REGULAR ARTICLE

Variation of secondary metabolite levels in maize seedlingroots induced by inoculation with Azospirillum, Pseudomonasand Glomus consortium under field conditions

Vincent Walker & Olivier Couillerot & Andreas Von Felten & Floriant BellvertJan Jansa & Monika Maurhofer & René Bally & Yvan Moënne-Loccoz & Gilles Comte

Received: 14 February 2011 /Accepted: 12 August 2011 /Published online: 20 September 2011# Springer Science+Business Media B.V. 2011

AbstractBackground and aims Many plant-beneficial micro-organisms can influence secondary plant metabolism,but whether these effects add up when plants are co-inoculated is unclear. This issue was assessed, underfield conditions, by comparing the early impacts of

seed inoculation on secondary metabolite profiles ofmaize at current or reduced mineral fertilizationlevels.Methods Maize seeds were inoculated singly withselected strains from bacterial genera Pseudomonasand Azospirillum or mycorrhizal genus Glomus, or withthese strains combined two by two or all three together.At 16 days, maize root methanolic extracts wereanalyzed by RP-HPLC and secondary metabolites(phenolics, flavonoids, xanthones, benzoxazionoids,etc.) identified by LC/MS.Results Inoculation did not impact on plant biomassbut resulted in enhanced total root surface, total rootvolume and/or root number in certain inoculatedtreatments, at reduced fertilization. Inoculation led toqualitative and quantitative modifications of rootsecondary metabolites, particularly benzoxazinoidsand diethylphthalate. These modifications dependedon fertilization level and microorganism(s) inoculated.The three selected strains gave distinct results whenused alone, but unexpectedly all microbial consortiagave somewhat similar results.Conclusions The early effects on maize secondarymetabolism were not additive, as combining strainsgave effects similar to those of Glomus alone. Thisis the first study demonstrating and analyzinginoculation effects on crop secondary metabolites inthe field.

Keywords Secondary metabolites . Benzoxazinoids .

Diethylphtalate . Mineral fertilization . Zea mays L.

Plant Soil (2012) 356:151–163DOI 10.1007/s11104-011-0960-2

Responsible Editor: Euan K. James.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11104-011-0960-2) containssupplementary material, which is available to authorized users.

V. Walker :O. Couillerot : F. Bellvert :R. Bally :Y. Moënne-Loccoz :G. Comte (*)Université de Lyon,69622 Lyon, Francee-mail: gilles.comte@univ-lyon1.fr

V. Walker :O. Couillerot : F. Bellvert :R. Bally :Y. Moënne-Loccoz :G. ComteUniversité Lyon 1,Villeurbanne, France

V. Walker :O. Couillerot : F. Bellvert :R. Bally :Y. Moënne-Loccoz :G. ComteCNRS, UMR5557, Ecologie Microbienne,Villeurbanne, France

A. Von Felten :M. MaurhoferInstitute of Integrative Biology, ETH,CH-8092 Zürich, Switzerland

J. JansaInstitute of Plant Agricultural Sciences, ETH,Eschikon 33,CH-8315 Lindau, Switzerland

Introduction

Conventional agriculture is highly dependent onchemical inputs including fertilizers in order tomaintain high yields. However, fertilizer sources arebecoming less and less plentiful and chemicalfertilizer usage can have adverse effects on ground-water quality, making these agricultural practicesunsustainable in the long term (Sánchez Pérez et al.2003). To maintain productivity while reducingchemicals inputs, selection of more nutrient-effectiveplant varieties is one strategy (Witcombe et al. 2008).Another strategy is to exploit the potential of plant-beneficial microbes as crop inoculants (Okon andLabandera-Gonzalez 1994; Lucy et al. 2004). Forcereal crops, prominent plant-beneficial microbesinclude plant growth-promoting rhizobacteria (PGPR)and arbuscular mycorrhizal fungi.

PGPR may lead to plant growth promotion in variousways. Many of them display associative nitrogenfixation, production of phytohormones and other signals,and/or ACC deaminase activity, as exemplified by certainAzospirillum strains (Dobbelaere et al. 2003; Prigent-Combaret et al. 2008; Richardson et al. 2009; Bashanand de-Bashan 2010). Among them, Azospirillumlipoferum CRT1 has been extensively studied forpromotion of maize growth under reduced chemicalfertilization conditions (Okon and Labandera-Gonzalez1994; El Zemrany et al. 2006; Baudoin et al. 2009).

Other PGPR, such as many fluorescent Pseudomonasstrains, have been mostly studied for their ability tosuppress phytoparasites via competition, antagonism orinduced resistance (Haas and Défago 2005; Weller et al.2007; Raaijmakers et al. 2009; Cornelis 2010), butsome of these Pseudomonas strains can also promoteplant growth (Couillerot et al. 2009). This is the case ofPseudomonas fluorescens F113 on maize (unpub-lished), a strain which may even enhance plant-beneficial properties of co-inoculated AzospirillumPGPR (Combes-Meynet et al. 2011). Co-inoculationof Pseudomonas and Azospirillum PGPR is a promisingplant-beneficial strategy (Marimuthu et al. 2002;Karthikeyan et al. 2009) but remains rarely studied.

Arbuscular mycorrhizal fungi establish mutualisticassociations with more than 80% of plant species,including cropped cereals. These fungi can improvemineral uptake (especially nitrogen and phosphorus)and tolerance to abiotic and biotic stress in theinoculated plant (Raju et al. 1990; Subramanian et

al. 1995; Rillig and Mummey 2006; Jansa et al. 2008;Garg and Chandel 2010). Several strains have beenstudied on maize, including Glomus intraradicesJJ291 (Jansa et al. 2005). Inoculation with a Pseudo-monas or Azospirillum PGPR does not have adverseeffects on root mycorrhization (Barea et al. 1998; MarVázquez et al. 2000; Russo et al. 2005), and evenresulted in enhanced plant-beneficial effects in severalco-inoculation experiments (Barea et al. 1983; Volpinand Kapulnik 1994; Mar Vázquez et al. 2000; Frey-Klett et al. 2007). However, we are not aware ofinvestigations dealing with three-component microbialconsortia in which Pseudomonas, Azospirillum andGlomus strains were combined, despite the usefulnessof combining them two by two. In a preliminaryexperiment, we observed that such three-componentconsortia promoted maize growth (unpublished), butthe work was done under greenhouse conditions,without considering the role of soil fertility level.

Recently, we found that inoculation of maize withAzospirillum strains resulted in significant changes insecondary metabolic profiles of roots and shootsunder greenhouse conditions, which corresponded toa modulation in the contents of benzoxazinoids(especially MBOA, DIMBOA-Glc) and phenoliccompounds (Walker et al. 2011). Benzoxazinoids aredefence molecules synthesized in Poaceae during rootsystem emergence (Park et al. 2004). Metabolicvariations induced in the plant by Pseudomonas(Singh et al. 2003) or Glomus inoculations (Schliemannet al. 2008b) have also been identified. On this basis, itis likely that inoculation with microbial consortia willalso have an impact on plant metabolic profiles, andtheir analysis should provide new knowledge on theeffect of these microbial consortia on plant physiology.So far, only one study has considered the impact of co-inoculation (i.e. with G. intraradices and either P.fluorescens or Rhizobium leguminosarum) on rootsecondary metabolism (Fester et al. 1999), and it wasdone under greenhouse conditions.

The objective of this work was to assess the effect ofa Pseudomonas, Azospirillum and Glomus microbialconsortium on root secondary metabolite profile ofmaize seedlings under field conditions and differentrates of mineral fertilization. To assess the contributionand microbial interactions of individual inoculantsstrains within the consortium, seeds were inoculatedwith the three-component consortium, with one of thethree two-component consortia obtained by combining

152 Plant Soil (2012) 356:151–163

strains two by two, or with a single strain, and otherseeds were used without inoculation (control). Thethree-component consortium and the non-inoculatedcontrol were also assessed at a different rate of mineralfertilization to take into account the importance of soilfertility level.

Materials and methods

Field experiment

Zea mays L. cultivar PR37Y15, a semi-late maizehybrid (Pioneer Semences, Aussonne, France), wassown in a field (44°49′11.60″N, 4°53′22.44″E) locat-ed at Pouzol Etoile near Valence (France), in theArvalis experimental station. The topsoil of thiscambisol contains 19.5% clay, 49.2% silt, 31.3% sand(silt loam texture), 1.77% organic matter, 106 mg Nkg−1, 62 mg P kg−1, 186 mg K kg−1, 140 mg Mgkg−1, 4,950 mg Ca kg−1, and has a pH of 7.7.

Strains inoculated were A. lipoferum CRT1 (Fagesand Mulard 1988), P. fluorescens F113 (Fenton et al.1992), and G. intraradices JJ291 (Jansa et al. 2005;deposited as strain BEG158 at www.kent.ac.uk/bio/beg). They were formulated as follows (without usingadditives). A. lipoferum CRT1 was coated onto seeds(on the day of sowing) using peat containing 1.0×109 CFU g−1 peat. This gave 7.8×106 CFU g−1 seedafter coating. P. fluorescens F113 was applied in thefurrow (under the seeds) using expanded perlite claybeads (particle size 1.0–2.5 mm) containing 5×107 CFU g−1 (which gave approximately 3.3×106 CFU under each seed). G. intraradices JJ291was prepared in standard plant cultures, followingcommercial procedures by Symbio-M (Lanškroun,Czech Republic). Mycorrhized roots were choppedand mixed with clinoptilolite zeolite of 0.5–2.5 mmparticle size (carrier). The resulting zeolite productcontained 7.9×105 gene copies (quantification methodunpublished) of the mitochondrial Large RibosomalSub-Unit (mLSU) of strain JJ291 per g, correspondingto a plant infectivity of about 1,000 mycorrhizalpropagules per g based on the most probable numbermethod. The Glomus zeolite product was mixed withPseudomonas clay beads prior to delivery (givingapproximately 1.0×106 mLSU gene copies of JJ291=1,300 mycorrhizal propagules under each seed).Inoculation was done with one, two or three microbial

partners, and sterile peat, clay beads and zeolite (allthree in the non-inoculated control) were used whenthe corresponding strain was not inoculated.

The experimental design was in randomized blocks(four blocks), and each elementary plot contained8 rows at least 10 m long, with 80 cm spacingbetween rows. Two treatments i.e. the Pseudomonas–Azospirillum–Glomus three-component consortiumand the non-inoculated control were studied at thecurrent rate of mineral fertilization in that region, i.e.200 kg N ha−1 (delivered as NH4NO3) and 36 kg Pha−1 [as Ca(H2PO4)2]. This is similar to fertilizationrates used in the main maize-growing areas of Europe.Additionally, the Pseudomonas–Azospirillum–Glomusthree-component consortium, each of the three two-component consortia (i.e. CRT1/F113, CRT1/JJ291 orF113/JJ291), each of the single inoculants and the non-inoculated control were studied under reduced mineralfertilization conditions, i.e. 120 kg N ha−1 and 0 kg Pha−1. No chemical treatment was applied to seeds,which were sown on 12 May 2009, at sowing densityof 76,600 seeds ha−1.

Sampling was carried out at 16 days after sowing,and 36 whole plants were taken per treatment. Amongthem, 16 plants (i.e. 4 per block) were used for bothroot dry weight (for statistical analysis, n=16) androot secondary metabolite profile (for statisticalanalysis, n=4; samples pooled per block beforeprofiling), 8 plants (i.e. 2 per block) for root systemarchitecture (for statistical analysis, n=8), 8 plants(i.e. 2 per block) for rhizosphere bacterial inoculantlevels (for statistical analysis, n=8).

Real-time PCR assessment of bacterial inoculants

For real-time PCR quantification of PGPR inoculants,rhizosphere DNA was extracted as described byCouillerot et al. (2010a). Briefly, roots and tightly-adhering soil were transferred in a 50-ml Falcon andflash-frozen in liquid nitrogen. Samples were thenlyophilized and homogenized by crushing in the tubesusing a spatula. Then, 250–300 mg were used for DNAextraction, using the FastDNA® SPIN® kit (BIO 101,Carlsbad, CA, USA). DNA concentrations wereassessed by OD measurements at 260 nm with Nano-Drop (Nanodrop Technologies, Wilmington, DE, USA).

A. lipoferum CRT1 was quantified as described inCouillerot et al. (2010a), using a LC-480 LightCycler(Roche Applied Science, Indianapolis, IN, USA).

Plant Soil (2012) 356:151–163 153

PCR was done in 20 μl containing 10 μl LightCyclerFastStart DNA Master SYBR Green I (Roche AppliedScience), 0.50 μM primers, 50 ng T4 gene 32 protein(Roche Applied Science) and 2 μl of template DNA.The cycling program included a 10-min incubation at95°C followed by 50 cycles consisting of 95°C for15 s, 68°C for 15 s and 72°C for 10 s.

P. fluorescens F113 was quantified as described inVon Felten et al. (2010), using a 7500 Fast Real-TimePCR System (Applied Biosystems, Foster City, CA,USA). PCR was done in 20 μl containing 0.5 μMprimers, 2 μl template DNA and 10 μl Fast SYBRGreen mix (Applied Biosystems). The two-stepcycling program included a 20-s initial pre-incubation at 95°C followed by 40 cycles of 95°Cfor 3 s and 60°C for 30 s.

For both A. lipoferum CRT1 and P. fluorescens F113quantification, CT values obtained were normalizedusing the internal standard APA9, as described, respec-tively, in Couillerot et al. (2010b) and Von Felten et al.(2010), in order to enable (1) normalization of DNAextraction efficiencies between rhizosphere samples and(2) comparison of quantifications performed withdifferent protocols (Park and Crowley 2005).

Maize morphology and biomass

For root growth analysis, soil adhering to roots of 16-day plants was discarded by washing with water.Individual root systems were dipped into liquidnitrogen, freeze-dried during 72 h (−54°C), and rootdry weight was determined using four plants pertreatment (also used for root secondary metaboliteprofiling).

Other plants were used to assessed root systemarchitecture, using WinRhizo® image analysis andsoftware (Regent instruments, Nepean, ON, USA), asdescribed (El Zemrany et al. 2006). Total root length,total root surface, total root volume and root numberwere recorded.

Secondary metabolic analysis

The freeze-dried root systems used for biomass deter-mination were introduced each into an Eppendorf tube,together with liquid nitrogen. Roots were then crushedusing a TissueLyser II ball mill (Qiagen, Courtaboeuf,France). Each root system was extracted using 3 ml ofmethanol for 200 mg of dried material. Extraction was

done twice, and extracts from the 4 plants were pooledand dried using Speedvac®-assisted evaporation. Eachsample was then resuspended in methanol to adjust to10 mg extract ml−1.

Chromatographic analysis was achieved with anAgilent 1200 series HPLC equipped with a degasser(G132A), a quaternary pump module (G1311A), anautomatic sampler (G1329A) and a Diode ArrayDetector (DAD G1315B), as described (Walker et al.2011). The separation was carried out at roomtemperature using a NUCLEODUR sphinx C18column (250×4.6 mm, 5 μm; Macherey-Nagel,Düren, Germany). For each sample, 20 μl of extractwas injected and the column was eluted at1 ml min−1, with an optimized gradient establishedusing solvents A (acetic acid 4 ‰ (v/v) in water) andB (acetic acid 4 ‰ (v/v) in acetonitrile) (CarloerbaReagents, Val de Reuil, France). The gradient usedwas an increase of solvent B (15 to 40% in 20 min),then isocratic conditions during 15 min, then increaseof solvent B (40 to 70% in 25 min) and finally anincrease of 30% of solvent B in 5 min. 3D data wererecorded and specific wavelengths at 254, 280, 310,and 366 nm were chosen for processing. Indeed, thesewavelengths allowed the detection of major secondarymetabolites (including phenolics, flavonoids, indoliccompounds, and benzoxazionoids), and they corre-spond typically to the absorbance maxima for manyof them (phenolics, flavonoids bands I and II,cinnamic acids, etc.). Chemstation Agilent softwarewas used for integration and comparison of chromato-grams. Each chromatogram was integrated afterstandardization of integration parameters. Backgroundpeaks (areas less than 1% of total peak areas) presenton chromatograms were not integrated.

Concentration of compounds was express as MBOAequivalents per g of dry roots. A standard range wasperformed using a commercial standard of 6-methoxbenzoxazolin-2-one (MBOA; Sigma-Aldrich,Saint Louis, MO, USA) and injected with the sameanalytical conditions as for root extracts. Five-pointranges have been used before to reach saturation of theabsorbance (i.e. 0, 0.0072, 0.072, 0.144, 0.288 and0.71 mM).

Metabolite purification and identification

Phthalate was purified in semi-preparative HPLC andidentified via GC/MS analysis. All extracts were

154 Plant Soil (2012) 356:151–163

pooled and concentrated before semi-preparativeHPLC. Purification of this compound was done usinga Varioprep Sphinx NUCLEODUR column (250×10 mm, 5 μm). The column was eluted at 2 ml min−1,with optimized gradient established using solvents A(in water) and B (in acetonitrile) (CarloerbaReagents). Compounds (50 μl per injection) wereeluted with a step by step gradient: increase of solventB (70 to 86%) in 10.5 min, then a rapid increase of14% of solvent B in 2.5 min. Phthalate was collectedand concentrated in order to perform a GC/MSanalysis. Then, 1 μl of purified molecule was injectedand eluted in GS/MS using a HP5 GC column(0.25 μm×30 m), with a nitrogen flow of15 ml min−1, a step by step temperature gradient withan increase from 60 to 320°C with 10°C min−1.Determination of chemical structure of phthalate wasperformed by comparison with mass spectra library(NIST and Wiley libraries).

All other compounds were identified by HPLC/MS, after separation using an Agilent 1100 seriesHPLC equipped with a degasser (G1322A), a binarypump module (G1312A), an automatic sampler(G1313A) and a Diode Array Detector (DADG1314A). The separation was carried out at roomtemperature using a NUCLEODUR sphinx C18column (250×4.6 mm, 5 μm Macherey-Nagel).HPLC was interfaced with a HP MSD 1100 series.Each chromatogram was automatically integratedusing Chemstation software (Agilent) and thenreprocessed manually for better standard integrationof minor peaks.

Mass spectrometry operating conditions weregas temperature 350°C at a flow rate of 10 lN2

min−1, nebulizer pressure 30 psi, quadripole temper-ature 30°C, capillary voltage 4000 V and fragmentor100. Full scan spectra from m/z 100 to 800 in bothpositive and negative ion modes were recorded.These parameters allowed the use of analyticalconditions similar to those used for HPLC-DADanalysis. Identification of each compound wasperformed by interpretation of mass spectra andcomparison with literature data.

Statistics

Inoculant cell numbers (expressed as log cells g−1

rhizosphere) were compared by ANOVA and Fisher’sLSD tests (P<0.05) at each sampling using SYSTAT

Version 12.0 software (Systat, Evanston, IL, USA).Biomass, root system architecture variables andmetabolite levels were studied by ANOVA followedwith Tukey’s tests (P<0.05), using SYSTAT. Retentiontime of each chromatographic peak (at 280 nm) wasaligned and its relative intensity (expressed as MBOAconcentration equivalents) recorded in a matrix toperform discriminant principal component analysis(PCA), using R software (v2.4 Open Source, http://www.r-project.org/).

Results

Effect of inoculations on early maize growth

At 16 days, there was a trend (not significant at P<0.05) for a 35% lower root biomass of maize with thethree-component consortium compared with the non-inoculated control when current fertilization level wasused (Fig. 1). Such a trend was not observed atreduced fertilization level. At reduced fertilizationlevel, there was no difference between the three-component consortium, the two-component consortiaand the single inoculations, except that inoculationwith G. intraradices JJ291 alone gave a lower rootbiomass in comparison with the three-componentconsortium, the bacterial two-component consortiumor inoculation with A. lipoferum CRT1 alone. Inaddition, there was a trend for lower root biomass ofmaize (by 24%) when inoculated with G. intraradicesJJ291 alone in comparison with the non-inoculatedcontrol at reduced fertilization level, but this trendwas not significant at P<0.05.

In the absence of inoculation, root system devel-opment was lower (i.e. lower total root length, totalroot surface, total root volume and/or root number)when the three-component consortium was used(trend significant at P<0.05 only for total rootsurface) or fertilization level was reduced (Fig. 1).At reduced fertilization, total root length was notinfluenced by inoculation, whereas total root surfaceand total root volume were higher when A. lipoferumCRT1 was used in combination with P. fluorescensF113, G. intraradices JJ291 or both, in comparisonwith the non-inoculated control. At reduced fertiliza-tion, inoculation also had a positive effect on rootnumber when P. fluorescens F113 and G. intraradicesJJ291 were used alone, together (but without an

Plant Soil (2012) 356:151–163 155

additive effect), or in combination with A. lipoferumCRT1. Overall, the lower root system developmentfound when reducing fertilization level was wellcompensated by most inoculation treatments.

Survival of bacterial inoculants

A. lipoferum CRT1 was below the detection limit of4×104 cells g−1 rhizosphere at 16 days after sowing,

Fig. 1 Effect of maizeinoculation treatments(indicated below the x-axis)on root system biomass andarchitecture at 16 days afterinoculation. Different lettersrepresent statistical differen-ces between treatments(ANOVA and Tukey’s tests,P<0.05)

156 Plant Soil (2012) 356:151–163

whereas P. fluorescens F113 was found at 105 to morethan 106 cells g−1 rhizosphere (Fig. 2). The cellnumber of P. fluorescens F113 was higher when otherstrain(s) had been co-inoculated. The two strainswere not detected when studying non-inoculatedplants.

Identification of maize secondary metabolites

Mass spectra recorded both in negative andpositive modes allowed identification of all majorsecondary metabolite found in maize tissues of thishybrid under greenhouse conditions (Walker et al.2011). In this work, an additional, apolar compoundwas prominent in maize inoculated with P. fluores-cens F113 alone (see below). This compound wasidentified according to GC/MS library as 1,2-benzenedicarboxylic acid, which is also termeddiethylphthalate (Fig. 3).

In non-inoculated plants, we were not able tofind diethylphthalate by HPLC but specific ionmonitoring by GC/MS allowed detection of smallsignals corresponding to this compound based onretention time, molecular mass and fragmentationpatterns. The latter was not produced by P.fluorescens F113 in laboratory cultures as indicatedby GC and HPLC analysis, even when using massspectrometry detection.

Effect of inoculations on secondary metabolite profileof maize roots

Chromatograms obtained from root methanolicextracts of maize cv. PR37Y15 showed a total of 26integrated peaks with the 10 treatments studied. Basedon retention times and peak areas at 280 nm, a datamatrix was built to carry out discriminant PCA. Thefactorial plan defined by axes 1 and 2 explained about29% data variability (Fig. 4). At reduced fertilizationlevel, discriminant analysis clearly separated the threesingle-inoculation treatments from one another andfrom the non-inoculated control. The three two-component consortia as well as the three-componentconsortium gave results that were rather similar to oneanother (and similar to those for G. intraradices JJ291alone) and distinct from those for the non-inoculatedcontrol. Fertilization level had a strong effect onsecondary metabolic profiles of maize, and at currentfertilization level the three-component consortiumwas distinct from the non-inoculated control.

Effect of inoculations on individual secondarymetabolites of maize at reduced fertilization level

Prevalence of secondary metabolites, which was esti-mated based on the ratio between dry total methanolicextract and dry root biomass, was higher upon consor-tium inoculation at current fertilization level (Fig. S1).In the non-inoculated control, this ratio was higher atreduced than at current fertilization level. At reducedfertilization level, it was higher with P. fluorescensF113 inoculation but lower when inoculation involvedboth P. fluorescens F113 and G. intraradices JJ291.

At reduced fertilization level, eight prevalent second-ary metabolites were identified whose amounts differedsignificantly between treatments based on the correlationcircle for discriminant PCA (data not shown), which was

Fig. 3 Chemical structure of 1,2-benzenedicarboxylic acid (i.e.diethylphthalate), which was detected in maize roots inoculatedwith P. fluorescens F113 only

Fig. 2 Survival of P. fluorescens F113 in the maize rhizosphereat 16 days after seed inoculation. The inoculant was used aloneor in combination with A. lipoferum CRT1 or G. intraradicesJJ291 (as indicated below the x-axis), and was assessed by real-time PCR. Different letters above the bars indicate significantdifferences between treatments (ANOVA and Fisher’s LSDtests, P<0.05)

Plant Soil (2012) 356:151–163 157

confirmed by ANOVA and Tukey’s tests (Fig. 5).According to UV spectra, they corresponded to fiveclasses of metabolites, i.e. benzoxazinoids (DIMBOA-Glc, DIMBOA, HDMBOA-Glc and MBOA), simplephenols (unknown compound 11), phthalates, cinnamicacids and xanthone-like compounds. Compared withthe non-inoculated control, single inoculation resultedin (1) lower DIMBOA-Glc and HDMBOA-Glc con-tents but similar MBOA content, (2) higher xanthonecontents (with A. lipoferum CRT1 or G. intraradicesJJ291), (3) the synthesis of diethylphthalate and lowercinnamic acid content (with P. fluorescens F113), and(4) strain-dependent DIMBOA levels, with especiallyan increase with P. fluorescens F113 (Fig. 5). Differ-ences in level of compound 11 were statisticallysignificant but modest. The effects of the microbialconsortia were (1) comparable to that of G. intraradicesJJ291 alone when considering contents in DIMBOA-Glc, DIMBOA and HDMBOA-Glc, (2) lower (al-though not always statistically significant) to thatof the non-inoculated control when consideringMBOA content, (3) negligible for contents incompound 11, diethylphthalate and cinnamic acid,and (4) consortium-dependent for the xanthonetype compound.

Effect of the three-component consortiumon individual secondary metabolites of maizeat current fertilization level

Mineral fertilization level had a significant impact onsecondary metabolic profiles based on discriminantPCA (Fig. 4) and on ANOVA and Tukey’s tests(Fig. 6). Reducing the fertilization level led mainly tolower DIMBOA-Glc and higher MBOA and DIM-BOA amounts (Fig. 6). In this context, the three-component consortium resulted in lower DIMBOA-Glc content (a compound only found at the currentfertilization level), lower cinnamic acid content andhigher HDMBOA-Glc content (only at the currentfertilization level), as well as lower MBOA contentand higher xanthone content (at both fertilizationlevels) (Fig. 6).

Discussion

This work aimed at understanding the effect ofmicrobial consortium inoculation on secondary met-abolic profiles of maize roots, in a context whereinoculation with plant-beneficial microorganisms is

Fig. 4 Discriminant PCA of maize root chromatographic data recorded at 280 nm

158 Plant Soil (2012) 356:151–163

proposed as a means to compensate for a reduction inthe amount of chemical fertilizers used for maizefarming (El Zemrany et al. 2006; Adesemoye et al.2009). The focus was put on maize seedlings, becausethe early interaction between plant-beneficial micro-organisms and seedlings is important for rhizosphereestablishment of bacterial inoculants (Beauchamp1993; Troxler et al. 1997) or successful plant-growthpromotion (Jacoud et al. 1999). At 16 days, root

biomass with the Pseudomonas–Azospirillum–Glomusthree-component consortium was higher at reducedthan at current fertilization level, but there was nodifference with the corresponding non-inoculated con-trol. However, maize root system at reduced fertiliza-tion displayed enhanced ramification (i.e. based onhigher total root length, total root surface, total rootvolume and/or root number) in several inoculationtreatments, which is important for plant uptake of

Fig. 6 Effect of seedinoculation with the three-component consortium ondiscriminating maizesecondary compounds atcurrent and reducedfertilization levels. For eachcompound, different lettersrepresent statistical differen-ces between treatments(ANOVA and Tukey’s tests,P<0.05)

Fig. 5 Effect of seed inoculations on discriminating maize secondary compounds at reduced fertilization level. Different lettersrepresent statistical differences between treatments between treatments (ANOVA and Tukey’s tests, P<0.05)

Plant Soil (2012) 356:151–163 159

water and soil nutrients (Dobbelaere et al. 2003;Richardson et al. 2009). These treatments includedthe three-component consortium, the two Azospirillum-based two-component consortia, and the Pseudomonassingle inoculation, and their application enabled com-pensation for the negative consequence of reducedfertilization levels on root system development. Indi-vidual microbial effects were not additive, yet com-bining inoculants tended to improve root systemdevelopment.

Certain microorganisms can lead to changes inplant secondary metabolism (Peipp et al. 1997; AitBarka et al. 2006). We previously found that singleinoculation with A. lipoferum CRT1 resulted inmodified secondary metabolic profiles of maize(Walker et al. 2011), especially for benzoxazinoids,but this was done under greenhouse conditions. Here,the same benzoxazinoids were detected in field-grownmaize seedlings, and the modifications triggered byinoculation with A. lipoferum CRT1 were comparableto those previously found in the greenhouse. Inaddition, the current work also demonstrated meta-bolic variations when inoculation was done with P.fluorescens F113 or with G. intraradices JJ291,indicating that extensive modifications in plantphysiology can be expected when maize is inoculatedwith a plant-beneficial microorganism. Benzoxazi-noids, which are synthesized during the emergence ofroots (Park et al. 2004), are defence molecules ofgreat importance in biotic interactions since they arerepellents for aphids (Nicol et al. 1992), displayantimicrobial properties (Sahi et al. 1990) and inhibitvir genes of Agrobacterium tumefaciens (Zhang et al.2000). In this study, we observed a specific variationof DIMBOA-Glc, DIMBOA and HDMBOA-Glccontents upon single inoculation, and further workwill be needed to determine whether this metabolicresponse is important for rhizosphere colonizationand/or expression of plant-beneficial effects byinoculated microbes. However, these metaboliceffects were inoculant specific, which was notsurprising since (1) the three microorganisms usedare very different from one another in terms oftaxonomy, ecology and modes of action, and (2) evendifferent strains of a same genus or species can havedifferent effects on maize physiology (Walker et al.2011).

Inoculation of P. fluorescens F113 alone inducedmajor accumulation of diethylphtalate, which corre-

sponded to a compound synthesized by maize and nota contaminant from plastic devices based on verifica-tion of analytical conditions, sample preparationsteps, and analysis of control plants. This compoundhas also been detected in Poaceae exudates (Xuan etal. 2006). Kapanen et al. (2007) showed thatpseudomonads used diethylphthalate as substrate,raising here the hypothesis that P. fluorescens F113could have modified root metabolism to securediethylphthalate as a carbon source. This wouldrequire its release as root exudate, and further workwill target this issue. This is reminiscent of thestimulation of amino acid root exudation triggered intomato by 2,4-diacetylphloroglucinol, a metaboliteproduced by the same bacterium (Phillips et al. 2004).

We found that single inoculation with G. intra-radices JJ291 also resulted in modified secondarymetabolic profiles for maize roots. Glomus affectedthe transcriptome of Medicago truncatula roots inartificial soil at 28 days after inoculation, includinggenes involved in secondary metabolism (Hohnjec et al.2005). Accordingly, Glomus-mediated changes in rootmetabolite patterns have been reported inM. truncatula(Schliemann et al. 2008a), leek (Schliemann et al.2008b) and barley (Peipp et al. 1997). In comparison,however, the current work is important because itshows that changes in root composition (1) couldoccur as early as at 16 days (and perhaps earlier),i.e. for seedlings rather than well-grown plantsmore than 40 days old, and (2) took place in non-sterile soil under field conditions rather than undercontrolled conditions.

The treatments studied at reduced fertilization pro-vided the opportunity to assess the significance ofcombining inoculants in terms of effects on rootsecondary metabolism. Even though each single inoc-ulation had a very distinct impact on metabolic profiles,there was not much difference (if any) between thedouble and triple inoculation(s), and these treatmentswere similar to single inoculation with Glomus. Thistook place even though there was a trend for a morepronounced effect of consortia versus single inocula-tions when considering root system architectureparameters. On the one hand, this suggests that Glomuseffects on maize physiology can be dominant, whichmay be in accordance with the fact that PGPR such asP. fluorescens F113 can function as a mycorrhiza helper(Barea et al. 1998). On the other hand, however,adding Glomus to this bacterial consortium had no

160 Plant Soil (2012) 356:151–163

approach of Walker et al. (2011) to probe the effect ofmicrobial interactions on plant host physiology, as wellas the potential of plant-beneficial microorganisms totrigger significant changes in root functioning (and inroot system architecture). Results at reduced fertilizationalso indicated that the effects induced on root secondarymetabolism by individual inoculants did not add up,although combining strains in microbial consortia wasuseful to maintain effective root system developmentdespite reduction in mineral fertilizer usage.

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

References

Adesemoye AO, Torbert HA, Kloepper JW (2009) Plantgrowth-promoting rhizobacteria allow reduced applicationrates of chemical fertilizers. Microb Ecol 58:921–929

Ait Barka E, Nowak J, Clement C (2006) Enhancement of chillingresistance of inoculated grapevine plantlets with a plantgrowth-promoting rhizobacterium, Burkholderia phytofir-mans strain PsJN. Appl Environ Microbiol 72:7246–7252

Barea JM, Bonis AF, Olivares J (1983) Interactions betweenAzospirillum and VA mycorrhiza and their effects ongrowth and nutrition of maize and ryegrass. Soil BiolBiochem 15:705–709

Barea JM, Andrade G, Bianciotto V, Dowling D, Lohrke S,Bonfante P, O'Gara F, Azcon-Aguilar C (1998) Impact onarbuscular mycorrhiza formation of Pseudomonas strainsused as inoculants for biocontrol of soil-borne fungal plantpathogens. Appl Environ Microbiol 64:2304–2307

Bashan Y, de-Bashan LE (2010) How the plant growth-promoting bacterium Azospirillum promotes plant growth—A critical assessment. Adv Agron 108:77–136

Baudoin E, Nazaret S, Mougel C, Ranjard L, Moënne-LoccozY (2009) Impact of inoculation with the phytostimulatory

PGPR Azospirillum lipoferum CRT1 on the geneticstructure of the rhizobacterial community of field-grownmaize. Soil Biol Biochem 41:409–413

Beauchamp CJ (1993) Mode of action of plant growth-promoting rhizobacteria and their potential use as biologicalcontrol agent. Phytoprotection 74:19–28

Combes-Meynet E, Pothier JF, Moënne-Loccoz Y, Prigent-Combaret C (2011) The Pseudomonas secondary metabolite2,4-diacetylphloroglucinol is a signal inducing rhizoplaneexpression of Azospirillum genes involved in plant-growthpromotion. Mol Plant-Microbe Interact 24:271–284

Cornelis P (2010) Iron uptake and metabolism in pseudomonads.Appl Microbiol Biotechnol 86:1637–1645

Couillerot O, Prigent-Combaret C, Caballero-Mellado J,Moënne-Loccoz Y (2009) Pseudomonas fluorescens andclosely-related fluorescent pseudomonads as biocontrolagents of soil-borne phytopathogens. Lett Appl Microbiol48:505–512

Couillerot O, Bouffaud ML, Muller D, Caballero-Mellado J,Moënne-Loccoz Y (2010a) Development of a real-timePCR method to quantify the PGPR strain Azospirillumlipoferum CRT1 on maize seedlings. Soil Biol Biochem42:2298–2305

Couillerot O, Poirier MA, Prigent-Combaret C, Mavingui P,Caballero-Mellado J, Moënne-Loccoz Y (2010b) Assess-ment of SCAR markers to design real-time PCR primersfor rhizosphere quantification of Azospirillum brasilensephytostimulatory inoculants of maize. J Appl Microbiol109:528–538

Dobbelaere S, Vanderleyden J, Okon Y (2003) Plant growthpromoting effects of diazotrophs in the rhizosphere. CritRev Plant Sci 22:107–149

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 phytosti-mulator Azospirillum lipoferum CRT1 and functionalimpact on maize crop, biodegradation of crop residues,and soil faunal indicators in a context of decreasingnitrogen fertilisation. Soil Biol Biochem 38:1712–1726

Fages J, Mulard D (1988) Isolement de bactéries rhizosphériqueset effet de leur inoculation en pot chez Zea mays. Agronomie8:308–314

Fenton AM, Stephens PM, Crowley J, O'Callaghan M, O'GaraF (1992) Exploitation of gene(s) involved in 2,4-diacetyl-phloroglucinol biosynthesis to confer a new biocontrolcapability to a Pseudomonas strain. Appl Environ Micro-biol 58:3873–3878

Fester T, Maier W, Strack D (1999) Accumulation of secondarycompounds in barley and wheat roots in response toinoculation with an arbuscular mycorrhizal fungus and co-inoculationwith rhizosphere bacteria. Mycorrhiza 8:241–246

Frey-Klett P, Garbaye J, Tarkka M (2007) The mycorrhizahelper bacteria revisited. New Phytol 176:22–36

Garg N, Chandel S (2010) Arbuscular mycorrhizal networks:process and functions. A review. Agron Sustain Dev30:581–599

Haas D, Défago G (2005) Biological control of soil-bornepathogens by fluorescent pseudomonads. Nat Rev Microbiol3:307–319

Hohnjec N, Vieweg MF, Pühler A, Becker A, Küster H (2005)Overlaps in the transcriptional profiles of Medicago

Plant Soil (2012) 356:151–163 161

effect on maize secondary metabolism. This indicatesthat interactions between microbial inoculants can becomplex and suggests that consortium effects may bedifficult to anticipate based on knowledge of singleinoculation effects.

In conclusion, this study confirms under fieldconditions the usefulness of the metabolic profiling

truncatula roots inoculated with two different Glomusfungi provide insights into the genetic program activatedduring arbuscular mycorrhiza. Plant Physiol 137:1283–1301

Jacoud C, Job D, Wadoux P, Bally R (1999) Initiation of rootgrowth stimulation by Azospirillum lipoferum CRT1 duringmaize seed germination. Can J Microbiol 45:339–342

Jansa J, Mozafar A, Frossard E (2005) Phosphorus acquisitionstrategies within arbuscular mycorrhizal fungal communi-ty of a single field site. Plant Soil 276:163–176

Jansa J, Smith FA, Smith SE (2008) Are there benefits ofsimultaneous root colonization by different arbuscularmycorrhizal fungi? New Phytol 177:779–789

Kapanen A, Stephen JR, Brüggemann J, Kiviranta A, WhiteDC, Itävaara M (2007) Diethyl phthalate in compost:ecotoxicological effects and response of the microbialcommunity. Chemosphere 67:2201–2209

Karthikeyan B, Jaleel CA, Azooz MM (2009) Individual andcombined effects of Azospirillum brasilense and Pseudo-monas fluorescens on biomass yield and ajmalicineproduction in Catharanthus roseus. Acad J Plant Sci2:69–73

Lucy M, Reed E, Glick BR (2004) Application of free livingplant growth-promoting rhizobacteria. Antonie van Lee-wenhoek 86:1–25

Mar Vázquez M, César S, Azcón R, Barea JM (2000)Interactions between arbuscular mycorrhizal fungi andother microbial inoculants (Azospirillum, Pseudomonas,Trichoderma) and their effects on microbial populationand enzyme activities in the rhizosphere of maize plants.Appl Soil Ecol 15:261–272

Marimuthu S, Subbian P, Ramamoorthy V, Samiyappan R(2002) Synergistic effect of combined application ofAzospirillum and Pseudomonas fluorescens with inorganicfertilizers on root rot incidence and yield of cotton. ZPflanzenkr Pflanzenschutz 109:569–577

Nicol D, Copaja SV, Wratten SD, Niemeyer HM (1992) Ascreen of worldwide wheat cultivars for hydroxamic acidlevels and aphid antixenosis. Ann Appl Biol 121:11–18

Okon Y, Labandera-Gonzalez CA (1994) Agronomic applica-tions of Azospirillum: an evaluation of 20 years worldwidefield inoculation. Soil Biol Biochem 26:1591–1601

Park J-W, Crowley DE (2005) Normalization of soil DNAextraction for accurate quantification of target genes byreal-time PCR and DGGE. BioTechniques 38:579–586

Park WJ, Hochholdinger F, Gierl A (2004) Release of thebenzoxazinoids defense molecules during lateral- andcrown root emergence in Zea mays. J Plant Physiol161:981–985

Peipp H, Maier W, Schmidt J, Wray V, Strack D (1997)Arbuscular mycorrhizal fungus-induced changes in theaccumulation of secondary compounds in barley roots.Phytochemistry 44:581–587

Phillips DA, Fox TC, King MD, Bhuvaneswari TV, Teuber LR(2004) Microbial products trigger amino acid exudationfrom plant roots. Plant Physiol 136:2887–2894

Prigent-Combaret C, Blaha D, Pothier JF, Vial L, Poirier M-A,Wisniewski-Dyé F, Moënne-Loccoz Y (2008) Physicalorganization and phylogenetic analysis of acdR as leucine-responsive regulator of the 1-aminocyclopropane-1-carbox-ylate deaminase gene acdS in phytobeneficial Azospirillum

lipoferum 4B and other Proteobacteria. FEMS MicrobiolEcol 65:202–219

Raaijmakers J, Paulitz T, Steinberg C, Alabouvette C, Moënne-Loccoz Y (2009) The rhizosphere: a playground andbattlefield for soilborne pathogens and beneficial micro-organisms. Plant Soil 321:341–361

Raju PS, Clark RB, Ellis JR, Maranville JW (1990) Effects ofspecies of VA-mycorrhizal fungi on growth and mineraluptake of sorghum at different temperatures. Plant Soil121:165–170

Richardson A, Barea JM, McNeill A, Prigent-Combaret C(2009) Acquisition of phosphorus and nitrogen in therhizosphere and plant growth promotion by microorganisms.Plant Soil 321:305–339

Rillig MC, Mummey DL (2006) Mycorrhizas and soilstructure. New Phytol 171:41–53

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 arbuscularmycorrhiza establishment in wheat and maize plants. BiolFertil Soils 41:301–309

Sahi SV, Chilton MD, Chilton WS (1990) Corn metabolitesaffect growth and virulence of Agrobacterium tumefaciens.Proc Natl Acad Sci USA 87:3879–3883

Sánchez Pérez JM, Antiguedad I, Arrate I, García-Linares C,Morell I (2003) The influence of nitrate leaching throughunsaturated soil on groundwater pollution in an agricul-tural area of the Basque country: a case study. Sci TotalEnviron 317:173–187

Schliemann W, Ammer C, Strack D (2008a) Metaboliteprofiling of mycorrhizal roots of Medicago truncatula.Phytochemistry 69:112–146

Schliemann W, Kolbe B, Schmidt J, Nimtz M, Wray V (2008b)Accumulation of apocarotenoids in mycorrhizal roots ofleek (Allium porrum). Phytochemistry 69:1680–1688

Singh UP, Sarma BK, Singh DP (2003) Effect of plant growth-promoting rhizobacteria and culture filtrate of Sclerotiumrolfsii on phenolic and salicylic acid contents in chickpea(Cicer arietinum). Curr Microbiol 46:131–140

Subramanian KS, Charest C, Dwyer LM, Hamilton RI (1995)Arbuscular mycorrhizas and water relations in maizeunder drought stress at tasselling. New Phytol 192:643–650

Troxler J, Zala M, Natsch A, Moënne-Loccoz Y, Défago G (1997)Autecology of the biocontrol strain Pseudomonas fluorescensCHA0 in the rhizosphere and inside roots at later stages ofplant development. FEMS Microbiol Ecol 23:119–130

Volpin H, Kapulnik Y (1994) Interaction of Azospirillumwith beneficial soil microorganisms. In: Okon Y (ed)Azospirillum/Plant associations. CRC Press, Boca Raton,pp 111–118

Von Felten A, Défago G, Maurhofer M (2010) Quantification ofPseudomonas fluorescens strains F113, CHA0 and Pf153in the rhizosphere of maize by strain-specific real-timePCR unaffected by the variability of DNA extractionefficiency. J Microb Methods 81:108–115

Walker V, Bertrand C, Bellvert F, Moënne-Loccoz Y, Bally R,Comte G (2011) Host plant secondary metabolite profilingshows complex, strain-dependent response of maize to theplant growth promoting rhizobacteria of the genus Azo-spirillum. New Phytol 189:494–506

162 Plant Soil (2012) 356:151–163

Weller DM, Landa BB, Mavrodi OV, Schroeder LK, De laFuente L, Blouin Bankhead S, Allende Molar R, BonsallRF, Mavrodi DV, Thomashow LS (2007) Role of 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonasspp. in the defense of plant roots. Plant Biol 9:4–20

Witcombe JR, Hollington PA, Howarth CJ, Reader S, SteeleKA (2008) Breeding for abiotic stresses for sustainableagriculture. Philos Trans R Soc Lond B 363:703–716

Xuan TD, Chung IIIM, Khanh TD, Tawata S (2006) Identifi-cation of phytotoxic substances from early growth ofBarnyard grass (Echinochloa crusgalli) root exudates. JChem Ecol 32:895–906

Zhang J, Boone L, Kocz R, Zhang C, Binns AN, Lynn DG(2000) At the maize/Agrobacterium interface: naturalfactors limiting host transformation. Chem Biol 7:611–621

Plant Soil (2012) 356:151–163 163