AJPS  Vol.6 No.10 , June 2015
Co-Inoculation of Soybean with Bradyrhizobium and Azospirillum Promotes Early Nodulation
Abstract: Soybean inoculation with elite strains of Bradyrhizobium to improve nodulation, N2 fixation, and grain yield is well established worldwide. However, when grown in soils where N is deficient, soybean undergoes an initial phase of N starvation that may last up to 20 days after seedling germination due to the lack of synchronism between the phase when seed N reserves are exhausted and the moment when plants begin to benefit from the nitrogen fixed by the bacteria. Practices that promote early nodulation may play a key role in reducing the N starvation period. Azospirillum is a plant growth promoting rhizobacteria (PGPR) that can stimulate root hair formation and root growth, creating more sites for early root infection and nodule formation by N2-fixing Bradyrhizobium spp. In this study, the effects of co-inoculating soybeans with Bradyrhizobium spp. and Azospirillum brasilense on nodulation precocity and N2 fixation were evaluated under greenhouse and field conditions. Nodule number and dry weight, as well as plant and root dry weight and N accumulated in shoots at 15, 18, 21, 24 and 30 days after emergence (DAE) were evaluated in response to inoculation with Bradyrhizobium spp. alone or when co-inoculated with Azospirillum sp. In the greenhouse, co-inoculated plants nodulated precociously as indicated by a significant increase (p < 0.05) in nodule biomass observed at (include) 21 DAE. More pronounced effects of co-inoculation were observed in the field as early as 18 DAE, suggesting that the presence of Azospirillum helps plants to overcome environmental stresses.
Cite this paper: Machaculeha Chibeba, A. , Fátima Guimarães, M. , Rodrigues Brito, O. , Nogueira, M. , Silva Araujo, R. and Hungria, M. (2015) Co-Inoculation of Soybean with Bradyrhizobium and Azospirillum Promotes Early Nodulation. American Journal of Plant Sciences, 6, 1641-1649. doi: 10.4236/ajps.2015.610164.

[1]   Chang, W.-S., Lee, H.-I. and Hungria, M. (2015) Soybean Production in the Americas (Chapter 41). In: Lugtenberg, B., Ed., Principles of Plant-Microbe Interactions, Springer, Switzerland, 393-400.

[2]   Hungria, M. and Campo, R. (2007) Inoculantes Microbianos: Situação no Brasil. In: Izaguirre-Mayoral, M.L., Labandera, C. and Sanjuan, J., Eds., Biofertilizantes en Iberoamérica: Visión Técnica, Científica y Empresarial, Cyted/ Biofag, Montevideo, 22-31.

[3]   Hungria, M., Barradas, C. and Wallsgrove, R. (1991) Nitrogen Fixation, Assimilation and Transport during the Initial Growth Stage of Phaseolus vulgaris L. Journal of Experimental Botany, 42, 839-844.

[4]   Abendroth, L.J., Elmore, R.W. and Ferguson, R.B. (2006) G06-1621 Soybean Inoculation: Understanding the Soil and Plant Mechanisms Involved (Part One of a Two-Part Series). Historical Materials from University of Nebraska-Lincoln Extension.

[5]   Atkins, C.A., Pate, J.S., Sanford, P.J., Dakora, F.D. and Matthews, I. (1989) Nitrogen Nutrition of Nodules in Relation to “N-Hunger” in Cowpea (Vigna unguiculata L. Walp). Plant Physiology, 90, 1644-1649.

[6]   Zilli, J., Valicheski, R.R., Rumjanek, N.G., Simões-Araújo, J.L., Freire Filho, F.R. and Neves, M.C.P. (2006) Eficiência Simbiótica de Estirpes de Bradyrhizobium Isoladas de Solo do Cerrado em Caupi. Pesquisa Agropecuária Brasileira, 41, 811-818.

[7]   Marschner, H. (1995) Nitrogen Fixation. In: Marschner, H., Ed., Mineral Nutrition of Higher Plants, 2nd Edition, Academic Press, London, 201-228.

[8]   Saharan, B. and Nehra, V. (2011) Plant Growth Promoting Rhizobacteria: A Critical Review. Life Science and Medical Research, 21, 1-30.

[9]   Hirsch, A., Fang, Y., Asad, S. and Kapulnik, Y. (1997) The Role of Phytohormones in Plant-Microbe Symbioses. Plant and Soil, 194, 171-184.

[10]   Ashraf, M.A., Rasool, M. and Mirza, M.S. (2011) Nitrogen Fixation and Indole Acetic Acid Production Potential of Pacteria Isolated from Rhizosphere of Sugarcane (Saccharum officinarum L.). Advances in Biological Research, 5, 348-355.

[11]   Tien, T., Gaskins, M. and Hubbell, D. (1979) Plant Growth Substances Produced by Azospirillum brasilense and Their Effect on the Growth of Pearl Millet (Pennisetum americanum L.). Applied and Environmental Microbiology, 37, 1016-1024.

[12]   Bottini, R., Fulchieri, M., Pearce, D. and Pharis, R.P. (1989) Identification of Gibberellins A1, A3, and Iso-A3 in Cultures of Azospirillum lipoferum. Plant Physiology, 90, 45-47.

[13]   Strzelczyk, E., Kampert, M. and Li, C. (1994) Cytokinin-Like Substances and Ethylene Production by Azospirillum in Media with Different Carbon Sources. Microbiological Research, 149, 55-60.

[14]   Gurska, J., Wang, W., Gerhardt, K.E., Khalid, A.M., Isherwood, D.M., Huang, X.-D., Glick, B.R. and Greenberg, B.M. (2009) Three Year Field Test of a Plant Growth Promoting Rhizobacteria Enhanced Phytoremediation System at a Land Farm for Treatment of Hydrocarbon Waste. Environmental Science and Technology, 43, 4472-4479.

[15]   Bashan, Y. and Holguin, G. (2002) Plant Growth-Promoting Bacteria: A Potential Tool for arid Mangrove Reforestation. Trees, 16, 159-166.

[16]   Bhattacharyya, P.N. and Jha, D.K. (2012) Plant Growth-Promoting Rhizobacteria (PGPR): Emergence in Agriculture. World Journal of Microbiology and Biotechnology, 28, 1327-1350.

[17]   Glick, B.R. (2012) Plant Growth-Promoting Bacteria: Mechanisms and Applications. Scientifica, 2012, Article ID: 963401.

[18]   Saikia, S.P., Dutta, S.P., Goswami, A., Bhau, B.S. and Kanjilal, P.B. (2010) Role of Azospirillum in the Improvement of Legumes. In: Khan, M.S., Zaidi, A. and Musarrat, J., Eds., Microbes for Legume Improvement, Springer-Verlag, Wien, 389-408.

[19]   Dobereiner, J. and Pedrosa, F.O. (1987) Nitrogen-Fixing Bacteria in Nonleguminous Crop Plants. Springer, Madison.

[20]   Hungria, M., Campo, R.J., Souza, E.M. and Pedrosa, F.O. (2010) Inoculation with Selected Strains of Azospirillum brasilense and A. lipoferum Improves Yields of Maize and Wheat in Brazil. Plant and Soil, 331, 413-425.

[21]   Hadas, R. and Okon, Y. (1987) Effect of Azospirillum brasilense Inoculation on Root Morphology and Respiration in Tomato Seedlings. Biology and Fertility of Soils, 5, 241-247.

[22]   Ribaudo, C.M., Krumpholz, E.M., Cassán, F.D., Bottini, R., Cantore, M.L. and Curá, J.A. (2006) Azospirillum sp. Promotes Root Hair Development in Tomato Plants through a Mechanism That Involves Ethylene. Journal of Plant Growth Regulation, 25, 175-185.

[23]   Bashan, Y., and Levanony, H. (1990) Current Status of Azospirillum Inoculation Technology: Azospirillum as a Challenge for Agriculture. Canadian Journal of Microbiology, 36, 591-608.

[24]   Benintende, S., Uhrich, W., Herrera, M., Gangge, F., Sterren, M. and Benintende, M. (2010) Comparación entre Coinoculación con Bradyrhizobium japonicum y Azospirillum brasilense e Inoculación simple con Bradyrhizobium japonicum en la Nodulación, Crecimiento y Acumulación de N en el Cultivo de Soja. Agriscientia, 27, 71-77.

[25]   Galal, Y.G.M. (1997) Dual Inoculation with Strains of Bradyrhizobium japonicum and Azospirillum brasilense to Improve Growth and Biological Nitrogen Fixation of Soybean (Glycine max L.). Biology and Fertility of Soils, 24, 317-322.

[26]   Hungria, M., Nogueira, M.A. and Araujo, R.S. (2013) Co-Inoculation of Soybeans and Common Beans with Rhizobia and Azospirilla: Strategies to Improve Sustainability. Biology and Fertility of Soils, 49, 791-801.

[27]   Okon, Y. and Labandera-Gonzalez, C.A. (1994) Agronomic Applications of Azospirillum: An Evaluation of 20 Years Worldwide Field Inoculation. Soil Biology and Biochemistry, 26, 1591-1601.

[28]   Badenoch-Jones, J., Flanders, D.J. and Rolfe, B.G. (1985) Association of Rhizobium Strains with Roots of Trifolium repens. Applied and Environmental Microbiology, 49, 1511-1520.

[29]   Hungria, M., Franchini, J.C., Campo, R.J., Crispino, C.C., Moraes, J.Z., Sibaldelli, R.N.R., Mendes, I.C. and Arihara, J. (2006) Nitrogen Nutrition of Soybean in Brazil: Contributions of Biological N2 Fixation and N Fertilizer to Grain Yield. Canadian Journal of Plant Science, 86, 927-939.

[30]   Vincent, J.M. (1970) A Manual for the Practical Study of the Root-Nodule Bacteria. Blackwell, Oxford.

[31]   Andrade, D.S. and Hamakawa, P.J. (1994) Estimativa do Número de Células Viáveis de Rizóbio no Solo e em Inoculantes por Infecção em Plantas. In: Hungria, M. and Araujo, R.S., Eds., Manual de Métodos Empregados em Estudos de Microbiologia Agrícola, EMBRAPA-SPI, Brasilia, 63-94.

[32]   Döbereiner, J., Baldani, V.L.D. and Baldani, J.I. (1995) Como Isolar e Identificar Bactérias Diazotróficas de Plantas não-Leguminosas. Embrapa-SPI, Itaguaí.

[33]   Feije, F. and Anger, V. (1972) Spot Tests in Inorganic Analyses. Analytica Chimica Acta, 149, 363-367.

[34]   Ferreira, D.F. (2011) Sisvar: A Computer Statistical Analysis System. Ciência e Agrotecnologia (UFLA), 35, 1039-1042.

[35]   Barbin, D. (2003) Planejamento e Análise Estatística de Experimentos Agron?micos. 2nd Edition, Mecenas, Londrina.

[36]   Bohrer, T.R.J. and Hungria, M. (1998) Avaliação de Cultivares de Soja quanto à Fixação Biológica do Nitrogênio. Pesquisa Agropecuária Brasileira, 33, 937-952.

[37]   Döbereiner, J. (1966) Evaluation of Nitrogen Fixation in Legumes by the Regression of Total Plant Nitrogen with Nodule Weight. Nature, 210, 850-852.

[38]   Hungria, M. and Bohrer, T. (2000) Variability of Nodulation and Dinitrogen Fixation Capacity among Soybean Cultivars. Biology and Fertility of Soils, 31, 45-52.

[39]   Souza, R.A., Hungria, M., Franchini, J.C., Maciel, C.D., Campo, R.J. and Zaia, D.A.M. (2008) Conjunto Mínimo de Parametros para Avaliação da Microbiota do Solo e da Fixação Biológica do Nitrogênio pela Soja. Pesquisa Agropecuária Brasileira, 43, 83-91.

[40]   Souza, R.A, Hungria, M., Franchini, J.C., Chueire, L.M.O., Barcellos, F.G. and Campo, R.J. (2008) Avaliação Qualitativa e Quantitativa da Microbiota do Solo e da Fixação Biológica do Nitrogênio pela Soja. Pesquisa Agropecuária Brasileira, 43, 71-82.

[41]   Cardoso, J.D., Gomes, D.F., Goes, K.C., Fonseca, N. S., Dorigo, O.F., Hungria, M. and Andrade, D.S. (2009) Relationship between Total Nodulation and Nodulation at the Root Crown of Peanut, Soybean and Common Bean Plants. Soil Biology and Biochemistry, 41, 1760-1763.

[42]   Aung, T.T., Tittabutr, P., Boonkerd, N., Herridge, D. and Teaumroong, N. (2013) Co-Inoculation Effects of Bradyrhizobium japonicum and Azospirillum sp. on Competitive Nodulation and Rhizosphere Eubacterial Community Structures of Soybean under Rhizobia-Established Eoil Conditions. African Journal of Biotechnology, 12, 2850-2862.

[43]   Cassán, F., Perrig, D., Sgroy, V., Masciarelli, O., Penna, C., and Luna, V. (2009) Azospirillum brasilense Az39 and Bradyrhizobium japonicum E109, Inoculated Singly or in Combination, Promote Seed Germination and Early Seedling Growth in corn (Zea mays L.) and Soybean (Glycine max L.). European Journal of Soil Biology, 45, 28-35.

[44]   Singh, C. and Rao, N.S. (1979) Associative Effect of Azospirilium brasilense with Rhizobium japonicum on Nodulation and Yield of Soybean (Glycine max). Plant and Soil, 53, 387-392.

[45]   Molla, A.H., Shamsuddin, Z.H., Halimi, M.S., Morziah, M. and Puteh, A.B. (2001) Potential for Enhancement of Root Growth and Nodulation of Soybean Co-Inoculated with Azospirillum and Bradyrhizobium in Laboratory Systems. Soil Biology & Biochemistry, 33, 457-463.

[46]   Kloepper, J.W., Lifshitz, R. and Zablotowicz, R.M. (1989) Free-Living Bacterial Inocula for Enhancing Crop Productivity. Trends in Biotechnology, 7, 39-44.

[47]   Itzigsohn, R., Kapulnik, Y., Okon, Y. and Dovrat, A. (1993) Physiological and Morphological Aspects of Interactions Between Rhizobium meliloti and Alfalfa (Medicago sativa) in Association with Azospirillum brasilense. Canadian Journal of Microbiology, 39, 610-615.

[48]   Groppa, M.D., Zawoznik, M.S. and Tomaro, M.L. (1998) Effect of Co-Inoculation with Bradyrhizobium japonicum and Azospirillum brasilense on Soybean Plants. European Journal of Soil Biology, 34, 75-80.

[49]   Bashan, Y. and Holguin, G. (1997) Azospirillum-Plant Relationships: Environmental and Physiological Advances (1990-1996). Canadian Journal of Microbiology, 43, 103-121.

[50]   Yahalom, E., Okon, Y. and Dovrat, A. (1990) Possible Mode of Action of Azospirillum brasilense Strain Cd on the Root Morphology and Nodule Formation in Burr Medic (Medicago polymorpha). Canadian Journal of Microbiology, 36, 10-14.

[51]   Hungria, M. and Vargas, M.A.T. (2000) Environmental Factors Affecting N2 Fixation in Grain Legumes in the Tropics, with an Emphasis on Brazil. Field Crops Research, 65, 151-164.

[52]   Hungria, M. and Kaschuk, G. (2014) Regulation of N2 Fixation and Assimilation in Nodulated and N-Fertilized Phaseolus vulgaris L. Exposed to High-Temperature Stress. Environmental and Experimental Botany, 98, 32-39.

[53]   Hungria, M., Nogueira, M.A. and Araujo, R.S. (2015) Soybean Seed Co-Inoculation with Bradyrhizobium spp. and Azospirillum brasilense: A New Biotechnological Tool to Improve Yield and Sustainability. American Journal of Plant Science, 6, 811-817.