaides L, Unêda-Trevisoli SH (2018) Avaliação de cultivares de soja submetidas a co-inoculação. Ciência Tecnol: Fatec-JB 10. Número especial
Lopes MJS, Dias-Filho MB, Gurgel ESC (2021) Successful plant growth-promoting microbes: Inoculation methods and abiotic factors. Front Sustain Food Syst 5:606454.
Maruta T, Ishikawa T (2017) Ascorbate peroxidases: Crucial roles of antioxidant enzymes in plant stress responses. In: Hossain M, Munné-Bosch S, Burritt D, Diaz-Vivancos P, Fujita M, Lorence A (Eds) Ascorbic acid in plant growth, development and stress tolerance. Springer, Cham.
Mota RP, Camargo R, Lemes EM, Lana RMQ, Almeida RF, Moraes ER (2019) Biosolid and sugarcane filter cake in the composition of organomineral fertilizer on soybean responses. Int J Recycl Org Waste Agric 8:131-137.
Müller TM (2016) Componentes de produtividade de grãos e de atividade enzimática na cultura do milho inoculado com Azospirillum brasilense em tratamento de sementes e aplicação foliar. 85 f. Tese (Doutorado) - Universidade Federal de Santa Maria. Santa Maria
Oliveira DP, Camargo R, Lemes EM, Lana RMQ, Matos ALIA, Magela MLM (2017) Organic matter sources in the composition of pelletized organomineral fertilizers used in sorghum crops. African J Agricult Research 12(32):2574-2581.
Pandey S, Fartyal D, Agarwal A, Shukla T, James D, Kaul T, Negi YK, Arora S, Reddy MK (2017) Abiotic stress tolerance in plants: Myriad roles of ascorbate peroxidase. Front Plant Sci 8:581.
Queiroz CGS, Alonso A, Mares-Guia M, Magalhães AC (1998) Chilling induced changes in membrane fluidity and antioxidant enzyme activities in Coffea arabica L. roots. Biol Plantarum 41(3):403- 413.
Rady MM (2012) A novel organo-mineral fertilizer can mitigate salinity stress effects for tomato production on reclaimed saline soil. S Afr J Bot 81:8-14.
Rejeb KB, Abdelly C, Savouré A (2014) How reactive oxygen species and face stress together. Plant Physiol Bioch 80:278-284.
Ribeiro RA, Ormeño-Orrillo E, Dall’agnol RF, Graham PH, Martinez-Romero E, Hungria M (2013) Novel rhizobium lineages isolated from root nodules of the common bean (Phaseolus vulgaris L.) in Andean and Mesoamerican areas. Res Microbiol 164(7):740-748.
Rocha LD (2018) Efeitos dos ácidos húmicos extraídos de lodo de esgoto em Zea mays L. sob restrição hídrica e estresse salino. 126 f. Tese (Doutorado) - Universidade Federal do Espírito Santo, Centro de Ciências Humanas e Sociais. Vitória
Santos HG, Jacomine PKT, Anjos LHC, Oliveira VA, Lumbreras JF, Coelho MR, Almeida JA, Araújo Filho JC, Oliveira JB, Cunha TJF (2018) Sistema Brasileiro de Classificação de Solos. 5. ed., rev. e ampl. − Brasília, DF: Embrapa
Scandalios JG (1993) Oxigen stress and superoxide dismutases. Plant Physiol 101(1):7-12.
Scandalios JG (2005) Oxidative stress: Molecular perception and transduction of signals triggering antioxidant gene defenses. Braz J Med Biol Res 38(7):995-1014.
Siegel BZ (1993) Plant peroxidases: An organism perspective. Plant Growth Regul 12:303-312
Silva LG, Camargo R, Lana RMQ, Delvaux JC, Fagan EB, Machado VJ (2020) Biochemical changes and development of soybean with use of pelletized organomineral fertilizer containing sewage sludge and filter cake. Acta Scientiarum Agronomy 42(1):e44249.
Slama I, M’Rabet R, Ksouri R, Talbi O, Debez A, Abdelly C (2017) Effects of salt treatment on growth, lipid membrane peroxidation, polyphenol content, and antioxidant activities in leaves of Sesuvium portulacastrum L. Arid Land Res Manag 31:1-14.
Smercina DN, Evans SE, Friesen ML, Tiemann LK (2019) To fix or not to fix: Controls on free-living nitrogen fixation in the rhizosphere. Appl Environ Microbiol 6;85(6):e02546-18.
Smirnoff N, Arnaud D (2019) Hydrogen peroxide metabolism and functions in plants. New Phytol 221:1197-1214.
Strothe S (1988) The role of free radicals in leaf senescence. Gerontology. 34(3):151-156.
Teisseire H, Guy V (2000) Copper-induced changes in antioxidant enzyme activities in fronds of duckweed (Lemna minor). Plant Sci 153(1):65-72. 10.1016/S0168-9452(99)00257-5
USEPA-United States Environmental Protection Agency (1992) Test methods for evaluating solid waste: Physical/chemical methods (SW 846). Washington, USA
Vasconcelos ACF, Zhang X, Ervin EH, Kiehl JC (2009) Enzymatic antioxidant responses to biostimulants in maize and soybean subjected to drought. Sci Agric 66(3):395-402.
Wani AS, Ahmad A, Hayat S, Tahir I (2016) Is foliar spray of proline sufficient for mitigation of salt stress in Brassica juncea cultivars? Environ Sci Pollut Res 23:13413–13423.
Xu KW, Penttinen P, Chen YX, Zou L, Zhou T, Zhang X, Hu C, Liu F (2013) Polyphasic characterization of rhizobia isolated from Leucaena leucocephala from Panxi, China. World J Microb Biot 29:2303-2315.
Xu ZZ, Zhou GS, Han G, Li Y (2011) Photosynthetic potential and its association with lipid peroxidation in response to high temperature at different leaf ages in maize. J Plant Growth Regul 30:41-50.