Nitrogen fixation and growth of Lens culinaris as affected by nickel availability: A pre-requisite for optimization of gromining

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Nitrogen fixation and growth of Lens culinaris as

affected by nickel availability: A pre-requisite for

optimization of gromining

Ramez Saad, A. Kobaissi, Christophe Robin, Guillaume Echevarria, E Benizri

To cite this version:

Ramez Saad, A. Kobaissi, Christophe Robin, Guillaume Echevarria, E Benizri. Nitrogen fixation and

growth of Lens culinaris as affected by nickel availability: A pre-requisite for optimization of

gromin-ing. Environmental and Experimental Botany, Elsevier, 2016, 131 (Environmental and Experimental

Botany), pp.1-9. �10.1016/j.envexpbot.2016.06.010�. �hal-01458433�

Nitrogen

fixation

and

growth

of

Lens

culinaris

as

affected

by

nickel

availability:

A

pre-requisite

for

optimization

of

agromining

R.

Saad

a,b,c

,

A.

Kobaissi

c

,

C.

Robin

d,e

,

G.

Echevarria

a,b

,

E.

Benizri

a,b,

*

a

UniversitédeLorraine,Laboratoire“SolsetEnvironnement”,UMR1120,Vandœuvre-lès-Nancy,F-54518,France

b

INRA,Laboratoire“SolsetEnvironnement”,UMR1120,Vandœuvre-lès-Nancy,F-54518,France

c

UniversitéLibanaise,Laboratoire“BiologieVégétaleetEnvironnement”,FacultédesSciences1,Beyrouth,Lebanon

d_{UniversitédeLorraine,Laboratoire“AgronomieetEnvironnement”Nancy-Colmar,UMR1121,Vandœuvre-lès-Nancy,F-54518,France} e_{INRA,Laboratoire“AgronomieetEnvironnement”Nancy-Colmar,UMR1121,Vandœuvre-lès-Nancy,F-54518,France}

ARTICLE INFO Articlehistory: Received11March2016

Receivedinrevisedform13June2016 Accepted16June2016

Availableonline23June2016 Keywords: Nitrogenfixation Lensculinaris Soilfertility Agromining Nickel ABSTRACT

Lowsoilfertilityinultramaficsoilslimitstheefficiencyofnickelphytoextraction.Developingmore efficientcroppingsystemsforagrominingcanbeachievedbytheassociationofahyperaccumulatorwith alegume byenhancingsoil fertility.However, legumecropscanresultsensitive toultramafic soil conditions, including nickelNiavailability. Weassessed here whetherLensculinaris is adapted to ultramafic environments by growing on soils displaying a wide range of Ni concentrations and consequentlyproducingfunctionalnodules.ThesoilwasenrichedwithdifferentNiconcentrations([Ni]) (0–90mgNikg
1).Natural15_{NabundancewasusedtoassessN}

2fixation(%Ndfa).Bioticparameterswere

investigated (nodule number, Ni, carbon and nitrogen concentrations, plant biomass...). Soil parameterswereinvestigated(total[Ni],DTPA-extractableNi,CandNconcentrations...).Mostof thephysicochemicalandbiologicalparametersweresignificantlyaffectedbytheincreasedsoil[Ni]. Nodulenumbersperplantwaslowerunderhigh[Ni]thancontrol(soilwithoutNi).Noduleslosttheir capacitiestofix N2underhigh Niaddition(90mg Nikg
1). Formany parameters,there wereno

significantdifferencesbetweencontrolandtreatmentsupto60mgofNikg
1addedtothesoil.Lentilis abletogrowonasoilcontainingamountsofNi-DTPAsimilartothosegenerallyfoundinserpentinesoils. Itcouldbeusedinassociationwithahyperaccumulatorplantasanitrogenproviderinordertooptimize Niagromining.

ã2016ElsevierB.V.Allrightsreserved.

1.Introduction

Serpentine soils (i.e. ultramafic soils) are derived from

ultramaficrocksandcontainsignificantamountsofmetallictrace

elements(MTE)includingcopper,chromium,iron,titanium,cobalt

andnickel(Berazaín,2007;Chaneyetal.,2008).Theaveragetotal

content of nickel (Ni) in these soils under temperate to

Mediterranean conditions can range between 1400 and

3500ppm (Kabata-Pendias, 2000; Bani et al., 2014).Ultramafic

soils are also characterized by high Mg concentrations and a

de_ficiencyinN,P,KandCa(Kabata-Pendias,2000;BoydandJaffré,

2009).Asaresultofthepresenceofheavymetals,veryhighMg:Ca

ratio (Bani et al., 2007) and deficiencies, serpentine soils are

entirelyill-suitedtoprofitableagriculturalproduction andeven

forests(Tumietal.,2012).Theirlow-fertilityandlow-productivity

make themunattractive for traditionalagricultureand manyof

theseareasareslowlyabandonedbyfarmers,withruralexodus

and landscapeclosure. However,serpentineoutcrops inEurope

cover over 10,000km2 _{and these ultramafic landscapes have}

potentialtoprovidemultipleecosystemservicesandcontributeto

Europe’s goals towards insuring production of renewable raw

materialsandrenewableenergy(Echevarriaetal.,2015).Theidea

ofphytominingmetalsemergedinthe90s(Chaneyetal.,2007)

andthegoalwastocultivateplantsabletoaccumulatetracemetals

from metal-rich soilsand transport them tothe shoots (>1%),

which could then be harvested as a bio-ore to recover highly

valuablemetals,e.g.nickel(Ni).Nickel-hyperaccumulatingplants

areconsideredidealcandidatesforagromining,whichisa

non-destructiveapproachtotherecoveryofhighvaluemetals(e.g.Ni)

frommetal-enrichedsoilsandores(vanderEntetal.,2013,2015).

InEurope,about40endemictaxaofNi-hyperaccumulatorsfrom

two families (Brassicaceae and Asteraceae) can be found. The

* Corresponding author at: Université de Lorraine, Laboratoire “Sols et Environnement”,UMR1120,Vandœuvre-lès-Nancy,F-54518,France.

E-mailaddress:emile.benizri@univ-lorraine.fr(E.Benizri).

http://dx.doi.org/10.1016/j.envexpbot.2016.06.010

0098-8472/ã2016ElsevierB.V.Allrightsreserved.

ContentslistsavailableatScienceDirect

Environmental

and

Experimental

Botany

processinvolvesrootuptakeoftracemetalsfromsoils,aswellas

theiractivetransporttotheshootswheretheycanbeextracted

afterharvest(Chaneyetal.,2008).Phytominingwasproventobe

efficientin the2000sand becamearealmarketopportunityin

2007(Chaneyetal.,2007;Tangetal.,2012).Fewworksdescribed

theimplementationofagroecosystemswhichcanleadtobetter

soil resource efficiency and to offer fully integrated, new

phytominingagriculture that couldcover thousands of km2 _in

Europe and benefit local communities with sustainable rural

development(Banietal.,2007,2015a,b).Therecentdevelopment

ofthephytominingconceptledtothedefinitionof“agromining”

(vanderEntetal.,2015)asawaytoprovidemultipleecosystem

services,suchasprovisioningservices(e.g.metal,fuel-biomass)

and supporting services (e.g. amelioration of the fertility of

ultramafic soilsover time). In this context, the use of the

Ni-hyperaccumulatorAlyssummuralewasproventobeeconomically

feasibleinEurope(Albania)basedonsuccessfulfieldexperiments

(Banietal.,2007,2015a,b).However,severalbottleneckshavebeen

identifiedandneedtobesolvedbeforeagrominingfullydevelops

inEurope.Inparticular,ultramaficsoilsshowedlow-fertilityand

low-productivity,thusfertilizeramendmentssuchasN,PandK

must be considered to significantly improve crop growth and

phytoextractionyield(Banietal.,2015a;Kiddetal.,2015).Moving

towards agromining would result in a more resource-friendly

agriculture,i.e.agro-ecologicalpracticesshouldbeintroducedasa

substitutetoconventionalfertilizationandpest-controlpractices.

As shown with cereal food crops, forage, silvo-pastoral,

agroforestryandhorticulturalsystems,theinclusionofalegume

ininter-croppingorco-croppingprovidesnitrogeninputsinthe

culturalsysteminadditiontoproducingvaluableyields(Lizarazo

etal.,2015;Peoplesetal.,2015).Indeed,itisknownthatlegumes,

used in either inter-cropping or co-cropping, influence the

N-economy of a system in two ways: they fix part of their

N-requirementfromatmosphericN2and,therefore,depleteavailable

soil-Nlesserthannon-legumes,andalsoprovidepartofthe

fixed-N upon mineralization of decaying plant residues to the

non-leguminous crop(Nyagumbo et al., 2015; Peopleset al., 2015).

Moreover,intraditionnalagronomy,legumecovercropsareused

toreduceorpreventerosion, producebiomassandadd organic

mattertothe soil,attractbeneficialinsects andthey canbreak

weed-,disease-andinsect-cycles.Therefore,inter-croppingor

co-croppinghyperaccumulator plantswithlegumes couldimprove

phytoextractionyieldthroughtheenrichmentofultramaficsoils

withnitrogenfixedfromtheair.ApartoftheNfixedbeingusually

transferred to companion plants (Rodrigues et al., 2015), the

hyperaccumulatorcouldbenefitfromthisbiologicalprocess,while

reducing fertilizer inputs and pesticides to the field. The

combinationofalegumespecieswithahyperaccumulatorplant

couldbe an innovative strategy for agromining; if the legume

tolerates high metal concentration, it could lead to higher

hyperaccumulatorbiomass and reduce theamount of fertilizer

supplied, combining higher economic performance and lower

environmentalimpact(Scaliseetal.,2015;Luceetal.,2015).Thus,

Nbiological_fixationcanactasaprimaryNsource(biofertilizer)

and provide an ecosystem service, thereby complementing or

replacing fertilizer inputs (Fustec et al., 2010; Mokgehle et al.,

2014).

Amongthefactorsthatreduceplantgrowthandcropyieldin

ultramafic soils (i.e. the Serpentine syndrome) we can list

(Whittaker 1954; Proctorand Woodell1975; Brooks1987): Mg

excessoverCa;K,PandCadeficiency;Nitoxicity.Growing

non-adaptedlegumesin ultramaficconditionscouldthenresultina

reduction of beneficial effect of co-cropping because of these

factors.Specificamendmentscanhelptheplantfacethenutrient

unbalances.Butthepresenceofmetals(inourcaseNi)insoilcould

limitthegrowthofnon-hyperaccumulatorspeciesandstimulate

theproduction ofethylene in plants(Abeleset al.,1992; Glick,

2005; Glick, 2014), which affects root growth and nodule

formation in legumes suchas beans, peas,clovers and vetches

(Ligeroetal.,1991;LeeandLarue,1992;HirschandFang,1994).

Hence,ourobjectivewastoinvestigatewhetherLensculinaris,a

legume, could be used in inter- or co-cropping with a

hyper-accumulatorplant,onanaturalultramaficNi-richsoil,inorderto

improve Ni phytoextraction by the hyperaccumulator plant

withoutanychemicalfertilizerinputs.Thefirststepistoconfirm

ifthis legumecangrow and formnodules onasoil containing

variedNiconcentrations.Weassessedwhetherthesenodulesfix

nitrogeninthepresenceofNi.Thisstudyisaprerequisiteforusing

Lens culinarisin inter-or co-croppingwitha hyperaccumulator

plant,onanaturalultramaficNi-richsoil,inordertoimproveNi

phytoextraction.Ourstrategywastogrowlentilsonsoilartificially

enrichedwithNiandthentoestimatenitrogenfixationbynatural

15_{N abundance becauseusing a serpentine soil doesnot allow}

assessing the effect of each of the factors that generate the

serpentinesyndrome.WechoseanagriculturalNi-enrichedsoilin

which wecarefullyadded Niinorder toreachthesame

DTPA-availableconcentrationsofNithanthosewecurrentlyfoundinthe

ultramafic soils (e.g. Albania) where we pretend to crop L.

culinaris+A. murale.Then, we willbe able to estimate if lentil

will be a good candidate for co-cropping or rotation with a

hyperaccumulatorplant.

2.Materialandmethods

2.1.Soilcharacteristicsandexperimentaldesign

We performeda two month microcosm study in controlled

conditions(photoperiod16h,temperature18Cnightand 22C

day, relative humidity 70%, PPFD: 350

_m

molm
2s
1)based on

threetreatments:lentilsowninsoilwithincreasingNi

concen-trations,ryegrasssowninsoilwithincreasingNiconcentrations

andlentilsowninsandalsowithincreasingNiconcentrations.The

soilusedinthisexperimentwasanagriculturaltopsoil,collected

fromthesurfacelayer(10–20cm),originatingfrom“LaBouzule”

Experimental Farm of Université de Lorraine (4844022.2700N,

619019.8500E,MeurtheetMoselle,France)andpreviously

cultivat-ed with nodulated pea, thus naturally containing Rhizobium

leguminosarum.Immediatelyaftercollection,thesoilwassieved

to<5mmtoremovecoarsefragmentsandstoredat4Cforless

than7daysuntilsoilphysicochemicalanalyseswerecarriedout.

Soil physicochemical properties were determined by the Soil

AnalysisLaboratoryofINRA(Arras,France).Itcontained36.7,51.5

and7.2%,clay,siltandsandrespectivelyhadaC/Nratioof9.2,aMg/

Caratioof0.08andanavailablephosphoruscontent(OlsenP)of63

mg



kg
1. Soil pH was 6.4 and was similar (less than 1 unit

difference)withtheultramaficsoilstargetedforfield

implemen-tation of agromining with legumeco-cropping in Albania and

Northwestern Spain (pH range from 6.0 to 7.0). Total Ni and

availableNi(DTPA-extractable)contentswererespectively67.5mg

Nikg
1and1.8mgNikg
1.Weartificiallyenrichedthesoilwith

nickelsulfate(NiSO4,7H2O)withsixdifferentconcentrations(0,

10, 20, 30, 60 and 90mg Nikg
1 dry soil). The soil was then

incubatedfor15dayspriortosowingplantstoavoidmajorchanges

inNiavailabilityduringplantgrowthandhavethemoccurbefore.

OurgoalwastokeepthegradientofavailableNiinarangethat

included30mgNikg
1,whichisthevaluethatwasmeasuredon

twofieldsiteswherelegumeswillbegrowntogetherwithAlyssum

murale.Microcosms(Polyvinylchloridetubes,4.7cmdiameterand

22cmheight)werefilledwith416.2gofsoil(ontheDWbasis).One

lentilorryegrassseedwassownineachmicrocosm.Lentilseeds

wereprovidedbySem-Patners(Lensculinarisvar.Beluga,France)

and ryegrass seeds by Forum (Lolium multiforum L. italicum.

France). A setof microcosms werefilled with sand,previously

enriched with the six nickel sulfate concentrations. Lentils

cultivatedonsandandinoculatedwithaRhizobiumstrain,were

usedascontrolsforquantificationofN2fixation(seebelow).Seeds

onsandysubstrateweredisinfectedbyimmersioninethanol95%

for30s,followedbyimmersionfor10mininahydrogenperoxide

solution(H2O2,3%).Theseedswerethenrinsedatleastfivetimes

withsteriledistilledwater.Beforeenrichedwithnickelsulfate,the

sandwaswashedwithhydrochloricacid(N/3,incubationfor12h

followed by several rinses with distilled water) in order to

mineralize the organic matter residues. Then, each sandy

microcosmwasinoculatedwithaRhizobiumstrain.Weusedthe

self-adheringpeat-basedinoculantforLentil(BeckerUnderwood,

Canada).Thisproductcontainsatleast109viablecellsofRhizobium

leguminosarumbiovarviciaestrain1435pergramandwebrought

1.2kg per 600kg seed, in accordance with manufacturer’s

instructions.

Theexperimenthadarandomizedcompleteblockdesignwith

threereplicates ofthefollowingtreatments:lentil andryegrass

cultivatedontheNi-enrichedagriculturalsoilandlentil

(inoculat-ed with Rhizobium) cultivated on Ni-enriched sand. The

Ni-contaminated agricultural soils were adjusted three times per

weekto70%ofsoilwaterholdingcapacitywithdistilledwater.

Lentilsonsandwereirrigatedwitha nutrientsolutionwithout

nitrogen(pH6.98)(Munns,1977).

2.2.Plantanalyses

After2monthsofculture,plantroots,stemsandleaveswere

collectedandseparated.Freshrootswerecarefullywashedwith

deionizedwaterandthenodulenumberperplantwascountedby

microscopicobservations.Then,rootsandshootspartswere

oven-driedat70Cfor72handdryweightsrecorded.Subsamples(0.5g)

ofdryandgroundplanttissuewereacid-digestedat95Cin2.5ml

ofconcentratedHNO3and5mlofH2O2(30%).Thefinalsolutions

werefiltered(0.45

m

mDigiFILTER)andcompletedupto25mlwith

deionized water. [Ni] in the solution was measured with an

InductivelyCoupledPlasma-AtomicEmissionSpectrometer

(ICP-AES,LibertyII,Varian).

Metalsareknowntocauseseveretoxicitytovariousmetabolic

activities of legumes, including physiological processes like

synthesisofchlorophyllpigments(BibiandHussain,2005;Ahmad

etal.,2008)andproteinsynthesis(Brahimaetal.,2010).Heavy

metalsareknownfortheirinhibitionofnitratereductaseactivity

(NRA) (Fatnassi et al., 2014). Consequently, total chlorophyll,

carotenoid concentrations and nitrate reductase activityin the

leaves were assessed as indicators of the Ni effects on plant

physiology. Chlorophyll and carotenoids were extracted by

incubation of leaves in acetone (80%) for 24h followed by

measurementsofopticaldensityusingaSmartSpecPlus

Spectro-photometer,BIO-RAD(wavelengthsof663,645and440nmfor

chlorophyll a, b and carotenoids, respectively). The respective

concentrationsofchlorophyllaandbwerecalculatedaccordingto

Arnon(1949).ThepotentialNRAwasestimatedinfreshleavesby

incubationin0.1Mphosphatebuffer(pH7.5),30mMKNO3,and5%

isopropanol at 28C for 2h, followed by the addition of

sulfanilamide 3M HCl and 0.02% naphthylethylene diamine

hydrochloride (NED HCl). The mixture was left for 20min for

maximumcolor developmentprior tooptical density

measure-mentwithaspectrophotometer(SmartSpecPlus

Spectrophotom-eter,BIO-RAD)at540nm(Jaworski,1971).ThetotalCandNinthe

plant parts were analyzed by combustion at 900C with an

analyzer C, N (vario MICRO cube, Elementar Analysensysteme

GmbH).

Weusedthe15_{Nnaturalabundance(}

_d

15_{N)technique(Kerley}

andJarvis,1999)toestimatetheproportionofN(%Ndfa)inlentils

derivedfrombiologicalnitrogenfixation(BNF).Weusedryegrass

(LoliumperenneL.)growninpotscontaminatedwithNiinthesame

wayasthelentils,asnon-legume(non-_fixing)controlplantsto

estimate

d

15_{NoftheNuptakefromsoil.Finally,weusedlentils}

grownonsandysubstrateandinoculatedwithaRhizobiumstrain

to estimate the

_d

15_{N of the legume when fixing 100% of N2}

(Amargeretal.,1979;Högberg,1997).The%Ndfawascalculated

usingthefollowingequation:

%Ndfa¼ ð

d

15 Nof Ryegrass

d

15 NLentilÞ ð

d

15_NofRyegrass

d

15_{NLentilonsand} Þ100

Then, the amount of Ndfa per plant was calculated by

multiplying the %Ndfa by the lentil biomass for each plant.

Nitrogenoriginatingfromtheseedswasquantified,bymeasuring

totalNand

d

15_N.

2.3.Soilanalyses

Soilmoisturewasdeterminedbyheatingsubsamplesto105C

untilaconstantweightwasachieved.Subsamples(0.5g)ofdrysoil

were acid-digested in 2ml of concentrated HNO3 and 6ml of

concentratedHClforquantificationofmajorandtraceelements.

Thefinalsolutionswerefiltered(0.45

_m

mDigiFILTER,SCPscience,

Canada)andcompletedupto50mlwithdeionizedwater.[Ni]in

thesolutionwasmeasuredwithanInductivelyCoupled

Plasma-Atomic Emission Spectrometer (ICP-AES, Liberty II, Varian).

AvailableNiinsoilsamplesfromeachmicrocosmwasextracted

witha DTPA–TEA solution(0.005MDiethyleneTriamine

Penta-acetic Acid, DTPA, 0.01M CaCl2, 0.1M triethanolamine,pH 7.3)

accordingtoLindsayandNorvell(1978)andthe[Ni]insolutions

wasmeasuredwithanICP-AES(LibertyII,Varian).TheCECwas

measuredaccordingtointernationalISOstandard23470.SoilpH

wasmeasuredusingapHmeterinasoil–watersuspension(soil:

waterratio=1:5).

Total and organic C and N were quantified with the C, N

Analyzer.SolubleC and Nweredeterminedon5gofdried soil

extractedwith25mlofCaCl2(1mM)byagitationat5rpmfor24h.

Themixturewas thenfilteredthroughaWhatmanfilter(nylon

membrane,0.45

_m

m)andsolubleCandNofCaCl2solutionswere

then analyzed with a TOC analyzer (TOC-VSCN equipment,

Shimadzu, Kyoto, Japan). Nitrate and ammonium ions were

quanti_fiedon15goffreshsoilmixedto75mlof0.016MKH2PO4

and stirred for30minat 17rpm. Thesoil suspensionwas then

a a' abc a' abc a' ab a' bc a' c a' 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 S R S R S R S R S R S R 0 10 20 30 60 90

Shoot

and r

o

ot

bi

om

ass

(g

.p

la

n

t

-1

)

Doses

of Ni (mg.kg

-1

₎

Fig.1.Shoot(greybars)androot(blackbars)biomass(gplant
1)oflentilsin relation to Ni additions (mgkg
1). Meansconfidence interval followed by differentlettersaresignificantlydifferentatp<0.05(Tukey’smultiplerangetest), (n=3).

centrifuged(20minat 5800g)and filtered througha Whatman

filter(nylonmembrane,0.45

m

m)beforeanalysisbyIon

Chroma-tography(Dionex1500iwithanAS4ASCcolumn;Sunnyvale,USA).

2.4.Statisticalanalyses

Variance analysiswascarried outonalldatausingone-way

ANOVA (Tukey test with a confidence interval of 95%). Also,

normality tests and k-sample comparison of variances were

analyzed.Thesestatisticalanalyseswerecarried outonXLSTAT

software(XLSTAT 2015.2.01.16520,http://www.xlstat.com).

Fur-thermore,alltheparametersstudiedweresubmittedtoPCAusing

StatBoxsoftware(Grimmersoft,Paris,France,http://www.statbox.

com).

3.Results

3.1.Plantanalyses

3.1.1.Shootandrootbiomass

Lentils from treatment without Ni (0mg Ni kg
1) had the

greatest shoot dry biomass (1.19g) when compared to other

treatments(Fig.1). Therewas nosignificant differencefor the

shootdrybiomassfrom0(control)upto30mgNikg
1.Treatment

with90mgNikg
1showedthesmallestshootbiomass(0.32g),

significantlylowerthanthatofthecontrol.Therootdryweight

wasnotaffectedbyNiaddition.

3.1.2.Carbonandnitrogencontentinshootsandroots

Total N and C in plant shoots were similarly affected by

increasingsoil[Ni](Fig.2).Thecontroltreatmenthadthehighest

values(0.02and0.54gplant
1oftotalNandtotalC,respectively)

andthe90mgNikg
1treatmentthelowestvalues(0.01and0.14g

plant
1 of total N and total C, respectively). There were no

significant differences among other treatments. There was no

significant effect of soil [Ni] on total N in theroots (data not

shown).Thetreatmentwith60mgNikg
1showed thehighest

amount of total C in roots (0.12g plant
1) with a significant

differencetoallothertreatments(datanotshown).

3.1.3.Niconcentrationsinshootsandroots

The total Ni concentrations in lentil shoots and roots

significantly increased when soil Ni-concentrations increased

(Fig.3).[Ni] was 34mg Ni kg
1DW in shoots for thehighest

soil[Ni]of90mgNikg
1.Unsurprisingly,rootsaccumulatedmuch

moreNithantheshoots,with172mgNikg
1inthesoiltreatment

contaminatedwith60mgNikg
1.TherewasnodetectableNiin

a' abc' abc' ab' bc' c' a bc abc ab bc c 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.0 0.1 0.2 0.3 0.4 0.5 0.6 N C N C N C N C N C N C 0 10 20 30 60 90

S

h

o

o

t to

ta

l

n

itro

g

e

n

(g

.p

la

n

t

-1

)

S

h

o

o

t to

ta

l

c

a

rb

o

n

(g

.p

la

n

t

-1

)

Doses

of Ni (mg.kg

-1

₎

Fig.2.Totalnitrogen(N:greybars)andcarbon(C:blackbars)inshootsoflentils(g plant
1)grownonsoilcontaminatedwithNi.Meansconfidenceintervalfollowed bydifferentlettersaresignificantlydifferentatp<0.05(Tukey’smultiplerange test),(n=3). c c c b ab a c b b b a a 0 50 100 150 200 250 0 5 10 15 20 25 30 35 40 S R S R S R S R S R S R 0 10 20 30 60 90 Tot a l N ickel i n t h e r oot (m g k g -1) Tot a l N ickel i n t h e shoot (m g k g -1) Doses of Ni (mg.kg-1₎

Fig.3.TotalNiconcentrationinshoots(S:greybars)androots(R:blackbars) (mgkg
1DW)oflentilsinrelationtoNiadditions(mgkg
1).Meansconfidence intervalfollowedbydifferentlettersaresignificantlydifferentatp<0.05(Tukey’s multiplerangetest),(n=3).

a a a a a a ab' b' b' _{b' b'} a' 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 0 5 10 15 20 25 30 35 0 10 20 30 60 90

Tot

a

l C

h

lo

ro

phy

ll

i

n

t

h

e shoot

(m

g

.g

-1

)

C

a

ro

te

noi

d

s

in

t

h

e shoot

(m

g

.g

-1

)

Doses

of Ni (mg.kg

-1

₎

Fig.4.Carotenoid(greysquares)andchlorophyll(blacktriangles)concentrationsin leaves oflentils (mgg
1 FW)grown onsoil contaminatedwith Ni.Means confidenceintervalfollowedbydifferentlettersaresignificantlydifferentatp<0.05 (Tukey’smultiplerangetest),(n=3).

a ab ab b b _b 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 10 20 30 60 90

N

it

rat

e R

e

duct

ase

A

c

tiv

ity

(µ

m

o

l

NO

2-g

-1

h

-1

)

Doses

of Ni (mg.kg

-1

₎

Fig.5. Variationofthenitratereductaseactivityinthefreshleaves(mmolNO2

g
1h
1)oflentilsgrownonsoilcontaminatedwithNi.Meansconfidenceinterval withincolumnsfollowedbythesameletterdonotdiffersignificantlyaccordingto Tukey’smultiplerangetest(a=0.05).

thecontrolshoots,althoughrootsdidaccumulatesmallquantities

(15.1mgNikg
1).

3.1.4.Pigmentsandnitratereductaseactivity

A significantly higher chlorophyll concentration (1.19mgg
1

FW)wasobservedinleavesofplantsgrownunderhighNiaddition

(90mg Ni kg
1 in comparison withtheother treatments). The

control was not different from other treatments (Fig. 4). The

carotenoidconcentrationinleaveswasnotaffectedsignificantlyby

soil[Ni],butitfollowedthesametrendasthechlorophyllgetting

somehowhigherwiththeincreaseof[Ni](Fig.4).NRAdecreased

slightlywhen[Ni]inthesoilincreased(Fig.5).Plantsgrowingon

soilscontaminatedwith30–90mgNikg
1hadsignificantlylower

NRA(0.70,0.71and0.68

_m

molNO2
g
1h
1,respectively)thanthe

control(0.87

_m

molNO2
g
1h
1).

3.1.5.Nodulenumber

Lentil plants growing on soil with 90mg Ni kg
1 had

significantly fewer nodules than the control (0mg Ni kg
1)

(Fig.6).Nosigni_ficantdifferencewasobservedforthisparameter

forthelowest[Ni].

3.1.6.N2fixation

The amountof nitrogenfixedfrom theair (Ndfa) decreased

when soil[Ni] increased(Fig. 7).Controlsoilshad significantly

greater Ndfa(12.2mgplant
1) incomparison toallother

treat-ments.Thehighest[Ni]completelycancelledNdfa(0mgplant
1).

Nosigni_ficantdifferenceswereshownamongother[Ni]of10,20,

30and60mgNikg
1.

3.2.Soilcharacteristics

3.2.1.TotalNiandDTPA-Ni

Soil[Ni]suppliedatthebeginningoftheexperimentstrongly

structuredthedistributionoftotalNiandDTPA-extractablenickel

amongthegradientofsoils(Fig.8).Thetotal[Ni]quantifiedinthe

soilatharvestisproportionaltothe[Ni]suppliedinthesoiland

reached a maximum of 138.8mg Nikg
1 withthe addition of

90mgNikg
1.DTPA-extractableNishowedthesametrendasthe

totalNiinthesoil(alsomeasuredafterharvest)andwascomprised

between50 and 55%of Niaddedand thehighest addition(i.e.

90mg Ni kg
1) led to a final DTPA concentration of 49.73mg

Nikg
1 (Fig. 8). The targeted concentrations observed in field

conditions for further implementationof legumes (i.e. 30mg

kg
1)werethuscomprisedintherangetestedhere.

3.2.2.pHandCEC

Regardlessofsoiltreatment,soilpHatharvestwassignificantly

lowerby0.13–0.22pHunitsthanthecontrolsoil(6.63)forthe60

and90mgNikg
1treatments,respectively(Fig.9).Inthesame

way,thetreatmentwith[Ni]at90mgkg
1hadthelowestCEC,the

difference withall othertreatments beingsignificant (datanot

shown).

3.2.3.Soilnitrogenandcarbon

Nosignificantdifferenceswereobservedbetweenanyofthe

treatmentsfor thepercentageoftotal andorganicsoilNandC

(datanotshown).Similarly,nosignificantdifferencewasdetected

forsolubleCinsoils(datanotshown).ThesolubleNconcentration

insoilsatharvestwasthehighest(58.8mgkg
1)withadditionof

90mgkg
1,comparedtocontrolandothertreatments(between

20.4 and 27.5mgkg
1). There was no significant difference

between control and treatments with additions from 10 to

60mgNikg
1. a ab ab ab ab b 0 50 100 150 200 250 300 350 400 0 10 20 30 60 90

N

odul

e num

ber

(

p

er

pl

ant

)

Doses

of Ni (mg.kg

-1

₎

Fig.6. Nodulenumber(perplant)oflentilsinrelationtoNiaddtions(mgkg
1_).

Meansconfidenceintervalfollowedbydifferentlettersaresignificantlydifferent atp<0.05(Tukey’smultiplerangetest),(n=3).

a b b b bc c 0 2 4 6 8 10 12 14 16 0 10 20 30 60 90

S

h

o

o

t N

d

fa

(m

g

.p

la

n

t

-1

)

Doses

of NiSO

4

.7H

2

O (mg.kg

-1

)

Fig.7. Shootnitrogenfixedfromtheair(Ndfa,mgplant
1)oflentilsgrownonsoil contaminatedwithNi.Meansconfidenceintervalfollowedbydifferentlettersare significantlydifferentatp<0.05(Tukey’smultiplerangetest),(n=3).

f e d c b a f' e' d' c' b' a' 0 10 20 30 40 50 60 0 20 40 60 80 100 120 140 160 DT P A -Ni To ta l N i DT P A -Ni To ta l N i DT P A -Ni To ta l N i DT P A -Ni To ta l N i DT P A -Ni To ta l N i DT P A -Ni To ta l N i 0 10 20 30 60 90

D

T

P

A

-N

i in

th

e

s

o

il

(m

g

.k

g

-1

)

Tot

al

N

i i

n

t

h

e

soi

l

(m

g.

kg

-1

)

Doses

of Ni (mg.kg

-1

₎

Fig.8.Totalnickel(total-Ni)(blackbars)andDTPA-extractablenickel(DTPA-Ni) (grey bars) in the soilat the harvest (mgkg
1). Meansconfidence interval followedbydifferentlettersaresignificantlydifferentatp<0.05(Tukey’smultiple rangetest),(n=3).

3.2.4.Ammoniumandnitrate

No ammonium was detected in soils at harvest for all

treatments (datanot shown). In contrast,high nitrate

concen-trationsweremeasured,withoutsignificantdifferenceamongall

treatments,except ahigher,but non-significant,nitrate

concen-trationfortheadditionof90mgkg
1(59.7mgkg
1)comparedto

thecontrol(34.6mgkg
1)(datanotshown).

4.Discussion

Hyperaccumulators areconsideredtobeidealcandidates for

application in agromining, a non-destructive approach for the

recoveryofhighvaluemetals(e.g.Ni)frommetal-enrichedsoils.

Howeveragrominingshouldfocusonspeciesshowingthehighest

levelsofhyperaccumulation.Unfortunately, theannualbiomass

productionofsomehyperaccumulatorspeciesis notsufficiently

high.Therefore,highbiomassyieldandmetalhyperaccumulation

areboth requiredinorder tomake agromining acommercially

viablealternative(Zhangetal.,2014;vanderEntetal.,2015).

As shown in classic agriculturalsystems, the inclusion of a

legumeininter-croppingorco-croppingprovidesnitrogeninputs

in theculturalsystem in addition toproducing valuableyields

(Lizarazoetal.,2015).So,ourobjectivewastoinvestigatewhether

Lens culinaris could be used in inter- or co-cropping with a

hyperaccumulatorplantinordertoimproveNiphytoextractionby

hyperaccumulatorplantswithoutanychemicalfertilizerinput.But

it is necessary to control if this legume can grow and form

functionalnoduleswithintherangeofnaturalNiconcentrations

displayed by ultramafic agricultural soils. Indeed, there are

numerousreportswhereelevatedamountsoftracemetalshave

beenfoundtolimit thegrowth ofboth rhizobiaand theirhost

legumes (Heckman et al., 1987; Broos et al., 2005) and

concomitantlyreducecropyields.But,itisalsoknownthatthe

degreeoftoleranceofplants(includinglegumes)totracemetals

have been divided into three categories: hypotolerance, basal

tolerance,andhypertolerance(Ernstetal.,2008),andthatsome

legumes can be found in different habitats polluted by excess

heavy metals. For example, Anthyllis vulneraria can be found

naturallyin ultramafic soils and is able to fix 80% of its total

nitrogen from atmosphere. The study of serpentinophytes in

North-EstPortugalrevealedthatsomefamilies,suchaslegumes,

showedhightolerancetoserpentinesoils(MenezesdeSequeira

andPintodaSilva,1992).DifferentspeciesofTrifolium(T.Bocconei,

T. cherleri, T. strictum), Anthyllis sampaiana, Lotus tenuis are

commonin theseserpentineenvironments.Thesameoccursin

ultramaficsoilsofAlbaniawherespeciesofTrifolium(T.nigriscens,

T.campestre,T.angustifolium)andofLotus(L.corniculatusandL.

angustissimus)are commonspecies or can even bedominating

speciesanddisplayextremelyhighNiconcentrationsinshoots,e.g.

T.nigrescens(Banietal.,2007,2013).

Inourstudy,lentilwhichwasneverthelessadverselyaffected

bytheincreaseofsoil[Ni],toleratedamoderateavailabilityofNi,

from7.5to30.8mgNi-DTPAkg-1_{(correspondingtoadditionsof}

10–60mgNikg
1)basedonresultsofplantbiomassweight,Cand

Ncontent,physiologicalparametersandnodulenumbers.Trace

metalscontaminatedsoilsseverelyaffectsthesurvivalof

Rhizobi-umstrainsasdescribedbyGusmãoetal.(2005).Rhizobiastrains

isolatedfromLensculinarisrevealedthelowestresistancetotrace

metals, in comparison to other strains isolated from different

nodulatedlegumes(Viciafaba,CicerarietinumandSullacoronaria),

whentestedonculturemediaenrichedwithtracemetals(Cd,Pb,

ZnandCu)(Fatnassietal.,2014).Thisfactexplainsthereduced

atmospheric N2fixation inourstudythatcouldberelatedtoa

decreasedsymbioticactivityofthemicrobesatthehighestsoilNi

availability.Theseoutcomesaffectedvariousparameterssuchas

theNdfa,butalsotheshootbiomass,whichwasalsonegatively

correlatedtoNiadditionstothesoil.Moreover,shootsandrootsof

enriched treatments accumulatedgreater [Ni], which was also

positively correlated to Ni-DTPA concentrations, total and

ex-changeableNiinsoilsatharvest.SuchaccumulationofNiinplant

partsisknowntocauseareductionoftheplantbiomass(Ahmad

etal.,2012)androotsstoredahigheramountofNithanshoots,as

reportedbypreviousstudiesonlegumes(Royetal.,2009).Asa

result of the decreased Ndfa, the 15_{N excess in lentil and the}

proportionofnitrogencontentinlentilcomingfromtheseed(data

notshown)increasedwhen[Ni]rosefrom0to90mgkg
1.Atthe

latterconcentrationtherewasabsolutelynomoreNinputtothe

systemfromatmospheric N2fixation,thusmakingthepotential

associationwithhyperaccumulatorsnonprofitableintermsofN

budget(competitionforNabsorptionbetweenbothspecies).

Physiologically and more precisely, [Ni] showed direct and

indirecttoxiceffectsonthevariousplantprocesses,whichwere

monitoredinthisstudy.Wefollowedtheeffectof[Ni]onpigments

concentrationsinleaves,butweeventuallyfoundthatcarotenoids

werenotsignificantlyaffectedbytheincreaseof[Ni]inthesoil.

However,Nihadacontradictoryeffectonchlorophyllcontentin

leaves. It is knownthat tracemetals have a negative effecton

chlorophyll content in plants (Zengin and Munzuroglu, 2005;

Fatobaetal.,2008).Ourhypothesisregardingtheincreaseofthe

chlorophyllcontentin freshleaves athighestNi concentrations

was that reduction of plant biomass results in a mechanical

concentrationofchlorophyllinleaves(Krupaetal.,1993;Prasad

andFreitas,2003).Nickeladdedtothesoildeleteriouslyaffected

nitratereductaseactivity(NRA)inshoots.ReductionofNRA,dueto

theinhibitionofnitratetranslocationfromrootstoshootsbyNi,

hasalsobeenreportedinmany previousstudies (e.g.Kevre_šan

etal.,1998;SharmaandSubhadra,2010).Consequently,decreased

amountsofleafnitrateimpliedlowerNRAlevels(Kevrešanetal.,

1998), which werealso affectedby thephotosynthetic process

(Bazzigalupietal.,1992).Metabolitesissuedfromthe

photosyn-thetic metabolism are essential tonitrate reduction. Therefore,

photosynthesis, related at the same time to leaf size and

chlorophyll quantities, was limited, thus decreasing both the

uptakeandreductionofnitrate(Bazzigalupietal.,1992).

Nitrogen and carbon in the shoots were also significantly

reduced by the Ni concentrations in the soil. Strong positive

correlationslinkedC andNcontentsinshootstoshootbiomass

and to Ndfa in shoots (Pearson correlations, 0.982 and 0.878,

respectively;datanotshown).Itisknownthathighconcentrations

ofmetalslimitnutrientandwateruptakefromthesoilandthus

reducebiomassproductionanddrymatteraccumulationinshoots

a _{a a} ab b c 6.2 6.3 6.3 6.4 6.4 6.5 6.5 6.6 6.6 6.7 6.7 0 10 20 30 60 90

pH

Doses

of Ni (mg.kg

-1

₎

Fig.9.pHofthesoilattheharvest. Meansconfidence intervalfollowedby differentlettersaresignificantlydifferentatp<0.05(Tukey’smultiplerangetest), (n=3).

and roots. Furthermore, the photosynthesis, carbohydrate and

proteinsynthesis arealsoreduceddue totheinducedoxidative

stressbytheheavymetal(Ahmadetal.,2012).Both,nitrogenfixed

fromtheairandnitrateassimilationfromthesoil,reportedtobe

reducedbytheheavymetaltoxicity,todiminishnitrogencontent

inbothshootsandroots(Ahmadetal.,2012).

Noammoniumconcentrationsweredetectedinoursoil.This

wasduetothemainconversionofammoniain soilintonitrate

throughnitrification(Robertson1997).Ingeneral,ammoniumis

foundinloweramountsinsoilsthannitrate(BurgerandJackson,

2003).

Soil pH and CEC decreased with increasing soil-Ni

concen-trationsandthiswasprobablyduetothecomplexationofNi2+_to

soilsurfacesitesandtotheNi2+_{exchangewithothercationsfrom}

CEC, in both cases releasing surface H+ ions. Furthermore, the

stabilityofNisorbedontoorganicparticlesandthepolymerization

ofcation-boundorganicmatterdiminishedtheCECinthesoilat

highNiconcentrations(Violanteetal.,2010).ThesolubleNpoolin

the soil and nitrate ions were reversely affected by increasing

concentrationsofNidue tothereduced abilityofthelentils to

absorbnutrientsathigherNiconcentrations(Ahmadetal.,2012).

Ononehand,weagreewiththiscommentbutontheotherhand,it

isneverpossibletoassessonesinglefactorinstudyingserpentine

stressbyusingserpentinesoilswhichcombineallstressesinone.

Thisis whywe based thestudy onDTPA-Ni ona soilthat we

anticipatedthinkingthatithadapHvalueclosetoneutralityto

avoidany pHeffect.Indeed, thepHvaluesafter croppingwere

slightlyhigher(whichbythewaymightexplaintheslightdecrease

inDTPA-Ni) butcomprised between6.5and 6.7 (consideredas

neutralpH).OurinterestwasthelevelofDTPA-Nitheplantwas

exposed to having all chemical fertility parameters in optimal

conditions,sothatwecouldhaveanideaofwhattheNistressison

thelegume.Thelevelofstresswasstillsignificant,althoughmany

serpentinesoilsmayexceedthisDTPA-Nivalueintopsoils.Sowe

canquestionthatideathatNiinserpentineisnotthestressing

factor.Weagree,buthereweusedanagriculturalsoilwhichis

normallyfertileandallstudiedparametersconfirmthephytotoxic

effectofNionlentilevenifthesoilpHwasneutral.

Innaturalultramaficsoilsstressestocropplantsarelinkedtoi)

thelowfertilityofthesesoils,ii)thetoxicityofNiand,iii)thehigh

ratio of Mg:Ca (Whittaker, 1954; Proctor and Woodell 1975;

Brooks,1987).Thetwolaststressescanbeovercomebyanadapted

chemicalfertilizationpattern,butitismoredifficulttochangeNi

availability, evenwith limestoneadditions (Kukier and Chaney,

2004). Our experience is thatDTPA-Ni and water-soluble Niin

temperateultramaficsoilsarestillhighenoughtocausetoxicity

symptomstonon-adaptedplantsatpHvaluesbelow8.0.Thisis

becausethemineralphasesthatbearNiaremoderatelysensitive

topHatthisrangeofvalues,i.e.below8.0(Massouraetal.,2006;

Banietal.,2014;Raousetal.,2013).RaisingthepHvalueof1unit

by liming to substantially decrease Ni availability cannot be

achievedwithoutanynegativesideeffectonPandKavailability

andrequiresfurtherfertilizerinput,thusbecominganeconomic

issue. Although liming has been proven to be bene_ficial toNi

phytoextractionyieldbyA.murale(KukierandChaney,2004),it

canbeexpensiveand canincreaseCaconcentrations inshoots,

thusincreasing thecostof hydrometallurgicaltreatment of the

biomassforNirecovery(Zhangetal.,2016).

Wedemonstrated that lentilscan tolerate strong

concentra-tionsofNi-DTPA inthesoilandit waspreviouslyreportedthat

lentilsareabletotoleratehighconcentrationsoftracemetals,such

ascopper,leadandcadmium(Fatnassietal.,2014).Thelentilscan

withstandawiderangeofDTPA-Niconcentrationsinthesoil(upto

30mg Nikg
1)thatare atleast oneorderof magnitude above

acceptablelevelsformostcrops(L’HuillierandEdighoffer,1996).

Thisrangeofconcentrationsis frequentlyobservedin naturally

richultramaficsoilalthoughslightlylowerthanmanyultramafic

soils(Banietal.,2009,2013).Therefore,thenaturallyultramafic

soils, which will be used in further experiments to test the

associationofLensculinariswithahyperaccumulatorplantunder

co-croppingorinrotation,displayDTPA-Niconcentrationswhich

are comprised in our tested range and the plant should be

successfullycroppedinfieldconditionsprovidedthatCa,KandMg

fertilityissuesaremanaged.

5.Conclusion

Our study con_firmed that lentils cantolerate relatively high

concentrationsofavailable nickelinthesoil(i.e.6timeshigher

thanbackgroundvaluesfoundinnonultramaficsoils),althougha

physiologicalstressisinducedwithadditionsofNiabove60mg

kg
1.Lentilscanstillproducenodulesthatfixnitrogenfromtheair

formostoftherangeofNiadditionstestedalthoughthenumberof

nodulesisstronglyaffectedbyNiadditions.Nitrogenfixationis

alsoaffectedinthesamewayandbecomesnullwhenavailable

concentrationsofNireach49.7mgkg
1DTPA-Ni.Thequantityof

availableNifoundintheserpentinesoil,whichwillbeusedinthe

futurework,iswithintherangeoftheconcentrationsinvestigated

inthisexperiment(i.e.30mgkg
1Ni-DTPA).Ourupcomingwork

will be to test Lens culinaris in inter- or co-cropping with a

hyperaccumulator plant to enhance its biomass production by

providingnitrogenandtoallowthishyperaccumulatortoextract

moreNi,however, thepotential associationwith

hyperaccumu-latorswillbecomenonprofitableintermsofNbudget

(competi-tionforNabsorptionbetweenbothspecies)ifweexceedavalueof

30mgkg
1Ni-DTPA.

Acknowledgment

We would like to acknowledge the technical team of

“LaboratoireSolset Environnement”for theirhelpand support

and the technical assistance of the joint research unit Forest

EcologyandEcophysiology(UMR1127INRA,EEF,Champenoux),

andespeciallyChristianHOSSANNfortheisotopicanalysisof15_N.

ThisworkwassupportedbytheFrenchNationalResearchAgency

through thenational“Investissements d’avenir” program,

refer-ence ANR-10-LABX-21—LABEX RESSOURCES21 and through the

ANR-14-CE04-0005project“Agromine”.Finally,wearethankfulto

theAssociationofSpecializationandScientificGuidance(ASSG)for

fundingthePhDscholarshipofRamezSaad.

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