Plant and Soil

, Volume 356, Issue 1–2, pp 151–163 | Cite as

Variation of secondary metabolite levels in maize seedling roots induced by inoculation with Azospirillum, Pseudomonas and Glomus consortium under field conditions

  • Vincent Walker
  • Olivier Couillerot
  • Andreas Von Felten
  • Floriant Bellvert
  • Jan Jansa
  • Monika Maurhofer
  • René Bally
  • Yvan Moënne-Loccoz
  • Gilles ComteEmail author
Regular Article


Background and aims

Many plant-beneficial microorganisms can influence secondary plant metabolism, but whether these effects add up when plants are co-inoculated is unclear. This issue was assessed, under field conditions, by comparing the early impacts of seed inoculation on secondary metabolite profiles of maize at current or reduced mineral fertilization levels.


Maize seeds were inoculated singly with selected strains from bacterial genera Pseudomonas and Azospirillum or mycorrhizal genus Glomus, or with these strains combined two by two or all three together. At 16 days, maize root methanolic extracts were analyzed by RP-HPLC and secondary metabolites (phenolics, flavonoids, xanthones, benzoxazionoids, etc.) identified by LC/MS.


Inoculation did not impact on plant biomass but resulted in enhanced total root surface, total root volume and/or root number in certain inoculated treatments, at reduced fertilization. Inoculation led to qualitative and quantitative modifications of root secondary metabolites, particularly benzoxazinoids and diethylphthalate. These modifications depended on fertilization level and microorganism(s) inoculated. The three selected strains gave distinct results when used alone, but unexpectedly all microbial consortia gave somewhat similar results.


The early effects on maize secondary metabolism were not additive, as combining strains gave effects similar to those of Glomus alone. This is the first study demonstrating and analyzing inoculation effects on crop secondary metabolites in the field.


Secondary metabolites Benzoxazinoids Diethylphtalate Mineral fertilization Zea mays L. 



This work was supported in part by the European Union (FW6 STREP project MicroMaize 036314). We are grateful to Pierre Castillon (Arvalis, Bazièges, France) and Arvalis staff at the Pouzol Etoile experimental station for implementation of the field trial. We thank Bachar Blal (Agrauxine, Quimper, France) and Aleš Látr (Symbio-M, Lanškroun, Czech Republic) for providing formulated microbial inoculants and MPN data, and Geneviève Défago (ETH Zürich) for discussions. This work made use of the platform DTAMB (IFR 41) in Université Lyon 1.

Supplementary material

11104_2011_960_MOESM1_ESM.doc (248 kb)
Fig. S1 Effect of maize inoculation treatments (indicated below the x-axis) on the ratio between dry total methanolic extract and dry root biomass at 16 days after inoculation. Different letters represent statistical differences between treatments (ANOVA and Tukey’s tests, P < 0.05) (DOC 247 kb)


  1. Adesemoye AO, Torbert HA, Kloepper JW (2009) Plant growth-promoting rhizobacteria allow reduced application rates of chemical fertilizers. Microb Ecol 58:921–929PubMedCrossRefGoogle Scholar
  2. Ait Barka E, Nowak J, Clement C (2006) Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN. Appl Environ Microbiol 72:7246–7252PubMedCrossRefGoogle Scholar
  3. Barea JM, Bonis AF, Olivares J (1983) Interactions between Azospirillum and VA mycorrhiza and their effects on growth and nutrition of maize and ryegrass. Soil Biol Biochem 15:705–709CrossRefGoogle Scholar
  4. Barea JM, Andrade G, Bianciotto V, 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 64:2304–2307PubMedGoogle Scholar
  5. 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
  6. 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 rhizobacterial community of field-grown maize. Soil Biol Biochem 41:409–413CrossRefGoogle Scholar
  7. Beauchamp CJ (1993) Mode of action of plant growth-promoting rhizobacteria and their potential use as biological control agent. Phytoprotection 74:19–28Google Scholar
  8. 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 24:271–284PubMedCrossRefGoogle Scholar
  9. Cornelis P (2010) Iron uptake and metabolism in pseudomonads. Appl Microbiol Biotechnol 86:1637–1645PubMedCrossRefGoogle Scholar
  10. Couillerot O, Prigent-Combaret C, Caballero-Mellado J, Moënne-Loccoz Y (2009) Pseudomonas fluorescens and closely-related fluorescent pseudomonads as biocontrol agents of soil-borne phytopathogens. Lett Appl Microbiol 48:505–512PubMedCrossRefGoogle Scholar
  11. Couillerot O, Bouffaud ML, Muller D, Caballero-Mellado J, Moënne-Loccoz Y (2010a) 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
  12. Couillerot O, Poirier MA, Prigent-Combaret C, Mavingui P, Caballero-Mellado J, Moënne-Loccoz Y (2010b) Assessment of SCAR markers to design real-time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize. J Appl Microbiol 109:528–538PubMedGoogle Scholar
  13. Dobbelaere S, Vanderleyden J, Okon Y (2003) Plant growth promoting effects of diazotrophs in the rhizosphere. Crit Rev Plant Sci 22:107–149CrossRefGoogle Scholar
  14. El Zemrany H, Cortet J, Lutz PM, 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
  15. Fages J, Mulard D (1988) Isolement de bactéries rhizosphériques et effet de leur inoculation en pot chez Zea mays. Agronomie 8:308–314CrossRefGoogle Scholar
  16. 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 58:3873–3878PubMedGoogle Scholar
  17. Fester T, Maier W, Strack D (1999) Accumulation of secondary compounds in barley and wheat roots in response to inoculation with an arbuscular mycorrhizal fungus and co-inoculation with rhizosphere bacteria. Mycorrhiza 8:241–246CrossRefGoogle Scholar
  18. Frey-Klett P, Garbaye J, Tarkka M (2007) The mycorrhiza helper bacteria revisited. New Phytol 176:22–36PubMedCrossRefGoogle Scholar
  19. Garg N, Chandel S (2010) Arbuscular mycorrhizal networks: process and functions. A review. Agron Sustain Dev 30:581–599CrossRefGoogle Scholar
  20. Haas D, Défago G (2005) Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat Rev Microbiol 3:307–319PubMedCrossRefGoogle Scholar
  21. Hohnjec N, Vieweg MF, Pühler A, Becker A, Küster H (2005) Overlaps in the transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus fungi provide insights into the genetic program activated during arbuscular mycorrhiza. Plant Physiol 137:1283–1301PubMedCrossRefGoogle Scholar
  22. 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
  23. 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
  24. Jansa J, Smith FA, Smith SE (2008) Are there benefits of simultaneous root colonization by different arbuscular mycorrhizal fungi? New Phytol 177:779–789PubMedCrossRefGoogle Scholar
  25. Kapanen A, Stephen JR, Brüggemann J, Kiviranta A, White DC, Itävaara M (2007) Diethyl phthalate in compost: ecotoxicological effects and response of the microbial community. Chemosphere 67:2201–2209PubMedCrossRefGoogle Scholar
  26. Karthikeyan B, Jaleel CA, Azooz MM (2009) Individual and combined effects of Azospirillum brasilense and Pseudomonas fluorescens on biomass yield and ajmalicine production in Catharanthus roseus. Acad J Plant Sci 2:69–73Google Scholar
  27. Lucy M, Reed E, Glick BR (2004) Application of free living plant growth-promoting rhizobacteria. Antonie van Leewenhoek 86:1–25CrossRefGoogle Scholar
  28. 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
  29. Marimuthu S, Subbian P, Ramamoorthy V, Samiyappan R (2002) Synergistic effect of combined application of Azospirillum and Pseudomonas fluorescens with inorganic fertilizers on root rot incidence and yield of cotton. Z Pflanzenkr Pflanzenschutz 109:569–577Google Scholar
  30. Nicol D, Copaja SV, Wratten SD, Niemeyer HM (1992) A screen of worldwide wheat cultivars for hydroxamic acid levels and aphid antixenosis. Ann Appl Biol 121:11–18CrossRefGoogle Scholar
  31. 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
  32. Park J-W, Crowley DE (2005) Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE. BioTechniques 38:579–586Google Scholar
  33. Park WJ, Hochholdinger F, Gierl A (2004) Release of the benzoxazinoids defense molecules during lateral- and crown root emergence in Zea mays. J Plant Physiol 161:981–985PubMedCrossRefGoogle Scholar
  34. Peipp H, Maier W, Schmidt J, Wray V, Strack D (1997) Arbuscular mycorrhizal fungus-induced changes in the accumulation of secondary compounds in barley roots. Phytochemistry 44:581–587CrossRefGoogle Scholar
  35. Phillips DA, Fox TC, King MD, Bhuvaneswari TV, Teuber LR (2004) Microbial products trigger amino acid exudation from plant roots. Plant Physiol 136:2887–2894PubMedCrossRefGoogle Scholar
  36. Prigent-Combaret C, Blaha D, Pothier JF, Vial L, Poirier M-A, 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 65:202–219Google Scholar
  37. Raaijmakers J, Paulitz T, Steinberg C, Alabouvette C, Moënne-Loccoz Y (2009) The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms. Plant Soil 321:341–361CrossRefGoogle Scholar
  38. Raju PS, Clark RB, Ellis JR, Maranville JW (1990) Effects of species of VA-mycorrhizal fungi on growth and mineral uptake of sorghum at different temperatures. Plant Soil 121:165–170CrossRefGoogle Scholar
  39. Richardson A, Barea JM, McNeill A, Prigent-Combaret C (2009) Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms. Plant Soil 321:305–339CrossRefGoogle Scholar
  40. Rillig MC, Mummey DL (2006) Mycorrhizas and soil structure. New Phytol 171:41–53PubMedCrossRefGoogle Scholar
  41. Russo A, Felici C, Toffanin A, Götz M, Collados C, Barea JM, 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
  42. Sahi SV, Chilton MD, Chilton WS (1990) Corn metabolites affect growth and virulence of Agrobacterium tumefaciens. Proc Natl Acad Sci USA 87:3879–3883PubMedCrossRefGoogle Scholar
  43. Sánchez Pérez JM, Antiguedad I, Arrate I, García-Linares C, Morell I (2003) The influence of nitrate leaching through unsaturated soil on groundwater pollution in an agricultural area of the Basque country: a case study. Sci Total Environ 317:173–187CrossRefGoogle Scholar
  44. Schliemann W, Ammer C, Strack D (2008a) Metabolite profiling of mycorrhizal roots of Medicago truncatula. Phytochemistry 69:112–146PubMedCrossRefGoogle Scholar
  45. Schliemann W, Kolbe B, Schmidt J, Nimtz M, Wray V (2008b) Accumulation of apocarotenoids in mycorrhizal roots of leek (Allium porrum). Phytochemistry 69:1680–1688PubMedCrossRefGoogle Scholar
  46. Singh UP, Sarma BK, Singh DP (2003) Effect of plant growth-promoting rhizobacteria and culture filtrate of Sclerotium rolfsii on phenolic and salicylic acid contents in chickpea (Cicer arietinum). Curr Microbiol 46:131–140PubMedCrossRefGoogle Scholar
  47. Subramanian KS, Charest C, Dwyer LM, Hamilton RI (1995) Arbuscular mycorrhizas and water relations in maize under drought stress at tasselling. New Phytol 192:643–650CrossRefGoogle Scholar
  48. Troxler J, Zala M, Natsch A, Moënne-Loccoz Y, Défago G (1997) Autecology of the biocontrol strain Pseudomonas fluorescens CHA0 in the rhizosphere and inside roots at later stages of plant development. FEMS Microbiol Ecol 23:119–130CrossRefGoogle Scholar
  49. Volpin H, Kapulnik Y (1994) Interaction of Azospirillum with beneficial soil microorganisms. In: Okon Y (ed) Azospirillum/Plant associations. CRC Press, Boca Raton, pp 111–118Google Scholar
  50. 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 Microb Methods 81:108–115CrossRefGoogle Scholar
  51. 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 the plant growth promoting rhizobacteria of the genus Azospirillum. New Phytol 189:494–506PubMedCrossRefGoogle Scholar
  52. Weller DM, Landa BB, Mavrodi OV, Schroeder LK, De la Fuente L, Blouin Bankhead S, Allende Molar R, Bonsall RF, Mavrodi DV, Thomashow LS (2007) Role of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp. in the defense of plant roots. Plant Biol 9:4–20PubMedCrossRefGoogle Scholar
  53. Witcombe JR, Hollington PA, Howarth CJ, Reader S, Steele KA (2008) Breeding for abiotic stresses for sustainable agriculture. Philos Trans R Soc Lond B 363:703–716CrossRefGoogle Scholar
  54. Xuan TD, Chung IIIM, Khanh TD, Tawata S (2006) Identification of phytotoxic substances from early growth of Barnyard grass (Echinochloa crusgalli) root exudates. J Chem Ecol 32:895–906PubMedCrossRefGoogle Scholar
  55. Zhang J, Boone L, Kocz R, Zhang C, Binns AN, Lynn DG (2000) At the maize/Agrobacterium interface: natural factors limiting host transformation. Chem Biol 7:611–621PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Vincent Walker
    • 1
    • 2
    • 3
  • Olivier Couillerot
    • 1
    • 2
    • 3
  • Andreas Von Felten
    • 4
  • Floriant Bellvert
    • 1
    • 2
    • 3
  • Jan Jansa
    • 5
  • Monika Maurhofer
    • 4
  • René Bally
    • 1
    • 2
    • 3
  • Yvan Moënne-Loccoz
    • 1
    • 2
    • 3
  • Gilles Comte
    • 1
    • 2
    • 3
    Email author
  1. 1.Université de LyonLyonFrance
  2. 2.Université Lyon 1VilleurbanneFrance
  3. 3.CNRS, UMR5557, Ecologie MicrobienneVilleurbanneFrance
  4. 4.Institute of Integrative BiologyETHZürichSwitzerland
  5. 5.Institute of Plant Agricultural SciencesETHLindauSwitzerland

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