Soil bacteria showing a potential of chlorpyrifos degradation and plant growth enhancement

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h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Soil

bacteria

showing

a

potential

of

chlorpyrifos

degradation

and

plant

growth

enhancement

Shamsa

Akbar

∗

,

Sikander

Sultan

DepartmentofMicrobiologyandMolecularGenetics,UniversityofthePunjab,Lahore,Pakistan

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r

t

i

c

l

e

i

n

f

o

Articlehistory: Received29April2015 Accepted21September2015 Availableonline20April2016 AssociateEditor:FernandoDini Andreote Keywords: Chlorpyrifos Bioremediation Phosphatesolubilization Achromobacter IAA

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b

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Background:Since1960s,theorganophosphatepesticidechlorpyrifoshasbeenwidelyused forthepurposeofpestcontrol.However,givenitspersistenceandtoxicitytowardslife forms,theeliminationofchlorpyrifosfromcontaminatedsiteshasbecomeanurgentissue. Forthisprocessbioremediationisthemethodofchoice.

Results:Twobacterialstrains,JCp4andFCp1,exhibitingchlorpyrifos-degradationpotential wereisolatedfrompesticidecontaminatedagriculturalfields.Theseisolateswereableto degrade84.4%and78.6%oftheinitialconcentrationofchlorpyrifos(100mgL−1)withina periodofonly10days.Basedon16SrRNAsequenceanalysis,thesestrainswereidentified asAchromobacterxylosoxidans(JCp4)andOchrobactrumsp.(FCp1).Thesestrainsexhibitedthe abilitytodegradechlorpyrifosinsterilizedaswellasnon-sterilizedsoils,andwereable todegrade93–100%oftheinputconcentration(200mgkg−1)within42days.Therateof degradationininoculatedsoilsrangedfrom4.40to4.76mg−1kg−1d−1withrateconstants varyingbetween0.047and0.069d−1.Thesestrainsalsodisplayedsubstantialplantgrowth promotingtraitssuchasphosphatesolubilization,indoleaceticacidproductionand ammo-niaproductionbothinabsenceaswellasinthepresenceofchlorpyrifos.However,presence ofchlorpyrifos(100and200mgL−1)wasfoundtohaveanegativeeffectonindoleaceticacid productionandphosphatesolubilizationwithpercentagereductionvaluesrangingbetween 2.65–10.6%and4.5–17.6%,respectively.Plantgrowthexperimentdemonstratedthat chlor-pyrifoshasanegativeeffectonplantgrowthandcausesadecreaseinparameterssuchas percentagegermination,plantheightandbiomass.Inoculationofsoilwith chlorpyrifos-degradingstrainswasfoundtoenhanceplantgrowthsignificantlyintermsofplantlength andweight.Moreover,itwasnotedthatthesestrainsdegradedchlorpyrifosatanincreased rate(5.69mg−1kg−1d−1)inplantedsoil.

Conclusion: Theresultsofthisstudy clearlydemonstratethatthechlorpyrifos-degrading strainshavethepotentialtodevelopintopromisingcandidatesforraisingtheproductivity ofcropsinpesticidecontaminatedsoils.

∗ _{Corresponding}_author.

E-mail:shamsa.akbar@yahoo.com(S.Akbar). http://dx.doi.org/10.1016/j.bjm.2016.04.009

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Introduction

Theextensiveuseofpesticidesthroughﬁeldapplication,crop spraying,handling,rinsingofcontainers,accidentalspills,etc. hasapotentialtoseverelycontaminatesoil.1_Most_of_the pesti-cidesthatareincommonusagetodayareknowntoadversely affect functional diversityofthe soilmicrobiota leading to lossofsoilfertilityandplantgrowth,whichinturnputthe sustainabilityofagriculturalsoilatseriousrisk.2 _To_add_to thecomplexityofthesituation,pesticideresiduesandtheir metabolitesofteninﬁltratethroughthesoilsurfaceintothe groundwaterandcausewidespreadcontaminationofaquatic ecosystems.3

Chlorpyrifos (CP) is a broad spectrum organophosphate insecticidethatisclassiﬁedasmoderatelytoxic.Since1960s, CPhas beenextensively used intheagricultural sectorfor controllinginsectinfestationsofcropssuchascotton, cere-als,vegetables and fruits.4 _Although_CP _is_considered _only moderately toxic, it is known to possess neurotoxic and immunotoxicpropertiesandhasbeenshowntobeharmful tobothanimalsandhumans.5_CP_has_also_been_reported_to causeareductioninthebacterial,fungalandactinomycete populationofthesoil6_and_is_known_to_inhibit_nitrogen min-eralizationinsoil.7DetectionofCPcontaminationinsurface waterbodiesandassociatedsedimentshasheightenedpublic concernonthetopic8,9 _and_warranted_urgent_attention_and treatmentoftheproblem.

Bioremediationisamethodthatexploitsthepotentialof microbialdegradationforprovidingacost-effectiveand reli-ableapproachtopesticideabatement.Severalsoilandaquatic environmentshavebeen successfullyreclaimedfrom pesti-cidecontaminationbyusingmicrobescapableofdegrading thepollutants.10_Hydrolysis,_either_chemically_or_as_a_result_of microbialactivity,degradesCPbyconvertingitto diethylthio-phosphoricacid(DETP)and3,5,6-trichloro-2-pyridinol(TCP).11 Pesticidedegradingbacteriafoundinsoilareknowntohave multifariousabilitiessuchasphytohormoneproduction, min-eral solubilization, N2-ﬁxation, etc., which are extremely crucialforpromotionofplantgrowth.Presenceofthe above-mentionedtraitsunderlinesandemphasizestheagronomic andenvironmentalsigniﬁcanceofsuchmicrobes.The poten-tialofmicrobestosimultaneouslydetoxifypollutantswhile enhancingplantgrowthhasbeenstudiedpreviouslyfor pes-ticidescarbofuranandthiamethoxam.12,13

Theobjective ofthisstudy wastoisolateand character-izeCP-degradingbacteriaandtodeterminethedegradation potentialofthesestrainsinbothsterileaswellasnon-sterile soil. Moreover, plant growth promoting potential of these bacteriawasalsoassessed; andthe strainswere testedfor theirabilitytobioremediatesoilandenhanceplantgrowthin contaminatedsoil.

Materials

and

methods

Chemicalsandmedia

Technicalgradechlorpyrifos(98%)wasobtainedfromtheAli Akbargroup,Pakistan.AnalyticalgradeCPand 3,5,6-trichloro-2-pyridinol (TCP) were purchased from Sigma–Aldrich (St

Louis,MO,USA).HPLCgradeorganicsolventswereprocured from Merck(Pakistan).Microbialisolations wereconducted in a mineral salt medium (MSM) (pH 6.8–7.0) containing (gL−1) K2HPO4, 1.5; KH2PO4, 0.5; NaCl, 0.5; (NH4)2SO4, 0.5; MgSO4·7H2O,0.2,and10mLof100×traceelementsolution. The100×traceelementsolutionwascomposedof(mgL−1) Na2EDTA·2H2O,500;FeCl2·4H2O,143;ZnCl2,4.7;MnCl2·4H2O, 3.0;H3BO3,30;CoCl2·6H2O,20;CuCl2·2H2O,1.0;NiCl2·6H2O,2.0; Na2MoO4·2H2O,3.0;andCaCl2·2H2O,100.

Enrichment,selectionandidentiﬁcationofchlorpyrifos degradingstrains

SoilsamplesfromagriculturalfieldsofJhangandFaisalabad, Punjab,Pakistan,wereusedforthepurposeofenrichmentof chlorpyrifosdegradingbacteria.Fivegramsofsoilwasadded toErlenmeyerflasks(250mL)containing50mLMSM supple-mentedwith50mgL−1CPandtheculturewasincubatedat 150rpm,30◦C.Theenrichmentprocesswasconductedover aperiodoffour weeks;followinganestablishedprotocol,14 at regular intervals of one week each of the culture was transferred into fresh growth medium. Subsequent to the lastenrichmentprocess,serialdilutionsoftheculturewere spread on MSM plates containing CP (50mgL−1) and the coloniesthusisolatedwerefurtherpurifiedbythestreakplate method.

Fordeterminingthedegradationpotential,cultureswere grown in triplicatein MSM containing 100mgL−1 CP fora periodof10daysandtheresidualCPconcentrationwas deter-mined byhighperformanceliquid chromatography(HPLC). CP quantiﬁcation was analyzed on a SYKAM HPLC (Ger-many)usinganS1122HPLCPump,S3210UVdetector,S1122 delivery system and Phenomenex C18 reversed-phase col-umn(150mm).Detectoroutputwasprocessedbytheclarity chromatography data system. Samples were eluted using methanol:H2O:acetic acid (80:20:0.5, v/v) at a ﬂow rate of 1.0mLmin−1. Detection was performed at230nm and the retentiontimesofCPandTCPweredeterminedtobe18.4and 16.1min,respectively.TwoisolateswiththehighestCP degra-dationpotential,designatedasJCp4andFCp1,wereselected forfurtherstudy.

Bacterialstrainswereidentifiedbyamplificationofthe16S rRNAgeneusinguniversalprimers27fand1492R. Amplifica-tion wascarriedout ina50␮Lreactionmixturecontaining 20pmol of primer (F and R) each, 25␮L PCR Master Mix (Thermoscientific)and20ngoftemplateDNA.ThePCR ther-mocyclingparameterswereasfollows:initialdenaturationat 94◦Cfor5min;followed by25cycles of94◦C (1min),50◦C (1min);72◦C(1min);andfinalextensionat72◦Cfor10min. TheamplifiedPCRproductswere sequencedand sequence homologieswereidentifiedusingnBLAST.GenBankaccession numberswereassignedfor16SrRNAgenesequencesofboth theisolates(KJ009240andKJ009242).

Determinationofauxiliarycharacteristics

Indoleaceticacid(IAA)production

LB-broth(50mL),supplementedwith50,100and150␮gmL−1 tryptophan, was inoculated with 50␮L of cell suspension (OD600=0.5)intriplicateandincubatedat35◦C,120rpmfor

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72h.At48and72hintervals,theculturewascentrifugedand 2mLofthesupernatantwasmixedwith4mLofSalkowski’s reagent. Intensity of colour was taken as OD535 and the amount of IAA produced was quantiﬁed using a standard curve.TheeffectofCPontheIAAproductioncapabilityofthe microbeswasevaluatedbysupplementingtheLB-brothwith 100or200mgL−1ofCPinpresenceof100␮gmL−1tryptophan.

Phosphatesolubilization

Theabilityofthebacteriatosolubilizephosphatewas deter-minedbyplating the bacteria on Pikovskaya agar medium asper the methoddescribed previously.15 _The_presence_of aclear zone around bacterialcolonies followingone week of incubation at 35◦C indicated phosphate solubilization. Phosphatesolubilizationwasanalyzedbycomputingthe Sol-ubilization Index (SI) which is the ratio of total diameter (colony+halo) tocolony diameter.The effect ofCP on the phosphate solubilization ability of the bacteria was deter-minedbysupplementingPikovskayaagarplateswith100and 200mgL−1chlorpyrifos.

Ammoniaproduction

McCartneybottlescontaining10mLpeptonewaterwere inoc-ulated withbacterial strains in triplicateand incubated at 35◦Cfor96h.TheNH3productionwasdetectedby observ-ingtheformationofyellowcolourinthebottleuponaddition ofNessler’sreagentandintensityofthecolourproducedwas noted.16 _The_effect _of _CP _on _ammonia _production _by bac-terialstrainsunderstudy wasevaluatedbythe additionof 100–200mgL−1CPtopeptonewater.

Biodegradationstudies

Inoculumpreparation

Seedcultureforeachisolatewasgrowninanutrient broth containing50mgL−1CP.Followinggrowth,thecultureswere centrifuged at4600×g for5min,washed and then diluted with MilliQ H2O. Colony forming units (cfumL−1) ofthese suspensionsweredeterminedbythedilutionplatecounting method.Forpesticidebiodegradationstudies,acell concen-trationcorrespondingto1.6×107_cfu_mL−1_was_used_so_as_to maintainuniformityincellnumbers.

Chlorpyrifosdegradationinliquidmedium

Erlenmeyerflasks(250mL) containingmineralsalt medium (100mL)supplementedwith100mgL−1ofCPwereinoculated withbacterialcellsuspensionintriplicates.Theflaskswere incubatedat30◦Cwithshakingat150rpmandan uninocu-latedflaskwasusedascontrol.Forgrowthstudyanalysisan aliquotof1mLculturewaswithdrawnatregularintervalsof 2dayseachfor10continuousdaysandgrowthwasevaluated asOD600.CPresidueextractionandestimationbyHPLCwas doneasdescribedpreviously.14

Biodegradationofchlorpyrifosinsoil

AnalysisofCPdegradationbyselectedstrainsisolatedfrom soilwas conductedin sterilized (S)and non-sterilized (nS) soilsamples. Therequiredsoilsamples(100g)were spiked withchlorpyrifostoaﬁnalconcentrationof200mgkg−1 by theadditionofanacetone-basedCPsolution.Thesolutionwas

initiallyaddedtoasmallportion(10g)ofsoil,which,following thesolventevaporationwasthenmixedwiththeremaining soilquantity.Thesoilsampleswereinoculatedandincubated at30◦C.Thetestwas performedintriplicateand uninocu-latedSandnSsoilswereusedascontrols.Sampleremoval, extractionandpesticideresidueestimationwerecarriedout asdescribedearlier.14

Plant–microbeinteractionandchlorpyrifosdegradation

Pot experimentswithVignaunguiculata(L.)Walp.were con-ductedinordertoexaminetheeffectsofbacterialinoculation onplantgrowthandpesticidedegradation.SeedsofV. ungui-culatawere surfacesterilizedbytreatmentwith0.1%HgCl2 solutionfor5minfollowedbywashingwithsterilized glass-distilled water.Soilsamples(1.5kg) werespikedwithCPto a concentrationof 200mgkg−1 using the same protocol as describedabove.Samplesweretheninoculatedwithmicrobial suspension togive ﬁnal concentration of1.6×107_cells_g−1_. Thetestwasperformedintriplicateanduninoculatedspiked and non-spiked soil samples were used as control. Steril-ized seedsofV.unguiculatawere sowninthe samplesoils, and then the soil was moistened with water. Care was taken to ensure that the pots were kept at ambient light andtemperature.Theseedemergenceprocesswasobserved daily and plants were allowed to grow for 6 weeks. The followingparametersofplantgrowthwererecorded:(a) per-centageofgermination,(b)shootlength(cm),(c)rootlength (cm),(d) leaflength(cm),(e)shootfresh weight(g), (f)root fresh weight (g), (g) shoot dry weight (g), and (h) root dry weight(g).

Dataanalysis

TheCPdegradationrateconstant(k)wasdeterminedusing the kinetic model Ct=C0×e−kt as described in a previous study.16 _Statistical _analysis _was _performed _using _the _IBM SPSS 20 program. Signiﬁcance (p<0.05) of differences was analyzedbyt-testorone-wayANOVAandassessedby post-hoccomparisonofmeansusingtheDuncan’smultiplerange test.

Results

Isolationandcharacterizationofchlorpyrifosdegrading strains

Fromtheenrichmentculture,severalpromising morpholog-ically differentcolonieswereisolated andpurified. Purified isolatesweregrowninamineralsaltmediumsupplemented withCP(100mgL−1).EstimationofCPdegradationpotential byHPLCdemonstratedthattheisolateswereabletodegrade 25–84.4%ofsupplementedCPwithinatimeframeof10days. MaximumCPdegradationpotentialwasexhibitedbyisolates JCp4andFCp1thatdegraded84.4%and76.8%oftheappliedCP, respectively.Basedonbiochemicalcharacterizationand16S rRNAgeneanalysis,theseisolateswere identifiedas Achro-mobactersp.andOchrobactrumsp.The16SrDNAsequencesof strainsJCp4andFCp1exhibitedclosest(99%)homologywith

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0 20 40 60 80 100 0 0.1 0.2 0.3 0.4 0.5 0 2 4 6 8 10 C oncen trati on (m gL –1 ) OD 600n m Incubation time (d) Control 0 20 40 60 80 100 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2 4 6 8 10 C once nt rati on (m gL –1 ) OD 600n m Incubation time (d) A. xylosoxidans JCp4 0 20 40 60 80 100 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 2 4 6 8 10 C oncen trati on (m gL –1 ) OD 600n m Incubation time (d) Ochrobactrum sp. FCp1

Fig.1–Degradationofchlorpyrifos(•-•)duringgrowth(-)ofbacterialstrainsinmineralsaltmediumatconcentrationof 100mgL−1.ErrorbarsrepresentSD,andvaluesaremeansofthreereplicates.

Achromobacterxylosoxidans(AB547225,FJ639331)and Ochrobac-trumsp.(KF479631,KF843719),respectively.

BacterialgrowthandbiodegradationofCPinliquid medium

ThebacterialstrainswereabletoutilizeCPasthesolesource ofCandexhibitedeffectivegrowthuptoaperiod6daysof incubationwithoutundergoinganinitiallagphase.Increase ingrowthwasveryslowbetween6thand8thday,and there-afterthedeclinephasewasinitiated(Fig.1).CPdegradation bybacterialstrainswasexhibitedasadecreaseinCP concen-trationthatwasproportionaltoincreaseinbacterialgrowth andatimedependentlossofCPwasobservedinbacterial cul-tures(Fig.1).A.xylosoxidansJCp4degraded84.4%ofCPatthe rateof7.9mgL−1d−1whileOchrobactrumsp.FCp1degraded 76.8%ofCPattherateof7.3mgL−1d−1.Inthecontrolﬂasks, CPdegradation,asabyproductofabioticlosses,was insigniﬁ-cant(6%)attheendof10days.HPLCanalysisalsorevealedthat A.xylosoxidansJCp4andOchrobactrumsp.FCp1wereableto mineralizeCPcompletelysuchthattherewasonlyatransient accumulationoftheCPmetabolicproductTCP (3,5,6-trichloro-2-pyridinol).

Determinationofauxiliarycharacteristics

The plant growth-promoting activities of bacterial strains, bothintheabsence aswell asinthepresenceofCP,were

determined (Table 1). The bacterial strains under study exhibited asubstantial productionofIAA following24hof incubation. Aconcentrationdependent increasein produc-tionofIAAwasobserved:JCp4andFCp1produced15.6and 15.8␮gmL−1 ofIAAat50␮gmL−1, 18.8 and 19.3␮gmL−1 at 100␮gmL−1,and23.3and25.0␮gmL−1ofIAAat150␮gmL−1 oftryptophan, respectively.When theeffectofpresenceor absence of CP on IAA production was compared, it was revealedthattherewasareductioninIAAproductionby2.65% (JCp4) and 4.3% (FCp1)when CP was present ata concen-trationof100mgL−1.AfurtherincreaseinCPconcentration (200mgL−1)suppressedIAAproductionandresultedinadrop inquantityofIAAproducedby10.6%(JCp4)and9.2%(FCp1) (Table1).Boththestrainsalsoshowedphosphate-solubilizing activitybyproducingaclearhaloaroundtheirgrowthinboth conditions. However, phosphate solubilization activity was reducedathigherconcentrationofCP.Furthermore,bacterial strainswerealsofoundpositiveforammoniaproductionin bothsituations(Table1).

Soilbioremediation

TheabilityofselectedbacterialstrainstodegradeCPinsoil was studied using sterilized (SS) as well as non-sterilized soil(nSS).TheCPdegradationdynamicsrevealedsigniﬁcant differences (p<0.05) between different treatments (Fig. 2, Table2).Moreover,CPdegradation insoilsuggestedatime dependent disappearancethat followedﬁrst order kinetics.

Table1–Plantgrowthpromotingactivitiesofcypermethrindegradingstrainsinpresenceandabsenceofchlorpyrifos.

Bacterialstrains Trp(␮gmL−1) IAA(␮gmL−1) CPconc.

(mgL−1)

IAA(␮gmL−1) Phosphatesolubilization NH3production

Zonesize(mm) SI Achromobacterxylosoxidans JCp4 50 15.6±0.4a _Control _18.8_±_0.04e ₁₇_±_0.57e _4.2e ₊₊₊ 100 18.8±0.26c ₁₀₀ _18.3_±_0.14d ₁₆_±_0.5d _4.0d ₊₊₊ 150 23.3±0.3e ₂₀₀ _16.8_±_0.24b ₁₁_±_1.0c _3.6b ₊₊ Ochrobactrumsp.FCp1 50 15.8±0.34b _Control _18.3_±_0.015d ₁₁_±_1.0c _3.8c ₊₊₊ 100 19.3±0.41d ₁₀₀ _17.5_±_0.02c _10.5_±_0.5b _3.6b ₊₊₊ 150 25.0±0.5f ₂₀₀ _16.6_±_0.014a ₁₀_±_0.0a _3.3a ₊₊

Trp=tryptophan;IAA=indoleaceticacid;SI=solubilizationindex.

Thevaluesindicatethemean±SDofthreereplicates.

+++=largeamountofammonia;++=moderateamountofammonia.

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0 40 80 120 160 200 42 35 28 21 14 7 0 CP c once nt ration (m gk g –1) Time (d) SS nSS PS 0 40 80 120 160 200 42 35 28 21 14 7 0 CP c once nt ration (m gk g –1) Time (d) SS+JCp4 nSS+JCp4 PS+JCp4 0 40 80 120 160 200 0 7 14 21 28 35 42 CP c once nt ration (m gk g –1) Time (d) SS+FCp1 nSS+FCp1 PS+FCp1

Fig.2–DegradationdynamicsofchlorpyrifosindifferentsoiltreatmentswithinitialCPconcentrationof200mgkg−1soil. Symbols:()sterilizedsoil(SS)orSSinoculatedwithCPdegradingstrains,(•)non-sterilizedsoil(nSS)ornSSinoculated withCPdegradingstrains,and()plantedsoil(PS)orPSinoculatedwithCPdegradingstrains.Valuesarethemeansof threereplicatesanderrorbarsrepresentSD.

Inuninoculated sterilized soil, the decrease in CP concen-trationwas insigniﬁcantand it was foundthat 80% ofthe appliedCP persisted till the end of the incubation period. In non-sterilized soildata clearly showed that indigenous microbespossessedthepotentialtodegradeCPintheamount appliedintheexperimentandutilized42.2%oftheinitialCP amountbytheendoftheincubationtime.However,theinitial phaseofCPdepletionwasfollowedbyalagperiodof7days duringwhichonly3%ofinitialCPwasdegraded.Following thisphase,CPdisappearancewasthenobservedtoincrease considerablywitharateconstantof0.0111d−1,averagerate of 2.01mgkg−1d−1 and T1/2 of62.45days. Bacterial strains exhibitedhigher CP degradationpotential innon-sterilized soils as compared to sterilized soil. A. xylosoxidans JCp4 degraded100%oftheappliedCPwithinatimeframeof42 dayswithaveragerateof4.57and4.76mgkg−1d−1 andrate constant of 0.0517 and 0.069d−1 in sterileand non-sterile soils,respectively. Half-life(T1/2)ofCP degradationbyJCp4 increasedfrom 10.07 daysin non-sterile soilto 12.93days insterilesoil.Ochrobactrumsp.FCp1utilized93%ofapplied CPwitharateconstant and averagerateof0.0477d−1 and 4.40mgkg−1d−1 in sterilized soil and T1/2 of 14.5 days. In contrast, in non-sterilized soil 100% of initially applied CP was degraded by FCp1 with rate constant of 0.064d−1,

averagerateof4.76mgkg−1d−1andT1/2of10.6days(Fig.2, Table2).

GrowthexperimentwithV.unguiculata(L.)Walp

Influenceofbacterialpresenceonplantgrowthandpesticide degradationwasstudiedusingplantgrowthexperiments.CP additiontosoilaffectedareductionincertainplant param-eters such as % germination (14.2%), shoot length (2cm), shoot freshweight(0.29g), rootfresh weight(0.05g),shoot dryweight(0.2g)androotdryweight(0.02g).Plantsgrownin CPsupplementedsoilsinoculatedwithCP-degradingbacterial strainsexhibitedsignificantenhancementingrowthinterms ofheightandweight.Anincreaseof6.5and7.8cminshoot lengthandof12.1and7.3cminrootlengthwasobservedin caseofJCp4andFCp1,respectively.Leaflengthwasenhanced upto2.1cm.Theincreaseinshootweightincreasewas stud-ied interms offreshand dry shootweight; anincreaseof 1.6and 1.17ginshootfreshweight andof0.4and 0.5gin shootdryweightwasobservedinplantsinoculatedwithJCp4 and FCp1,respectively.Rootweightwasalsoenhanced sig-nificantly;JCp4andFCp1enhancedrootfreshweightby1.36 and1.54gandrootdryweightby0.15and0.16g,respectively (Fig.3,Table3).

Table2–Kineticstudiesofchlorpyrifosdegradationinvariousmicrobiologicallyactivesoils.Theinitialchlorpyrifos concentrationwas200mgkg−1soil.

Soiltreatment Regressionequation k(d−1)A _V_(mg_kg−1_d−1₎B _T

1/2(d)D R2C SS ln(Ct/C0)=−0.004t−0.015 0.0053a 0.84a 130.7l 0.983 nSS ln(Ct/C0)=−0.013t+0.025 0.0111b 2.01b 62.45k 0.985 PS ln(Ct/C0)=−0.016t+0.042 0.0128a 2.27a 54.15d 0.982 SS+JCp4 ln(Ct/C0)=−0.063t+0.231 0.0517e 4.57h 12.93e 0.941 nSS+JCp4 ln(Ct/C0)=−0.089t+0.257 0.0688i 4.76j 10.07a 0.922 PS+JCp4 ln(Ct/C0)=−0.092t+0.194 0.088c 5.69b 7.86a 0.944 SS+FCp1 ln(Ct/C0)=−0.058t+0.159 0.0476g 4.40g 14.56f 0.953 nSS+FCp1 ln(Ct/C0)=−0.078t+0.173 0.064h 4.76j 10.83b 0.972 PS+FCp1 ln(Ct/C0)=−0.076t+0.141 0.079b 5.69b 8.77c 0.962

SS,sterilizedsoil;nSS,non-sterilizedsoil;PS,plantedsoil;+,inoculatedwith. A_Linear_equation_between_ln(Ct/C

0)ofchemicaldataandtimeyieldedregressionequationandregressioncoefﬁcient(R2). B _Degradation_rate_constant_(k)_calculated_using_Ct₌_C

0×e−kt.CtresidualconcentrationofCP(mgkg−1). C _Chlorpyrifos_{disappearance}_rate.

D_{Disappearance}_time_calculated_by_T

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Table3–MeasurementofgrowthparametersofV.unguiculataindifferentmicrobiologicallyactivesoilssupplemented withCP(200mgkg−1).

Growthparameters Control CP+Cowpea CP+Cowpea+JCp4 CP+Cowpea+FCp1

%germination 87.5±0.0ab ₇₅_±_0.0a _91.6_±_6.4b _91.6_±_7.2b

Shootlength(cm) 15.1±0.36b _13.1_±_0.3a _21.6_±_0.35c _22.9_±_0.36d

Rootlength(cm) 16.1±0.36a ₁₆_±_0.31a _28.2_±_0.78c _23.4_±_0.82b

Leaflength(cm) 3.03±0.15a _3.07_±_0.15a _5.15_±_0.13b _5.20_±_0.1b

Shootfreshweight(g) 1.23±0.07b _0.94_±_0.11a _2.83_±_0.10d _2.40_±_0.09c

Rootfreshweight(g) 0.6±0.03a _0.55_±_0.07a _1.96_±_0.10b _2.14_±_0.06c

Shootdryweight(g) 0.3±0.035b _0.18_±_0.006a _0.7_±_0.03c _0.8_±_0.01d

Rootdryweight(g) 0.1±0.008a _0.08_±_0.007a _0.25_±_.013b _0.26_±_.003b

Thevaluesindicatethemean±SDofthreereplicates.

Differentlettersinsamerowsindicatesigniﬁcantlydifferentvalues.

Control

CP

CP+JCp4

CP+FCp1

Fig.3–GrowthexperimentwithV.unguiculatainsoil supplementedwithCP(200mgkg−1).Control,withoutCP andbacterialinoculation;CP,noinoculum;CP+JCp4, inoculatedwithA.xylosoxidansJCp4andCP+FCp1, inoculatedwithOchrobactrumsp.FCp1.

Withinsixweeks48%ofCPwasdegradedinun-inoculated

soils at the rate of 2.27mgkg−1d−1 and rate constant of

0.0128d−1.Thehalf-lifeofCPinun-inoculatedplantedsoils

wascalculatedtobe54.15days.Ininoculatedsoils100%of

applied CP was degraded within 35d with average rate of

5.7mgkg−1d−1. A. xylosoxidans JCp4 degraded 93.3% CP in

planted soil with rate constant of 0.088d−1 and T1/2 was

observedtobe7.86days.Ochrobactrumsp.FCp1utilized89%

CPwithin28dayswithrateconstantof0.079d−1andT1/2of

8.77days(Fig.2,Table2).

Discussion

Chlorpyrifosisavery popularpesticidethat isused exten-sively for the purpose of pest control in vegetable and cottonﬁelds.However,it iswell knownthatCPistoxicfor mammalsandcanleadtocontaminationofsoilandwater resources which makes its removal from the environment anextremelyurgentissue.Bioremediationisaprocessthat utilizesthe degradation potential ofmicrobesto provide a cost-effectiveandreliableapproachforpesticideabatement. Tothispurpose,CPdegradingbacterialstrainswereisolated fromcontaminatedagriculturalsoilsamples.Twopromising CP-degradingisolatesthatwereidentiﬁedwereA.xylosoxidans JCp4andOchrobactrumsp.FCp1whichwerefoundtodegrade 84.4% and 78.6% of CP (100mgL−1), respectively, within a timeperiodof10days.Inafewpreviouslypublishedreports,

Achromobacter17_and _Ochrobactrum18,19 _have_been _studied_for theirroleinpesticidebiodegradationbut,tothebestofour knowledge,thisistheﬁrststudythataimedtoanalyze simul-taneously CP degradation and plant growth promotion by thesebacterialstrains.

For analyzing CP degradation, an inoculum size of 1.6×107_cfu_g−1_was_used._{Incidentally,}_it_was_noted_that_the inoculum density used was capable of removing CP pro-ficiently without undergoing a lag phase. As reported in previousstudiesinoculumdensityisanessentialfactor influ-encing the efficient degradation ofapplied pesticides.In a previousreportitwasfoundthataninoculumdensitygreater than106cfug−1wascapableofdegradingchlorpyrifoswithout lagphase.20_One_possible_explanation_for_requiring_a_high ini-tialinoculumdensityforpesticidedegradationcouldbethat ahighernumberofbacteriaarerequiredtostartandsustain fastdegradationduringincubation.Soildegradationstudies revealed that the selectedCP-degrading strains were capa-ble ofdegrading CPbothinliquidmediaaswellasinsoil. Previousstudiesonpesticidedegradationhavereportedthat microbesthatarecapableofdegradingthesecompoundsin culture mediaare also capableofdegradation insoil.3,21,22 Thedegradation kineticsweredetermined usingfirst order rateequation Ct=C0×e−kt becausethepesticide disappear-ancewasnotedtobetime-dependent.Therateconstantsfor CPdegradationbybacterialstrainsinsoilrangedfrom0.047 to0.069d−1withT1/2of10–14.5days.Similarfirstorderrate equations have beenused tostudy degradation kineticsof pesticidesinsoilinafewpreviouslypublishedreports.23,24 Comparable degradationkinetic values havebeen reported inearlierstudiesthatanalyzeddegradationofCP.For exam-ple,Cyconetal.notedthatSerratiamarcescenswascapableof degradingCPatrateconstantrangingfrom0.017to0.052d−1 withT1/2of13.6–37daysindifferenttypesofsoils.25

CP degradation was observed to be higher in uninocu-lated non-sterilized soils (42.2%)as compared to sterilized soils(17.2%).ThisﬁndingdemonstratesthatCPdegradationis mediatedbysoilmicroﬂoraandthatindigenousmicrobesare capableofdegradingCP.Inearlierstudies,authorsobserved an 80% increase in diazinon degradation in non-sterilized soilsascomparedtosterilizedsoils.24_Degradation_of_CP_was high in inoculated non-sterile soils as compared to inoc-ulated sterile soilsindicating the bioremediation potential ofCPdegradingstrains.AnincreaseinCP-degradationwas observedinnon-sterilesoilssupplementedwithCP-degrading

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strainsA.xylosoxidansJCp4(9.6%)andOchrobactrumsp.FCp1 (7.75%).TheobservedenhancementinCPutilizationcanbe attributedto the fact that inoculationof soilwith degrad-ing bacteria leadsto an increasein its catabolic potential. Also, the ability of the indigenous microbes to utilize the appliedpesticidecompound mostlikelyplayeda synergis-ticroleintheirbiodegradation.Increasedrateofdepletionof pesticidesincontaminatedsoilsbyaugmentationof poten-tialdegrading strains hasbeen observedinearlier studies. Forinstance,Burkholderiasp.FDS-1enhancedthe fenitroth-ionutilizationratewheninoculatedinFTcontaminatedsoil containingnativemicroﬂora.26_In_a_similar_vein,_S._marcescens degradeddeltamethrinmoreefﬁcientlyinnon-sterilesoilsas comparedtosterilesoils.27

Determinationofplantgrowthpromotingcharacteristics revealedpresenceofsubstantialphosphatesolubilization,IAA andammoniaproductionbyCPdegradingstrains.Bacterial stainsbelongingtogeneraAchromobacter28_and_Ochrobactrum15 havebeen isolatedand studiedfortheir plantgrowth pro-motingtraits. Usually bacteria from plantrhizosphere and bulkagriculturalsoilpossessplantgrowthpromotingtraits; some of these microbes also contain pesticide degrading capacitiesasaresultofcontinuousexposuretothese com-pounds.Inearlierstudies,diverse bacterialstrainsisolated fromasoilenrichmentcultureresponsibleforthiamethoxam degradationwerealsofoundtopossessIAAproductionand N2-fixationabilities.13ChlorpyrifosdegradingAcinetobacter cal-coaceticus alsoshowed PGP traits.29 _Significant _decrease _in PGPactivitiesofCP-resistantbacteriawasobservedin pres-enceofchlorpyrifos.CPhasbeenpreviouslyreportedtoexert an inhibitory effect on soil microbial activities.2 _Similarly, anotherstudyhasalsoreportedadecreaseinplant-growth promotingtraitsofbacteriainpresenceofpesticidesabove therecommendeddose.30_Pesticides_affect_the_soil microbio-logicalactivitiesbyalteringthepopulationofcellulolyticand phosphatesolubilizingmicroorganismsandtherebychanging thenitrogenbalanceandammonificationofsoil.31

ThegrowthofV.unguiculatawasinhibitedwhensoilwas supplemented with 200mgkg−1 of CP. CP addition to soil alsoreduced certainplant parameterssuch as% germina-tion(14.2%),shootlength(13.2%),shootfreshweight(23.5%), rootfreshweight(8.3%),shootdryweight(40%)androotdry weight(20%).Bidlanetal.hasreportedthatuseoflindaneat aconcentrationof100␮gL−1inhibitedthegermination pro-cessingreengramandradish.32_Different_pesticides_such_as aldrin,carbofuran,fenamiphosandphoratehavebeenfound tohaveadverseeffectsonplantgrowthparameterssuchas plantheight,weightandrootnodulation.33_The_negative_effect ofpesticidesonplantgrowthcanbeattributedtoinhibitionof electronﬂowinthephotosyntheticchain.OPsandDDThave beenfoundtoaffectphotosynthesisbyuncouplingthe photo-syntheticelectronﬂowsuchthatATPsynthesisisblocked.34 Enhancedplant(V. unguiculata)growth wasobservedwhen soilswereinoculatedwithCPbacterialstrains.Following inoc-ulationwithA.xylosoxidansJCp4andOchrobactrumsp.FCp1, increaseinplantgrowthwasmonitoredbytracking param-eterssuchas%germination(4.68%),plantheight(59.6%and 48.6%),plantfreshweight(193.8%and176.6%)andplantdry weight(137.5%and165%).Theenhancedrateofseed germi-nationandplantgrowthcanbeattributedtoIAAproduction,

phosphatesolubilizationanddecreaseintoxicityofpesticides owingtoitsincreaseddegradation.

DegradationofCPwasalsostudiedinplantedsoils inoc-ulated with bacterial strains. In CP supplementedsoils, A. xylosoxidansJCp4andOchrobactrumsp.FCp1utilized93.3%and 88.9%ofappliedCPin28daysandwerefoundtodegradeCP completelyafter35days.TheincreaseofCPdegradationin plantedsoilcouldbeattributedtothegreaterpesticide degrad-ing activity of the inoculatedmicrobes. Plant rootssecrete exudatesthatcanserveasasourceofcarbonformicrobial growthwhichinturndegradespesticideatanacceleratedrate. Inseveralstudies,theplantseedlingshavebeenshieldedfrom thetoxiceffectsofvariouspesticidesbybio-augmentingthe soilwithpesticidedegradingbacteria.32,35

Inconclusion,theCP-degradingbacterialisolatesobtained as a result of this study exhibited strong CP-degradation potentialandwereabletobioremediatesoilwithCP concen-trationashighas200mgkg−1.These bacterialstrainswere foundtopossessnotonlypesticidedegradingcapacitiesthat lowerthetoxiceffectsofthesepesticidesonplantsbutalso variousother traitsthathelpedinplantgrowthpromotion. Thesetraitsincludedproductionofphytohormonessuchas IAAthatenhancecellgrowth,solubilizationofphosphatefor rootuptakeandN2-ﬁxationforplantuptake.Collating efﬁ-cientbiodegradationpotentialalongwithmultiplebiological properties,theseisolateshavethepotentialtodevelopinto valuablecandidatesfordevelopmentofbioremediation strate-gies.

Conﬂict

of

interest

Theauthorsdeclarethatnoconﬂictofinterest.

Acknowledgements

TheﬁnancialsupportforthePhDworkwasprovidedbyHigher Education Commission (HEC), Islamabad, Pakistan through IndigenousscholarshipforworkinPakistanandInternational ResearchSupportInitiativeProgram(IRSIP)fundstovisitthe FacultyofAgricultureandEnvironment,UniversityofSydney.

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