Applied Soil Ecology 61 (2012) 147 157

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Applied Soil Ecology

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Growth toassocia an

C. Jugea, alifa Agriculture anb Dpartement Comto0A6

a r t i c l

Article history:Received 8 DeReceived in reAccepted 2 Ma

Keywords:BradyrhizobiumAzospirillumArbuscular mycorrhizaeRhizosphere inSoybeanBiochemical re

and a as b) and

(Glycine max [L.] Merr.), nodulation, mycorrhization and plant metabolites concentrations, as comparedto Bradyrhizobium alone, after 28 and 56 days of growth under controlled conditions. All microbial com-binations increased soybean root biomass, but not shoot biomass. The highest total biomass of soybean(root + shoot) was observed with the double association Azospirillum and Bradyrhizobium (B + A). Thistreatment reduced the total number of nodules but seems to increase their capacity to x nitrogen, as

1. Introdu

Legumesganisms: noprovide theand arbuscworks whic2008; Fortinexplored thcommon bHause and in root exupounds in et al., 2007bial bacteriestablishme

CorresponE-mail add

0929-1393/$ http://dx.doi.oteractions

sponses

shown by their high starch concentration during establishment. The presence of mycorrhizae (B + M) hada transitory adverse effect on depth of nodulation as compared to Bradyrhizobium alone, which couldindicate competition between these two symbionts during establishment (day 28). The triple associa-tion (B + A + M) reduced shoot growth, as well as the number of small nodules. The higher concentrationof the stress-induced amino acid proline in nodules and leaves in response to B + A + M indicates thatsoybean plants are under stress when in presence of the combination of three symbionts. During estab-lishment, the concentration of coumestrol in soybean roots was lower with microbial combinations thanwith Bradyrhizobium alone, which could indicate a common regulating signal between soybean and bothAzospirillum and mycorrhiza. Our results show that complex interactions and competition between thethree microorganisms induced differential growth and nodulation responses, which can be linked tometabolic changes.

Crown Copyright 2012 Published by Elsevier B.V. All rights reserved.

ction

live in symbiosis with two major rhizosphere microor-dule-forming bacteria capable to x atmospheric N2 to

plant with nitrogen (N) (Prvost and Antoun, 2007),ular mycorrhizal fungi forming external hyphal net-h transport P and water to their host (Smith and Read,

et al., 2008; Khasa et al., 2009). Numerous studies havee common features of these two symbioses, which shareiochemical plant symbiotic signals (Vierheilig, 2004;Schaarschmidt, 2009). For instance, avonoids founddates of host plants are known as key signaling com-a number of plantmicrobe interactions (Steinkellner). Flavonoids act both as chemoattractants for rhizo-a and elicitors of fungal hyphal growth in mycorrhizant (Steinkellner et al., 2007). Recent major discoveries

ding author. Tel.: +1 418 210 5005; fax: +1 418 648 2402.ress: annick.bertrand@agr.gc.ca (A. Bertrand).

of common symbiotic genes activation (Gherbi et al., 2008; Hayashiet al., 2010) have denitively established that the rhizobiumsymbiosis has evolved from the ancestral arbuscular mycorrhizalsymbiosis (Parniske, 2008; Oldroyd et al., 2009). Moreover, therecent description of symbiotic signals that stimulate the formationof arbuscular mycorrhizae, the MYC factors, conrmed that theyare ancestral molecular analogs of the lipo-chitooligosaccharidicNOD factors of the rhizobial symbiosis (Maillet et al., 2011).Numerous authors have studied the interaction between legumes,rhizobial and arbuscular mycorrhizal partners (reviewed by Chalket al., 2006). With soybean cultivated in pots, earlier studies havereported both antagonistic (Bethlenfalvay et al., 1985) and syner-gistic effects of the two symbionts (Bethlenfalvay et al., 1988).

Other rhizospheric microbes, such as plant growth promotingrhizobacteria (PGPR) can also interact with plants and improvetheir growth (Hayat et al., 2010). Among them, diazotrophicrhizobacteria from the genus Azospirillum are able to reduce atmo-spheric N2 into ammonia, and have been shown to promoteplant growth. Associations of rhizobium and Azospirillum specieswith several legumes, including soybean, generally promote seed

see front matter. Crown Copyright 2012 Published by Elsevier B.V. All rights reserved.rg/10.1016/j.apsoil.2012.05.006 and biochemical responses of soybeantions with Bradyrhizobium, Azospirillum

D. Prvosta, A. Bertranda,, M. Bipfubusaa, F.-P. Chd Agri-Food Canada, 2560 Hochelaga Blvd, Qubec City, QC, Canada G1V 2J3

de Phytologie, Facult des Sciences de lagriculture et de lalimentation, Pavillon Paul-

e i n f o

cember 2011vised form 3 April 2012y 2012

a b s t r a c t

Bradyrhizobium (B), Azospirillum (A) spheric microorganisms often appliedthe effects of double (B + A and B + M/ locate /apsoi l

double and triple microbiald arbuscular mycorrhizae

ourb

is, Universit Laval, 2425 rue de lAgriculture, Qubec, QC, Canada G1V

rbuscular mycorrhizal fungi (M) are plant benecial rhizo-iofertilizers. The objectives of the present work were to study

triple (B + A + M) microbial combinations on soybean yield

148 C. Juge et al. / Applied Soil Ecology 61 (2012) 147 157

germination, plant growth, root ramication and nodulation (Khanet al., 2010). In addition, microbial associations of Azospirillum withAM fungi have been shown to improve yields of legumes (Toro et al.,1996; Saini et al., 2004), as well as those of gramineous crops likemaize (Miyley (Negi etof the planto these rhias source o(Saharan anroot surfaceplant a bettmote plant 2003).

Biochemare complelites betwestatus of thbiosis withthe leaves tsucrose. In to the bacteresponse, tproduce frothe nodulesform of ureinia and theet al., 2000)ammonia avtion by high(Serraj et alphotosynthin support assimilationation in nodavailabilitytionship beIt is howevtion by theits net photthat are theother nutrie

In eld Azospirillumto increase 2004), incluHowever, inonistic effecpartners (Rpractical pobium, Azospbio-inoculaLucy et al.,expanding ueral commeunderstandand arbuscuogy.

The objeof double aand mycororrhizationmicroorganassessmentin nodules avonoids

will help to understand plantmicrobe signaling when multiplemicroorganisms are involved.

2. Materials and methods

icrob

o ba: Brc ag

andillumhere

cteriadardnse cvits, a wed (Poake

arbrain r Te

um cixed1 (i.

seedr (25ce ofing treepngenilized

of Gnmen, ConC da

uxedliners oweek

eatm

expour ur i

A. canicumremeatmred oed a dulaial tr

ant btion

he treatma streparo assules

Noduauchi et al., 2008), sorghum (Saini et al., 2004) and bar- al., 1990). In this non-symbiotic association, the rolet is to provide the constituents of their root exudateszospheric bacteria. The bacteria can then utilize thesef energy and in counterpart provide NH3 to the plantd Nehra, 2011). Furthermore, Azospirillum can colonize

and induce root morphological changes that give theer access to nutrients (Okon and Kapulnik, 1986) or pro-growth through diverse mechanisms (Dobbelaere et al.,

ical interactions between plants and microorganismsx and the accumulation and translocation of metabo-en stems and roots or nodules are indicators of thee symbiosis (Bertrand et al., 2011). In soybean in sym-

Bradyrhizobium, photosynthates are transported fromo the nodules through the phloem under the form ofthe nodule, carbon (C) from sucrose is made availableroids to sustain N2 xation (Crawford et al., 2000). In

he bacteroids release to the plant the ammonia theym N2 xation. In soybean, nitrogen is exported from

to the other plant parts through the xylem under thedes, which are degraded in the leaves to release ammo-n assimilated by the plant into amino acids (Crawford. The regulation of this pathway is complex and involvesailability to the plant and feedback inhibition of N2 xa-

concentration of nitrogenous compounds in the leaves., 1999a,b). Many reports conrm that a major portion ofates are translocated to the nodules and utilized thereof N2 xation, nodule growth and maintenance, and

of ammonia (Streeter, 1980). On the other hand, N2 x-ules is rapidly inhibited under conditions that reduce C

(Curioni et al., 1999). The mutualistic biochemical rela-tween soybean and mycorrhiza is not fully understood.er known that, in return to enhanced mineral nutri-

fungi, the host plant channels between 4 and 20% ofosynthetic C to its mycobionts through the arbuscules

sites of exchange for phosphorus, carbon, water, andnts (Lerat et al., 2003).studies, synergistic relationships between Rhizobium,

and/or arbuscular mycorrhizal fungi have been shownbiomass of legume and of non-legume crops (Saini et al.,ding soybean (Bagyaraj et al., 1979; Hayat et al., 2010).

laboratory experiments, synergistic as well as antag-ts have been observed between the three rhizosphericequena et al., 1997; Valdenegro et al., 2001). From aint of view, the three plant benecial microbes Rhizo-irillum and arbuscular mycorrhizal fungi are used asnts all over the world (Brockwell and Bottomley, 1995;

2004; Hamel and Plenchette, 2007). Considering these of these microorganisms in agriculture and the gen-rcial importance of soybean, there is a need to better

the interactive effects of Bradyrhizobium, Azospirillumlar mycorrhizal fungi on soybean growth and physiol-

ctives of the present work were to study the effectsnd triple inoculations with B. japonicum, Azospirillumrhizal fungi on soybean yield, nodulation and myc-. To better characterize soybean response to theseisms, C and N metabolism were investigated by the

of carbohydrates, amino acids and ureides contentsand in leaves. Furthermore, measurements of threein soybean roots, daidzein, genistein and coumestrol,

2.1. M

TwulationQubecultureAzospirrhizospuid bain stancanadeLazaroinoculper seewere s

Thelare stPremieinoculwas m2 mL L

Tencultivaprovinaccordpots (TInc., Taof stersporesenviroPGR1522/17

photoning, semillilitadded

2.2. Tr

Thewith fand foicum +B. japomeasubial tremeasuincludand nomicrob

2.3. Plevalua

At teach tunder were ssured t(2) nod2010).ial inoculation and plant growth conditions

cterial microorganisms were used for soybean inoc-adyrhizobium japonicum, strain 14M2b, isolated fromricultural soil under soybeanmaize rotations (Agri-

Agri-Food Canada (AAFC) culture collection), and canadense, strain DS2, a new species isolated from corn

in London, Ontario (AAFC, Mehnaz et al., 2007). Liq-l inocula were prepared by growing B. japonicum cells

yeast mannitol broth (YMB) (Vincent, 1970), and A.ells in standard malate N-free medium (Mehnaz and2006). Dilutions of individual or combined bacterialre used to inoculate soybean seed at a rate of 106 cellsrvost et al., 2010). About two hundred soybean seedsd in 2 mL of appropriate bacterial inocula overnight.uscular mycorrhizal fungus used was Glomus irregu-DAOM 197198 (AAFC Ottawa, Canada) obtained fromch, Rivire-du-Loup, QC, in the form of sterile liquidontaining 400 spores mL1. The mycorrhizal inoculum

to the plant growth substrate at the concentration ofe. 800 spores L1) before sowing.s of soybean [Glycine max (L.) Merr.] cv. Korus, an early50 Crop Heat Units) recommended for cultivation in the

Qubec, Canada (CRAAQ, 2003), previously inoculatedo the bacterial treatments, were sown in 24-cm deepots TPOT9, 10 cm-diam. 24 cm-depth, Stuewe & Sonst, OR, USA). Pots were previously lled with a mixture

silicevermiculite (1:1 (v/v)) inoculated or not withlomus irregulare. Plants were grown under controlledtal conditions in a growth chamber (Conviron Modeltrolled Environments Limited, Winnipeg, Canada) aty/night temperatures with a 16 h-photoperiod and a

density of 500 mol m2 s1. Two weeks after seed-gs were thinned to three plants per pot. Two hundredf N-free Hoaglands solution at 1/2 P concentration wasly to each pot. Plants were watered daily.

ents and experimental design

eriment was set up as a complete randomized designreplicates, two dates of harvest (28 and 56 days)noculation treatments: B. japonicum (B); B. japon-nadense (B + A); B. japonicum + G. irregulare (B + M);

+ A. canadense + G. irregulare (B + A + M). Biochemicalnts were made on plants from four pots for each micro-ent while growth and nodulation characteristics weren plants from four other pots. The experiment thustotal of 64 pots: 4 replicates 2 (1 pot for growth

tion measurements + 1 pot for biochemical analyses) 4eatments 2 dates of harvest.

iomass, nodulation, Azospirillum and mycorrhizal

wo harvest dates, roots and shoots from four pots forent were separated and roots were gently washed

eam of cold water to remove the substrate. Nodulesated from roots by hand. Three parameters were mea-ess nodulation characteristics: (1) number of nodules;

size; and (3) root depth for nodulation (Prvost et al.,les were separated in two classes according to their size

C. Juge et al. / Applied Soil Ecology 61 (2012) 147 157 149

(small: 5 mm). Nodulation depth was deter-mined by measuring the distance between the crown and thelowest nodules on the root system (Prvost et al., 2010). Shoots,roots and nodules were dried for 48 h at 70 C for the determina-tion of dry wfrom the lopresence of

Azospirilby a techniments of rooof sterile ph101 to 10

cyclohexim30 C.

Specic (hyphae, ara procedurecessive bathFungal struderived frocation of inhyphae (Jug

2.4. Tissue e

Leaves, ntreatment. Ea same pot aApproximaules, and 0.using a FreeCity, MO, USLyophylizedMill (MM 3at room tem

The exttion of carbmethanol:ctissue/extraity. Tubes wand the supwater was ashaken andphase was ness on a ro80 C. Thetwice with 1leaves and n

For avoextracted iing 15 min 3000 g an80 C.

2.5. Carboh

Mono-, dtied by hanalytic sy(WATERS, Mpump, a Moindex detecHPX-87P co0.5 mL min

determined

et al. (2011). Total soluble sugars are the sum of rafnose, sucrose,glucose, fructose and pinitol.

Starch was quantied as glucose equivalent with the p-hydroxybenzoic acid hydrazide method of Blakeney and Mutton

aftelucotarchce to

ino

nty-s ACQcal s, MAtra re). Thn (2.e UV

desty w

the the

are p

eide

HPL 510 ectollan

M (10 mmtoinparianto

avon

dzeinted a

conA). Tmn TY P

we min

B: m1 mi25% in.

strolies os andty TQill (2enist

atisti

data f ha

nd usftwaure (ion oeight (DW). Two samples, one from the upper and onewer part of the root system, were taken to evaluate the

Azospirillum and the colonization by the AM fungus.lum cells on root surface were detected and quantiedque adapted from Pacovsky et al. (1985). Briey, frag-ts without nodules were shaken for 2030 min in 50 mLosphate buffer saline (PBS). Diluted suspensions from4 were plated on N-free medium supplemented withide. Colonies were counted after 3 days of incubation at

coloration of internal mycorrhizal fungal structuresbuscules and spores) was done using Trypan blue by

adapted from Phillips and Hayman (1970), using suc-s allowing the roots to become permeable to the dye.

ctures were measured by a grid-line intersect methodm Tennant (1975) which is adapted for the quanti-traradical fungal structures and of low density runnere et al., 2009).

xtractions for biochemical analyses

odules and roots were sampled from four pots for eachach tissue was pooled from the three plants growing innd thoroughly mixed to obtain a homogeneous sample.tely 1 g fresh weight (FW) of leaves, 0.5 g FW of nod-5 g FW of roots were sampled and lyophylized for 72 hzone Bulk Tray Drier (model 7806020, Labconco, KansasA) and a 12-L Freeze Drier (model 7754040, Labconco).

samples were ground to a ne powder using a Mixer01, Retsch GmbH & Co. KG, Haan, Germany) and storedperature under dry conditions.

raction of leaves and nodules for the quantica-ohydrates, amino acids and ureides was done usinghloroform:water (12:5:3, v/v/v) in a 1:6 (w/v) ratio ofctant, heated 30 min at 65 C to stop enzymatic activ-ere subsequently centrifuged for 10 min at 1500 g

ernatant was collected. For phase separation, 0.25 mL ofdded to a 1-mL subsample of supernatant, tubes were

centrifuged for 3 min at 21,000 g and the aqueouscollected. A 1-mL subsample was evaporated to dry-tary evaporator, solubilized in water and kept frozen at

non-soluble residues left after extraction were washed0 mL of methanol and used for starch determination inodules.noid analysis, approximately 0.1 g of root samples wasn 5 mL of 90% methanol in an ultrasonic bath dur-at 60 C. Samples were then centrifuged for 15 min atd 1 mL of the supernatant was collected and kept at

ydrate analysis

i-, tri-, and tetrasaccharides were separated and quan-igh performance liquid chromatography (HPLC). Thestem was controlled by WATERS Empower softwareilford, MA, USA) and was composed of a Model 515del 717plus autosampler and a Model 2410 refractivetor (WATERS). Sugars were separated on a Bio-Radlumn eluted isocratically at 85 C at a ow rate of1 with H2O. Peak identity and sugar quantity were

by comparison to standards, as described in Bertrand

(1980)amylogMO). Sreferen

2.6. Am

TweWateranalytiMilfordTag UlbamatcolumTunablditionsquantitainingand ofments

2.7. Ur

TheModelUV DetUSA). AODS-Awith 1of allanby comand all

2.8. Fl

DaiseparasystemMA, USC8 coluACQUIditions0.5 mLphase B for 1.2), 50of 6.5 mcoumeidentitcoside(AcquiGleldhstrol, g

2.9. St

All dates otors, aSAS soprocedformatr gelatinization at 100 C and digestion for 90 min withsidase (Sigma A7255, Sigma Chemical Co., St. Louis,

amounts were determined spectrophotometrically by a glucose standard curve.

acids analysis

one amino acids were separated and quantied usingUITY Ultra Performant Liquid Chromatography (UPLC)

ystem controlled by the Empower II software (WATERS,, USA). The amino acids were derivatized using AccQeagent (6-aminoquinolyl-N-hydroxysuccinimidyl car-e amino acids were separated on an AccQ Tag Ultra

1 mm 100 mm) and detected with Waters ACQUITY detector at 260 nm under the chromatographic con-cribed in Cohen (2007). Peak identity and amino acidere determined by comparison to a standard mix con-21 amino acids. The concentrations of total amino acidsamino acids that responded signicantly to the treat-resented in Section 3.

s analysis

C system for the analysis of the ureides consisted in apump, a Model 717plus Autosampler and a Model 2487r set at a absorbance of 200 nm (WATERS, Milford, MA,toin and allantoic acid were separated on a YMC-Pack50 mm 6 mm I.D.) column eluted isocratically at 30 C

acetic acid at a ow rate of 1 mL min1. Peak identity and allantoic acid, and their quantity were determinedson to standards. Total ureides are the sum of allantoinic acids.

oids analysis

, genistein and coumestrol and their conjugates werend quantied using Waters ACQUITY UPLC analyticaltrolled by the Empower II software (WATERS, Milford,he avonoids were separated on an Acquity UPLC BEH(2.1 mm 100 mm, 1.7 m) and detected with WatersDA detector set at 240 nm. The chromatographic con-re as follows: column temperature, 35 C ow rate,1, mobile phase A, 0.2% formic acid in water, mobile

ethanol. The gradient was of 25% B for 0.4 min, 2540%n (curve 6), hold for 1.5 min, 4050% B for 1.0 min (curveB for 0.01 min (curve 1), hold for 2.5 min, for a total runPeak identity and quantity of daidzein, genistein and

were determined by comparison to standards. Peaksf their respective conjugates (glucosides, malonyl glu-

acetyl glucosides) were determined by TQ detection detector, WATERS) using the SIR method described in007). Total avonoids are the sum of daidzein, coume-ein and their respective conjugates.

cal analysis

collected were analyzed by ANOVA, including the tworvest and the four microbial treatments as xed fac-ing the General Linear Model (GLM) procedure of there package. Normality was tested using the UnivariateSAS Institute, Cary, NC, USA, 19992001). A log trans-f data was used, when necessary, to satisfy ANOVA

150 C. Juge et al. / Applied Soil Ecology 61 (2012) 147 157

Fig. 1. Biomasgrowth. Soybezobium and Azwith the threeferences betwdifferent letteeach bar, n = 4

assumptionat P < 0.05. Dof harvest w

3. Results

3.1. Shoot a

Plant estbiomass angrowth (Figbiomass weB associatecantly lowe(Fig. 1A). Rbial combinSuppl. Tabl35% with B was measu

3.2. Nodula

During epresence o(P < 0.01, Suand root de

end of the experiment (day 56). Signicant differences in numberand size of nodules were observed on day 56 in response to thedifferent combinations of microorganisms. Treatments B + A + Mand B + M had signicantly less nodules than Bradyrhizobium alone

). Sizent ther

and

ospir

spirilen B +own)as 3 bac

ycorr

xpely a fe, atts notcorrhwer)nd v

B + Aper M tr(Fig. 2Btreatmthree o(Fig. 2C

3.3. Az

Azobetwenot shlum w3 108

3.4. M

As e28. Onsurface(resultthe myand locules ain theon deeB + A + s of shoot (A) and roots (B) of soybean after 28 days and 56 days ofan was inoculated with either Bradyrhizobium alone (B); Bradyrhi-ospirillum (B + A); Bradyrhizobium and mycorrhizal fungi (B + M) or

microorganisms (B + A + M). The symbol **** indicates signicant dif-een the two dates of harvest at P < 0.001. For each date, bars withrs indicate differences between microbial treatments at P < 0.05. For

SE.

s. Mean separation was performed using Duncans testifferences between microbial treatments for each dateere determined using the least squares means.

nd root biomass

ablishment was slow with only around 1 g DW of shootd 0.5 g DW of root biomass per pot after 28 days of. 1A and B). After 56 days of growth, the highest shootre observed with Bradyrhizobium alone (B) and with

d with Azospirillum (B + A). Shoot biomass was signi-r with the triple association B + A + M than with B or B + Aoot biomass was signicantly increased by all micro-ations as compared to Bradyrhizobium alone (P < 0.05,e 1). Root biomass was increased by 77% with B + A, by+ M and by 48% with B + A + M. The highest root biomassred with B + A (Fig. 1B).

tion of soybean

stablishment (day 28), nodulation was shallower in thef arbuscular mycorrhizal fungus (B + M and B + A + M)ppl. Table 1, Fig. 2A). This effect was however transitorypth for nodulation was similar for all treatments at the

in the top 1deeper part

3.5. Carboh

The conglucose, pibetween dasucrose wathan 75% oshown). At of soluble streatmentstreatment.

The confrom arounSuppl. TablA major sigin nodules bSuppl. Tabl(day 28), sttreatmentsTable 1). Onfor all treat

3.6. Carboh

The conbled betweend of the ewas observconcentrati56 similarlydecreased dconcentratie distribution of nodules was different for the B + A + Mwhich had signicantly more large nodules than the

treatments and less small nodules than B and B + M D).

illum establishment

lum numbers on roots did not differ signicantly A and B + A + M treatments at both sampling dates (data. On day 28, the mean population density of Azospiril- 104 cells g1 root whereas it increased to a mean ofteria g1 root on day 56.

hization of soybean roots

cted, no intraradical colonization was observed on dayw appressoria were visible at several places on the rootesting the early beginning of mycorrhizal colonization

shown). On day 56, in response to the B + M treatment,izal fungus was well established in the two parts (upper

of the root systems, where intraradical hyphae, arbus-esicles were clearly visible. The presence of Azospirillum

+ M treatment signicantly reduced mycorrhizationroots compared with B + M (Fig. 3). In response to theeatment, the percentage of mycorrhization was higher2 cm (25% of mycorrhizal roots) as compared with the

of the root system (5% of mycorrhizal roots).

ydrates in nodules

centration of soluble sugars (sum of rafnose, sucrose,nitol and fructose) signicantly increased in nodulesy 28 and day 56 (P < 0.001, Suppl. Table 1, Fig. 4A1) ands the major soluble sugar in nodules, representing moref soluble sugars (average of 15.3 mg g1 DW, data notthe end of the experiment (day 56), the concentrationugars differed signicantly between B + M and B + A + M

with the highest concentration measured in the latter

centration of pinitol decreased signicantly in nodulesd 5 mg g1 on day 28 to 3 mg g1 on day 56 (P < 0.001,e 1), where it was similar for all treatments (Fig. 4B1).nicant decrease of starch concentration was observedetween day 28 and day 56 for all treatments (P < 0.001,

e 1, Fig. 4C1). It is noticeable that during establishmentarch concentration was much higher in response to the

with Azospirillum, B + A and B + A + M (P < 0.001, Suppl. day 56, starch levels were low (20 mg g1) and similarments.

ydrates in leaves

centration of total soluble sugars in leaves nearly dou-en day 28 and day 56 (P < 0.001, Suppl. Table 1). At thexperiment a signicantly higher level of soluble sugarsed in response to the B + A + M treatment (Fig. 4A2). Theons of pinitol increased in leaves between day 28 and

for all treatments (Fig. 4B2) while starch concentrationuring the same period (Fig. 4C2). On day 56, the loweston of starch in leaves was obtained in response to the

C. Juge et al. / Applied Soil Ecology 61 (2012) 147 157 151

Fig. 2. Root dgrowth. Soybethree microordate, bars with

B + M treatmalone, whic

3.7. Amino

Total amcantly fromincrease waamino acid all treatmenon day 56 (dacids was streatment a

Glutamaules and itof treatmenestablishmeless abundaday 28 andestablishmehigher in retreatments.

The condiffered siglishment (Pglutamate, treatments epth for nodulation (A), total nodules number (B), number of small nodules (C) and nan was inoculated with either Bradyrhizobium alone (B); Bradyrhizobium and Azospirilluganisms (B + A + M). The symbols *** and **** indicate signicant differences between th

different letters indicate differences between microbial treatments at P < 0.05. For each

ent and it differed signicantly from Bradyrhizobiumh had the highest concentration.

acids concentrations in nodules

ino acids concentration in nodules increased signi- day 28 to 56 (P < 0.001, Suppl. Table 1, Fig. 5A1). Thiss mainly due to asparagine, one of the most abundantin nodules, which concentration increased similarly forts, from 10 mol g1 DW on day 28 to 40 mol g1 DWata not shown). On day 56, the concentration of aminoignicantly higher in nodules in response to the B + As compared to the B and the B + M treatments.te was the second most abundant amino acid in nod-s concentration was signicantly lower in presencets containing Azospirillum (B + A and B + A + M) duringnt (P < 0.01, Suppl. Table 1) (Fig. 5B1). Glutamine wasnt and its concentration decreased in nodules between

day 56 for all microbial treatments (Fig. 5C1). Duringnt (day 28), glutamine concentration was signicantlysponse to the B + A co-inoculation than for the other

centrations of gamma-aminobutyric acid (GABA)nicantly between treatments on day 28, during estab-

< 0.01, Suppl. Table 1) and, as it was observed forGABA concentration was lower in response to the twocontaining Azospirillum, B + A and B + A + M (Fig. 6A1).

On the othetreatmentstime, prolinto B + A + M

Fig. 3. Mycorbetween 0 andulated with eimicroorganismthe two microferent letters ibar, n = 4 SE.umber of large nodules (D) in soybean after 28 days and 56 days ofm (B + A); Bradyrhizobium and mycorrhizal fungi (B + M) or with thee two dates of harvest at P < 0.01 and P < 0.001 respectively. For eachbar, n = 4 SE.

r hand, the concentration of proline differed between only at the end of the experiment (day 56). At thate concentration was signicantly higher in response

than to B + M and B treatments (Fig. 6B1).

rhization of soybean roots after 56 days of growth at root depths 12 cm and between 12 and 25 cm from the crown. Soybean was inoc-ther Bradyrhizobium and mycorrhizal fungi (B + M) or with the threes (B + A + M). The symbol * indicates signicant differences between

bial treatments at P < 0.1. For each microbial treatment, bars with dif-ndicate differences between the two root depths at P < 0.05. For each

152 C. Juge et al. / Applied Soil Ecology 61 (2012) 147 157

Fig. 4. Concengrowth. Soybethree microorrespectively. F

3.8. Amino

Concenthigher thandoubled froAt the end in the concobtained wwhich wertrations of total sugars (A), pinitol (B) and starch (C) in nodules (A1, B1, and C1) andan was inoculated with either Bradyrhizobium alone (B); Bradyrhizobium and Azospirilluganisms (B + A + M). The symbols *, **, *** and **** indicate signicant differences betweor each date, bars with different letters indicate differences between microbial treatmen

acids concentrations in leaves

rations of amino acid in leaves were more than 10 times in nodules. Total amino acids level in leaves almostm day 28 to day 56 (P < 0.001, Suppl. Table 1, Fig. 5A2).of the experiment, we observed signicant differencesentrations of total amino acids: the highest level wasith B + M and the lowest with B and B + A + M treatments,e similar. Glutamate concentration differed between

treatmentssured in B plants (Fig.between da

GABA wdoubled betions, withday 56 (Fig.leaves, and in leaves (A2, B2, and C2) of soybean after 28 days and 56 days ofm (B + A); Bradyrhizobium and mycorrhizal fungi (B + M) or with theen the two dates of harvest at P < 0.1, P < 0.05, P < 0.01 and P < 0.001ts at P < 0.05. For each bar, n = 4 SE.

only on day 28 with the highest concentration mea-+ A + M which signicantly differed from B + A-treated

5B2). Glutamine concentration increased signicantlyy 28 and day 56 for all treatments (Fig. 5C2).as the major amino acid in leaves and its concentrationtween day 28 and day 56 for all microbial combina-

a signicant higher level for the B + M treatment on 6A2). Proline concentration also increased with time inat the end of the experiment, proline concentration was

C. Juge et al. / Applied Soil Ecology 61 (2012) 147 157 153

Fig. 5. Concengrowth. Soybethree microorrespectively. F

signicantlyand B + M, a

3.9. Ureides

In nodulallantoin) inSuppl. Table

In leaveestablishmecentration o28 and day tration in letreatment tintermediattrations of total amino acids (A), glutamate (B), and glutamine (C) in nodules (A1, B1, Can was inoculated with either Bradyrhizobium alone (B); Bradyrhizobium and Azospirilluganisms (B + A + M). The symbols *, **, *** and **** indicate signicant differences betweor each date, bars with different letters indicate differences between microbial treatmen

higher in response to B + A + M than in response to Bs was observed in nodules (Fig. 6B2).

concentrations in nodules and in leaves

es, the concentration of total ureides (allantoic acid andcreased signicantly from day 28 to day 56 (P < 0.001,

1, Fig. 7A).s, total ureides concentration was very low duringnt and was similar for all treatments (Fig. 7B). The con-f ureides increased more than 10 times between day56. At the end of the experiment, total ureides concen-aves was signicantly higher in response to the B + Mhan for B and B + A + M while the concentration wase in response to B + A.

3.10. Flavo

The concoumestrolfor all mic(P < 0.001, avonoids treatment opared to B afor all treatm56 (Fig. 8B)

During ewas signiccombinatiocentration and B + A at1) and in leaves (A2, B2, C2) of soybean after 28 days and 56 days ofm (B + A); Bradyrhizobium and mycorrhizal fungi (B + M) or with theen the two dates of harvest at P < 0.1, P < 0.05, P < 0.01 and P < 0.001ts at P < 0.05. For each bar, n = 4 SE.

noids concentrations in roots

centrations of total avonoids (daidzein, genistein, and their major conjugates) decreased signicantlyrobial treatments between the two dates of harvestSuppl. Table 1, Fig. 8A). The concentrations of totalwere signicantly lower in response to the B + A + Mn day 28 and to the B + A treatment on day 56 as com-lone. The concentration of daidzein decreased similarlyents from about 6 to 3 mg g1 DW between day 28 and

.stablishment (day 28), the concentration of coumestrolantly higher in response to the B treatment than for anyn of microorganisms (P < 0.05, Suppl. Table 1). The con-of coumestrol differed signicantly between B + A + M

the end of the experiment (Fig. 8C).

154 C. Juge et al. / Applied Soil Ecology 61 (2012) 147 157

Fig. 6. Concen B1) anSoybean was (B + microorganismeach date, bar

4. Discussi

Bradyrhiplant beneto improveconditions, microorgantions of miccompared tever not in

Fig. 7. Concenalone (B); Bradindicate signibetween micrtrations of gamma-aminobutyric acid (GABA) (A) and proline (B) in nodules (A1, inoculated with either Bradyrhizobium alone (B); Bradyrhizobium and Azospirillum

s (B + A + M). The symbols **, *** and **** indicate signicant differences between the tw

s with different letters indicate differences between microbial treatments at P < 0.05. For

on

zobium, Azospirillum and arbuscular mycorrhizae arecial rhizosperic microorganisms having the potential

biomass production. In this study under controlledwe wanted to assess the synergistic effect of theseisms on soybean growth and physiology. All combina-roorganisms increased the root biomass of soybean aso Bradyrhizobium alone. The shoot biomass was how-creased in response to multiple inoculations and was

even lowerganisms tobetween thtion paramto C and N m

We obsetion of soluexperimentsynthates tof two stor

trations of total ureides in nodules (A) and in leaves (B) of soybean after 28 days and 56yrhizobium and Azospirillum (B + A); Bradyrhizobium and mycorrhizal fungi (B + M) or wcant differences between the two dates of harvest at P < 0.1, P < 0.01 and P < 0.001 respe

obial treatments at P < 0.05. For each bar, n = 4 SE.d in leaves (A2, B2) of soybean after 28 days and 56 days of growth.A); Bradyrhizobium and mycorrhizal fungi (B + M) or with the three

o dates of harvest at P < 0.05, P < 0.01 and P < 0.001 respectively. For

each bar, n = 4 SE.

when soybean was inoculated with the three microor-gether. To better understand the complex interactionse plant and the microorganisms, we assessed nodula-eters as well as the concentration of metabolites related

etabolism in leaves and nodules.rved, for all treatments, an increase in the concentra-ble sugars in nodules during the time course of the

which is consistent with an increased import of photo-o support N2 xation. Furthermore, the large decreaseage carbohydrates, starch and pinitol, during the same

days of growth. Soybean was inoculated with either Bradyrhizobiumith the three microorganisms (B + A + M). The symbols *, *** and ****ctively. For each date, bars with different letters indicate differences

C. Juge et al. / Applied Soil Ecology 61 (2012) 147 157 155

Fig. 8. Concenof coumestrol growth. Soybebium and Azosthe three micrdifferences beFor each datetreatments at

period shownodules to nitrogen xnumber of Furthermorduring thatexported froused as souconcomitanindicators between pl

and metabolism differed however according to the microorganismsinvolved in the association. In the next sections, we will discussseparately the effect on soybean of the different combinations of

rganisms as compared to Bradyrhizobium alone.

fects

highassois in of leg

andghesof Azik, 1microo

4.1. Ef

Theto the result lation (Volpinthe hieffect Kapulntrations of total avonoids (A), of daidzein and its conjugates (B) andand its conjugates (C) in roots of soybean after 28 days and 56 days ofan was inoculated with either Bradyrhizobium alone (B); Bradyrhizo-pirillum (B + A); Bradyrhizobium and mycorrhizal fungi (B + M) or withoorganisms (B + A + M). The symbols ** and **** indicate signicanttween the two dates of harvest at P < 0.05 and P < 0.001 respectively., bars with different letters indicate differences between microbialP < 0.05. For each bar, n = 4 SE.

s that these two sources of energy are used by thesupport their growth and maintenance as well as foration. As a result, shoot and root biomasses and thenodules increased markedly between these two dates.e, the concentration of ureides increased in leaves

period. Ureides are the nal products of N2 xationm soybean nodules to the shoots to be catabolized andrce of N (Crawford et al., 2000). Their accumulations,t with those of amino acids in leaves are additionalof efcient symbioses and metabolite translocationants and microorganisms. Soybean growth, nodulation

of lateral roet al., 2001nodulation in responseBradyrhizobvation of Mnodules in presults couour experimbean inoculthat co-ino(Chebotar erhization, pestablishmelevels of sbean inoculresults coumicrobial a

4.2. Effects

The duamus irregulshoot yieldincreased, wmycorrhiza

In our ean adverseof experimbetween mthe root sitavailable foan establishsecond oneby the commlar mycorrhrespond toaltered signreducing thwas howevdepth of noBradyrhizobmental connodulation

The preson N metabGABA and higher conchypothesis rhizobia wi1992; Sprenof Azospirillum (B + A)

est biomass (shoot + root) was observed in responseciation of Azospirillum and Bradyrhizobium (B + A). Thisaccordance with the generally observed growth stimu-umes by co-inoculations of Azospirillum and Rhizobium

Kapulnik, 1994). The microbial B + A treatment inducedt root yield compared to all other treatments. Thisospirillum is well documented in legumes (Volpin and994) and in soybean, where stimulations of root length,ot growth and of root hairs have been reported (Molla). On the other hand, in the presence of Azospirillum,was reduced as shown by a lower number of nodules

to both B + A and B + A + M treatments compared withiuum alone. This is in contradiction with the obser-olla et al. (2001), of an increase in the number ofresence of Azospirillum. The discrepancy between theseld be due to the Azospirillum strain that was used inent (A. canadense) since a previous study with soy-

ated with Azospirillum and other PGPR species showedculation effects on nodulation were strain dependantt al., 2001). Azospirillum however favored root mycor-articularly in the upper part of the root system. Duringnt, the lowest levels of soluble sugars and the highest

tarch and pinitol were observed in nodules of soy-ated with both Bradyrhizobium and Azospirillum. Theseld be interpreted as a positive effect of this specicssociation on nodule metabolism (Walsh et al., 1987).

of mycorrhizal fungus (B + M)

l combination of Bradyrhizobium japonicum (B) and Glo-are (M) in the present study did not improve soybean

as compared to B alone. However, root yield washich conrms the root enhancing effects of arbuscular

e observed on various host plants (Hodge et al., 2009).xperiment, the presence of a mycorrhizal fungus had

impact on depth of nodulation after the rst monthent which could indicate a competition for the rootsycorrhizae and Bradyrhizobium during establishment;es occupied by one of the symbionts being no furtherr the second one. Recent studies showed that in alfalfa,ed symbiosis can systemically exert an inhibition on the

(Catford et al., 2003). This inhibition could be explainedon origin and physiological features between arbuscu-

izal and rhizobial symbioses since both microorganisms the same plant signal and since the plant could havealing when one of the symbiont is established, thuse establishment of the second one. This inhibitory effecter transitory since at the end of the experiment bothdulation and the number of nodules were similar forium alone and B + M, showing that under our experi-ditions, the mycorhizal fungus did not impair soybeanon the long term.ence of the mycorrhizal fungus seems to have an impactolism in nodules in which we observed high levels ofof its precursor glutamate during establishment. Thisentration of N compounds in the nodules support thethat the arbuscular mycorrhizal fungus supplementsth P, thus sustaining bacterial N2 xation (Bethlenfalvay,t and James, 2007). Furthermore, Sulieman and Schulze

156 C. Juge et al. / Applied Soil Ecology 61 (2012) 147 157

(2010) showed that GABA feeding into the xylem sap increases N2-xing activity in nodules of Medicago truncatula. They concludedthat this non-protein amino acid might be involved in upregu-lating nodule activity as a constituent of the amino acid cycle inlegumes nomediate intaccumulatehigh conceB + M couldof the expepounds (amto B + M as leaves becaules duringN compountion of N2 et al., 2011)with B + M acated plantconsumes mecial effecgrowth adv

When coAzospirillumAzospirillumitive effectsreserves camycorrhiza

4.3. Effects

Inoculatadverse impreduced thereduced thecould indicpresent. Thof a shallowillum was pthe potentiisms duringet al., 2004rhizae did nwas similarmodied thof large nodincrease in xation sincefcient th2010). It diin our expeof sugars sileaves of soshoot biomstressed in concentratiexperimentto accumulabiotic streability to stand Cress, 1

4.4. Plant m

Root avrecognizing

reported by Antunes et al. (2006) we observed that daidzein, genis-tein and coumestrol are key signal compounds associated with theestablishment of symbiosis with soybean, with daidzein being themost abundant. In our study, the level of total avonoids decreased

the t treaizedwithccumoncluitionnce tkly p

bacen dargans pastrolred tlar lum arganlatorn. Co

al., 1k et f couulti

y theoncepirill

wled

than. M.-was sReseaf Cennatunt) toledg

lleag

dix A

plem in th5.00

nces

, P.M.noids

symbihner)j, D.Jculareld. N, A., Pohydrns of alvayis. II. At Physalvay

and eand phalvayprospdules (Sulieman, 2011). GABA has also been shown toeractions between plants and microorganisms and to

in response to biotic stress (Roberts, 2007) and thentration of GABA in soybean nodules in response to

be linked to its role as a signaling molecule. At the endriment, we observed higher concentrations of N com-ino acids and ureides) in soybean leaves in responsecompared to the other treatments. This indicates thatme a stronger sink of N compounds provided by the nod-

the time course of the experiment. The accumulation ofds in the leaves could however cause feedback inhibi-xation in the nodules (King and Purcell, 2005; Bertrand. The lower level of starch in leaves of plants inoculatedt day 56 could signify that the fungus uses the translo-

reserve carbohydrates to build its own biomass andore C for its maintenance respiration than for its ben-

t on plant, thus depriving in part the plant from directantage of the double symbiosis (Kaschuk et al., 2009).mparing the association of Bradyrhizobium with either

or mycorrhizal fungus, our results indicate that induced less competition for nodulation and more pos-

on plant metabolism, through the accumulation ofrbohydrates in nodules (Walsh et al., 1987), than thel fungus.

of triple microbial combination (B + A + M)

ion with the three microorganisms together had anact on shoot biomass of soybean. The triple association

root depth for nodulation during establishment and number of nodules at the end of the experiment whichate a competition when the three microorganisms areis hypothesis is further supported by the observationer mycorrhization of the root system when Azospir-resent. It is well known that mycorrhizal roots haveal to affect and be affected by free-living microorgan-

the initiation and formation of mycorrhizae (Johansson). It is however noticeable that the presence of mycor-ot inhibit Azospirillum population for which cell density

for both B + A and B + A + M. The triple combination alsoe distribution of nodule size by increasing the numberules while decreasing the number of small nodules. Thisthe number of large nodules could be benecial for N2e large soybean nodules have been reported to be more

an small ones (King and Purcell, 2001; Prvost et al.,d not however translate into higher soybean biomassriment. This could be explained by an inefcient usence soluble sugars were more abundant in nodules andybean inoculated with the triple association. The lowerass could also be linked to the fact that the plants werepresence of the three symbionts. We observed a highon of proline in both nodules and leaves at the end of the

in response to this treatment. Proline has been shownate in plants in response to a wide range of biotic andsses and to act as a compatible solute while having theabilize cell structure and to detoxify free-radicals (Hare997).

icrobe signaling

onoids are the molecular signals involved in the general process between plant and microorganisms. As was

duringganismrecognstudy slight aThey cin addresistato quicto bothbetwemicrooplant acoumecompaa simijaponicmicrooa regusoybea(Xie et(Kosslalevel othree mlated blower cof Azos

Ackno

Weand Drstudy Crops port osur la ernmeacknowand co

Appen

Supfound,2012.0

Refere

Antunesavotite (kirc

Bagyaravesithe

Bertrandcarbstrai

BethlenfbiosPlan

Bethlenfhostuse

Bethlenfand ime course of the experiment in response to all microor-tments. This could be an indication that the plant had

all microorganisms as benecial partners. In an early mycorrhizal soybean, Morandi et al. (1984) observedulations of daidzein and coumestrol in soybean roots.ded that the slight accumulation of these avonoids,

to be benecial to micorrhizal fungi, increases planto subsequent pathogenic attacks by enabling the plantsroduce larger quantities of avonoids which are toxicteria and nematodes. Since root avonoids decreasedy 28 and day 56, we could conclude that none of theisms used in our experiment were recognized by thethogenic. During establishment, the concentration of

was lower for all multiple microbial combinations aso Bradyrhizobium alone. Antunes et al. (2006) observedower level of coumestrol in soybean when both B.nd Glomus clarum were present as compared to eachism alone and concluded that this avonoid could bey molecule for the establishment of both symbioses inumestrol has been shown to stimulate hyphal growth995) as well as to induce nod genes in Bradyrhizobium

al., 1987). In our experiment we observed a similar lowermestrol in presence of Azospirillum suggesting that theple associations, B + A, B + M and B + A + M could be regu-

same avonoid signals in soybean. To our knowledge, antration of coumestrol in soybean roots in the presenceum has never been reported.

gements

k Carole Gauvin-Trudel, Sandra Delaney, Jose BourassaO. Duceppe for their excellent technical assistance. Thisupported by the project no 830 of the AAFC Soils andrch & Development Centre, Qubec. The nancial sup-tre SVE (strategic cluster of the Fonds de recherchere et les technologies (FQRNT) from the Qubec Gov-

A. Bertrand, D. Prvost and F.-P. Chalifour is gratefullyed. We dedicate this article in memory of our friendue Dr. Horst Vierheilig (19602011).

. Supplementary data

entary data associated with this article can bee online version, at http://dx.doi.org/10.1016/j.apsoil.

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Growth and biochemical responses of soybean to double and triple microbial associations with Bradyrhizobium, Azospirillum ...1 Introduction2 Materials and methods2.1 Microbial inoculation and plant growth conditions2.2 Treatments and experimental design2.3 Plant biomass, nodulation, Azospirillum and mycorrhizal evaluation2.4 Tissue extractions for biochemical analyses2.5 Carbohydrate analysis2.6 Amino acids analysis2.7 Ureides analysis2.8 Flavonoids analysis2.9 Statistical analysis

3 Results3.1 Shoot and root biomass3.2 Nodulation of soybean3.3 Azospirillum establishment3.4 Mycorrhization of soybean roots3.5 Carbohydrates in nodules3.6 Carbohydrates in leaves3.7 Amino acids concentrations in nodules3.8 Amino acids concentrations in leaves3.9 Ureides concentrations in nodules and in leaves3.10 Flavonoids concentrations in roots

4 Discussion4.1 Effects of Azospirillum (B+A)4.2 Effects of mycorrhizal fungus (B+M)4.3 Effects of triple microbial combination (B+A+M)4.4 Plant microbe signaling

AcknowledgementsAppendix A Supplementary dataReferences