Applied Microbiology and Biotechnology

, Volume 97, Issue 10, pp 4639–4649 | Cite as

Comparison of prominent Azospirillum strains in AzospirillumPseudomonasGlomus consortia for promotion of maize growth

  • Olivier Couillerot
  • Augusto Ramírez-Trujillo
  • Vincent Walker
  • Andreas von Felten
  • Jan Jansa
  • Monika Maurhofer
  • Geneviève Défago
  • Claire Prigent-Combaret
  • Gilles Comte
  • Jesus Caballero-Mellado
  • Yvan Moënne-Loccoz
Environmental Biotechnology

Abstract

Azospirillum are prominent plant growth-promoting rhizobacteria (PGPR) extensively used as phytostimulatory crop inoculants, but only few studies are dealing with Azospirillum-containing mixed inocula involving more than two microorganisms. We compared here three prominent Azospirillum strains as part of three-component consortia including also the PGPR Pseudomonas fluorescens F113 and a mycorrhizal inoculant mix composed of three Glomus strains. Inoculant colonization of maize was assessed by quantitative PCR, transcription of auxin synthesis gene ipdC (involved in phytostimulation) in Azospirillum by RT-PCR, and effects on maize by secondary metabolic profiling and shoot biomass measurements. Results showed that phytostimulation by all the three-component consortia was comparable, despite contrasted survival of the Azospirillum strains and different secondary metabolic responses of maize to inoculation. Unexpectedly, the presence of Azospirillum in the inoculum resulted in lower phytostimulation in comparison with the PseudomonasGlomus two-component consortium, but this effect was transient. Azospirillum's ipdC gene was transcribed in all treatments, especially with three-component consortia, but not with all plants and samplings. Inoculation had no negative impact on the prevalence of mycorrhizal taxa in roots. In conclusion, this study brought new insights in the functioning of microbial consortia and showed that AzospirillumPseudomonasGlomus three-component inoculants may be useful in environmental biotechnology for maize growth promotion.

Keywords

PGPR Azospirillum Pseudomonas Glomus Microbial consortia Inoculum survival Maize 

Supplementary material

253_2012_4249_MOESM1_ESM.pdf (232 kb)
ESM 1(PDF 231 kb)

References

  1. Adesemoye A, Torbert H, Kloepper J (2009) Plant growth-promoting rhizobacteria allow reduced application rates of chemical fertilizers. Microbial Ecol 58:921–929CrossRefGoogle Scholar
  2. Barea JM, Andrade G, Bianciotto VV, Dowling D, Lohrke S, Bonfante P, O'Gara F, Azcon-Aguilar C (1998) Impact on arbuscular mycorrhiza formation of Pseudomonas strains used as inoculants for biocontrol of soil-borne fungal plant pathogens. Appl Environ Microbiol 64:2304–2307Google Scholar
  3. Basaglia M, Casella S, Peruch U, Poggiolini S, Vamerali T, Mosca G, Vanderleyden J, De Troch P, Nuti MP (2003) Field release of genetically marked Azospirillum brasilense in association with Sorghum bicolor L. Plant Soil 256:281–290CrossRefGoogle Scholar
  4. Bashan Y (1998) Inoculants of plant growth promoting bacteria for use in agriculture. Biotech Adv 16:729–770CrossRefGoogle Scholar
  5. Bashan Y, Moreno M, Troyo E (2000) Growth promotion of the seawater-irrigated oilseed halophyte Salicornia bigelovii inoculated with mangrove rhizosphere bacteria and halotolerant Azospirillum spp. Biol Fertil Soils 32:265–272CrossRefGoogle Scholar
  6. Baudoin E, Couillerot O, Spaepen S, Moënne-Loccoz Y, Nazaret S (2009a) Applicability of the 16S–23S rDNA internal spacer for PCR detection of the phytostimulatory PGPR inoculant Azospirillum lipoferum CRT1 in field soil. J Appl Microbiol 108:25–38CrossRefGoogle Scholar
  7. Baudoin E, Nazaret S, Mougel C, Ranjard L, Moënne-Loccoz Y (2009b) Impact of inoculation with the phytostimulatory PGPR Azospirillum lipoferum CRT1 on the genetic structure of the rhizobacterial community of field-grown maize. Soil Biol Biochem 41:409–413CrossRefGoogle Scholar
  8. Belimov AA, Kojemiakov AP, Chuvarliyeva CV (1995) Interaction between barley and mixed cultures of nitrogen fixing and phosphate-solubilizing bacteria. Plant Soil 173:29–37CrossRefGoogle Scholar
  9. Biró B, Köves-Péchy K, Vörös I, Takács T, Eggenberger P, Strasser RJ (2000) Interrelations between Azospirillum and Rhizobium nitrogen-fixers and arbuscular mycorrhizal fungi in the rhizosphere of alfalfa in sterile, AMF-free or normal soil conditions. Appl Soil Ecol 15:159–168CrossRefGoogle Scholar
  10. Cassan F, Perrig D, Sgroy V, Masciarelli O, Penna C, 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.). Eur J Plant Pathol 45:28–35Google Scholar
  11. Charyulu PBBN, Foucassie F, Barbouche AK, Rondro Harisoa L, Omar AMN, Marie R, Balandreau J (1985) Field inoculation of rice using in vitro selected bacterial and plant genotypes. In: Klingmüller W (ed) Azospirillum III: genetics, physiology, ecology. Springer-Verlag, Heidelberg, pp 163–179Google Scholar
  12. Combes-Meynet E, Pothier JF, Moënne-Loccoz Y, Prigent-Combaret C (2011) The Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol is a signal inducing rhizoplane expression of Azospirillum genes involved in plant-growth promotion. Mol Plant Microbe Interact 24:271–284CrossRefGoogle Scholar
  13. Corich V, Giacomini A, Concheri G, Ritzerfeld B, Vendramin P, Struffi P, Basaglia M, Squartini A, Casella S, Nuti MP, Peruch U, Poggiolini S, de Troch P, Vanderleyden J, Fedi S, Fenton A, Moënne-Loccoz Y, Dowling DN, O'Gara F (1995) Environmental impact of genetically modified Azospirillum brasilense, Pseudomonas fluorescens and Rhizobium leguminosarum released as soil/seed inoculants. In: Jones DD (ed) Proceedings of the third international symposium on the biosafety results of field tests of genetically-modified plants and microorganisms. University of California, Monterey, pp 371–388Google Scholar
  14. Couillerot O, Poirier MA, Prigent-Combaret C, Mavingui P, Caballero-Mellado J, Moënne-Loccoz Y (2010a) Assessment of SCAR markers to design real-time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize. J Appl Microbiol 109:528–538Google Scholar
  15. Couillerot O, Bouffaud ML, Muller D, Caballero-Mellado J, Moënne-Loccoz Y (2010b) Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings. Soil Biol Biochem 42:2298–2305CrossRefGoogle Scholar
  16. Couillerot O, Combes-Meynet E, Pothier JF, Bellvert F, Challita E, Poirier MA, Rohr R, Comte G, Moënne-Loccoz Y, Prigent-Combaret Y (2011) The role of the antimicrobial compound 2,4-diacetylphloroglucinol in the impact of biocontrol Pseudomonas fluorescens F113 on Azospirillum brasilense phytostimulators. Microbiology 157:1694–1705CrossRefGoogle Scholar
  17. Creus CM, Graziano M, Casanovas EM, Pereyra MA, Simontacchi M, Puntarulo S, Barassi CA, Lamattina L (2005) Nitric oxide is involved in the Azospirillum brasilense-induced lateral root formation in tomato. Planta 221:297–303CrossRefGoogle Scholar
  18. Dobbelaere S, Croonenborghs A, Amber T, Ptacek D, Vanderleyden J, Dutto P, Labandera-Gonzalez C, Caballero-Mellado J, Aguirre JF, Kapulnik Y, Shimon B, Burdman S, Kadouri D, Sarig S, Okon Y (2001) Responses of agronomically important crops to inoculation with Azospirillum. Austral J Plant Physiol 28:1–9Google Scholar
  19. Dobbelaere S, Croonenborghs A, Thys A, Ptacek D, Okon Y, Vanderleyden J (2002) Effect of inoculation with wild type Azospirillum brasilense and A. irakense strains on development and nitrogen uptake of spring wheat and grain maize. Biol Fertil Soils 36:284–297CrossRefGoogle Scholar
  20. Dobbelaere S, Vanderleyden J, Okon Y (2003) Plant growth-promoting effects of diazotrophs in the rhizosphere. Crit Rev Plant Sci 22:107–149CrossRefGoogle Scholar
  21. Döbereiner J, Marriel IE, Nery M (1976) Ecological distribution of Spirillum lipoferum Beijerinck. Can J Microbiol 22:1464–1472CrossRefGoogle Scholar
  22. El Zemrany H, Cortet J, Peter Lutz M, Chabert A, Baudoin E, Haurat J, Maughan N, Felix D, Défago G, Bally R, Moënne-Loccoz Y (2006) Field survival of the phytostimulator Azospirillum lipoferum CRT1 and functional impact on maize crop, biodegradation of crop residues, and soil faunal indicators in a context of decreasing nitrogen fertilisation. Soil Biol Biochem 38:1712–1726CrossRefGoogle Scholar
  23. El-Komy HMA (2005) Coimmobilization of Azospirillum lipoferum and Bacillus megaterium for successful phosphorus and nitrogen nutrition of wheat plants. Food Technol Biotech 43:19–27Google Scholar
  24. Er F, Kilic M, Brohi AR, Ogut M (2004) Nodulation and growth of beans [Phaseolus vulgaris L.] inoculated with genetically modified Rhizobium etli strains and Azospirillum brasilense. Agrochimica 48:124–131Google Scholar
  25. Fages J, Mulard D (1988) Isolement de bactéries rhizosphériques et effet de leur inoculation en pots chez Zea mays. Agronomie 8:309–314CrossRefGoogle Scholar
  26. Fenton AM, Stephens PM, Crowley J, O'Callaghan M, O'Gara F (1992) Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain. Appl Environ Microbiol 58:3873–3878Google Scholar
  27. Fuentes-Ramirez L, Caballero-Mellado J (2006) Bacterial biofertilizers. In: Siddiqui ZA (ed) PGPR: biocontrol and biofertilization. Springer-Verlag, Heidelberg, pp 143–172CrossRefGoogle Scholar
  28. Garbaye J (1994) Helper bacteria: a new dimension to the mycorrhizal symbiosis. New Phytol 128:197–210CrossRefGoogle Scholar
  29. Gaur R, Shani N, Kawaljeet, Johri BN, Rossi P, Aragno M (2004) Diacetylphloroglucinol-producing pseudomonads do not influence AM fungi in wheat rhizosphere. Curr Sci 88:453–457Google Scholar
  30. Han SO, New PB (1998) Variation in nitrogen fixing ability among natural isolates of Azospirillum. Microbial Ecol 36:193–201CrossRefGoogle Scholar
  31. Herschkovitz Y, Lerner A, Davidov Y, Okon Y, Jurkevitch E (2005a) Azospirillum brasilense does not affect population structure of specific rhizobacterial communities of inoculated maize (Zea mays). Environ Microbiol 7:1847–1852CrossRefGoogle Scholar
  32. Herschkovitz Y, Lerner A, Davidov Y, Rothballer M, Hartmann A, Okon Y, Jurkevitch E (2005b) Inoculation with the plant-growth-promoting rhizobacterium Azospirillum brasilense causes little disturbance in the rhizosphere and rhizoplane of maize (Zea mays). Microbial Ecol 50:277–288CrossRefGoogle Scholar
  33. Jacoud C, Faure D, Wadoux P, Bally R (1998) Development of a strain-specific probe to follow inoculated Azospirillum lipoferum CRT1 under field conditions and enhancement of maize root development by inoculation. FEMS Microbiol Ecol 27:43–51CrossRefGoogle Scholar
  34. Jacoud C, Job D, Wadoux P, Bally R (1999) Initiation of root growth stimulation by Azospirillum lipoferum CRT1 during maize seed germination. Can J Microbiol 45:339–342Google Scholar
  35. James EK (2000) Nitrogen fixation in endophytic and associative symbiosis. Field Crops Res 65:197–209CrossRefGoogle Scholar
  36. Jansa J, Mozafar A, Frossard E (2005) Phosphorus acquisition strategies within arbuscular mycorrhizal fungal community of a single field site. Plant Soil 276:163–176CrossRefGoogle Scholar
  37. Jansa J, Smith FA, Smith SE (2008) Are there benefits of simultaneous root colonization by different arbuscular mycorrhizal fungi? New Phytol 177:779–789CrossRefGoogle Scholar
  38. Joe MM, Sivakumar PK (2010) Seed priming with co-flocs of Azospirillum and Pseudomonas for effective management of rice blast. Arch Phytopathol Plant Protect 43:1551–1563CrossRefGoogle Scholar
  39. Khan MA, Khokhar SN, Ahmed R, Afzal A (2009) Wheat growth and yield in response to coinoculation of Rhizobium, Azospirillum and Pseudomonas under rainfed conditions. Int J Biol Biotechnol 6:257–263Google Scholar
  40. Khorshidi YR, Ardakani MR, Ramezanpour MR, Khavazi K, Zargari K (2011) Response of yield and yield components of rice (Oryza sativa L.) to Pseudomonas fluorescens and Azospirillum lipoferum under different nitrogen levels. American-Eurasian J Agric & Environ Sci 10:387–395Google Scholar
  41. 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
  42. Lucy M, Reed E, Glick BR (2004) Applications of free living plant growth-promoting rhizobacteria. Anton Leeuw Int J G 86:1–25CrossRefGoogle Scholar
  43. Mar Vázquez M, César S, Azcón R, Barea JM (2000) Interactions between arbuscular mycorrhizal fungi and other microbial inoculants (Azospirillum, Pseudomonas, Trichoderma) and their effects on microbial population and enzyme activities in the rhizosphere of maize plants. Appl Soil Ecol 15:261–272CrossRefGoogle Scholar
  44. Naiman AD, Latronico A, Garcia de Salamone IEG (2009) Inoculation of wheat with Azospirillum brasilense and Pseudomonas fluorescens: impact on the production and culturable rhizosphere microflora. Eur J Plant Pathol 45:44–51Google Scholar
  45. Okon Y, Labandera-Gonzalez CA (1994) Agronomic applications of Azospirillum: an evaluation of 20 years worldwide field inoculation. Soil Biol Biochem 26:1591–1601CrossRefGoogle Scholar
  46. Oliveira ALM, Stoffels M, Schmid M, Reis VM, Baldani JI, Hartmann A (2009) Colonization of sugarcane plantlets by mixed inoculations with diazotrophic bacteria. Eur J Soil Biol 45:106–113CrossRefGoogle Scholar
  47. Park JW, Crowley DE (2005) Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE. Biotechniques 38:579–586CrossRefGoogle Scholar
  48. Pedraza RO, Bellone CH, Carrizo de Bellone S, Boa Sorte PMF, Teixeira KRdS (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 Plant Pathol 45:36–43Google Scholar
  49. Prigent-Combaret C, Blaha D, Pothier JF, Vial L, Poirier MA, Wisniewski-Dyé F, Moënne-Loccoz Y (2008) Physical organization and phylogenetic analysis of acdR as leucine-responsive regulator of the 1-aminocyclopropane-1-carboxylate deaminase gene acdS in phytobeneficial Azospirillum lipoferum 4B and other Proteobacteria. FEMS Microbiol Ecol 65:202–219CrossRefGoogle Scholar
  50. Pulido LE, Cabrera A, Medina N (2003) Biofertilization using rhizobacteria and AMF in the production of tomato (Lycopersicon esculentum Mill.) and onion (Allium cepa L.) seedlings: root colonization and nutritional status. Cultivos Tropicales 24:5–13Google Scholar
  51. Raverkar K, Konde B (1988) Effect of Rhizobium and Azospirillum lipoferum inoculation on the nodulation, yield and nitrogen uptake of peanut cultivars. Plant Soil 106:249–252CrossRefGoogle Scholar
  52. Remans R, Ramaekers L, Schelkens S, Hernandez G, Garcia A, Reyes J, Mendez N, Toscano V, Mulling M, Galvez L, Vanderleyden J (2008) Effect of RhizobiumAzospirillum coinoculation on nitrogen fixation and yield of two contrasting Phaseolus vulgaris L. genotypes cultivated across different environments in Cuba. Plant Soil 312:25–37CrossRefGoogle Scholar
  53. Rodriguez Caceres EA (1982) Improved medium for isolation of Azospirilllum spp. Appl Environ Microbiol 44:990–991Google Scholar
  54. Rodriguez-Salazar J, Suarez R, Caballero-Mellado J, Iturriaga G (2009) Trehalose accumulation in Azospirillum brasilense improves drought tolerance and biomass in maize plants. FEMS Microbiol Lett 296:52–59CrossRefGoogle Scholar
  55. Russo A, Felici C, Toffanin A, Götz M, Collados C, Barea J, Moënne-Loccoz Y, Smalla K, Vanderleyden J, Nuti M (2005) Effect of Azospirillum inoculants on arbuscular mycorrhiza establishment in wheat and maize plants. Biol Fertil Soils 41:301–309CrossRefGoogle Scholar
  56. Singh SR, Zargar MY, Singh U, Ishaq M (2010) Influence of bio-inoculants and inorganic fertilizers on yield, nutrient balance, microbial dynamics and quality of strawberry (Fragariax ananassa) under rainfed conditions of Kashmir valley. Indian J Agric Sci 80:275–281Google Scholar
  57. Steinberg C, Gamard P, Faurie G, Lensi R (1989) Survival and potential denitrifying activity of Azospirillum lipoferum and Bradyrhizobium japonicum inoculated into sterilized soil. Biol Fertil Soils 7:101–107CrossRefGoogle Scholar
  58. Thonar C, Erb A, Jansa J (2011) Real-time PCR to quantify composition of arbuscular mycorrhizal fungal communities—marker design, verification, calibration, and field validation. Mol Ecol Resour 12:219–232CrossRefGoogle Scholar
  59. von Felten A, Défago G, Maurhofer M (2010) Quantification of Pseudomonas fluorescens strains F113, CHA0 and Pf153 in the rhizosphere of maize by strain-specific real-time PCR unaffected by the variability of DNA extraction efficiency. J Microbiol Meth 81:108–115CrossRefGoogle Scholar
  60. Wagg C, Jansa J, Stadler M, Schmid B, van der Heijden MGA (2011) Mycorrhizal fungal identity and diversity relaxes plant–plant competition. Ecology 92:1303–1313CrossRefGoogle Scholar
  61. Walker V, Bertrand C, Bellvert F, Moënne-Loccoz Y, Bally R, Comte G (2011) Host plant secondary metabolite profiling shows complex, strain-dependent response of maize to plant growth-promoting rhizobacteria of the genus Azospirillum. New Phytol 189:494–506CrossRefGoogle Scholar
  62. Walker V, Couillerot O, Von Felten A, Bellvert F, Jansa J, Maurhofer M, Bally R, Moënne-Loccoz Y, Comte G (2012) Variation of secondary metabolite levels in maize seedling roots induced by inoculation with Azospirillum, Pseudomonas and Glomus consortium under field conditions. Plant Soil 356:151–163Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Olivier Couillerot
    • 1
    • 2
    • 3
  • Augusto Ramírez-Trujillo
    • 4
  • Vincent Walker
    • 1
    • 2
    • 3
  • Andreas von Felten
    • 5
  • Jan Jansa
    • 6
  • Monika Maurhofer
    • 5
  • Geneviève Défago
    • 5
  • Claire Prigent-Combaret
    • 1
    • 2
    • 3
  • Gilles Comte
    • 1
    • 2
    • 3
  • Jesus Caballero-Mellado
    • 4
  • Yvan Moënne-Loccoz
    • 1
    • 2
    • 3
  1. 1.Université de LyonLyonFrance
  2. 2.Université Lyon 1VilleurbanneFrance
  3. 3.Ecologie MicrobienneCNRS, UMR5557VilleurbanneFrance
  4. 4.Centro de Ciencias Genómicas (CCG)Universidad Nacional Autónoma de MéxicoCuernavacaMéxico
  5. 5.Institute of Integrative BiologyETH ZurichZürichSwitzerland
  6. 6.Institute of Agricultural SciencesETH ZurichLindauSwitzerland

Personalised recommendations