RESEARCH Open Access Root exudate profiling of maize ... · RESEARCH Open Access Root exudate...

da Silva Lima et al Chemical and Biological Technologies in Agriculture 2014 123httpwwwchembioagrocomcontent1123

RESEARCH Open Access

Root exudate profiling of maize seedlingsinoculated with Herbaspirillum seropedicae andhumic acidsLiacutevia da Silva Lima1 Faacutebio Lopes Olivares1 Rodrigo Rodrigues de Oliveira2 Maria Raquel Garcia Vega2Nataacutelia Oliveira Aguiar1 and Luciano Pasqualoto Canellas1

Abstract

Background Co-inoculation of maize with Herbaspirillum seropedicae and humic substances increases the sizes ofplant-associated bacterial populations and enhances grain yields under laboratory and field conditions Root exudationis a key mechanism in the regulation of plant-bacterial interactions in the rhizosphere humic matter supplementationis known to change the exudation of H+ ions and organic acids from maize roots Our starting premise was thatH seropedicae and humic acids would modify maize seedling exudation profiles We postulated that a betterunderstanding of these shifts in exudate profiles might be useful in improving the chemical environment to promotebetter performance of plant growth-promoting bacteria delivered as bioinoculants Thus root exudates of maize werecollected and analyzed by gas chromatography-mass spectrometry (GC-MS) and proton nuclear magnetic resonance(1H NMR)

Results Nitrogenous compounds fatty acids organic acids steroids and terpenoid derivatives were the mainstructural moieties found in root exudates Significant changes in exudation patterns occurred 14 days after theinitiation of experiments Quantities of fatty acids phenols and organic acids exuded by seedlings treated with humicacids alone differed from the quantities exuded in other treatments Seedlings treated with H seropedicae orH seropedicae in combination with humic acids exuded a diversity of nitrogenous compounds most of which hadheterocyclic structures Twenty-one days after initiating the experiment seedlings treated with H seropedicae aloneexuded elevated quantities of steroids and terpenoid derivatives related to precursors of gibberellic acids (kaurenoicacids)

Conclusions Changes in root exudation profiles induced by our treatments became most marked 14 and 21 daysafter initiation of the experiment on those days we observed (i) increased fatty acid exudation from seedlings treatedonly with humic acids and (ii) increased exudations of nitrogenated compounds and terpenes from seedlings treatedonly with H seropedicae Improved knowledge on the effects of bacterial inoculants and supplementation withhumates on plant exudate composition may contribute substantially to improved understanding of plant metabolicresponses and lead to new approaches in the use of selected compounds as additives in bioinoculant formulationsthat will modulate the cross-talk between bacteria and plants thereby improving crop yields

Keywords Plant growth-promoting bacteria Endophytic interaction Humic substances

Correspondence lucianocanellasgmailcom1Nuacutecleo de Desenvolvimento de Insumos Bioloacutegicos para a Agricultura(NUDIBA) Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF)Av Alberto Lamego 2000 Campos dos Goytacazes 28013-602 Rio deJaneiro BrazilFull list of author information is available at the end of the article

copy 2014 da Silva Lima et al licensee Springer This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (httpcreativecommonsorglicensesby40) which permits unrestricted use distribution andreproduction in any medium provided the original work is properly credited

da Silva Lima et al Chemical and Biological Technologies in Agriculture 2014 123 Page 2 of 18httpwwwchembioagrocomcontent1123

BackgroundRoot exudates function in processes of plant adaptationThey have roles in nutrient cycling in the rhizosphereand in responses to pathogens and symbiotic micro-organisms [1] The quantities of organic compoundsexuded by roots are variable but they are frequently asignificant proportion of the carbon fixed photosynthet-ically by plants [2] Plant type species age and environ-mental factors including biotic and abiotic stressors allaffect exudation profiles [34] The plant rhizospheremodulates microbial community structure and functionprimarily through the release of chemical compounds [5]The dominant organic compounds exuded by roots

reflect central components of cell metabolism includingfree sugars (eg glucose sucrose) amino acids (eg gly-cine glutamate) and organic acids (eg citrate malateand oxalate) [2] Maize exudates comprise sugars (70)phenolics (18) organic acids (7) and amino acids (3)[6] Other compounds like fatty acids sterols enzymesvitamins and plant growth regulators (eg auxins gibber-ellins and cytokinins) are generally released in only verysmall quantities [7] Although only small quantities of sec-ondary metabolites are exuded by roots their plant role insignaling to the microbial community is substantial [8]Recently we determined that the combined inoculation

of maize with the endophytic diazotrophic bacteriumHerbaspirillum seropedicae and humic acids increasedroot colonization and promoted plant growth and grainyields [9] Canellas and Olivares [10] reviewed the use ofhumic substances as plant growth promoters they consid-ered the effects of these substances on plant metabolismand their use as carriers in procedures for the bioinocula-tion of plant growth-promoting bacteria in field cropsystemsCanellas et al [11] reported changes in maize exud-

ation profiles after humic acid application that includedenhanced secretion of inorganic ions (ie H+ ions) andshort-chain organic acids [12] The addition of humicacids of different size fractions may have substantial effectson the quantities of bioavailable carbon deposited bymaize plant roots these additions produce significantchanges in the structure and activity of soil microbial com-munities [13] Hence chemical changes induced by humicmatter augmentation in the rhizosphere may enhancecolonization of maize plants by inoculated endophytic dia-zotrophic bacteria carried in the humic substancesH seropedicae is a plant growth-promoting diazo-

trophic β-proteobacterium found mainly in associationwith grasses and other non-leguminous plants [14]Roesch et al [15] used molecular tools to assess diazo-trophic bacterial diversity within rhizosphere soils rootsand stems of field-grown maize and observed a predomin-ance of α-proteobacteria and β-proteobacteria sequencesin the rhizosphere soil and stem samples Herbaspirillum

was one of the dominant genera in the interiors of maizeplants but was rarer in soil The members of this genushave been tested in the formulation of biofertilizers withvariable success in field crop trials ([16-19] and referencestherein [20-23]) The whole genome sequence of H sero-pedicae has been published [24] The species capacity forN fixation production of auxin and other phytohormonesand the colonization of diverse plant species has been pre-viously demonstrated [25-27]Root colonization is a basic first step for successful in-

oculation An expansion of studies on metabolite ex-change between plants and bacteria and the geneticresponses of plants will fill knowledge gaps in our un-derstanding of the colonization process The role ofroot-exuded flavonoids in legume-Rhizobium interac-tions has been examined in detail These exudationsgenerate a finely tuned cross-molecular dialogue involv-ing the secretion of lipochitooligosaccharides and themodulation of bacterial cell wall surface polysaccharides(extracellular polysaccharide (EPS) and lipopolysaccharide(LPS)) that result in plant root nodulation [28] There isless information available on the role of plant metabolitesin successful interactions with non-nodulating plantgrowth-promoting bacteria Gough et al [29] showed thatflavonoids promote endophytic colonization of Arabidop-sis thaliana (L) Heynh roots by H seropedicae andTadra-Sfeir et al [30] demonstrated that naringenin (a fla-vonoid in the flavonone class) is involved in the geneexpression of cell wall components (EPS LPS) and auxinsIn addition to its role in the genetic modulation of cellwall assembly H seropedicae has an operon associatedwith the degradation of aromatic compounds [31] Anability to degrade flavonoids would likely confer an im-portant competitive advantage in rhizosphereroot coloni-zation of the host plant by providing both a carbon sourceand associated detoxification mechanisms Balsanelli et al[32] showed that surface lipopolysaccharides produced byH seropedicae strain Smr1 are required for attachmentand endophytic colonization of maize plants They [32]found that the H seropedicae attachment process ispartially mediated by a root lectin that specifically bindsN-acetyl glucosamine residuesRecently Marks et al [33] demonstrated the potential

of using bacterial metabolites to enhance the perform-ance of biofertilizers thereby opening the possibility ofchemical manipulation of carriers to benefit bacterialdelivery to field crops Furthermore metabolites exudedby host plants may help in guiding genomic studiesof plant-bacterial interactions In the present workwe examined main changes in exudation profiles ofmaize seedling roots induced under laboratory con-ditions by (i) single applications of humic acids or(ii) H seropedicae or (iii) combinations of the bacteria andhumic acids


MethodsHumic substancesHumic-like substances were extracted as described pre-viously [11] In brief ten volumes of 05 mol Lminus1 MNaOH were mixed with one volume of earthworm com-post under a N2 atmosphere After 12 h the suspensionwas centrifuged at 5000 times g humic acids (HA) wereextracted thrice in this manner and the final HA pelletwas de-ashed by combining it with ten volumes of a di-luted mixture of HF-HCl solution (5 mL Lminus1 HCl [12 M] +5 mL Lminus1 HF [48 vv]) After centrifugation (5000 times g)for 15 min the sample was repeatedly washed with wateruntil a negative test against AgNO3 was obtained Subse-quently the sample was dialyzed against deionized waterusing a 1000-Da cutoff membrane (Thomas ScientificSwedesboro NJ USA) The dialyzate was lyophilized Wethen prepared a HA solution by solubilizing HA powderin 1 mL of 01 M mol Lminus1 NaOH followed by pH adjust-ment to 65 with 01 M HCl

Microorganism usedH seropedicae strain HRC 54 was originally isolatedfrom sugarcane roots [34] It has been used as part ofthe sugarcane inoculant developed by Embrapa (BrazilianEnterprise for Agricultural Research) The pre-inoculumwas obtained after growth in DYGS liquid medium [35]for 24 h at 30degC on an orbital shaker rotating at 150 rpmSubsequently 20 μL of the suspension was transferred toJNFb liquid medium supplemented with NH4Cl (1 g Lminus1)and then grown for 36 h at 34degC on an orbital shaker ro-tating at 150 rpm Cells were pelleted by centrifugation(4000timesg for 15 min) and resuspended in sterilized waterat cell densities of 108 colony-forming units (cfu) mLminus1The inoculant was prepared by diluting 200 mL of bacter-ial suspension in 800 mL of humic acid solution at pH 65to produce a final humic acid concentration of 50 mg C Lminus1

and a final bacterial concentration of 2 times 107 cells mLminus1The composition of JNFb medium (per liter) was as fol-

lows malic acid (50 g) K2HPO4 (06 g) KH2PO4 (18 g)MgSO47H2O (02 g) NaCl (01 g) CaCl2 (002 g) 05bromothymol blue in 02 N KOH (2 mL) vitamin solution(1 mL) micronutrient solution (2 mL) 164 FeEDTA so-lution (4 mL) and KOH (45 g) One-hundred milliliters ofvitamin solution contained 10 mg of biotin and 20 mg ofpyridoxol-HCl The micronutrient solution contained (perliter) the following CuSO4 (04 g) ZnSO47H2O (012 g)H3BO3 (14 g) Na2MoO42H2O (10 g) and MnSO4H2O(15 g) pH was adjusted to 58 For bacterial counts weused the same media with a semisolid consistency obtainedby adding 19 g Lminus1 of agar [14] The bacterial populationwas determined by the most probable number technique(MPN) positive growth was recognized by the formation ofa thick white pellicle replication was threefold and densitywas expressed as the log of cell number gminus1 root fresh mass

after growth on JNFb N-free semisolid medium (followingDoumlbereiner et al [35]) The presence of H seropedicae wasconfirmed by collecting a piece of pellicle with a platinumloop mounting it on a slide under a coverslip and makingobservations under phase contrast microscopy to deter-mine cell shape and movement and colony appearance inJNFb solid medium as described by Doumlbereiner et al [35]When cell shape in the pellicle material differed from thatof H seropedicae we identified the microbes as native bac-teria associated with maize roots

ExperimentalTreatment of plantsMaize seeds (Zea mays L var Dekalb 7815) were surface-sterilized by soaking in 05 NaClO for 30 min followedby rinsing and then soaking in water for 6 h Afterwardthe seeds were sown on wet filter paper and germinated inthe dark at 28degC Four days after germination 30 maizeseedlings with root length approximately 05 cm weretransferred into 22-L vessels previously filled with 2 L ofone-fourth-strength Furlani nutrient solution (containing3527 μMCa 2310 μMK 855 μMMg 45 μMP 587 μM S25 μM B 77 Fe 91 μM Mn 063 μM Cu 083 μM Mo229 μM Zn 174 μM Na and 75 μM EDTA) with inor-ganic N content adjusted to a low concentration (100 μmolLminus1 [NO3

minus +NH4+]) These low levels of N and P were

used to simulate the low availability in highly weatheredtropical soils and to avoid the inhibition of the diazotrophicbacteria The seedlings were fixed into a perforated Teflonsupport with holes of 15-mm diameter in which seeds havebeen fitted The system was continuously aerated by a lowflux pump normally used in aquarium systems Four treat-ments (n = 3 pots per treatment) were prepared by supple-menting the nutrient solution with the following 1 HA(50 mg C Lminus1) 2 plant growth-promoting bacteria (PGPB)H seropedicae strain HRC 54 (final bacterial suspension of2 times 107 cells mLminus1) 3 humic acids plus H seropedicae(HA + PGPB) and 4 control (C) without any additionsSeedlings were collected 7 14 and 21 days after inocula-tion After 1 week and each week thereafter one half ofthe nutrient solution in each pot was replaced with freshnutrient solution through the end of the experiment Theexperiment was repeated thrice independently

Exudate collectionMaize seedlings were removed from the pots their rootswere immersed in glass tubes filled with 50 mL of001 mol Lminus1 KOH for 5 min to remove organic anionsadhering to the root surfaces We then thoroughlywashed the roots with tap water followed by a final rinsein distilled water Complete root systems of seedlingsfrom a single pot were inserted in a glass tube (65-cminner diameter (id) times 15-cm tall) filled with 80 mL ofultrapure water in which to collect the root exudates


After 2 h we collected the suspensions containing rootexudates and filtered them through 022-μm filter mem-branes to remove root detritus and microbial cells Thefiltered samples were kept frozen until we concentratedthem by liquid chromatography using 10 cm of reversephase (RP) C18 LiChroprepreg RP-18 (15 to 25 μm MerckMillipore Billerica MA USA) as the stationary phase inan open glass column (25-cm id times 20-cm tall) Theaqueous suspension of exudate was forced through thecolumn under low pressure provided by an aquariumpump Compounds were eluted from the column withmethanol under gravity and the solvent was removedunder low temperature (4degC) under vacuum (RocketEvaporator System Genevac Stone Ridge NY USA) Wedrove our exudate capture to exclude sugars and aminoacids and collected mainly products of the secondary me-tabolism using the RP C18 column

NMR sample preparation and data collectionFor nuclear magnetic resonance (NMR) analysis wedissolved exudate extracts in DMSO-d6 (700 μL) Allspectra were recorded at room temperature on a BrukerAvance DRX 500 spectrometer equipped with a 5-mminverse detection probe (Bruker GmbH RheinstettenGermany) operating at 50013 MHz for 1 h For eachsample we recorded 360 scans (FIDs) with the followingparameter settings 64 k data points pulse width 85 μs(90deg) spectral width of 4401 Hz acquisition time of74 s and a relaxation delay of 10 s For spectrum pro-cessing we used 64 k points and applied an exponentialmultiplication associated with a line broadening of 03 HzSpectra were referenced to tetramethylsilane (TMS) at00 ppm To obtain exudate profiles for each of the treat-ments in the study we pooled extracts from treatmentreplicates and dissolved the dried methanolic extracts in700 μL of DMSO-d6 Dissolved extracts were transferredto a 5-mm NMR tube for analysis

Principal component analysisThe proton nuclear magnetic resonance (1H NMR) spec-tra were reduced to ASCII files using OACD softwarethe resulting data matrix was imported into The Un-scrambler 101 software (wwwcamocom) Signals corre-sponding to the solvent TMS and noise from watersuppression were removed from the data set prior tostatistical analysis Principal component analysis (PCA)was performed by auto scaling the variables using nor-malization and calculation of the first derivative as atransformation procedure

Gas chromatography-mass spectrometryAfter NMR analysis we analyzed the exudates by gaschromatography-mass spectrometry (GC-MS) GC separa-tions were performed on a GCMS QP2010 Plus instrument

(Shimadzu Tokyo Japan) equipped with an Rtx-5MSWCOT capillary column (30 m times 025 mm film thickness025 μm) (Restek Bellefonte PA USA) The exudateswere derivatized by refluxing 030 mg of sample for 1 h at70degC with an excess of MeOH and acetyl chloride driedunder a stream of N2 followed by silylation with 100 μLof NN-bis[trimethylsilyl]trifluoroacetamide1 trimethyl-chlorosilane (Superchrom Milan Italy) in closed vials at60degC for 30 min Chromatographic separation was achievedunder the following temperature regimen 60degC for1 min (isothermal) rising by 7degC minminus1 to 100degC and thenby 4degC minminus1 to 320degC followed by 10 min at 320degC (iso-thermal) Helium was the carrier gas supplied at 190 mLminminus1 the injector temperature was 250degC and the splitinjection mode had a split flow of 30 mL minminus1 Massspectra were obtained in EI mode (70 eV) scanning in therange of mz 45 to 850 with a cycle time of 1 s Compoundidentification was based on comparisons of mass spectrawith the NIST library database (httpwwwnistgovsrdnist1acfm) published spectra and real standards Due tothe large variety of compounds with different chromato-graphic responses that we detected external calibrationcurves for quantitative analysis were built by mixing me-thyl esters andor methyl ethers of the following molecularstandards tridecanoic acid octadecanol 16-hydroxyhex-adecanoic acid docosandioic acid β-sitosterol and cin-namic acid Increasing quantities of standard mixtureswere loaded into a quartz boat and moistened with 05 mLof tetramethylammonium hydroxide (TMAH) solution(25 in methanol)

ResultsRoot tissue colonization by H seropedicae strain HRC54We examined the population dynamics of H seropedicaestrain HRC 54 associated with maize roots 7 14 21 and30 days after inoculation (Figure 1) For all inoculationtreatments (PGPB and HA + PGPB) the root-associatedbacterial numbers were higher than those of uninocu-lated plants (controls) Cell shape and colony appearanceconfirmed the presence of Herbaspirillum in the pellicleharvested from the highest dilution thereby indicatingthe effectiveness of inoculationMaize plants treated with only HA had higher bacter-

ial numbers associated with roots than control plantsEven after seed surface disinfection diazotrophic bacteriawere recoverable from treated plants these microbialpopulations were naturally occurring N fixers associ-ated with maize seeds They were clearly differentfrom H seropedicae (under phase contrast microscopy) incell shape and colony form when grown in JNFb solidmedium (data not shown)We compared treatments PGPB and HA+ PGPB ob-

serving higher numbers of root-associated viable H serope-dicae cells in the latter This result is qualitatively similar

Figure 1 Number of bacterial cells (log cells gminus1 fresh tissue) on roots of maize seedlings during different growth times Treatmentscontrol plants log 109 cells mLminus1 of Herbaspirillum seropedicae strain HRC 54 humic acids isolated from vermicompost (50 mg Lminus1) and bacteriaplus humic acids The values represent the mean plusmn standard deviation

Figure 2 The yield of exudates at 7 14 and 21 days aftertreatments The values represent the mean plusmn standard deviation


to those obtained previously [9] indicating that HA per seincrease the numbers of H seropedicae cells colonizingroot tissues and help maintain large populations in inocu-lated plants over protracted time periods (Figure 1) Weidentified similar tendencies for natural diazotrophswhose population sizes were enhanced by HA application(in comparison with populations in control plants)

Mass of root exudatesThe yields of exudates retained by the procedures usedare depicted in Figure 2 Over the course of the experi-ment the quantities of exudates collected across sam-pling occasions and treatments ranged broadly between025 and 400 mg gminus1 root dry weight making it difficultto identify any treatment effects

1H NMR and PCANuclear magnetic resonance (1H NMR) coupled withmultivariate analysis (PCA) enabled rapid discriminationamong root exudate samples The 1H NMR spectra ofthe exudate compounds eluted from the RP C18 columnwith methanol on days 7 14 and 21 are depicted inFigures 345 respectively the main 1H chemical shiftsare detailed in Table 1 Visual inspection of the 1H NMRspectra revealed a predominance of signals in the carbo-hydrate region (25 to 45 ppm) followed in rank orderby signals in the aliphaticorganic acid (00 to 30 ppm)

and aromatic (50 to 80 ppm) regions NMR spectrarevealed different exudate profiles between treated andcontrol plants On day 7 the control spectrum con-tained several chemical shifts in the aliphatic regionwith a small signal at 096 ppm and a short intensesignal at 124 ppm The sugar region had a main signalat 371 ppm and other signals of low intensity at 341and 393 ppm In the aromatic region it was possible to

Figure 3 Spectra of root exudate at 7 days


observe small signals in the 669 to 674 ppm region andat 70 ppm The signals at 124 ppm may have beenrelated to the presence of CH3 compounds of aliphaticacids and βCH3 in amino acids of maize extracts Thestrong absorption at 371 ppm may be attributed to thepresence of glycosylated compounds (β-Glc and α-Glc)The signals at 67 ppm are typical of hydroxybenzoicacids such as cinnamic and protocatechuic acids Tryp-tophan histidine and gallic acids have signals near70 ppm Treatment of maize seedlings with HA changedthe region of aliphatic absorption and additional signalswere observed at 084 088 and 110 ppm These signalswere also recorded for exudates from plants treated with

H seropedicae (treatment PGPB) which had an add-itional signal at 164 ppm that was absent in controlexudates Additional signals were present at 108 120and 20 ppm in exudates from treatment HA + PGPBThe exudate spectrum from seedlings treated with HAalone had an additional absorption at 393 ppm attri-butable to sucrose (348 384 390 422 and 542 ppm)andor lysine (since a typical signal was also observed at164 ppm [δCH2]) Exudates from seedlings in treatmentHA + PGPB had additional though very small signals inthe aromatic region in the range 634 to 662 ppm and avery small signal at 852 ppm Compounds similar toniacin have signals in this region



The profiles of exudates collected on day 14 were verydifferent from those collected on day 7 The intensity ofthe signal at 123 ppm was enhanced in all spectra withthe highest intensity in the exudates collected from seed-lings in treatment PGPB We observed very weak signalsrelated to aromatic compounds in the control exudatespectrum on day 14 with just one visible signal at570 ppm In contrast signals for this region were verystrong in treatment HA + PGPB exudates In treatmentHA there was a diversity of signal shoulders in regions

499 to 513 565 to 571 and 669 to 680 ppm and indi-vidual signals at 699 and 718 ppm Treatment PGPBexudates had clearer signals in this region than treat-ment HA treatment PGPB had well-resolved signals at564 585 607 677 693 and 720 ppm This spectrumwas marked by weak main signals at 354 and 372 ppmThere were additional signals in the aliphatic regionat 148 177 and 203 which were not present in thespectra from the controls and treatment HA The char-acteristics of the exudate spectra were similar between



treatments PGPB and HA + PGPB ie a strong modifi-cation in the aromatic region including a sharp signal at698 ppm However in treatment HA + PGPB therewere changes in the sugar region including an increasedintensity in the signal at 372 ppmOn day 21 after inoculation seedlings were less depen-

dent on seed reserves and the exudate profiles were

modified by the treatments (Figure 2 Table 1) The spec-tra were more complex than those in earlier exudatesthey were characterized by an abundance of signalsControl exudates had a wide range of small signals inthe aliphatic region In the control spectra from days 7and 14 the main signal in this region was at 208 ppmAdditional signals occurred in the sugar region between

Table 1 1H chemical shiftControl HA PGPB HA + PGPB

7 14 21 7 14 21 7 14 21 7 14 21

Aliphatic 096 010 010 to 072 084 038 068 084 038 001 to 059 108 077 078

124 038 077 093 085 070 093 085 062 110 085 082 to 089

085 080 to 139 110 123 071 110 123 064 120 123 094

123 144 124 073 to 100 124 148 065 124 149 095

147 to 150 164 103 to 122 164 177 070 20 229 107 to 110

154 to 168 125 203 073 305 235 117 to 128

176 128 to 154 077 318 292 to 317 141 to 154

178 157 080 to 086 209

181 160 to 206 090 to 124 224

184 209 134 246 to 257

186 to 239 210 135 293

242 214 to 222 139 295

243 225 144 299 to 328

247 228 148 to 151

252 230 154

256 234 to 242 157

257 247 160 to 169

269 252 176

275 to 336 257 177

260 to 269 180

272 to 323 184

187 to 242

247

252

256 to 336

Sugars 347 339 338 to 472 340 327 to 361 350 340 to 356 344 346 340

371 373 492 374 456 366 354 366 to 500 365 373 342

393 393 499 to 513 368 372 426 456 353

371 to 465 456 458 356 to 382

467 to 499 465 464 388

466

Aromatic 669 to 674 570 545 671 565 to 571 500 to 616 651 564 501 to 680 634 to 662 565 566

70 561 701 588 619 to 654 665 585 685 852 (very low) 587 572

563 669 to 680 657 to 690 594 687 596 577

566 699 694 to 761 667 689 to 704 60 582

568 718 771 677 707 670 682

570 to 572 773 693 710 to 949 673 683

575 787 720 964 698 834

583 800 969 to 999 716

588 to 593 805

597 834

598 845

634 849

642 to 860 974

969 to 978


da Silva Lima et al Chemical and Biological Technologies in Agriculture 2014 123 0 of 18httpwwwchembioagrocomcontent1123

303 and 345 ppm including a sharp signal at 318 ppmThe main absorption in this region occurred at 373ppm as in earlier exudates Signals between 455 and467 ppm were very minor or absent in exudates from days7 and 14 but much higher and with greater definition byday 21 which was also the case for signals in the aromaticregion (650 to 719 ppm) Additional small signals oc-curred at 969 976 and 978 ppm The exudate profilefrom treatment HA was generally quite similar to that ofthe control but significant differences were apparent Forexample there was no sharp high-intensity signal at225 ppm in the control but this was observable in treat-ment HA exudates which also had a very intense signal at557 ppm (this was the main signal in treatment HA +PGPB spectra but a very minor one in treatment PGPB andin the control) On day 21 the spectra of exudates wereclosely similar between treatment PGPB and the controlSimilarities and dissimilarities among treatments and ex-

perimental days were examined by PCA analysis (Figure 6)PC1 (89) and PC2 (10) accounted for 99 of the totalvariance in the 1H NMR data set Symbols in the PCA plotare identified by treatment codes and days of exudate col-lection Three groups of symbols are apparent in theCartesian two-dimensional space of axes PC1 and PC2these correspond to the three occasions of exudate collec-tion The day 7 group is located in the positive sector ofthe PC1 axis and in the negative sector of the PC2 axisPositive values of PC1 and the loading coefficient respon-sible for this distinction are large for the carbohydrate 1HNMR region (3 to 4 ppm) HA + PGPB symbols for theday 14 fall in the negative sector of PC2 but other treat-ment symbols for this collection day fall in the positivesector of this axis HA and PGPB symbols cluster togetherthe control treatment had high values of aliphatic moieties(0 to 2 ppm) On day 21 the HA + PGPB and HA treat-ments had very different exudate compositions with ele-vated aromatic and aliphatic contents respectively Thecontrol and PGPB treatment clustered togetherOverall the PCA analysis aided interpretation and com-

parisons in the data set Thus on day 7 exudate composi-tions were not significantly different among treatmentsbut significant differences were observed on days 14 and21 This trend was corroborated by GC-MS analysis Thechromatograms are available in the supplementary data(Additional files 1 2 and 3) Main exudates retained inthe RP C18 column and identified by GC-MS are indi-cated in Table 2 On day 7 the chromatograms wereclosely similar the main compounds were nitrogenouscarbohydrate derivatives and fatty acids (as methyl esters)Nevertheless there were some differences among treat-ments on day 7 For example exudation of carbohydratecompounds by root seedlings in treatment HA + PGPBexceeds those in other treatments (Table 2) The chro-matogram peaks of these carbohydrate compounds for

treatments PGPB and HA were twice as high as that forthe control we identified galacto gluco and mannose py-ranoside structures in these peaks D-glucopyranosidemoieties associated with aromatic rings were also foundin control exudates but 16-β-glucose was found only inexudates from plants in treatment HA The quantities anddiversities of nitrogenous compounds on day 7 were alsogreater in treatment HA and PGPB exudates than in thecontrols (Table 2) Benzilamines polyamines pyrrol deriv-atives amino acid complexes and nucleotide derivativeswere included in this group of compounds Benzilaminesand polyamines were exuded only in treatments HA andHA+ PGPB Nitrogenous compounds from the phenyl-alanine pathway were present these included pyrimidi-nedione pyrrole-3-carboxylic acid and a derivative fromp-coumaric hydrolysis with a retention time at 2891 min(identified as caffeine) Structures derived from adenosinewere found in all treatments but those from purine werefound only in treatment PGPB Overall treatment HA +PGPB induced the largest and most diverse exudation ofnitrogenous compoundsFatty acids comprised the second largest class of com-

pounds exuded on day 7 Unlike nitrogenous compoundstreatments PGPB and HA+ PGPB induced exudations ofonly small quantities of fatty acids (in comparison withtreatment HA and the control) Chromatogram peak areasfor fatty acids were elevated by 52 in treatment HA incomparison with the control on day 7 The main fatty acidin the HA exudate was hexadecanoic acid (palmitic acid)Stearic acid (octadecenoic) was exuded only by controlplants while tridecanoic acid and eicosanoic acid (alsoknown as arachidic acid) which are minor constituents ofcorn oil were found only in treatment HA exudates Pal-mitic and stearic acids were found in treatment PGPB ex-udates Although GC-MS is not particularly appropriatefor short-chain organic acid identification we did observethe presence of propenoic acid associated with long-chaincarbon compounds in control and treatment HA exudateswe also detected succinic acid associated with aromaticmoieties in treatment HA + PGPB Benzenedicarboxylicacid was the main aromatic exudate The steroid isosteviolwas found in all exudates Other steroids such as cyclola-nostane and androstane were found in exudates producedin treatments HA PGPB and HA + PGPBThe main classes of compounds were similar among

days 7 14 and 21 (Table 2) but the diversity of com-pounds diminished progressively as the seedlings grewThere were differences among the treatments on day 14nitrogenous compounds were still abundant in the HAPGPB and HA + PGPB treatment exudates the concen-trations were threefold to fourfold higher than those inthe control Nucleotide derivatives were observed on day7 but not later Products from phenylalanine and pyri-dines were exuded in treatments PGPB and HA+ PGPB

Figure 6 PCA scores and PCA loading plot (A) PCA scores indicating good separation in different groups according to treatments and daysof treatments (B) PCA loading plot Position of the variables along the PCs indicates their importance for that PC


while phenyl cyanate and pyrrol derivatives were found intreatment HA exudates The compounds present in exu-dates from treatments PGPB and HA + PGPB were closelysimilar (Table 2) although benzene 1-isocyanato-4-meth-oxy was exuded only in treatment PGPB and carbonicacid monoamine N-(24-dimethoxyphenyl)- and butylester only in treatment HA + PGPB Exudations of carbo-hydrates were elevated in treatments HA and PGPBThese exudates contained compounds we had identifiedon day 7 However quantities of carbohydrate derivatives

were reduced in treatment HA + PGPB and absent in con-trol seedling exudatesHexadecanoic and octadecanoic acids were the main

fatty acids occurring as methyl esters in control exu-dates These compounds also occurred in other treat-ments but the diversity was greatest in PGPB and HA +PGPB treatments However on day 7 the quantities anddiversity were highest in treatment HA As on day 7products of the isosteviol reaction occurred in all exu-dates another steroid (androstane derivative) was also

Table 2 Compounds identified by GC-MS analysis

Retentiontime (min)

Compound class Control HA H seropedicae HA +H seropedicae

7 14 21 7 14 21 7 14 21 7 14 21

Nitrogenous compounds

9575 3-Butenoic acid 3-methoxy-4-nitro-methyl ester (E)

nd nd nd nd 382 nd nd nd nd nd nd nd

14754 Benzene 1-isocyanato-4-methoxy nd nd nd nd 082 nd nd 083 nd 0 nd nd

20618 34-Dimethoxy-6-amino toluene 854 275 0 893 478 608 741 500 970 1214 543 335

21787 1H-Pyrrole-3-carboxylic acid5-formyl-1-(methoxymethyl)- methyl ester

422 nd nd nd 121 nd nd nd 265 nd nd nd

21795 345-Trimethoxybenzylamine nd nd nd 511 nd nd 347 118 118 456 121 nd

24873 25-Dimethoxyphenyl isocyanate 1141 741 0 1678 1318 2430 1436 1064 4874 1882 2419 1691

25952 24-Dimethoxyphenylamine N-ethoxycarbonyl- 048 nd nd 0 nd nd nd 026 057 068 070 nd

26019 9H-Purin-6-amine NN9-trimethyl 215 nd nd 130 nd nd 197 nd nd 184 nd nd

26982 4-(Diethylamino)salicylaldehyde nd nd nd nd nd nd nd nd 287 nd nd nd

27134 234-Trimethoxyphenylacetonitrile nd nd nd nd nd nd nd nd 094 nd nd nd

28503 Bicyclo[221]heptane-2-acetamideN-(13-dihydro-56-dimethoxy-3-oxo-4-isobenzofuranyl)

304 nd nd 469 nd 530 351 nd 1317 526 nd 332

28703 345-Trimethoxyphenyl cyanate nd nd nd nd 223 nd nd nd nd nd nd nd

28709 Tris(246-dimethylamino)pyrimidine nd nd nd nd nd nd nd 175 nd nd 334 nd

28915 Caffeine nd nd nd 048 nd nd nd nd nd nd nd nd

29340 Acetamide N-[2-(34-dimethoxyphenyl)ethyl] nd nd nd nd nd nd nd 089 406 nd nd nd

29346 3-Pyridinecarboxamide 4-(methoxymethyl)-6-methyl-2-propoxy-

nd nd nd nd nd 053 nd nd nd nd nd 149

29537 13-Benzenediamine NNprime-diethyl nd nd nd nd 041 nd nd nd nd nd nd nd

30534 Carbonic acid monoamideN-(24-dimethoxyphenyl)- butyl ester

nd nd 955 nd nd nd nd nd nd nd nd nd

30774 3-(2-Ethoxycarbonyl-ethyl)-4-methyl-1H-pyrrole-2-carboxylic acid ethyl ester



nd nd nd nd nd nd nd nd nd nd 693 nd

30801 Propanamide 3-amino-N-(25-dimethoxyphenyl)-3-(hydroxyimino)-

nd nd nd nd nd nd nd 1499 nd nd nd nd

26134 Phenol 4-(aminomethyl)-2-methoxy nd nd nd nd 033 nd nd nd nd nd nd nd

30741 15-Bis[veratrylaminomethyl]-26-dihydroxynaphthalene

nd 125 nd nd nd nd nd nd nd nd nd nd

30952 3H-Purin-6-amine NN3-trimethyl nd nd nd nd nd nd 061 nd nd 058 nd nd

31834 Benzeneethanamine N-acetyl-2345-tetramethoxy nd nd nd nd nd nd nd 283 nd nd nd nd

32132 Acetamide N-(345-trimethoxyphenethyl) nd nd nd nd nd nd nd nd 091 nd nd 040

35077 Uridine 2prime-deoxy-3-methyl-3prime5prime-di-O-methyl- nd nd nd nd nd nd nd nd nd 019 nd nd

35926 Cytidine N-methyl- 2prime3prime5prime-trimethyl ether 062 nd nd 144 nd nd 152 nd nd 055 nd nd

36019 Thymidine 2prime-deoxy-NOO-trimethyl- 091 nd nd 162 nd nd 224 nd nd 337 nd nd

37792 N-[12-Aminododecyl]aziridine 052 nd nd 058 nd nd 0 nd nd 095 nd nd

41125 Adenosine 2prime-deoxy-NNOO-tetramethyl- nd nd nd 065 nd nd 160 nd nd 211 nd nd

42007 Adenosine 3prime-amino-3prime-deoxy-NN-dimethyl nd nd nd 020 nd nd 046 nd nd 066 nd nd

nd nd nd nd nd nd nd nd nd nd nd nd


Table 2 Compounds identified by GC-MS analysis (Continued)

Carbohydrate compounds

17168 16-Anhydro-β-D-glucose trimethyl ether nd nd nd 135 nd nd nd 077 nd 083 nd nd

18 α-D-Galactopyranoside methyl2346-tetra-O-methyl

174 nd nd 317 nd nd 073 nd nd 424 nd nd

17688 346-Tri-O-methyl-D-glucose nd nd nd nd 054 nd nd 083 nd nd nd nd

181280 α-Methyl 4-methylmannoside nd nd nd nd nd nd nd 055 nd nd nd nd

21355 24567-Pentamethoxyheptanoic acidmethyl ester


20206 β-D-Glucopyranose 16-anhydro nd nd nd nd nd nd nd nd 102 nd nd nd

28814 1234-Tetramethylmannose nd nd nd nd 185 nd nd 175 nd nd 058 nd

28631 α-D-Mannopyranoside methyl2346-tetra-O-methyl

nd nd 333 nd nd nd 0 nd nd nd nd 053

37425 α-D-Glucopyranoside phenyl 2346-tetra-O-methyl

nd nd nd nd 4161 3193 nd 2257 nd nd 562 nd

Fatty acids as methyl ester

26320 Tridecanoic acid 12-methyl- methyl ester 077 nd nd 0 nd nd 106 126 nd 080 162 nd

26323 Methyl tetradecanoate nd nd nd 122 nd nd nd nd nd 0 nd nd

27419 2-Hexadecenoic acid methyl ester (E) nd nd nd 032 nd nd nd nd nd 037 nd nd

27901 Tridecanoic acid 12-methyl- methyl ester nd nd nd 028 nd nd nd nd nd 0 nd nd

30133 Methyl hexadec-9-enoate 106 nd nd 294 nd nd 084 nd nd 047 nd nd

30181 9-Octadecenoic acid (Z)- methyl ester 0 nd nd 0 nd nd nd nd nd 013 nd nd

30625 Hexadecanoic acid methyl ester 422 226 0 728 nd nd 405 nd 104 322 331 196

30327 6-Octadecenoic acid methyl ester nd nd nd nd nd nd nd nd nd nd 082 nd

34053 9-Octadecenoic acid methyl ester (E) 127 nd nd nd nd nd nd 073 nd nd 084 nd

34164 8-Octadecenoic acid methyl ester (E) 067 nd nd nd nd nd nd nd nd nd nd nd

34548 Octadecanoic acid methyl ester 095 111 0 136 057 nd 215 161 028 140 160 067

38161 Eicosanoic acid methyl ester nd nd nd 016 nd nd nd nd nd nd nd nd

Organic acids

26507 Benzoic acid 45-dimethoxy-2-(4-methoxybenzenesulfonylamino)-


40052 Succinic acid di(5-methoxy-3-methylpent-2-yl) ester

nd nd nd nd nd nd nd nd nd 036 nd nd

43743 Cyclopropanecarboxylic acid22-dimethyl-3-(2-methyl-1-propenyl)-2-methyl-4-oxo-3-(2-propenyl)-2-cyclopenten-1-yl ester


Aromatics and phenol derivatives

15817 2-Methoxy-4-vinylphenol nd nd nd nd nd nd nd nd 03 0 nd nd

19337 Phenol 46-di(11-dimethylethyl)-2-methyl nd nd nd 024 nd nd nd nd nd 023 nd nd

24653 Isoelemicin nd nd nd nd nd nd nd nd 049 0 nd nd

37592 Benzene (1-methyldodecyl) 031 nd nd nd nd nd 198 nd nd 080 nd nd

41779 12-Benzenedicarboxylic acid diisooctyl ester nd nd 6971 nd nd nd 149 nd nd 044 nd nd

41784 12-Benzenedicarboxylic acidmono(2-ethylhexyl) ester

155 nd nd nd nd 237 nd nd nd 0 nd 749

21512 Phenol 26-dimethoxy-4-(2-propenyl) nd nd nd nd 025 nd nd nd nd nd nd nd

35007 Phenol 5-methoxy-234-trimethyl nd 0 nd nd 022 nd nd nd nd nd nd nd

37803 Benzene (1-methylnonadecyl) nd 078 nd nd nd nd nd 115 nd nd 136 nd



22015 Benzoic acid 4-hydroxy-35-dimethoxy nd nd nd nd 021 nd nd nd nd nd nd nd

22016 1234-Tetramethoxybenzene nd nd nd nd nd nd nd nd nd nd 027 nd

23399 Benzoic acid 34-dimethoxy- methyl ester nd nd nd nd 072 141 nd nd nd nd nd 165

25152 2-Propenoic acid 3-(4-methoxyphenyl)-methyl ester

nd nd nd 117 103 nd nd 065 nd nd 066 nd

26277 Benzenepropanoic acid 34-dimethoxy-methyl ester


26328 Benzoic acid 345-trimethoxy- methyl ester nd nd nd 0 nd nd nd nd 270 nd nd nd

29738 2-Propenoic acid 3-(34-dimethoxyphenyl)-methyl ester

049 nd nd 0 261 502 nd 075 424 023 124 321

36154 2-Propenoic acid 2-methyldodecyl ester 0 nd nd 0 nd nd nd nd nd 012 nd nd

Terpenoids and steroids

43074 17β-Hydroxy-6α-pentyl-4-oxa-5β-androstan-3-one nd nd nd 074 nd nd 093 nd nd nd nd nd

43412 Dihydroxyisosteviol 388 nd nd 394 nd nd 545 395 nd 369 073 nd

42582 Dihydro-isosteviol methyl ester nd 204 nd nd 0 942 nd nd nd nd nd 1703

43332 Methyl dihydroisosteviol nd nd nd nd 147 nd nd nd nd nd nd nd

43665 Isosteviol methyl ester nd 288 nd nd 481 nd nd 490 1718 nd 468 nd

39849 5-Cholene 324-dihydroxy- nd nd nd nd nd nd nd nd nd 012 nd nd

43430 Patchoulene nd nd nd 036 nd nd nd nd nd nd nd nd

45538 Androstan-17-one 3-ethyl-3-hydroxy- (5α) nd 078 nd nd 289 nd nd 103 050 nd 230 nd

42354 5α6α-Epoxy-17-oxo-6β-pentyl-4-nor-35-secoandrostan-3-oicacid methyl ester

nd nd nd nd nd nd nd nd nd nd nd 031

43091 5β-Pregnan-17α21-diol-320-dione nd nd nd nd nd nd nd nd nd nd nd 2588

43434 Pregnan-20-one (5α17α) nd nd 248 nd nd nd nd nd nd nd nd nd

Alcohols

3974 Ethanol 2-(2-methoxyethoxy) nd nd nd nd nd nd nd nd 118 033 nd nd

6715 1-Hexanol 2-ethyl 165 748 271 039 113 086 nd nd 339 nd 119

12925 Ethanol 2-phenoxy nd nd nd nd nd nd nd nd 063 nd nd nd

42010 9-t-Butyltricyclo[4211(25)]decane-910-diol nd nd nd nd nd nd nd nd nd nd 103 nd

Unidentified

30542 nd nd nd nd nd nd nd nd nd nd nd 314


31825 nd nd nd nd nd nd nd nd nd nd nd nd

32184 nd nd nd nd 079 nd nd nd nd nd nd nd


38362 nd nd nd nd nd nd nd nd nd 042 nd nd


40033 nd nd nd nd nd nd nd nd nd nd 043 nd

40230 nd nd nd 020 nd nd nd nd nd 037 nd nd





43746 nd nd nd nd nd nd nd 043 nd 0 nd nd




44532 nd nd nd 0 nd nd nd nd nd nd nd 045

45275 nd nd nd 074 nd nd 035 nd nd 089 nd nd

46026 nd nd nd nd nd nd 0 nd nd 011 nd nd

46277 nd nd nd nd nd nd 0 nd nd nd nd 172

46606 nd nd nd nd nd 053 0 nd nd 0 nd nd

Compound exudates from maize seedlings at different growth stages (7 14 and 21 days) induced or not (control) by humic acids (HA) Herbaspirillum seropedicae(H seropedicae) and HA and H seropedicae nd not detected


found in the exudates though in only small quantities inthe controlCompounds identified on day 21 are shown in Table 2

Exudates collected on that day contained a smaller di-versity of compounds than earlier collections Hexanolwas found in control exudates and ethanol phenoxyproducts in treatment PGPB likely due to carbohydratehydrolysis reactions Nitrogenous compounds were themain exudate products retained by the RP C18 columncarbonic acid monoamide N-(24-dimethoxyphenyl)-and butyl ester were found only in control exudates onday 21 25-Dimethoxyphenyl isocyanate occurred in ele-vated quantities in exudates of treatments HA PGPB andHA + PGPB a derivative from acetamide and dimethoxyamino toluene was also abundant A nitrogenous com-pound identified as 4-(diethylamino)salicylaldehyde wasfound in treatment PGPB exudates on day 21 but not ondays 7 and 14 Finally products from acetamide were typ-ical of exudates in treatments PGPB and HA+ PGPB theywere found on day 21 at 2934 and 3213 min

DiscussionOur aim in the present work was to determine the ef-fects of single and combined applications of humic acidand the endophytic diazotrophic bacterium H seropedicaeon exudation profiles of maize roots This was a first step inidentifying compounds that may be involved in rhizosphereinteractions and modulation of root colonization andendophytic establishment of bacteriaWe demonstrated enhanced root colonization by H

seropedicae in the presence of humic acids (Figure 1)Previous work showed that the elevated bacterial popu-lation associated with maize roots may be explainable inpart by humic acid sorption at the plant cell wall surfacewhich is associated with increased attachment and endo-phytic colonization by H seropedicae [910] Moreoverthe main infection sites for H seropedicae in grasses arethe points of lateral root emergence which are inducedby humic acid and may contribute to enhanced estab-lishment of the endophytic population [11]Canellas et al [12] and Puglisi et al [13] previously ob-

served changes in exudation profiles of organic acids inmaize seedlings treated with humic acid However iden-tifying causal relationships in changed exudate profiles is

complex and results may not always be directly attribut-able to treatment effects on the quantities and the com-position of root exudates Juo and Storzky [36] observeddecreases in protein and carbohydrate release with in-creasing plant age using an electrophoretic approachThey suggested that the RP C18 column used to retainthe exudates may have limited their quantitative analysisWe noticed on day 21 (across all treatments) that exud-ate yields had declined (Figure 2) relative to earlier col-lections on days 7 and 14

1H NMR spectroscopy (Figures 345) demonstrateddifferences in exudate profiles under the influence of thetreatments and PCA analysis revealed significant changesfrom day 14 onward (Figure 6) Roncatto-Maccari et al[37] observed that 3 days after maize inoculation thepopulation of H seropedicae mainly comprises single cellsattached to the root surface They (loc cit) found thatafter 12 days the cells were connected and forming aggre-gates they detected (by scanning electron microscopy) ahalo of mucilage surrounding the bacterial colony on theroots of inoculated maize Although plant-bacterial signal-ing is considered a rapid phenomenon changes in exudateNMR spectra in our experiment occurred only after exten-sive colonization on the root axis (ie on day 14)One group of candidate biomolecules emerging from

this prospective study comprised a number of com-pounds that have been identified as possible inducingagents of the quorum sensing (QS) system which is amechanism responsible for modulating population dens-ity biofilm formation and the specific genetic machineryfor niche persistence and colonization at the root levelThese compounds include oligopeptides and substitutedgamma-butyrolactones 3-hydroxypalmitic acid methylester 34-dihydroxy-2-heptylquinoline and a furanosylborate diester ([5] and references therein) Interestinglyour analyses of the overall transcriptome profile of Hseropedicae strain Smr1 (as part of a broad examinationof gene expression in planktonic and biofilm lifestyles)have shown that biofilm maturation is coupled with dif-ferentially regulated genes involved in aromatic metabol-ism and multidrug transport efflux activation (data notshown) These potential quorum sensing inducers maybe key molecules in the well-known rhizosphere compe-tence of H seropedicae [1425] Functional studies linking


these compounds to chemotaxis and biofilm induction areurgently requiredAnother interesting future study that may develop

from our prospective explorations might be an evalu-ation of the ability of H seropedicae to use secondarymetabolites as carbon sources that are exclusively or pri-marily exuded from maize roots treated with this bacter-ium The presence of a catabolic machinery for specificcleavage of these compounds (especially compoundswith antimicrobial properties) would represent a nutri-tional advantage for the colonization of the host plantrhizosphereWe identified hexadecanoic acid (palmitic acid) in

exudates of all treatments on days 7 and 14 but by day21 these compounds were detected only in treatmentPGPB Non-quinoline derivatives of other heterocyclicaromatic N compounds belonging to the pyrimidineclass were collected exclusively in treatments PGPB andHA + PGPB (Table 2) The most remarkable differencein root exudates among treatments was in N com-pounds these increased with time in all treatments otherthan the controls in which quantities decreased Some ni-trogenous compounds like benzenamine (C6H5CONH2)24-dimethoxy 34-dimethoxy-6-amino toluene and 25-dimethoxyphenyl isocyanate were found in the exudatesthey are probably derivatives of aromaticphenolic com-pounds that react with TMAH used in methoxylationsince the phenolic compounds which are the main sec-ondary metabolites synthesized by maize react promptlywith NH2 Pyrimidinediones comprise a class of chemicalcompounds characterized by a pyrimidine ring substitutedwith two carbonyl groups 24(1H3H)-pyrimidinedione135-trimethyl is typical of uracil derivatives ie nucleo-bases in the nucleic acid of RNA Pyrrole carboxylic acidderivatives which may originate from the L-tryptophanpathway have been described as antibacterial agents Fi-nally we found one polyamine derivative of spermineSuch derivatives have been implicated in a wide range ofmetabolic processes in plants ranging from cell divisionand organogenesis to protection against stress they actas plant and microbial growth stimulants and chemo-attractants [5]Stearic and palmitic acids were the main fatty acids ex-

uded by maize seedling roots These fatty acids are pre-dominant in most plants they are synthesized by acetyl-CoA carboxylase and fatty acid synthase Quantities ofthese fatty acids exuded by the controls differed betweendays 14 and 21 Stearic and palmitic acids were exudedby maize seedlings treated with H seropedicae withor without HA (Tables 2) Terpenoids derived from thekaurenoic acid pathway including gibberellic acidswere found in all treatments However their diversitywas highest in treatments PGPB and HA + PGPB Asignificant increase in transcript levels of ent-kaurene

oxidase genes that are involved in gibberellin synthesisoccurs in maize seedlings treated with H seropedicae[38] The agreement between gene expression in thegibberellin biosynthesis pathway (Zmko1) and the exud-ation profile in the same biological model supports oursuggestion that the gibberellin synthesis pathway in roottissues is modulated by H seropedicae colonizationRoot colonization by H seropedicae in graminaceous

plants requires movement of the bacteria to the root sur-face followed in sequence by adsorption and attachment tothe cell wall surface [2527] Attached bacteria may persistat the root surface as aggregates or biofilms and they occa-sionally trigger plant responses that induce lateral rootsites which serve as entryways for endophytic establish-ment in the plant host Humic acids are heterogeneous ir-regular and amphiphilic structures with high chargedensities and hydrophobic cores that facilitate adsorptionphenomena and lateral root induction [39] As H seropedi-cae is a flagellated cell there is a potential for induction offlagellar activity by plant-released compounds (chemotacticsubstances) as previously described for other bacteria [40]From the inventory of exuded compounds it may be pos-

sible to select candidate molecules as quorum sensing me-diators of biofilm maturation as substrates for catabolicmechanisms to supply energy or as detoxifiers thereby in-creasing the rhizosphere competence of HerbaspirillumFurthermore changes in rhizosphere hydrophobicity byfatty acid exudates may be an important mechanism forpreserving cell viability We detected relatively largeamounts of gibberellic acid precursor in exudates isolatedfrom maize seedlings treated with H seropedicae this bac-terium induced changes in nitrogenous compounds as well

ConclusionThe number of unidentified compounds was muchhigher in treatments PGPB and HA + PGPB than in thecontrol The limitations of a conventional GC-MS ap-proach such as low mass resolution and the need forprevious volatilization of compounds suggest that theinstrument should be combined with other analyticaltools like liquid chromatography coupled to high massdefinition spectroscopy We found that root exudatesfrom maize seedlings mostly comprised fatty acids andnitrogenated compounds We observed changes in rootexudate profiles in maize seedlings treated with HA andH seropedicae These changes were most pronouncedon days 14 and 21 when there was enhanced fatty acidexudation in treatment HA and elevated nitrogenate andterpene exudation from treatment PGPBThis is the first study to date that has evaluated the

combined application of a plant growth-promoting bac-teria and humic acid on maize root exudation profilesThis study was carried out on controlled conditions in ahydroponic culture Overall the chemical changes we


observed in the rhizosphere may improve the survivalpersistence and biological activity of H seropedicae inthe presence of humic substances and provide a pool ofcandidate molecules for use as additives in inoculanttechnology formulation which will contribute substan-tially to progress in arable agriculture However thefindings of this hydroponic study need to be confirmedunder real soil conditions

Additional files

Additional file 1 Figure S1 GC of root exudate at 7 days



Competing interestsThe authors declare that they have no competing interests

Authors contributionsLSL conducted the laboratory experiments and did the first inventory ofexudates FLO and LPC wrote and discussed the manuscript RRO performedand interpreted the NMR data MRGV reviewed the nomenclature ofcompounds and the biosynthesis route NOA was responsible for themultivariate analysis All authors read and approved the final manuscript

AcknowledgementsThe work was supported by Conselho Nacional de DesenvolvimentoCientiacutefico e Tecnoloacutegico (CNPq) Fundaccedilatildeo de Amparo agrave Pesquisa do Estadodo Rio de Janeiro (FAPERJ) Instituto Nacional de Ciecircncia e Tecnologia (INCT)para a Fixaccedilatildeo Bioloacutegica de Nitrogecircnio Internacional Foundation of Science(IFS) and OCWP

Author details1Nuacutecleo de Desenvolvimento de Insumos Bioloacutegicos para a Agricultura(NUDIBA) Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF)Av Alberto Lamego 2000 Campos dos Goytacazes 28013-602 Rio deJaneiro Brazil 2Laboratoacuterio de Ciecircncias Quiacutemicas (LCQUI) UniversidadeEstadual do Norte Fluminense Darcy Ribeiro (UENF) Av Alberto Lamego2000 Campos dos Goytacazes 28013-602 Rio de Janeiro Brazil

Received 12 June 2014 Accepted 30 October 2014

References1 Vivanco JM Baluška F (2012) Secretions and exudates in biological systems

12 Signaling and communication in plants Springer Berlin p 2902 Jones DL Nguyen C Finlay RD (2009) Carbon flow in the rhizosphere

carbon trading at the soilndashroot interface Plant Soil 3215ndash333 Uren NC (2007) Types amounts and possible functions of compounds

released into the rhizosphere by soil-grown plants In Pinton R Varanini ZNannipieri P (ed) The rhizosphere biochemistry and organic substances atthe soilndashplant interface CRC Press Boca Raton pp 1ndash21

4 Mimmo T Hann S Jaitz L Cescoa S Gessa CE Puschenreiter M (2011) Timeand substrate dependent exudation of carboxylates by Lupinus albus L andBrassica napus L Plant Physiol Biochem 491272ndash1278

5 Faure D Vereecke D Leveau JHJ (2009) Molecular communication in therhizosphere Plant Soil 321279ndash303

6 Azaizeh HA Marschner H Roumlmheld V Wittenmayer L (1995) Effects ofvesicular abruscular micorrhizal fungus and other soil microorganisms ongrowth mineral nutrient acquisition and root exudation of soil-grown maizeplants Mycorrhiza 5321ndash327

7 Neumann G Roumlmheld V (2007) The release of root exudates as affected bythe plant physiological status In Pinton R Varanini Z Nannipieri P (ed) Therhizosphere biochemistry and organic substances at the soilndashplantinterface CRC Press Boca Raton pp 24ndash72

8 Hassan S Mathesius U (2012) The role of flavonoids in rootndashrhizospheresignalling opportunities and challenges for improving plantndashmicrobeinteractions J Exp Bot 633429ndash3444

9 Canellas LP Martiacutenez-Balmori D Meacutedici LO Aguiar NO Campostrini E RosaRC Faccedilanha A Olivares FL (2013) A combination of humic substances andHerbaspirillum seropedicae inoculation enhances the growth of maize(Zea mays L) Plant Soil 366119ndash132

10 Canellas LP Olivares FL (2014) Physiological responses to humic substancesas plant growth promoter Chem Biol Technol Agr 13

11 Canellas LP Olivares FL Okorokova-Faccedilanha AL Faccedilanha AR (2002) Humicacids isolated from earthworm compost enhance root elongation lateralroot emergence and plasma membrane H+-ATPase activity in maize rootsPlant Physiol 1301951ndash1957

12 Canellas LP Teixeira Junior LRL Dobbss LB Silva CA Medici LO ZandonadiDB Faccedilanha AR (2008) Humic acids cross interactions with root and organicacids Ann Appl Biol 153157ndash166

13 Puglisi E Pascazio S Suciu N Cattani I Fait G Spaccini R Crecchio C PiccoloA Trevisan M (2013) Rhizosphere microbial diversity as influenced by humicsubstance amendments and chemical composition of rhizodepositsJ Geochem Expl 12982ndash94

14 Olivares FL Baldani VLD Reis VM Baldani JI Doumlbereiner J (1996) Occurrenceof the endophytic diazotrophs Herbaspirillum spp in roots stems and leavespredominantly of Gramineae Biol Fertil Soils 21197ndash200

15 Roesch LFW Camargo FAO Bento FM Triplett EW (2007) Biodiversity ofdiazotrophic bacteria within the soil root and stem of field-grown maizePlant Soil 30291ndash104

16 Pan B Bai YM Leibovitch S Smith DL (1999) Plant-growth-promotingrhizobacteria and kinetin as ways to promote corn growth and yield in ashort-growing-season area Eur J Agr 11179ndash186

17 Riggs PJ Chelius MK Iniguez AL Kaeppler SM Triplett EW (2001) Enhancedmaize productivity by inoculation with diazotrophic bacteria Austr J PlantPhysiol 28829ndash836

18 Oliveira ALM Urquiaga S Doumlbereiner J Baldani JI (2002) The effect ofinoculating endophytic N2-fixing bacteria on micropropagated sugarcaneplants Plant Soil 242205ndash215

19 Lucy ME Reed E Glick BR (2004) Applications of free-living plant growth-promoting rhizobacteria A van Leeuw J Microb 861ndash25

20 Kennedy IR Choudhury ATMA Kecskeacutes ML (2004) Non-symbiotic bacterialdiazotrophs in crop-farming systems can their potential for plant growthpromotion be better exploited Soil Biol Biochem 361229ndash1244

21 Shaharoona B Arshad M Zahir ZA Khalid A (2006) Performance ofPseudomonas spp containing ACC-deaminase for improving growth andyield of maize (Zea mays L) in the presence of nitrogenous fertilizer SoilBiol Biochem 382971ndash2975

22 Mehnaz S Kowalik T Reynolds B Lazarovits G (2010) Growth promotingeffects of corn (Zea mays) bacterial isolates under greenhouse and fieldconditions Soil Biol Biochem 421848ndash1856

23 Montantildeez A Sicardi M (2013) Effects of inoculation on growth promotionand biological nitrogen fixation in maize (Zea mays L) under greenhouseand field conditions Bas Res J Agric Sci Rev 2102ndash110

24 Pedrosa FO Monteiro RA Wassem R Cruz LO Ayub RA Colauto NBFernandez M Fungaro MHP Grisard E Hungria M Madeira HM HumbertoMF Nodari RO Osaku CA Petzl-Erler ML Terenzi H Vieira LGE Steffens MBRWeiss VA Pereira LFP Almeida MIM Alves LR Marin A Araujo LM BalsanelliE Baura VA Chubatsu LS Faoro H Favetti A Friedermann G et al (2011)Genome of Herbaspirillum seropedicae strain SmR1 a specialized diazotrophicendophyte of tropical grasses PLoS Genet 7(5)e1002064

25 James EK Olivares FL (1998) Infection and colonization of sugarcane and othergraminaceous plants by endophytic diazotrophs Crit Rev Pl Sci 1777ndash119

26 Baldotto LEB Olivares FL (2008) Phylloepiphytic interaction betweenbacteria and different plant species in a tropical agricultural system Can JMicrob 54918ndash931

27 Monteiro RA Balsanelli E Wassem R Anelis M Brusamarello-Santos LCCSchmidt MA Tadra-Sfeir MZ Pankievicz VCS Cruz LM Chubatsu LS PedrosaFO Souza EM (2012) Herbaspirillum-plant interactions microscopicalhistological and molecular aspects Plant Soil 356175ndash196

28 Hungria M (1994) Sinais Moleculares envolvidos na nodulaccedilatildeo dasleguminosas por rizoacutebio Rev Bras Ci Solo 18339ndash364

29 Gough C Galera C Vasse J Webster G Cocking EC Deacutenarieacute J (1997) Specificflavonoids promote intercellular root colonization of Arabidopsis thaliana byAzorhizobium caulinodans ORS571 (1997) Mol PlantndashMicr Interac 10560ndash570


30 Tadra-Sfeir MZ Souza EM Faoro H Műller-Santos M Baura VA Tuleski TRRigo LU Yates MG Wassem R Pedrosa FO Monteiro RA (2011) Naringeninregulates expression of genes involved in cell wall synthesis inHerbaspirillum seropedicae Appl Environ Microbiol 772180ndash2183

31 Marin AM Souza EM Pedrosa FO Souza LM Sassaki GL Baura VA Yates MGWassem R Monteiro RA (2012) Naringenin degradation by the endophyticdiazotroph Herbaspirillum seropedicae SmR1 Microbiol 159167ndash75

32 Balsanelli E Tuleski TR de Baura VA Yates MG Chubatsu LS Pedrosa FO deSouza EM Monteiro RA (2013) Maize root lectins mediate the interactionwith Herbaspirillum seropedicae via N-acetyl glucosamine residues oflipopolysaccharides PLoS One 8(10)e77001 doi101371journalpone0077001

33 Marks BB Nogueira MA Hungria M Megias M (2013) Biotechnologicalpotential of rhizobial metabolites to enhance the performance ofBradyrhizobium spp and Azospirillum brasilense inoculants with soybean andmaize AMB Express 32

34 Baldani JI Pot B Kirchhof G Falsen E Baldani VLD Olivares FL Hoste BKersters K Hartmann A Gillis M Doumlbereiner J (1996) Emended descriptionof Herbaspirillum inclusion of [Pseudomonas] rubrisubalbicans a mild plantpathogen as Herbaspirillum rubrisubalbicans comb Nov and classificationof a group of clinical isolates (EF group 1) as Herbaspirilum species 3 Int JSyst Bact 46802ndash810

35 Doumlbereiner J Baldani VLD Baldani JI (1995) Como isolar e identificarbacteacuterias diazotroacuteficas de plantas natildeo leguminosas Embrapa AgrobiologiaSeropeacutedica

36 Juo P-S Storkzky G (1970) Electrophoretic separation of proteins from rootsand root exudates Can J Bot 48713ndash718

37 Roncato-Maccari LDB Ramos HJO Pedrosa FO Alquini Y Chubatsu LS YatesMG Rigo LU Stefens MBR Souza EM (2003) Endophytic Herbaspirillumseropedicae expresses nif genes in gramineous plants FEMS Microbiol Ecol4539ndash47

38 Amaral FP Bueno JCF Hermes VS Arisi ACM (2014) Gene expressionanalysis of maize seedlings (DKB240 variety) inoculated with plant growthpromoting bacterium Herbaspirillum seropedicae Symbiosis doi101007s13199-014-0270-6

39 Piccolo A (2002) The supramolecular structure of humic substances A novelunderstanding of humus chemistry and implications in soil science Adv Agr7557ndash134

40 Ashy AM Watson MD Shaw CH (1987) A Ti-plasmid determined function isresponsible for chemotaxis of Agrobacterium tumefaciens towards the plantwound acetosyringone FEMS Microbial Lett 41189ndash198

doi101186s40538-014-0023-zCite this article as da Silva Lima et al Root exudate profiling of maizeseedlings inoculated with Herbaspirillum seropedicae and humic acidsChemical and Biological Technologies in Agriculture 2014 123

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Abstract

Background

Results

Conclusions

Background

Methods

Humic substances

Microorganism used

Experimental

Treatment of plants

Exudate collection

NMR sample preparation and data collection

Principal component analysis

Gas chromatography-mass spectrometry

Results

Root tissue colonization by H seropedicae strain HRC54

Mass of root exudates

1H NMR and PCA

Discussion

Conclusion

Additional files

Competing interests

Authors contributions

Acknowledgements

Author details

References

da Silva Lima et al Chemical and Biological Technologies in Agriculture 2014 123 of 18httpwwwchembioagrocomcontent1123

BackgroundRoot exudates function in processes of plant adaptationThey have roles in nutrient cycling in the rhizosphereand in responses to pathogens and symbiotic micro-organisms [1] The quantities of organic compoundsexuded by roots are variable but they are frequently asignificant proportion of the carbon fixed photosynthet-ically by plants [2] Plant type species age and environ-mental factors including biotic and abiotic stressors allaffect exudation profiles [34] The plant rhizospheremodulates microbial community structure and functionprimarily through the release of chemical compounds [5]The dominant organic compounds exuded by roots

reflect central components of cell metabolism includingfree sugars (eg glucose sucrose) amino acids (eg gly-cine glutamate) and organic acids (eg citrate malateand oxalate) [2] Maize exudates comprise sugars (70)phenolics (18) organic acids (7) and amino acids (3)[6] Other compounds like fatty acids sterols enzymesvitamins and plant growth regulators (eg auxins gibber-ellins and cytokinins) are generally released in only verysmall quantities [7] Although only small quantities of sec-ondary metabolites are exuded by roots their plant role insignaling to the microbial community is substantial [8]Recently we determined that the combined inoculation

of maize with the endophytic diazotrophic bacteriumHerbaspirillum seropedicae and humic acids increasedroot colonization and promoted plant growth and grainyields [9] Canellas and Olivares [10] reviewed the use ofhumic substances as plant growth promoters they consid-ered the effects of these substances on plant metabolismand their use as carriers in procedures for the bioinocula-tion of plant growth-promoting bacteria in field cropsystemsCanellas et al [11] reported changes in maize exud-

ation profiles after humic acid application that includedenhanced secretion of inorganic ions (ie H+ ions) andshort-chain organic acids [12] The addition of humicacids of different size fractions may have substantial effectson the quantities of bioavailable carbon deposited bymaize plant roots these additions produce significantchanges in the structure and activity of soil microbial com-munities [13] Hence chemical changes induced by humicmatter augmentation in the rhizosphere may enhancecolonization of maize plants by inoculated endophytic dia-zotrophic bacteria carried in the humic substancesH seropedicae is a plant growth-promoting diazo-

trophic β-proteobacterium found mainly in associationwith grasses and other non-leguminous plants [14]Roesch et al [15] used molecular tools to assess diazo-trophic bacterial diversity within rhizosphere soils rootsand stems of field-grown maize and observed a predomin-ance of α-proteobacteria and β-proteobacteria sequencesin the rhizosphere soil and stem samples Herbaspirillum

was one of the dominant genera in the interiors of maizeplants but was rarer in soil The members of this genushave been tested in the formulation of biofertilizers withvariable success in field crop trials ([16-19] and referencestherein [20-23]) The whole genome sequence of H sero-pedicae has been published [24] The species capacity forN fixation production of auxin and other phytohormonesand the colonization of diverse plant species has been pre-viously demonstrated [25-27]Root colonization is a basic first step for successful in-

oculation An expansion of studies on metabolite ex-change between plants and bacteria and the geneticresponses of plants will fill knowledge gaps in our un-derstanding of the colonization process The role ofroot-exuded flavonoids in legume-Rhizobium interac-tions has been examined in detail These exudationsgenerate a finely tuned cross-molecular dialogue involv-ing the secretion of lipochitooligosaccharides and themodulation of bacterial cell wall surface polysaccharides(extracellular polysaccharide (EPS) and lipopolysaccharide(LPS)) that result in plant root nodulation [28] There isless information available on the role of plant metabolitesin successful interactions with non-nodulating plantgrowth-promoting bacteria Gough et al [29] showed thatflavonoids promote endophytic colonization of Arabidop-sis thaliana (L) Heynh roots by H seropedicae andTadra-Sfeir et al [30] demonstrated that naringenin (a fla-vonoid in the flavonone class) is involved in the geneexpression of cell wall components (EPS LPS) and auxinsIn addition to its role in the genetic modulation of cellwall assembly H seropedicae has an operon associatedwith the degradation of aromatic compounds [31] Anability to degrade flavonoids would likely confer an im-portant competitive advantage in rhizosphereroot coloni-zation of the host plant by providing both a carbon sourceand associated detoxification mechanisms Balsanelli et al[32] showed that surface lipopolysaccharides produced byH seropedicae strain Smr1 are required for attachmentand endophytic colonization of maize plants They [32]found that the H seropedicae attachment process ispartially mediated by a root lectin that specifically bindsN-acetyl glucosamine residuesRecently Marks et al [33] demonstrated the potential

of using bacterial metabolites to enhance the perform-ance of biofertilizers thereby opening the possibility ofchemical manipulation of carriers to benefit bacterialdelivery to field crops Furthermore metabolites exudedby host plants may help in guiding genomic studiesof plant-bacterial interactions In the present workwe examined main changes in exudation profiles ofmaize seedling roots induced under laboratory con-ditions by (i) single applications of humic acids or(ii) H seropedicae or (iii) combinations of the bacteria andhumic acids









































7 14 21 7 14 21 7 14 21 7 14 21

Aliphatic 096 010 010 to 072 084 038 068 084 038 001 to 059 108 077 078

124 038 077 093 085 070 093 085 062 110 085 082 to 089

085 080 to 139 110 123 071 110 123 064 120 123 094

123 144 124 073 to 100 124 148 065 124 149 095

147 to 150 164 103 to 122 164 177 070 20 229 107 to 110

154 to 168 125 203 073 305 235 117 to 128

176 128 to 154 077 318 292 to 317 141 to 154

178 157 080 to 086 209

181 160 to 206 090 to 124 224

184 209 134 246 to 257

186 to 239 210 135 293

242 214 to 222 139 295

243 225 144 299 to 328

247 228 148 to 151

252 230 154

256 234 to 242 157

257 247 160 to 169

269 252 176

275 to 336 257 177

260 to 269 180

272 to 323 184

187 to 242

247

252

256 to 336

Sugars 347 339 338 to 472 340 327 to 361 350 340 to 356 344 346 340

371 373 492 374 456 366 354 366 to 500 365 373 342

393 393 499 to 513 368 372 426 456 353

371 to 465 456 458 356 to 382

467 to 499 465 464 388

466


70 561 701 588 619 to 654 665 585 685 852 (very low) 587 572

563 669 to 680 657 to 690 594 687 596 577

566 699 694 to 761 667 689 to 704 60 582

568 718 771 677 707 670 682

570 to 572 773 693 710 to 949 673 683

575 787 720 964 698 834

583 800 969 to 999 716

588 to 593 805

597 834

598 845

634 849

642 to 860 974

969 to 978















Retentiontime (min)


7 14 21 7 14 21 7 14 21 7 14 21















304 nd nd 469 nd 530 351 nd 1317 526 nd 332


























































Organic acids





























049 nd nd 0 261 502 nd 075 424 023 124 321















Alcohols





Unidentified













































Additional files




























































Abstract

Background

Results

Conclusions

Background

Methods

Humic substances

Microorganism used

Experimental

Treatment of plants

Exudate collection




Results



1H NMR and PCA

Discussion

Conclusion

Additional files

Competing interests


Acknowledgements

Author details

References









































7 14 21 7 14 21 7 14 21 7 14 21

Aliphatic 096 010 010 to 072 084 038 068 084 038 001 to 059 108 077 078

124 038 077 093 085 070 093 085 062 110 085 082 to 089

085 080 to 139 110 123 071 110 123 064 120 123 094

123 144 124 073 to 100 124 148 065 124 149 095

147 to 150 164 103 to 122 164 177 070 20 229 107 to 110

154 to 168 125 203 073 305 235 117 to 128

176 128 to 154 077 318 292 to 317 141 to 154

178 157 080 to 086 209

181 160 to 206 090 to 124 224

184 209 134 246 to 257

186 to 239 210 135 293

242 214 to 222 139 295

243 225 144 299 to 328

247 228 148 to 151

252 230 154

256 234 to 242 157

257 247 160 to 169

269 252 176

275 to 336 257 177

260 to 269 180

272 to 323 184

187 to 242

247

252

256 to 336

Sugars 347 339 338 to 472 340 327 to 361 350 340 to 356 344 346 340

371 373 492 374 456 366 354 366 to 500 365 373 342

393 393 499 to 513 368 372 426 456 353

371 to 465 456 458 356 to 382

467 to 499 465 464 388

466


70 561 701 588 619 to 654 665 585 685 852 (very low) 587 572

563 669 to 680 657 to 690 594 687 596 577

566 699 694 to 761 667 689 to 704 60 582

568 718 771 677 707 670 682

570 to 572 773 693 710 to 949 673 683

575 787 720 964 698 834

583 800 969 to 999 716

588 to 593 805

597 834

598 845

634 849

642 to 860 974

969 to 978















Retentiontime (min)


7 14 21 7 14 21 7 14 21 7 14 21















304 nd nd 469 nd 530 351 nd 1317 526 nd 332


























































Organic acids





























049 nd nd 0 261 502 nd 075 424 023 124 321















Alcohols





Unidentified













































Additional files




























































Abstract

Background

Results

Conclusions

Background

Methods

Humic substances

Microorganism used

Experimental

Treatment of plants

Exudate collection




Results



1H NMR and PCA

Discussion

Conclusion

Additional files

Competing interests


Acknowledgements

Author details

References














Retentiontime (min)


7 14 21 7 14 21 7 14 21 7 14 21















304 nd nd 469 nd 530 351 nd 1317 526 nd 332


























































Organic acids





























049 nd nd 0 261 502 nd 075 424 023 124 321















Alcohols





Unidentified













































Additional files




























































Abstract

Background

Results

Conclusions

Background

Methods

Humic substances

Microorganism used

Experimental

Treatment of plants

Exudate collection




Results



1H NMR and PCA

Discussion

Conclusion

Additional files

Competing interests


Acknowledgements

Author details

References





























Organic acids





























049 nd nd 0 261 502 nd 075 424 023 124 321















Alcohols





Unidentified













































Additional files




























































Abstract

Background

Results

Conclusions

Background

Methods

Humic substances

Microorganism used

Experimental

Treatment of plants

Exudate collection




Results



1H NMR and PCA

Discussion

Conclusion

Additional files

Competing interests


Acknowledgements

Author details

References











049 nd nd 0 261 502 nd 075 424 023 124 321















Alcohols





Unidentified













































Additional files




























































Abstract

Background

Results

Conclusions

Background

Methods

Humic substances

Microorganism used

Experimental

Treatment of plants

Exudate collection




Results



1H NMR and PCA

Discussion

Conclusion

Additional files

Competing interests


Acknowledgements

Author details

References





























Additional files




























































Abstract

Background

Results

Conclusions

Background

Methods

Humic substances

Microorganism used

Experimental

Treatment of plants

Exudate collection




Results



1H NMR and PCA

Discussion

Conclusion

Additional files

Competing interests


Acknowledgements

Author details

References






















Abstract

Background

Results

Conclusions

Background

Methods

Humic substances

Microorganism used

Experimental

Treatment of plants

Exudate collection




Results



1H NMR and PCA

Discussion

Conclusion

Additional files

Competing interests


Acknowledgements

Author details

References

RESEARCH Open Access Root exudate profiling of maize ... · RESEARCH Open Access Root exudate...

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