Comparative bio-efficacy of nuclear polyhedrosis virus (NPV) and Spinosad against American bollwormm, Helicoverpa armigera (Hubner)

RevistaBrasileiradeEntomologia63(2019)277–282

REVISTA

BRASILEIRA

DE

Entomologia

AJournalonInsectDiversityandEvolution w w w . r b e n t o m o l o g i a . c o m

Biological

Control

and

Crop

Protection

Comparative

bio-efficacy

of

nuclear

polyhedrosis

virus

(NPV)

and

Spinosad

against

American

bollwormm,

Helicoverpa

armigera

(Hubner)

Ahmad

Nawaz

a,∗,1

_,

_Habib

_Ali

b,∗,1

_,

_Muhammad

_Sufyan

a

_,

_Muhammad

_Dildar

_Gogi

a

_,

Muhammad

Jalal

Arif

a

_,

_Mazhar

_Hussain

_Ranjha

a,b

_,

_Muhammad

_Arshid

a

_,

_Muhammad

_Waseem

a

_,

Tariq

Mustafa

a,b

_,

_Muhammad

_Qasim

c

_,

_Muhammad

_Rizwan

a

_,

_Madiha

_Zaynab

g

_,

_Khalid

_Ali

_Khan

d,e,f

_,

Hamed

A.

Ghramh

d,e,f

a_{UniversityofAgriculture,DepartmentofEntomology,IntegratedPestManagementLaboratory,Faisalabad,Pakistan} b_{UniversityofAgriculture,Faisalabad,Pakistan}

c_{ZhejiangUniversity,InstituteofInsectSciences,Zhejiang,P.R.China}

d_{KingKhalidUniversity,ResearchCenterforAdvancedMaterialsScience(RCAMS),Abha,SaudiArabia} e_{KingKhalidUniversity,FacultyofScience,UnitofBeeResearchandHoneyProduction,Abha,SaudiArabia} f_{KingKhalidUniversity,FacultyofScience,BiologyDepartment,Abha,SaudiArabia}

g_{FujianAgricultureandForestryUniversity,CollegeoflifeScience,Fuzhou,Fuzhou,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received12June2019 Accepted3September2019 Availableonline17September2019 AssociateEditor:RegianeBueno Keywords: Americanbollworm Bio-pesticide Insecticide Microbialcontrol NPV

a

b

s

t

r

a

c

t

Americanbollworm(ABW),Helicoverpaarmigera(Hubner),isconsideredasamajorpestofcotton, Gossyp-iumhirsutum,allovertheglobe.Duetoitsdestructivefeedingnatureandcontinuousconsumptionof thesamechemicals,itdevolvedresistantagainstmanyinsecticides.Therefore,acombinedapplication ofbio-andsynthetic-pesticideneedtoevaluateagainstthispest.Theentomopathogenicviruseslike nuclearpolyhedrosisvirus(NPV),amemberofbaculoviruses,canbethepotentialcandidatesforbetter controlagainstABW.ThepresentstudywasconductedtoassessthecomparativeefficacyofNPVand Spinosad240SC(withtheconcentrationof250mL·ha−_{l)againstABWinthecontrolledenvironment.}

TheABWwastreatedwithdifferentconcentrationsofNPVandSpinosadseparatelyandinacombination ofNPVwith0.1%Spinosad.Theresultsrevealedthathighestconcentrationsshowedhighestmortality (95%)followedby95%,92%,84%,82%and78%mortalityat1×109_,1×108_,1×107_,1×106_and1×105

POBs,respectively.Spinosadwhenmixedindietgive100%mortalityat0.8%followedby50.87%,42.10%, 29.82%,26.31%and22.80%mortalityat0.4%,0.2%,0.1%,0.5%and0.025%respectively.Theresultsofthis studyrevealedthatmicrobialcontrolofABWthroughNPVisaneffectivetool.Therepeateduseof syn-theticpesticidescausedtheresurgenceofmanyinsectpests,andthisstudyresultswouldprovideuseful insighttobuildaframeworkforfutureinvestigationsforthemanagementofmanymajorinsectpests.

©2019SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

American bollworm (ABW), Helicoverpa armigera (Hubner) (Lepidoptera:Noctuidae),hasbeenobservedmostdangerouspest andyielddeclinerofthefieldcropsandvegetablesinPakistansince the1990sand early21stcentury. Ithasbecomethemost haz-ardouspestduetoitspolyphagousnature,migratorybehavior,high

∗ Correspondingauthors.

E-mails:ahmad.nawaz1793@uaf.edu.pk(A.Nawaz),habibali1417@yahoo.com (H.Ali).

1_{Theseauthorshavecontributedequallytothiswork.}

fecundityrate,multiplegenerationsandcandeveloptheresistance againstinsecticides.Itisestimatedthatannually,2.5millionbales losseswerecausedbythisinsectpesttothecottoncrop(Karim, 2000;Muhammadetal.,2009).

Differentlevelsofresistance(moderatetohigh)toawiderange ofinsecticideshavebeenreportedworldwide,includingPakistan, among thefield populationof ABW(SayyedandWright,2006; DamalasandEleftherohorinos,2011;IshtiaqandSaleem,2011). Therefore,themanagementofthispestforanextendedperiodwith reducedriskofinsecticidesisthedireneedoftimeanda prag-maticallysuggestedoption.Amongthevariousbiologicalcontrol agentsusedasanalternativetosyntheticinsecticides(Alietal.,

https://doi.org/10.1016/j.rbe.2019.09.001

0085-5626/©2019SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

278 A.Nawazetal./RevistaBrasileiradeEntomologia63(2019)277–282 Table1

EffectofdifferentconcentrationsofSpinosadonlarvaeofAmericanbollworm, Heli-coverpaarmigeraafterdifferenttimeintervalsindietmixedmethod.

Concentrations% 24h 48h 72h 0.8 16.81A_{±0.39 33.81}A_{±0.54 50.87}A_±0.81 0.4 15.30A_{±0.25 26.02}B_{±0.49 42.10}AB_±0.66 0.2 10.15AB_{±0.43 20.31}C_{±0.26 29.82}BC_±0.36 0.1 8.39AB_{±0.39 15.23}CD_{±0.31 26.31}C_±0.31 0.05 3.51B_{±0.26 11.86}D_{±0.51 22.80}C_±0.44 0.025 3.79B_{±0.33 11.61}D_{±0.47 22.80}C_±0.37 CONTROL 2.07B_{±0.22 2.38}E_{±0.37 3.71}D_±0.28

2015,2018a,b,c,d,e),thepreferenceforentomopathogenicNuclear PolyhedrosisVirus(NPV)hasbeenincreasedintherecentyears. Furthermore,newchemistry insecticideshave beenestablished fromnaturalsources,disturbthenormalphysiologicalfunctionsof thetargetedspeciesinpestmanagementprograms(Qasimetal., 2018a;Schneideretal.,2003; Thompsonetal.,2000; Dhadialla et al., 1998) by targeting insect development mechanism like moultingaswellaschloridechannelactivator.Anotheradvantage ofnewchemistryinsecticideoverthebroad-spectruminsecticides isthattheyareselectivetotargetpestspeciesandlessdangerous tonaturalenemies(Grafton-Cardwelletal.,2005).

Among microbial insecticides, the insect pathogenic viruses suchasbaculoviruseswereusedsuccessfullyforbiologicalcontrol underintegratedpestmanagement(IPM)program.Ithasbeenused formorethan20yearswithgreatsuccess(Zhang,1989)indifferent partsoftheworldformanagingdifferentinsectpestspeciesbased ontheirseveraladvantages.Thesemicrobialagentsarehighly host-specificandare knowntobeentirelysafefor humans,animals andnon-targetbeneficialorganismssuchasbees,predatoryinsects andparasitoids(MonobrullahandNagata,1999;Nakaietal.,2003; Islametal.,2018a,b).Keepinginviewthefactsasmentionedabove, thepresentstudywascarriedouttoevaluatetheeffectivenessof NPVagainstABWundercontrolledconditions.

Materialsandmethods

ThestudywasconductedinIntegratedPestManagement(IPM) Lab,DepartmentofEntomology,UniversityofAgriculture, Faisal-abad,Pakistanduring2016–2018.

Preparationofartificialdiet

To prepare the artificialdiet for lepidopterans, the product F9772(Lot#110216-01)wasusedaccordingtothemanufacturer’s recommendations. Moreover, following ingredients were used; agar(19.0g), distilledwater (875mL)(stirredtheagaruntilthe agarwasdissolvedcompletely)and144.0gdrymixingredients (Sucrose,SoyFlour,50%,WheatGerm,stabilized,SaltMix,Wesson, USDAVitaminPremix,Fiber,SorbicAcid,MethylParaben,Ascorbic Acid).Agarsolutionwasboiledandstiruntilasmooth,andeven consistencywasachieved.Pourthedietintosmallcups approxi-matelyone-thirdofacup,andlefttocoolandsolidifyreadyforthe additionoflarvae.

Rearingofinsects

The larvae of ABW were collected from the host field and rearedinthelaboratoryundercontrolledconditions(temperature 25±2◦Candrelativehumidity(RH)75%).Thecubesofthe artifi-cialdietwerepreparedandshiftedtoplasticvials.Asinglelarva wasallowedtofeedonthedietineachplasticvial.Foradults,a 10%sugarsolutionwasprovideduntiladultdies,foregglayingon muslincloth.Thelaideggswerecollectedafter24hinapetridishin

agrowthchamberforeclosion.Aftereclosion,larvaewereshifted toanartificialdietforfurtherexperiments.

IsolationofNPVfrominfectedABWfromfiled

InfectedlarvaeofABWwerecollectedfromthefield.For iso-lationofthevirus, astandard procedurewasadoptedin which homogenization, filtration and centrifugation were done. The occlusionbodieswerepurified(Tompkins,1991).ThePolyhedral occlusionbodies(POB)permillilitrewerecountedusing hemocy-tometerunderthelightmicroscope.

Bioassay

AccessingtheefficacyofNPVonABW

ThehomogenizedpopulationofABWwasreared,andselected secondandthirdinstar(20larvae)wereallowedtofeedonthe arti-ficialdietmixwithNPVformulation.Sixdifferentformulationsof NPV(1×105_,1×106_,1×107_,1×108_,1×109_and1×1010_POBs) were used to assess the efficacy of NPV. The experiment was repeatedthrice,andcontrolwaskeptuntreated.Mortalitydatawas takenaftertheintervalofevery24hsubjectedtodifferentdose formulationuntilallthelarvaedied.

AssessingtheefficacyofSpinosad

Spinosad240SC(Arysta Life Science,Pakistan) wasassessed againstABWattherateof80–100mL·acre−1.Sixconcentrations ofspinosad(0.8%,0.4%.0.2%,0.1%,0.05%and0.025%)weremade andtestedagainsttheABWlarvaethroughdietmixmethod.The experimentwasrepeatedthrice,alongwiththecontroltreatment. Mortalitydataforalltreatmentsweretakenattheintervalof24h, 48hand72h.

AssessingtheefficacyofcombinetoxicityofNPVandSpinosad Various concentrations of NPV were also mixed with 0.1% spinosadto checkthe combinedtoxicity of NPV and Spinosad. Twentylarvae wereallowedtofeedona diet mixedwithNPV andSpinosadincombination.Mortalitydataweretakenafter24h subjectedtoconcentrationsofNPVandSpinosad.

Statisticalanalysis

MortalitydataweresubjectedtoAbbott’sformula(Flemingand Retnakaran,1985).DatawereanalyzedusingStatisticasoftware and meanswereseparated by usingTukey’s HSDtest. The Co-Toxicitytestwascalculatedbythefollowingformula.

CTF= OC-OE/OE×100

CTFmeanscotoxicityfactor,OCmeansobservedpercentage mortalityandOEistheresultofthecombinedapplication.TheOE istheexpectedpercentageofmortalityandrepresentsthesumof percentagemortalityproducedbyeachofthetreatmentusedin combination.ThevalueCTFvariesbetween20and−20.Valueof CTFabove20revealedthattherewasasynergeticeffectbetween twomixtures.ThevalueofCTFin−20showedthattherewasan antagonisticrelationshipbetweentwomixturesandthevalueof CTFbetween20and−20showedthattherewasanadditiveeffect oncomparativeefficacy.

Results

ANOVAparametersindicatedthatallNPVconcentrationswere significantlyeffectiveagainstthesecondand thirdinstarlarvae ofABW(F=592,P=0.000).Resultsshowedthathigher concentra-tionshadgiventhehighestmortalitywhentreatedwithNPValone indietmixedmethod.Itwasalsoobservedthattheeffectiveness

A. Nawaz et al. / Revista Brasileira de Entomologia 63 (2019) 277–282 279 Table2

MortalitypercentageofAmericanbollworm,HelicoverpaarmigeraagainstdifferentconcentrationsofNPValoneandinacombinationof0.1%Spinosad.

Concentrations DAT1±S.E. DAT2±S.E. DAT3±S.E. DAT4±S.E. DAT5±S.E. DAT6±S.E. DAT7±S.E. DAT8±S.E. DAT9±S.E. DAT10±S.E. 1×1010_{alone 20.07}A_{±0.25 28.39}A_{±0.39 36.14}A_{±0.80 45.61}A_{±0.82 57.89}A_{±0.88 66.67}A_{±0.90 79.63}A_{±1.11 92.15}A_{±2.11 95.00}A_{±2.11 95.00}A_±2.05 1×109_{alone 15.39}B_{±0.35 20.05}AB_{±0.42 32.71}AB_{±0.77 42.10}A_{±0.87 49.12}B_{±0.79 50.08}B_{±0.85 74.07}A_{±1.95 84.31}A_{±2.01 98.03}A_{±2.02 100.00}A_±1.99 1×108_{alone 10.06}C_{±0.40 16.67}BC_{±0.25 27.54}BC_{±0.31 33.42}B_{±0.60 42.10}C_{±0.65 50.81}B_{±0.79 59.25}B_{±0.81 66.59}B_{±0.80 78.43}B_{±1.25 92.15}B_±2.07 1×107_{alone 10.55}C_{±0.30 13.45}BC_{±0.22 24.12}CD_{±0.49 31.57}B_{±0.59 38.59}CD_{±0.59 44.49}B_{±0.91 53.70}B_{±0.86 62.74}B_{±0.91 68.62}C_{±0.97 84.31}C_±1.75 1×106_{alone 6.45}C_{±0.45 11.62}BC_{±0.49 20.70}CD_{±0.25 28.07}BC_{±0.36 36.84}CD_{±0.25 44.58}B_{±0.83 50.01}B_{±0.79 58.82}B_{±0.88 62.74}C_{±0.92 82.35}C_±1.90 1×105_{alone 6.38}C_{±0.54 10.08}CD_{±0.50 17.28}D_{±0.51 24.56}C_{±0.22 35.08}D_{±0.51 42.53}B_{±0.76 50.82}B_{±0.90 56.86}B_{±0.79 60.78}C_{±0.88 78.43}C_±1.20 Control 2.09D_{±0.39 2.04}D_{±0.30 3.06}E_{±0.33 4.44}D_{±0.29 5.31}E_{±0.42 5.60}C_{±0.29 7.29}C_{±0.45 8.61}C_{±0.25 10.02}D_{±0.49 10.05}D_±0.39 1×1010_{+Spin. 16.81}A_{±0.39 33.81}A_{±0.54 50.87}A_{±0.81 71.93}A_{±1.11 94.73}A_{±1.97 95.00}A_{±2.09 95.00}A_{±2.01 95.00}A_{±2.11 95.00}A_{±2.02 95.00}A_±2.11 1×109_{+Spin. 15.30}A_{±0.25 26.02}B_{±0.49 42.10}AB_{±0.66 64.91}AB_{±0.95 83.82}A_{±1.50 90.74}A_{±1.91 95.00}A_{±2.11 95.00}A_{±2.04 95.00}A_{±1.97 95.00}A_±1.91 1×108_{+Spin. 10.15}AB_{±0.43 20.31}C_{±0.26 29.82}BC_{±0.36 42.10}BC_{±0.88 51.85}B_{±0.89 64.81}B_{±0.89 73.52}B_{±1.89 84.31}B_{±1.93 98.03}A_{±2.11 100.00}A_±2.08 1×107_{+Spin. 8.39}AB_{±0.39 15.23}CD_{±0.31 26.31}C_{±0.31 40.35}BC_{±0.76 48.24}B_{±0.66 53.70}B_{±0.66 50.87}C_{±0.91 62.74}C_{±0.58 65.85}B_{±0.88 74.51}B_±1.39 1×106_{+Spin. 3.51}B_{±0.26 11.86}D_{±0.51 22.80}C_{±0.44 35.08}C_{±0.57 46.49}B_{±0.91 51.85}B_{±0.81 45.50}C_{±0.88 57.86}C_{±0.81 62.74}B_{±0.79 70.58}B_±1.85 1×105_{+Spin. 3.79}B_{±0.33 11.61}D_{±0.47 22.80}C_{±0.37 35.18}C_{±0.61 46.49}B_{±0.88 50.16}B_{±0.77 45.20}C_{±0.81 56.65}C_{±0.88 60.78}B_{±0.91 68.62}B_±0.91 Control 2.07B_{±0.22 2.38}E_{±0.37 3.71}D_{±0.28 5.09}D_{±0.37 7.70}C_{±0.21 9.08}C_{±0.44 9.76}D_{±0.30 10.09}D_{±0.33 10.05}C_{±0.27 10.06}C_±0.21

280 A. Nawaz et al. / Revista Brasileira de Entomologia 63 (2019) 277–282 Table3

EffectofSpinosadonmeanlarvalmortalityofAmericanbollworm,HelicoverpaarmigeralarvaetreatedwithdifferentconcentrationofNPVinacombinationof0.1%Spinosadfromday1–3.

Concentrations Day-1 Day-2 Day-3

Actualmortality Expectedmortality CTF Effect Actualmortality Expectedmortality CTF Effect Actualmortality Expectedmortality CTF Effect

NPV6 20.07A_{±0.25 28.39 −9.94 Additive 25.35}A_{±0.39 43.62 −18.27 Additive 44.63}A_{±0.61 62.45 −17.82 Additive}

NPV5 15.39B_{±0.35 23.78 −8.67 Additive 20.26}AB_{±0.26 35.28 −15.02 Additive 42.98}A_{±0.69 59.02 −16.04 Additive}

NPV4 10.06C_{±0.40 18.45 −10.06 Additive 15.17}AB_{±0.49 31.9 −16.73 Additive 32.16}B_{±0.55 53.85 −21.69 Antagonistic}

NPV3 10.55C_{±0.30 18.94 −10.51 Additive 15.17}AB_{±0.33 28.68 −13.51 Additive 30.40}B_{±0.49 50.43 −20.03 Antagonistic}

NPV2 6.45C_{±0.45 14.84 −9.03 Additive 10.08}BC_{±0.51 26.85 −16.77 Additive 17.83}C_{±0.33 47.01 −29.18 Antagonistic}

NPV1 6.38C_{±0.54 14.77 −11.54 Additive 10.08}BC_{±0.41 25.31 −15.23 Additive 17.83}C_{±0.41 43.59 −25.76 Antagonistic}

A.Nawazetal./RevistaBrasileiradeEntomologia63(2019)277–282 281 of NPV increased withtime. Mean percent mortality

compari-sonshowedthatmaximummortality(95.00±2.05%)wasseenat 1×1010_{POBwhichwas9.95timeshigherthancontrol,followedby} 95.00±1.99%at1×109_{POBwhichwas9.95timeshigherthan} con-trol.However,mostdilutedNPVdose(1×105_{POB)causedenough} larvalmortality,reachingupto78.43±1.20%whichwas7.80times higherthancontrol(10.05±0.39%)(Table2).

Overall,analysed dataindicated thatallspinosad concentra-tionsweresignificantlylethaltothesecondandthirdinstarlarvae ofABW(F=27.4, P=0.000). Meanpercentmortalitycomparison showedthat maximum mortality(50.87±0.81%)was observed at(0.8%)whichwas13.71timeshigherthancontrolfollowedby 42.10±0.66%at(0.4%)whichwas11.34timeshigherthan con-trol.Though,mostdiluteddose(0.025%)causedonly22.80±0.37% mortalityofABWlarvae,whichwas6.14timeshigherthancontrol (3.71±0.28%)(Table1).

ANOVAparametersindicated thatthecombined effectofall NPVconcentrationsandspinosadprovedtobesignificantagainst thesecondorthirdinstarlarvaeofABW(F=680,P=0.000).Mean percent mortality comparison showed that maximum mortal-ity (95.00±2.06%) wasobserved at NPV6+ Spinosad (1×1010 POB+0.1%)whichwas9.91timeshigherthancontrolfollowedby (95.00±1.98%)atNPV5+Spinosad(1×109_{POB+0.1%)whichwas} 9.91timeshigherthancontrol.Eventhoughthecombinationof NPV1+Spinosad(1×105_{POB+0.1%)wasalsotoomuchlethalto} ABWlarvae,causing80.71±1.91%mortality,whichwas7.99times higherthancontrol(10.09±0.31%)(Table2).

CombinedapplicationofdifferentconcentrationsofNPVwith0.1% Spinosad

WhendifferentconcentrationsofNPV(1×105_,1×106_,1×107_, 1×108_{, 1×10}9 _{and 1×10}10_{POBs) were mixed with 0.1% of} Spinosad,themortalityoflarvaeincreased.Itwasobservedthat themortalityoflarvaewashigherinthecombinedapplicationas comparedtotheindividualapplicationofbothNPVandSpinosad. ItwasalsoobservedthathigherconcentrationsofNPVwithalow concentrationofSpinosadgiveanadditiveeffect(CTFinbetween −20and+20).WhileinthecaseoflowNPVandSpinosad concentra-tiongivesanantagonisticimpact(CTF≥−20).Accordingtoresults ofdayoneandtwo,allNPVconcentrationsshowedtheadditive effectwithSpinosad(CTFinbetween−20and+20).Afterthreedays ofapplication,twohighestconcentrationsofNPVgavean addi-tiveimpactwhiletherestoftheconcentrationsgaveantagonistic effects(CTF≥−20)(Table3).

Discussion

Whileusingthebio-agentinIPM,therearesomelimitations suchastheseagentsareslowinaction,lowpersistencein envi-ronment and need of a repeatedapplication to infectthehost population. Thecombined useof microbial agents mayhelp in resolvingtheseproblemsand increasingthepathogenicity, per-sistenceandrateofinfection(Alietal.,2015,2016).Theissueof insecticideresistancecanalsobeminimizedbyusingtheseagents astheyuseddifferenttacticstokillorcontrolhost,ascomparedto otherconventionalinsecticides(Balaetal.,2018).ABWisthepest ofgreatimportancethatattacksmanyvaluablecrops.

TheresistanceofABWagainstmanyinsecticideshasbeennoted, andit wastheissueinfocusformany years.Entomopathogens cancontrolthepesthavingresistanceagainstinsecticides(Cloyd, 2014).Therotationofcropsandpesticidescanalsobehelpfulto minimizetheproblemofresistance(ZahnandMorse,2013).When two ormorebio-agentsare usedincombination, theyincrease theeffectiveness,andhostspectrumofeachotherandreducethe

mortalitytime(Kalantarietal.,2014).Itcanbeimaginedthatthe mutualactionofmicrobesmayincreasethevirulenceasexpected alone.ThepolyhedralbodiesofNPVattachtothemidgutofhost andmultiply,therebydestroyingthegutcells.Themidgutisthe firstbindingsiteofPOBs,wheretheymultiplyandtheninfection transferfromcelltocell,causingthedeathofthehost(Liuetal., 2006;Arifetal.,2018;Qasimetal.,2018b;Shakeeletal.,2018).

Amongtheseconcentrations,aftertheexposuretotreateddiet, mortality was recorded. High concentrations of both NPV and Spinosadgivesthehighest mortality.Such asNPV gavehighest mortality(95%,95%and92.15%)at1×1010_,1×109 _and1×108 POBsconcentrationsandtheseresultswereparalleltothoseresults ofArrizubietaetal.(2016),whoreportedthatamixtureofNPV andinsecticidesagainstH.armigerawaseffectiveunderlaboratory condition.Theseresultsshowedthateffectivenessandtenacityof mixturecausedsignificantmortalityoflarvae.Spinosadgavethe highestmortality(95%,95%and95%)and0.8%,0.4%and0.2% con-centrationsand theseresultsweresupportedbyJatand Ameta (2013).ThestudyresultsconcludedthatFlubendiamide480SCand Spinosad45SC(at200mL·ha−1)werefoundtobeeffectiveagainst thelarvaeofABW,andtheseinsecticidescaused89.94%and74.67% mortality,respectively.

Thevirusesmultiplywithinthebodyofthehostanddestroy thehostcelltissues.Thepathogenicityandrateofresponsedepend uponthedose,temperatureandlarvalbodysize,anditvariesfrom 4to14days(Jonesetal.,1994).Thusthepathogenicityvarieswith treatmentandtime,asthedoseincreasethecontrolefficacyalso increases.

Combinationoftwoormorebio-agentcanbeanoptiontodelay theresistanceandincreasetheeffectiveness(Kalantarietal.,2014). Inthecurrentstudy,bothadditiveandantagonisticinteractions wereobserved,andtheseresultsweresupportedwiththefindings ofWangetal.(2009)whofoundthatlethalandsub-lethaldosesof spinosadincreasethemortalityanddelaythedevelopmental pro-cess.TheadditiveimpactwassupportedbytheresultsofWakil etal.(2012),whofoundthattherewasanadditiveeffectbetween NPVandAzadiractumagainstABW.Moreover,Qayyumetal.(2015)

alsoreportedtheadditiveimpactofNPVandBacillusthuringiensis againstABW.Inourstudy,theantagonisticeffectwassupported bytheoutcomesofTrangetal.(2002)astheyobservedthe antago-nisticeffectofNPVandImidacloprid.Theantagonisticeffectmight beduetothedecreaseinfeedingpotentialorpHchangeofgut (El-HelalyandEl-bendary,2013).

Conclusion

WespeculatedthatmicrobialcontrolthroughNPVandSpinosad isaneffectiveapproachagainstABW.Therepeateduseofsynthetic pesticidesagainstmajorpestscouldbethemainreasonofpest resurgence,andthisstudyresultswouldprovideusefulinsightto buildaframeworkforfutureinvestigationsandtoreducetheuse oftoxicchemicalsforthecontrolofinsectpestsofmajorcrops.

Conflictofinterest

Allauthorsdeclarenoconflictofinterests.

Acknowledgements

WeareprofoundlygratefultotheHigherEducation Commis-sion(HEC) Pakistan who provided thefinancialsupport tothis project.WearealsothankfultoProfessorYoumingHou,Plant Pro-tectionCollege,FujianAgricultureandForestryUniversity,Fuzhou, China, for critical reading and suggestions for this manuscript. WealsoacknowledgethesupportoftheKingKhalidUniversity

282 A.Nawazetal./RevistaBrasileiradeEntomologia63(2019)277–282

(RCAMS/KKU/010-19)ResearchCenterforAdvancedMaterials Sci-ence(RCAMS)atKingKhalidUniversity,KingdomofSaudiArabia.

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