Plant and Soil

, Volume 424, Issue 1–2, pp 405–417 | Cite as

Microorganisms reveal what plants do not: wheat growth and rhizosphere microbial communities after Azospirillum brasilense inoculation and nitrogen fertilization under field conditions

  • Luciana P. Di Salvo
  • Lucía Ferrando
  • Ana Fernández-Scavino
  • Inés E. García de SalamoneEmail author
Regular Article



Azospirillum brasilense is one of plant growth promoting bacteria used to improve plant growth and grain yield of cereal crops. The level of inoculation response is defined by complex plant-microorganism interactions, many of them still unknown. Thus, we evaluated both agronomic response and microbial ecology of wheat crop under A. brasilense inoculation and nitrogen fertilization at field conditions in order to improve inoculation efficiency.


Treatments were: control, nitrogen fertilization and inoculation with 40M and 42M strains. Functional and structural diversity of rhizosphere bacterial communities were evaluated by community-level physiological and terminal restriction fragment length polymorphism profiles. Besides, aerial biomass, grain yield and counts of microaerophilic diazotrophic rhizobacteria were determined.


Plant ontogeny modified the number of culturable microaerophilic diazotrophic rhizobacteria. Although agronomic response did not show differences, plant ontogeny and the agricultural practices modified both physiology and genetic structure of rhizosphere microbial communities. Interestingly, these differences due to the treatments were observed at jointing stage but not at grain-filling stage of wheat.


Our results demonstrate how different management decisions can change plant- microorganism relationships. While wheat could not show differences between some agricultural treatments, under the soil surface microbial communities could show them.


Triticum aestivum CLPP Crop production, Functional diversity Structural diversity T-RFLP 



This work was partially supported by FONCYT 2008 PICT 1864 from the MINCyT, UBACyT project 20020090100255, Universidad de Buenos Aires (UBA) in Argentina. The authors received financial support from the collaborative projects PROMAI UBA and AUGM for travel expenses of L.P.D.S., L.F. and A.F.S. We are grateful to the agronomist Patricio Perdoménico and personal of “La Aurora”, Villa Moll, Buenos Aires, Argentina. We are also grateful to editors and anonymous reviewers for their comments and suggestions. We would like to dedicate this work to the memory of Dr. Katia RS Teixeira, Brazilian researcher of the EMBRAPA, Rio de Janeiro, Brazil, who always will be in our hearts.


  1. Abril A, Biasutti C, Maich R, Dubbini L, Noe L (2006) Inoculación con Azospirillum spp. en la región semiárida-central de la Argentina: factores que afectan la colonización rizosférica. Ciencia Suelo 24:11–19Google Scholar
  2. Allison SD, Martiny JBH (2008) Resistance, resilience, and redundancy in microbial communities. Proc Natl Acad Sci U S A 105:11512–11519CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bashan Y, de-Bashan LE (2010) How the plant growth-promoting bacterium Azospirillum promotes plant growth—a critical assessment. Adv Agron 108:77–136CrossRefGoogle Scholar
  4. Bastida F, Zsolnay A, Hernández T, García C (2008) Past, present and future of soil quality indices: A biological perspective. Geoderma 147:159–171CrossRefGoogle Scholar
  5. Bossio DA, Girvan MS, Verchot L, Bullimore J, Borelli T, Albrecht A, Scow KM, Ball AS, Pretty JN, Osborn AM (2005) Soil microbial community response to land use change in an agricultural landscape of Western Kenya. Microb Ecol 49:50–62CrossRefPubMedGoogle Scholar
  6. van Bruggen AHC, Semenov AM (2000) In search of biological indicators for soil health and disease suppression. App Soil Ecol 15:13–24CrossRefGoogle Scholar
  7. Caballero-Mellado J, Onofre-Lemus J, Estrada-de los Santos P, Martínez-Aguilar L (2007) The tomato rhizosphere, an environment rich in nitrogen-fixing Burkholderia species with capabilities of interest for agriculture and bioremediation. Appl Environ Microbiol 73:5308–5319CrossRefPubMedPubMedCentralGoogle Scholar
  8. Casanovas EM, Fasciglione G, Barassi CA (2015) Azospirillum spp. and related PGPRs inocula use in intensive agriculture. In: Cassán FD, Okon Y, Creus CM (eds) Handbook for Azospirillum: Technical Issues and Protocols. Springer, Berlin, pp 447–468Google Scholar
  9. Casaretto E, Labandera C (2008) Respuesta de maíz a la inoculación con Azospirillum. In: Cassán FC, García de Salamone IE (eds) Azospirillum sp.: cell physiology, plant interactions and agronomic research in Argentina, Asociación Argentina de Microbiología, Buenos Aires, Argentina, pp. 261–268Google Scholar
  10. Cassán FD, Díaz-Zorita M (2016) Azospirillum sp. in current agriculture: From the laboratory to the field. Soil Biol Bioch 103:117–130CrossRefGoogle Scholar
  11. Cassán FD, Okon Y, Creus MC (2015) Handbook for Azospirillum: Technical Issues and Protocols. Springer, BerlinCrossRefGoogle Scholar
  12. Conn VM, Franco CMM (2004) Analysis of the endophytic actinobacterial population in the roots of wheat (Triticum aestivum L.) by terminal restriction fragment length polymorphism and sequencing of 16S rRNA. Appl Environ Microbiol 70:1787–1794CrossRefPubMedPubMedCentralGoogle Scholar
  13. Cummings SP (2009) The application of plant growth promoting rhizobacteria (PGPR) in low input and organic cultivation of graminaceous crops; potential and problems. Environ Biot 5:43–50Google Scholar
  14. Den Herder G, Van Isterdael G, Beeckman T, De Smet I (2010) The roots of a new green revolution. Trends Plant Sci 15:600–607CrossRefGoogle Scholar
  15. Díaz-Zorita M, Fernández-Canigia MV, Bravo OA, Berger A, Satorre EH (2015) Field evaluation of extensive crops inoculated with Azospirillum sp. In: Cassán FD, Okon Y, Creus CM (eds) Handbook for Azospirillum: Technical Issues and Protocols. Springer, Berlin, pp 435–445Google Scholar
  16. Di Cello F, Bevivino A, Chiarini L, Fani R, Paffetti D, Tabacchioni S, Dalmastri C (1997) Biodiversity of a Burkholderia cepacia population isolated from the maize rhizosphere at different plant growth stages. Appl Environ Microbiol 63:4485–4493PubMedPubMedCentralGoogle Scholar
  17. Di Rienzo JA, Casanoves F, Balzarini MG, Gonzalez L, Tablada M, Robledo CW (2011) InfoStat versión 2011. Grupo InfoStat, FCA, Universidad Nacional de Córdoba, ArgentinaGoogle Scholar
  18. Di Salvo LP, García de Salamone IE (2012) Evaluation of soil-microbial communities by their CLPP. Standardization of a laboratory technique to replace commercial available microplates. Austral Ecol 22:129–136Google Scholar
  19. Di Salvo LP, Silva E, Teixeira KR, Esquivel-Cote R, Pereyra MA, García de Salamone IE (2014) Physiological and biochemical characterization of Azospirillum brasilense strains commonly used as plant growth-promoting rhizobacteria. J Basic Microbiol 54:1310–1321CrossRefPubMedGoogle Scholar
  20. Döbereiner J (1998) Isolation and identification of aerobic nitrogen-fixing bacteria from soil and plants. In: Alef K, Nannipieri P (eds) Methods in applied soil microbiology and biochemistry. Academic Press, California, pp 134–141Google Scholar
  21. Döbereiner J, Pedrosa F (1987) Nitrogen-fixing bacteria in non-leguminous crop plant. Spring, MichiganGoogle Scholar
  22. Dunbar J, Ticknor LO, Kuske CR (2001) Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities. Appl Environ Microbiol 67:190–197CrossRefPubMedPubMedCentralGoogle Scholar
  23. FAOSTAT (2012) Food and Agricultural commodities production.
  24. Ferrando L, Fernández Mañay J, Fernández Scavino A (2012) Molecular and culture-dependent analyses revealed similarities in the endophytic bacterial community composition of leaves from three rice (Oryza sativa) varieties. FEMS Microbiol Ecol 80:696–708CrossRefPubMedGoogle Scholar
  25. Fuentes-Ramírez LE, Bustillos-Cristales R, Tapia-Hernández A, Jiménez-Salgado T, Wang ET, Martínez-Romero E, Caballero-Mellado J (2001) Novel nitrogen-fixing acetic acid bacteria, Gluconacetobacter johannae sp. nov. and Gluconacetobacter azotocaptans sp. nov., associated with coffee plants. Int J Syst Evol Microbiol 51:1305–1314CrossRefPubMedGoogle Scholar
  26. Gamalero E, Glick BR (2011) Mechanisms used by plant growth-promoting bacteria. In: Maheshwari DK (ed) Bacteria in agrobiology: Plant nutrient management. Springer, Berlin, pp 17–46Google Scholar
  27. García de Salamone IE (2012a) Use of soil microorganisms to improve plant growth and ecosystem sustainability. In: Caliskan M (ed) The molecular basis of plant genetic diversity. INTECH, Rijeka, Croatia, pp. 233–258. Open access:
  28. García de Salamone IE (2012b) Microorganismos promotores del crecimiento vegetal. Informaciones Agronómicas de Hispanoamérica 5:12–16.$FILE/3%20Art.pdf
  29. García de Salamone IE, Döbereiner J (1996) Maize genotype effects on the response to Azospirillum inoculation. Biol Fert Soils 21:193–196CrossRefGoogle Scholar
  30. García de Salamone IE, Di Salvo LP, Escobar Ortega JS, Boa Sorte MP, Urquiaga S, Dos Santos Teixeira KR (2010) Field response of rice paddy crop to inoculation with Azospirillum: physiology of rhizosphere bacterial communities and the genetic diversity of endophytic bacteria in different parts of the plants. Plant Soil 336:351–362CrossRefGoogle Scholar
  31. García de Salamone IE, Funes JM, Di Salvo LP, Escobar Ortega JS, D'Auria F, Ferrando L, Fernandez Scavino A (2012) Inoculation of paddy rice with Azospirillum brasilense and Pseudomonas fluorescens: Impact of plant genotypes on the rhizosphere microbial communities and field crop production. Appl Soil Ecol 61:196–204CrossRefGoogle Scholar
  32. Germida JJ, Siciliano SD (2001) Taxonomic diversity of bacteria associated with the roots of modern, recent and ancient wheat cultivars. Biol Fert Soils 33:410–415CrossRefGoogle Scholar
  33. Gewin V (2010) An underground revolution. Nature 466:552–553CrossRefPubMedGoogle Scholar
  34. Gillis M, Tran Van V, Bardin R, Goor M, Hebbar P, Willems A, Segers P, Kersters K, Heulin T, Fernandez MP (1995) Polyphasic taxonomy in the genus Burkholderia leading to an emended description of the genus and proposition of Burkholderia vietnarniensis sp. nov. for N2-fixing isolates from rice in Vietnam. Int J Syst Bacteriol 45:274–289CrossRefGoogle Scholar
  35. Gil-Sotres F, Trasar-Cepeda C, Leiros MC, Seoane S (2005) Different approaches to evaluating soil quality using biochemical properties. Soil Biol Biochem 37:877–887CrossRefGoogle Scholar
  36. Gómez E, Garland J, Conti M (2004) Reproducibility in the response of soil bacterial community-level physiological profiles from a land use intensification gradient. Appl Soil Ecol 26:21–30CrossRefGoogle Scholar
  37. Grandy AS, Robertson GP, Thelen KD (2006) Do productivity and environmental trade-offs justify periodically cultivating no-till cropping systems? Agron J 98:1377–1383CrossRefGoogle Scholar
  38. Herschkovitz Y, Lerner A, Davidov Y, Rothballer M, Hartmann A, Okon Y, Jurkevitch E (2005) Inoculation with the plant-growth-promoting rhizobacterium Azospirillum brasilense causes little disturbance in the rhizosphere and rhizoplane of maize (Zea mays). Microb Ecol 50:277–288CrossRefPubMedGoogle Scholar
  39. Hooper DU, Bignell DE, Brown VK, Brussard L, Dangerfield JM, Wall DH, Wardle DA, Coleman DC, Giller KE, Lavelle P, Van der Putten WH, De Ruiter PC, Rusek J, Silver WL, Tiedje JM, Wolters V (2000) Interactions between aboveground and belowground biodiversity in terrestrial ecosystems: patterns, mechanisms, and feedbacks. Bioscience 50:1049–1061CrossRefGoogle Scholar
  40. Houlden A, Timms-Wilson TM, Day MJ, Bailey MJ (2008) Influence of plant development stage on microbial community structure and activity in the rhizosphere of three field crops. FEMS Microbiol Ecol 65:193–201CrossRefPubMedGoogle Scholar
  41. Hungria M, Campo RJ, Souza EM, Pedrosa FO (2010) Inoculation with selected strains of Azospirillum brasilense and A. lipoferum improves yields of maize and wheat in Brazil. Plant Soil 331:413–425CrossRefGoogle Scholar
  42. Karthikeyan N, Prasanna R, Nain L, Kaushik BD (2007) Evaluating the potential of plant growth promoting cyanobacteria as inoculants for wheat. Eur J Soil Biol 43:23–30CrossRefGoogle Scholar
  43. Kazi N, Deaker R, Wilson N, Muhammad K, Trethowan R (2016) The response of wheat genotypes to inoculation with Azospirillum brasilense in the field. Field Crop Res 196:368–378CrossRefGoogle Scholar
  44. Kent AD, Triplett EW (2002) Microbial communities and their interactions in soil and rhizosphere ecosystems. Ann Rev Microbiol 56:211–236CrossRefGoogle Scholar
  45. Kobližek M, Béjà O, Bidigare RR, Cheristensen S, Benitez-Nelson B, Vetriani C, Kolber MK, Falkowski PG, Kolber ZS (2003) Isolation and characterization of Erythrobacter sp. strains from the upper ocean. Arch Microbiol 180:327–338CrossRefPubMedGoogle Scholar
  46. Kirk JL, Beaudette LA, Hart M, Moutoglis P, Klironomos JN, Lee H, Trevors JT (2004) Methods of studying soil microbial diversity. J Microbiol Meth 58:169–188CrossRefGoogle Scholar
  47. Li WJ, Xu P, Zhang LP, Tang SK, Cui XL, Mao PH, Xu LH, Schumann P, Stackebrandt E, Jiang CL (2003) Streptomonospora alba sp. nov., a novel halophilic actinomycete, and emended description of the genus Streptomonospora Cui et al. 2001. Int J Syst Evol Microbiol 53:1421–1425CrossRefPubMedGoogle Scholar
  48. Lu Y, Rosencrantz K, Liesack W, Conrad R (2006) Structure and activity of bacterial community inhabiting rice roots and the rhizosphere. Environ Microbiol 8:1351–1360CrossRefPubMedGoogle Scholar
  49. Lupwayi NZ, Rice WA, Clayton GW (1998) Soil microbial diversity and community structure under wheat as influenced by tillage and crop rotation. Soil Biol Biochem 30:1733–1741CrossRefGoogle Scholar
  50. de Man JC (1983) MPN tables, corrected. Eur J Appl Microbiol Biotech 17:301–305CrossRefGoogle Scholar
  51. Marchesi JR, Sato T, Weightman AJ, Martin TA, Fry JC, Hiom SJ, Wade WG (1998) Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. App Environ Microbiol 64:795–799Google Scholar
  52. Mendes LW, Kuramae EE, Navarrete AA, van Veen JA, Tsai SM (2014) Taxonomical and functional microbial community selection in soybean rhizosphere. ISME J 8:1577–1587CrossRefPubMedPubMedCentralGoogle Scholar
  53. Mendes LW, Tsai SM, Navarrete AA, de Hollander M, van Veen JA, Kuramae EE (2015) Soil-borne microbiome: linking diversity to function. Microb Ecol 70:255–265CrossRefPubMedGoogle Scholar
  54. Miethling R, Wieland G, Backhaus H, Tebbe CC (2000) Variation of microbial rhizosphere communities in response to crop species, soil origin, and inoculation with Sinorhizobium meliloti L33. Microb Ecol 41:43–56CrossRefGoogle Scholar
  55. Mills AL, Garland JL (2002) Application of physiological profiles to assess community properties. In: Hurst CJ, Crawford RL, Knudsen GR, McInerney MJ, Stenzenback LD (eds) Manual of environmental microbiology. ASM Press, Washington DC, pp 135–146Google Scholar
  56. Minz D, Ofek M (2011) Rhizosphere microorganisms. In: Rosenberg E, Gophna U (eds) Beneficial microorganisms in multicellular life forms. Springer, Verlag, pp 105–121Google Scholar
  57. Naiman AD, Latrónico A, García de Salamone IE (2009) Inoculation of wheat with Azospirillum brasilense and Pseudomonas fluorescens: impact on the production and culturable rhizosphere microflora. Eur J Soil Biol 45:44–51CrossRefGoogle Scholar
  58. Pedraza RO, Bellone CH, Carrizo de Bellone S, Fernandes Boa Sorte PM, Teixeira KRS (2009) Azospirillum inoculation and nitrogen fertilization effect on grain yield and on the diversity of endophytic bacteria in the phyllosphere of rice rainfed crop. Eur J Soil Biol 45:36–43CrossRefGoogle Scholar
  59. Peixoto RS, Coutinho HLC, Madari B, Machado PLOA, Rumjanek NG, Van Elsas JD, Seldin L, Rosado AS (2006) Soil aggregation and bacterial community structure as affected by tillage and cover cropping in the Brazilian Cerrados. Soil Till Res 90:16–28CrossRefGoogle Scholar
  60. Philippot L, Raaijmakers JM, Lemanceau P, Van der Putten W (2013) Going back to the roots: the microbial ecology of the rhizosphere. Nat Rev 11:789–799Google Scholar
  61. Preston-Mafham J, Boddy L, Randerson PF (2002) Analysis of microbial community functional diversity using sole-carbon-source utilisation profiles - a critique. FEMS Microbiol Ecol 42:1–14PubMedGoogle Scholar
  62. Roesch LFW, Olivares FL, Passaglia LMP, Selbach PA, Saccol de Sá EL, Camargo FAO (2006) Characterization of diazotrophic bacteria associated with maize: effect of plant genotype, ontogeny and nitrogen-supply. World J Microb Biot 22:967–974CrossRefGoogle Scholar
  63. Saleem M, Moe LA (2014) Multitrophic microbial interactions for eco- and agro-biotechnological processes: theory and practice. Trends Biotechnol 32:529–537CrossRefPubMedGoogle Scholar
  64. Sanguin H, Sarniguet A, Gazengel K, Moënne-Loccoz Y, Grundmann GL (2009) Rhizosphere bacterial communities associated with disease suppressiveness stages of take-all decline in wheat monoculture. New Phytol 184:694–707CrossRefPubMedGoogle Scholar
  65. Sawamura H, Yamada M, Endo K, Soda S, Ishigaki T, Ike M (2010) Characterization of microorganisms at different landfill depths using carbon-utilization patterns and 16S rRNA gene based T-RFLP. J Biosci Bioeng 109:130–137CrossRefPubMedGoogle Scholar
  66. Semmartin M, Di Bella C, García de Salamone IE (2010) Grazing-induced changes in plant species composition affect plant and soil properties of grassland mesocosms. Plant Soil 328:471–481CrossRefGoogle Scholar
  67. Shyu C, Soule T, Bent SJ, Foster JA, Forney LJ (2007) MiCA: A Web-Based Tool for the Analysis of Microbial Communities Based on Terminal-Restriction Fragment Length Polymorphisms of 16S and 18S rRNA Genes. J Microb Ecol 53:562–570CrossRefGoogle Scholar
  68. Sisti CPJ, Santos HP, Kochhann R, Alves BJR, Urquiaga S, Boddey RM (2004) Change in carbon and nitrogen stocks in soil under 13 years of conventional and zero tillage in southern Brazil. Soil Till Res 76:39–58CrossRefGoogle Scholar
  69. Smalla K, Oros-Sichler M, Milling A, Heuer H, Baumgarte S, Becker R, Neuber G, Kropf S, Ulrich A, Tebbe CC (2007) Bacterial diversity of soils assessed by DGGE, T-RFLP and SSCP fingerprints of PCR-amplified 16S rRNA gene fragments: Do the different methods provide similar results? J Microbiol Meth 69:470–479CrossRefGoogle Scholar
  70. Steddom K, Menge JA, Crowley D, Borneman J (2002) Effect of repetitive applications of the biocontrol bacterium Pseudomonas putida 06909-rif/nal on citrus soil microbial communities. Biol Control 92:857–862Google Scholar
  71. Tilman D, Cassman KG, Matson PA, Naylor R, Polasky S (2002) Agricultural sustainability and intensive production practices. Nature 418:671–677CrossRefPubMedGoogle Scholar
  72. Torsvik V, Øvreås L (2002) Microbial diversity and function in soil: from genes to ecosystems. Curr Opin Microbiol 5:240–245CrossRefPubMedGoogle Scholar
  73. Wardle DA, Bardgett RD, Klironomos JN, Setälä H, Van der Putten WH, Wall DH (2004) Ecological linkages between aboveground and belowground biota. Science 304:1629–1633CrossRefPubMedGoogle Scholar
  74. Xu Y, Wang G, Jin J, Liu J, Zhang Q, Liu X (2009) Bacterial communities in soybean rhizosphere in response to soil type, soybean genotype, and their growth stage. Soil Biol Biochem 41:919–925CrossRefGoogle Scholar
  75. Xue D, Yao HY, Ge DY, Huang CY (2008) Soil microbial community structure in diverse land use systems: a comparative study using Biolog, DGGE, and PLFA analyses. Pedosphere 18:653–663CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  • Luciana P. Di Salvo
    • 1
  • Lucía Ferrando
    • 2
  • Ana Fernández-Scavino
    • 2
  • Inés E. García de Salamone
    • 3
    Email author
  1. 1.CONICET - Cátedra de Microbiología Agrícola, Facultad de AgronomíaUniversidad de Buenos Aires. Av. San Martín 4453Ciudad Autónoma de Buenos AiresArgentina
  2. 2.Cátedra de Microbiología, Facultad de QuímicaUniversidad de la RepúblicaMontevideoUruguay
  3. 3.Facultad de Agronomía, Departamento de Biología Aplicada y AlimentosUniversidad de Buenos AiresCiudad Autónoma de Buenos AiresArgentina

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