Advertisement

Annals of Microbiology

, Volume 65, Issue 4, pp 2187–2200 | Cite as

Composition and activity of endophytic bacterial communities in field-grown maize plants inoculated with Azospirillum brasilense

  • Emilyn Emy Matsumura
  • Vinícius Andrade Secco
  • Renata Stolf Moreira
  • Odair José Andrade Pais dos Santos
  • Mariangela Hungria
  • André Luiz Martinez de Oliveira
Original Article

Abstract

The application of agricultural practices in which non-leguminous plants are inoculated with growth-promoting diazotrophic bacteria is gaining importance worldwide. Nevertheless, an efficient strategy for using this inoculation technology is still lacking, and a better comprehension of the environmental factors that influence a plant’s ability to support its associative bacterial community is indispensable to achieving standardized inoculation responses. To address the effects of nitrogen (N)-fertilization on the diversity of both the total and metabolically active endophytic bacterial communities of field-grown maize plants, we extracted total DNA and RNA from maize plants inoculated with Azospirillum brasilense strain Ab-V5 that were growing in Oxisol and treated with regular and low levels of N-fertilizers (RN and LN, respectively). Four clonal libraries were constructed and sequenced and the dominant populations analyzed. Partial description of the bacterial diversity indicated that plants receiving RN- and LN-treatments can maintain bacterial communities with similar diversity indexes for the total endophytic bacterial community, although the communities of Novosphingobium and Methylobacterium were unevenly distributed. Fertilization management had a stronger effect on the dominant populations of the metabolically active bacterial community, and 16S rRNA gene libraries from RN plants suggested a lower diversity of such populations in comparison with libraries from LN plants. The agronomic parameters obtained at the end of the crop season indicated that the inoculation treatment was efficient in promoting plant growth. However, the combination of regular treatments with N-fertilizers and plant inoculation did not have an additive effect and actually tended to decrease crop productivity.

Keywords

Bacterial diversity Plant growth-promoting bacteria Non-legume inoculation 16S RNA gene library 

Notes

Acknowledgments

The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for granting Emilyn Emy Matsumura and Vinicius Andrade Secco MSc and IC fellowships, respectively. This work was partially financed by the Instituto Nacional de Ciência e Tecnologia da Fixação Biológica do Nitrogênio (INCT-FBN) and the Ministério da Ciência e Tecnologia (MCT), the CNPq and the Fundo Setorial do Agronegócio (CT-AGRO) process no. 557746/2009-4.

Conflict of interest

None.

Supplementary material

13213_2015_1059_MOESM1_ESM.doc (67 kb)
ESM 1 (DOC 67 kb)
13213_2015_1059_MOESM2_ESM.doc (60 kb)
ESM 2 (DOC 60 kb)
13213_2015_1059_MOESM3_ESM.doc (39 kb)
ESM 3 (DOC 39 kb)
13213_2015_1059_MOESM4_ESM.doc (47 kb)
ESM 4 (DOC 47 kb)

References

  1. Araújo W, Marcon J, Maccheroni W Jr, van Elsas JD, van Vuurde JWL, Azevedo JL (2002) Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl Environ Microbiol 68:4906–4914PubMedCentralCrossRefPubMedGoogle Scholar
  2. Arruda L, Beneduzi A, Martins A, Lisboa B, Lopes C, Bertolo F, Passaglia LMP, Vargas LK (2013) Screening of rhizobacteria isolated from maize (Zea mays L.) in Rio Grande do Sul State (South Brazil) and analysis of their potential to improve plant growth. Appl Soil Ecol 63:15–22CrossRefGoogle Scholar
  3. Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ (2005) At least 1 in 20 16S rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies. Appl Environ Microbiol 71:7724–7736PubMedCentralCrossRefPubMedGoogle Scholar
  4. Baudoin E, Nazaret S, Mougel C, Ranjard L, Moënne-Loccoz Y (2009) Impact of inoculation with the phytostimulatory PGPR Azospirillum lipoferum CRT1 on the genetic structure of the rhizobial community of field-grown maize. Soil Biol Biochem 41:409–413CrossRefGoogle Scholar
  5. Baudoin E, Lerner A, Mirza MS, El Zemrany H, Prigent-Combaret C, Jurkevich E, Spaepen S, Vanderleyden J, Nazaret S, Okon Y, Moënne-Loccoz Y (2010) Effects of Azospirillum brasilense with genetically-modified auxin biosynthesis gene ipdC upon the diversity of the indigenous microbiota of the wheat rhizosphere. Res Microbiol 161:219–226CrossRefPubMedGoogle Scholar
  6. Berendsen RL, Pieterse CMJ, Bakker PAHM (2012) The rhizosphere microbiome and plant health. Trends Plant Sci 17:478–486CrossRefPubMedGoogle Scholar
  7. Bouffaud ML, Poirier MA, Muller D, Loccoz YM (2014) Root microbiome relates to plant host evolution in maize and other Poaceae. Environ Microbiol 16(9):2804–2814Google Scholar
  8. Carvalhais LC, Dennis PG, Fedoseyenko D, Hajirezaei MR, Borriss R, von Wirén N (2010) Root exudation of sugars, amino acids, and organic acids by maize as affected by nitrogen, phosphorus, potassium, and iron deficiency. J Plant Nutr Soil Sci 000:1–9. doi: 10.1002/jpln.201000085
  9. Castro-Sowinski S, Herschkovitz Y, Okon Y, Jurkevitch E (2007) Effects of inoculation with plant growth-promoting rhizobacteria on resident rhizosphere microorganisms. FEMS Microbiol Lett 276:1–11CrossRefPubMedGoogle Scholar
  10. Chelius MK, Triplett EW (2001) The diversity of Archaea and Bacteria in association with the roots of Zea mays L. Microb Ecol 41:252–263CrossRefPubMedGoogle Scholar
  11. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM (2009) The ribosomal database project: improved alignments and new tools for RNA analysis. Nucleic Acids Res 37:141–145CrossRefGoogle Scholar
  12. Correa OS, Romero AM, Montecchia MS, Soria MA (2006) Tomato genotype and Azospirillum inoculation modulate the changes in bacterial communities associated with roots and leaves. J Appl Microbiol 102:781–786CrossRefGoogle Scholar
  13. De-Bashan LE, Hernandez JP, Nelson KN, Bashan Y, Maier RM (2010) Growth of quailbush in acidic, metalliferous desert mine tailings: effect of Azospirillum brasilense Sp6 on biomass production and rhizosphere community structure. Microb Ecol 60:915–927PubMedCentralCrossRefPubMedGoogle Scholar
  14. Döbereiner J (1992) Recent changes in concepts of plant-bacteria interactions: endophytic N2 fixing bacteria. Cienc Cult 44:310–313Google Scholar
  15. Ewing B, Green P (1998) Base-calling of automated sequencer traces using Phred II. Error probabilities. Genome Res 8:186–194CrossRefPubMedGoogle Scholar
  16. FAO (Food and Agriculture Organization) (2012) World agriculture towards 2030/2050: the 2012 revision. ESA working paper no. 12–03. Available at: http://www.fao.org/docrep/016/ap106e/ap106e.pdf. Accessed 26 Feb 2014
  17. Friesen ML, Porter SS, Stark SC, von Wettberg EJ, Sachs JL, Martinez-Romero E (2011) Microbially mediated plant functional traits. Annu Rev Ecol Evol Syst 42:23–46CrossRefGoogle Scholar
  18. Gaiero JR, McCall CA, Thompson KA, Day NJ, Best AS, Dunfield KE (2013) Inside the root microbiome: bacterial root endophytes and plant growth promotion. Am J Bot 100:1738–1750CrossRefPubMedGoogle Scholar
  19. Glick BR (2012) Plant growth-promoting bacteria: mechanisms and applications. Scientifica 2012, article ID 963401. doi: 10.6064/2012/963401
  20. Gurtler V, Stanisich VA (1996) New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region. Microbiology 142:3–16CrossRefPubMedGoogle Scholar
  21. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  22. Hartmann A, Schimid M, van Tuinen D, Berg G (2009) Plant-driven selection of microbes. Plant Soil 321:235–257CrossRefGoogle Scholar
  23. 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
  24. Hrynkiewicz K, Baum C, Niedojadlo J, Dahm H (2009) Promotion of mycorrhiza formation and growth of willows by the bacterial strain Sphingomonas sp. 23 L on fly ash. Biol Fert Soils 45:385–394CrossRefGoogle Scholar
  25. Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequences alignments. Bioinformatics 20:2317–2319CrossRefPubMedGoogle Scholar
  26. Hungria M, Campo RJ, Souza EM, Pedrosa FO (2010) Inoculation with selected strains of Azospirillum brasilense and A. lipoferum improves yield of maize and wheat in Brazil. Plant Soil 331:413–425CrossRefGoogle Scholar
  27. Ikeda S, Okubo T, Kaneko T, Inaba S, Maekawa T, Eda S, Sato S, Tabata S, Mitsui H, Minamisawa K (2010) Community shifts of soybean stem-associated bacteria responding to different nodulation phenotypes and N levels. ISME J 4:315–326CrossRefPubMedGoogle Scholar
  28. Ikeda AC, Bassani LL, Adamoski D, Stringari D, Cordeiro VK, Glienke C, Steffens MBR, Hungria M, Galli-Terasawa LV (2012) Morphological and genetic characterization of endophytic bacteria isolated from roots of different maize genotypes. Microb Ecol 65:154–160CrossRefPubMedGoogle Scholar
  29. Indiragandhi P, Anandham R, Kim KA, Yim WJ, Madhaiyan M, Sa TM (2008) Induction of defense responses in tomato against Pseudomonas syringae pv. tomato by regulating the stress ethylene level with Methylobacterium oryzae CBMB20 containing 1-aminocyclopropane-1-carboxylate deaminase. World J Microbiol Biotechnol 24:1037–1045CrossRefGoogle Scholar
  30. Jourand P, Giraud E, Béna G, Sy A, Willems A, Gillis M, Dreyfus B, Lajudie P (2004) Methylobacterium nodulans sp. nov., for a group of aerobic, facultatively methylotrophic, legume-root-nodule-forming and nitrogen-fixing bacteria. Int J Syst Evol Microbiol 54:2269–2273CrossRefPubMedGoogle Scholar
  31. Knauth S, Hurek T, Brar D, Reinhold-Hurek B (2005) Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice. Environ Microbiol 7:1725–1733CrossRefPubMedGoogle Scholar
  32. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948CrossRefPubMedGoogle Scholar
  33. Lerner A, Herschkovitz Y, Baudoin E, Nazaret S, Moënne-Loccoz Y, Okon Y, Jurkevitch E (2006) Effect of Azospirillum brasilense inoculation on rhizobacterial communities analyzed by denaturing gradient gel electrophoresis and automated ribosomal intergenic spacer analysis. Soil Biol Biochem 38:1212–1218CrossRefGoogle Scholar
  34. Liu Y, Zuo S, Zou Y, Wang J, Song W (2013) Investigation on diversity and population succession dynamics of endophytic bacteria from seeds of maize (Zea mays L., Nongda108) at different growth stages. Ann Microbiol 63:71–79CrossRefGoogle Scholar
  35. Madhaiyan M, Poonguzhali S, Sa T (2007) Metal tolerating methylotrophic bacteria reduces nickel and cadmium toxicity and promotes plant growth of tomato (Lycopersicon esculentum L.). Chemosphere 69:220–228CrossRefPubMedGoogle Scholar
  36. Masciarelli O, Urbani L, Reinoso H, Luna V (2013) Alternative mechanism for the evaluation of the Indole-3-acetic acid (IAA) production by Azospirillum brasilense strains and its effects on the germination and growth of maize seedlings. J Microbiol 51:590–597CrossRefPubMedGoogle Scholar
  37. Montañez A, Blanco AR, Barlocco C, Beracochea M, Sicardi M (2012) Characterization of cultivable putative endophytic plant growth promoting bacteria associated with maize cultivars (Zea mays L.) and their inoculation effects in vitro. Appl Soil Ecol 58:21–28CrossRefGoogle Scholar
  38. Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700PubMedCentralPubMedGoogle Scholar
  39. Pariona-Llanos R, Ferrara FIS, Gonzales HHS, Barbosa HR (2010) Influence of organic fertilization on the number of culturable diazotrophic endophytic bacteria isolated from sugarcane. Eur J Soil Biol 46:387–393CrossRefGoogle Scholar
  40. Partida-Martínez LP, Heil M (2011) The microbe-free plant: fact or artifact? Front Plant Sci 2:100. doi: 10.3389/fpls.2011.00100
  41. Pérez-Montaño F, Alías-Villegas C, Bellogín RA, del Cerro P, Espuny MR, Jiménez-Guerrero I, López-Baena FJ, Ollero FJ, Cubo T (2013) Plant growth promotion in cereal and leguminous agricultural important plants: from microrganisms capacities to crop production. Microbiol Res 169:325–336CrossRefPubMedGoogle Scholar
  42. Prakamhang J, Minamisawa K, Teamtaisong K, Bookerd N, Teaumroong N (2009) The communities of endophytic diazotrophic bacteria in cultivated rice (Oryza sativa L.). Appl Soil Ecol 42:141–149CrossRefGoogle Scholar
  43. Reinhold-Hurek B, Hurek T (2011) Living inside plants: bacterial endophytes. Curr Opin Plant Biol 14:435–443CrossRefPubMedGoogle Scholar
  44. Rodrigues Neto J, Malavolta Júnior VA, Victor O (1986) Meio simples para isolamento e cultivo de Xantomonas campestris pv. citri tipo B. Summa Phytopathol 12:16Google Scholar
  45. Roesch LFW, Camargo FAO, Bento FM, Triplett EW (2008) Biodiversity of diazotrophic bacteria within the soil, root and stem of field-grown maize. Plant Soil 302:91–104CrossRefGoogle Scholar
  46. Rösch C, Bothe H (2005) Improved assessment of denitrifying, N2-fixing, and total-community bacteria by terminal restriction fragment length polymorphism analysis using multiple restriction enzymes. Appl Environ Microbiol 71:2026–2035PubMedCentralCrossRefPubMedGoogle Scholar
  47. Ryan RP, Germaine K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent developments and applications. FEMS Microbiol Lett 278:1–9CrossRefPubMedGoogle Scholar
  48. Saleem M, Lamkemeyer T, Schützenmeister A, Madlung J, Sakai H, Piepho HP, Nordheim A, Hochholdinger F (2010) Specification of cortical parenchyma and stele of maize primary roots by asymmetric levels of auxin, cytokinin, and cytokinin-regulated proteins. Plant Physiol 152:4–18PubMedCentralCrossRefPubMedGoogle Scholar
  49. Salvagiotti F, Cassman KG, Specht JE, Walters DT, Weiss A, Dobermann A (2008) Nitrogen uptake, fixation and response to fertilizer N in soybeans: a review. Field Crop Res 108:1–13CrossRefGoogle Scholar
  50. Sambrook J, Russel DW (2001) Molecular cloning: A laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  51. Schlüter U, Mascher M, Colmsee C, Scholz U, Braütigam A, Fahnenstich H, Sonnewald U (2012) Maize source leaf adaptation to nitrogen deficiency affects not only nitrogen and carbon metabolism but also control of phosphate homeostasis1. Plant Physiol 160:1384–1406PubMedCentralCrossRefPubMedGoogle Scholar
  52. Seghers D, Wittebolle L, Top EM, Verstraete W, Siciliano SD (2004) Impact of agricultural practices on the Zea mays L. endophytic community. Appl Environ Microbiol 73:1475–1482CrossRefGoogle Scholar
  53. Singh JS, Pandey VC, Singh DP (2011) Efficient soil microorganisms: a new dimension for sustainable agriculture and environmental development. Agric Ecosyst Environ 140:339–353CrossRefGoogle Scholar
  54. Singleton DR, Furlong MA, Rathbun SL, Whitman WB (2001) Quantitative comparisons of 16S rRNA gene sequence libraries from environmental sequences. Appl Environ Microbiol 67:4374–4376PubMedCentralCrossRefPubMedGoogle Scholar
  55. Sy A, Giraud E, Jourand P, Garcia N, Willems A, DeLajudie P, Prin Y, Neyra M, Gillis M, Boivin-Masson C, Dreyfus B (2001) Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes. J Bacteriol 183:214–220PubMedCentralCrossRefPubMedGoogle Scholar
  56. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729PubMedCentralCrossRefPubMedGoogle Scholar
  57. Videira SS, Araújo JLS, Rodrigues LS, Baldani VLD, Baldani JI (2009) Occurrence and diversity of nitrogen-fixing Sphingomonas bacteria associated with rice plants grown in Brazil. FEMS Microbiol Lett 293:11–19CrossRefPubMedGoogle Scholar
  58. Videira SS, Silva MCP, Galisa PS, Dias ACF, Nissinen R, Baldani VLD, van Elsas JD, Baldani JI, Salles JF (2013) Culture-independent molecular approaches reveal a mostly unknown high diversity of active nitrogen-fixing bacteria associated with Pennisetum purpureum – a bioenergy crop. Plant Soil 373:737–754CrossRefGoogle Scholar
  59. Wahab AMA, Zahran HH, Abd-Alla MH (1996) Root-hair formation and nodulation of four grain legumes as affected by the form and the application time of nitrogen fertilizer. Folia Microbiol 41:303–308CrossRefGoogle Scholar
  60. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naïve bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267PubMedCentralCrossRefPubMedGoogle Scholar
  61. Yu J, Pressoir G, Briggs WH et al (2006) A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. Nat Genet 38:203–208CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg and the University of Milan 2015

Authors and Affiliations

  • Emilyn Emy Matsumura
    • 1
  • Vinícius Andrade Secco
    • 2
  • Renata Stolf Moreira
    • 3
  • Odair José Andrade Pais dos Santos
    • 2
  • Mariangela Hungria
    • 4
  • André Luiz Martinez de Oliveira
    • 2
  1. 1.Departamento de MicrobiologiaUniversidade Estadual de LondrinaLondrinaBrazil
  2. 2.Departamento de Bioquímica e BiotecnologiaUniversidade Estadual de LondrinaLondrinaBrazil
  3. 3.Departamento de Biologia Animal e VegetalUniversidade Estadual de LondrinaLondrinaBrazil
  4. 4.Laboratório de Biotecnologia do SoloEmbrapa SojaLondrinaBrazil

Personalised recommendations