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Crop Protection 29 (2010) 677e683

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Crop Protection

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Efficacy of entomopathogenic nematodes (Nematoda: Rhabditida) and insecticidemixtures to control Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae)in corn crops

Aldomario S. Negrisoli*, Mauro S. Garcia, Carla R.C. Barbosa Negrisoli, Daniel Bernardi,Alexandre da SilvaLaboratório de Biologia de Insetos e Controle Biológico, FAEM/UFPel, 354, 96010-900 Pelotas, Brazil

a r t i c l e i n f o

Article history:Received 20 August 2009Received in revised form11 January 2010Accepted 4 February 2010

Keywords:Heterorhabditis indicaSteinernema carpocapsaeS. glaseriFall armywormPesticidesBiological control

* Corresponding author. Tel.: þ55 53 3275 9031.E-mail address: asnegrisoli@gmail.com (A.S. Negri

0261-2194/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.cropro.2010.02.002

a b s t r a c t

The main insect pest in Brazilian corn is fall armyworm, Spodoptera frugiperda (Smith, 1797) (Lepidop-tera: Noctuidae). Entomopathogenic nematodes (EPNs) can be used to control this pest, and can beapplied together with various insecticides. Thus, the objective of this work was to evaluate the efficacy ofmixtures of EPNs and insecticides to control S. frugiperda in corn crops. In laboratory bioassays threespecies of EPNs were tested (Heterorhabditis indica, Steinernema carpocapsae and Steinernema glaseri)together with 18 registered insecticides to control S. frugiperda in corn. Efficacy of association betweeninsecticides and EPNs on S. frugiperda larvae was evaluated against the insect's third instar, 2 and 4 daysafter applications in laboratory. Experiments in the field were performed in two consecutive years, withlocated application of H. indica and S. carpocapsae (250 IJs/cm2) mixed with chlorpyrifos (0.3 L/ha) andlufenuron (0.15 L/ha) on the corn husk. In laboratory, after two days exposure the interaction betweenchlorpyrifos and H. indica was synergistic, while interaction with cypermethrin, spinosad, methox-yfenozide and deltamethrin þ triazofos was additive, as was interaction between lufenuron, chlorpyrifosand cypermethrin with S. carpocapsae. In contrast, the interaction between chlorpyrifos (Vexter� andLorsban�) and lufenuron with S. glaseri was synergistic. In the field, the best treatment was the mixtureof H. indica with lufenuron (0.15 L/ha), with 62.5% and 57.5% larval mortality in the two evaluation yearsin the field, respectively.

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1. Introduction

Among the major insect pests in corn crops in Brazil, fallarmyworm, Spodoptera frugiperda (Smith, 1797) (Lepidoptera:Noctuidae) is considered the most important one. Insect infesta-tions occur from plant emergence until ear formation, can result inyield reductions of up to 34%infestation (Cruz et al., 1995, 1997).

Natural occurrence of Steinernematidae and Heterorhabditidaein soil samples collected in 64 localities (corresponding to 10.9%)associated with populations of S. frugiperda in corn and sorghumcrops and pastures in Mexico, have demonstrated these entomo-pathogens' potential to control this pest in such agro-ecosystems(Molina-Ochoa et al., 2003). Various foliage application technolo-gies to control S. frugiperdawere tested using Heterorhabditis indicaPoinar, Karunakar and David, 1992 and Steinernema sp and the

soli).

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compatibility of these EPNs with tensioactives, resulting in lowefficacy in the pest control. (Garcia et al., 2008).

In chemical control of S. frugiperda, the use of high concentra-tions of the same active ingredient allied with to the absence ofrefuge areas leads to high selective pressure, resulting in develop-ment of resistance to these chemicals. On the other hand, thecombination of a stressor and a natural enemy may increase ordecrease insect adaptability to the agrochemicals, depending onthe intrinsic characteristic of the system (Gould et al., 1991; Johnsonet al., 1997; Koppenhöfer and Kaya,1998). Thus, the objective of thiswork was to evaluate the efficacy of control of S. frugiperda in thelaboratory and field, by associating insecticides and EPNs.

2. Materials and methods

Insect rearing, EPN multiplication and bioassays were per-formed at the Laboratório de Biologia de Insetos e ControleBiológico of the Departmento de Fitossanidade at the Faculdadede Agronomia “Eliseu Maciel” of the Universidade Federal dePelotas/UFPel, Brazil.

Table 1Characteristics of insecticides registered in Brazil for Spodoptera frugiperda in corn (Empygdio and Teixeira, 2006).

Name Formulation T.C.a M.A.b Chemical group Concentration(Kg or L ha�1)b

Spray volume(L ha�1)c

Technical Commercial

Beta cyfluthrin Turbo ECe II SCM Pyrethroid 0.050 200e300Cypermethrin Galgotrin EC I SCM Pyrethroid 0.025 150Chlorpyrifos Lorsban 480 BR EC II CI Organophosphate 0.300 100e300Chlorpyrifos Vexter EC II CI Organophosphate 0.300 100e300Deltamethrin Decis 25 EC EC III SCM Pyrethroid 0.100 10e20d

Deltamethrin þTriazofos

Deltaphos EC EC I SCM þ CI Pyrethroid þOrganophosphate

0.175 300

Diflubenzuron Dimilin WP IV Csi Benzoylurea 0.050 200e400Gamacyhalothrin Stallion 150 CS CS III SCM Pyrethroid 0.012 300Lambdacyhalothrin Karate Zeon

250 CSCS III SCM Pyrethroid 0.015 250

Lufenuron Match EC IV Csi Benzoylurea 0.150 150e400Methoxyfenozide Intrepid 240 SC SC IV Mmh Diacylhydrazine 0.075 200e400Methil parathion Folidole 600 EC II CI Organophosphate 0.337 100Permethrin Talcord 250 CE EC I SCM Pyrethroid 0.050 e

Permethrin Valon 384 EC EC II SCM Pyrethroid 0.0325 e

Espinosad Tracer SC III NS Espinosines 0.050 100e300Triazofos Hostathion

400 BREC II CI Organophosphate 0.250 100e400

Triflumuron Certero SC IV Csi Benzoylurea 0.025 200e300Alpha cypermethrin Fastac SC III SCM Pyrethroid 0.025 150e300

a T.C. ¼ Toxicological class of the a.i.b M.A. ¼ Mode of action, Ea ¼ ecdise accelerator; SCM ¼ sodium channel modulator; NS ¼ “non-systemic”; CI ¼ cholinesterase inhibitor Csi ¼ chitine synthesis inhibitor.c Corresponding to terrestrial application.d Corresponding to aereal application.e Recommended for S. frugiperda in corn when the study was performed.

A.S. Negrisoli et al. / Crop Protection 29 (2010) 677e683678

Larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae) werereared in a controlled room at 28� 2 �C, 60� 10% relative humidity(RH) and 12 h photoperiod (Parra, 1998). Fall armyworm werereared in a controlled room at 25� 2 �C, 70� 10% relative humidity(RH) and 12 h photoperiod, according to themethodology proposedby Greene et al. (1976).

Entomopathogenic nematodes (EPNs) H. indica Poinar, 1976strain IBCB-n5 (isolated in Itapetininga, São Paulo, Brazil), Stei-nernema carpocapsae (Weiser, 1955) strain Sta Rosa and S. glaseri(Steiner,1929) strain Sta Rosa (isolated in Santa Rosa do Viterbo, SaoPaulo, Brazil), weremultiplied on last instar G. mellonella, accordingto methodology described by Kaya and Stock (1997). Every day IJswere collected and stored in (zip-lock type) plastic bags containingpolyurethane sponge blocks. In order to reduce contaminantproliferation EPNs were maintained in an environmentallycontrolled chamber at 12 �C and 24 h darkness, using distilledwaterwith 0.1 % sodium hypochlorite solution during the whole process.

2.1. Efficacy of insecticide and EPN mixture to control Spodopterafrugiperda in laboratory

Third instar S. frugiperda were submitted to the treatments inplastic boxes (150 mL) containing wet sand (10%, w/w) as substrateand 5g of artificial diet, and inoculated with the following treat-ments: 1- insecticide at the half recommended dose; 2- entomo-pathogenic nematodes; 3- a combination of each insecticide (halfrecommended dose); 4- control (water) and each nematode,considering ten larvae as one replicate, with a total of ten replicatesfor each treatment. Localized application of the treatments wasmade with a micro-pipette (100 mL capacity), corresponding toa dose that will be normally applied in 1 ha, expressed in mL/cm2

and considering the highest volume of mixture recommended(Table 1), totaling 2.0 mL per vial. Nematode concentration wasadjusted to 100 IJs per container (2.6 IJs/cm2). Each containerreceived one larva and mortality was registered 2 and 4 days afterapplication. The experimental statistical design was factorial,mortality values were submitted to analysis of variance and

differences between treatment means were estimated by Tukey'sHDS test at P < 0.05 probability.

2.2. Efficacy of insecticide and EPN mixture, to control Spodopterafrugiperda in the field

The experiments were performed in the county of Capão doLeão, near the Federal University of Pelotas, Rio Grande do Sul,Brazil, during 2007/2008 (first assay) and 2008/2009 (secondassay). The efficacy of control of S. frugiperda in corn plantations inthe field by the combination of two nematode species (H. indica andSteinernema carpocapse, 250 IJs/cm2) and two insecticides (Match�

0.15 L/ha and Lorsban� 0.3 L/ha) previously selected in the labo-ratory, was evaluated.

The experimental area was prepared by incorporating organicmanure in the soil (87.5 m3/ha), and weeds were controlledmechanically. Corn (cv. AS 32 Agroeste�) planting was performedwith the aid of a manual planter (two seeds per hollow) andchemical fertilizer (250 kg/ha of 5e20e20 formulation), witha distance of 0.5 m between rows and plants. After emergence,plots (4 � 4 m) were delimited with wood sticks, with a total of 64plants per plot, considering two lines on each side of the area asborder lines. One month after plant emergence soil-cover fertil-ization was done with urea (70 Kg/ha).

After plants gained an average of eight leaves, one third instarS. furgiperda larva was placed in each plant. After 24 h, plants weremanually sprayed with the aid of a pressurized sprayer (PCP-1PGuarany�) containing spraying mixture (close to soaking point)prepared with the following treatments: 1- insecticide at half therecommended dose; 2- entomopathogenic nematodes and 3- combi-nation of each insecticide (half doses); 4- control (water) and eachnematode. Treatments were applied between 5:00 and 6:30 p.m.During the first experimental period (2007/2008), the apex of eachplant was covered with a fine cloth bag after spraying and tied withafine rope toprevent larvae escaping.During the secondexperimentalperiod (2008/2009), plant apexes were not covered. Treatments wereapplied in 10 and 30 plants selected randomly inside each plot in

Table 2Interaction between insecticides and entomopathogenic nematodes Heterorhabditis indica over mortality (mean � SE) of Spodoptera frugiperda larvae two and four days aftertreatment in laboratory (temperature 22 � 1 �C, relative humidity 70 � 10% and photoperiod of 12 h).

2 days after treatment 4 days after treatment

Treatments Pob Ic Treatments Pob Ic

Lorsban 86.0 � 6.00 ad Synergistic Lorsban 90.0 � 4.47 ae AntagonistGalgotrin 72.0 � 3.74 ab Additive Stallion 90.0 � 7.75 a AntagonistTracer 70.0 � 7.75 abc Additive Galgotrin 88.0 � 4.90 ab AntagonistIntrepid 66.0 � 9.80 abc Additive Match 88.0 � 4.90 ab AntagonistDeltaphos 64.0 � 4.00 abc Additive Fastac 88.0 � 2.00 ab AntagonistKarate Zeon 64.0 � 6.00 abc Antagonist Controla 88.0 � 2.00 ab e

Controla 60.0 � 5.48 abcd e Vexter 86.0 � 6.78 ab AntagonistCertero 54.0 � 7.48 bcde Antagonist Dimilin 84.0 � 2.45 ab AntagonistDimilin 52.0 � 8.60 bcde Antagonist Karate Zeon 82.0 � 5.83 ab AntagonistStallion 52.0 � 3.74 bcde Antagonist Intrepid 78.0 � 3.74 ab AntagonistMatch 48.0 � 15.3 bcde Antagonist Turbo 76.0 � 5.10 ab AntagonistFastac 46.0 � 10.7 bcde Antagonist Valon 76.0 � 5.10 ab AntagonistTalcord 44.0 � 5.10 bcde Antagonist Tracer 74.0 � 7.48 ab AntagonistVexter 42.0 � 9.70 bcde Antagonist Certero 68.0 � 9.17 ab AntagonistHostathion 42.0 � 7.35 bcde Antagonist Talcord 68.0 � 9.17 ab AntagonistTurbo 40.0 � 4.47 cde Antagonist Hostathion 68.0 � 8.00 ab AntagonistValon 42.0 � 8.00 cde Antagonist Deltaphos 66.0 � 4.00 ab AntagonistDecis 32.0 � 10.6 de Antagonist Decis 58.0 � 3.74 b AntagonistFolidol 24.0 � 6.78 e Antagonist Folidol 58.0 � 3.74 b Antagonist

a Entomopathogenic nematode without insecticide.b Po: Percentage of observed mortality.c Interaction between treatments: antagonist (X2 > 3.84 e Po < Pe), additive (X2 < 3.84), synergistic (X2 > 3.84 e Po > Pe), being 3,84 correspondent to 1 liberty degree at

P � 0.05.d Means followed by the same letter are not statistically different by the Tukey test at P � 0.05.

A.S. Negrisoli et al. / Crop Protection 29 (2010) 677e683 679

experiment1and2respectively, consideringeachplot asone replicate.Each treatment had five replicates. The experimental statistical designwas completely randomized and larval mortality was evaluated fourdays after treatments.

2.3. Data analysis

S. frugiperda mortality data was submitted to analysis of vari-ance and differences between treatment means were estimated byTukey's HDS test at P < 0.05 probability. In order to evaluate theeffect of the interaction between insecticides and nematodes

Table 3Interaction between insecticides and entomopathogenic nematodes Steinernema carpocaafter treatment in laboratory (temperature 22 � 1 �C, relative humidity 70 � 10% and ph

2 days after treatment

Treatments Pob Ic

Match 90.0 � 4.47 ae AdditiveLorsban 90.0 � 3.16 a AdditiveGalgotrin 82.0 � 3.74 a AdditiveControla 78.0 � 5.83 a e

Karate Zeon 70.0 � 3.16 abc AntagonistIntrepid 62.0 � 5.48 abcd AntagonistVexter 60.0 � 8.37 abcd AntagonistDimilin 56.0 � 10.9 abcd AntagonistFastac 46.0 � 2.45 bcde AntagonistStallion 40.0 � 7.07 cde AntagonistDecis 38.0 � 9.70 de AntagonistHostathion 34.0 � 7.48 de AntagonistTracer 34.0 � 5.10 de AntagonistDeltaphos 32.0 � 5.83 de AntagonistCertero 26.0 � 6.78 e AntagonistValon 26.0 � 5.10 e AntagonistFolidol 22.0 � 7.35 e AntagonistTalcord 22.0 � 3.74 e AntagonistTurbo 22.0 � 3.74 e Antagonist

a Entomopathogenic nematode without insecticide.b Po: Percentage of observed mortality.c Interaction between treatments: antagonist (X2 > 3.84 e Po < Pe), additive (X2 < 3.84

P � 0.05.d Means followed by the same letter are not statistically different by the Tukey test at

a binomial test was used; the observed and expected mortalitypercentages of Robertson and Preslier (1992) were compared,modified by Nishimatsu and Jackson (1998). Expected mortalitypercentage was obtained through the formula: Pe [ Po D (1LPo)(P1) D (1LPo)(1LP1)(P2), where Pe represents the expectedmortality for the combination of EPNs and insecticides; Po repre-sents mortality in control treatment (insect's natural mortality); P1represents mortality after treatment only with insecticide, and P2represents mortality after treatment with nematodes only.

Thevalue for chi-square (X2)was calculated through the formula:X2 [ (Lo L Le)/Le D (Do L De)/De, where Lo is the number of living

psae over mortality (mean � SE) of Spodoptera frugiperda larvae two and four daysotoperiod of 12 h).

4 days after treatment

Treatments Pob Ic

Lorsban 98.0 � 2.00 ad AntagonistStallion 98.0 � 2.00 a AntagonistGalgotrin 96.0 � 1.87 ab AntagonistMatch 94.0 � 2.45 ab AntagonistFastac 94.0 � 2.45 ab AntagonistControla 92.0 � 4.06 ab AntagonistVexter 86.0 � 5.10 abc AntagonistDimilin 86.0 � 5.10 abc AntagonistKarate Zeon 84.0 � 6.78 abc AntagonistIntrepid 78.0 � 4.90 abc AntagonistTurbo 76.0 � 8.60 abc AntagonistValon 74.0 � 2.45 abc AntagonistTracer 74.0 � 2.45 abc AntagonistCertero 72.0 � 8.60 abc AntagonistTalcord 72.0 � 8.60 abc AntagonistHostathion 64.0 � 2.45 bcd AntagonistDeltaphos 64.0 � 2.45 bcd AntagonistDecis 56.0 � 9.80 cd AntagonistFolidol 36.0 � 10.8 d Antagonist

), synergistic (X2 > 3.84 e Po > Pe), being 3,84 correspondent to 1 liberty degree at

P � 0.05.

Table 4Interaction between insecticides and Steinernema glaseri over mortality (mean � SE) of Spodoptera frugiperda larvae, two and four days after treatment in laboratory(temperature 22 � 1 �C, relative humidity 70 � 10% and photoperiod of 12 h).

2 days after treatment 4 days after treatment

Treatments Pob Ic Treatments Pob Ic

Vexter 88.0 � 5.83 ad Synergistic Vexter 100 � 0.0 ae AdditiveLorsban 68.0 � 17.1 ab Synergistic Lorsban 96.0 � 2.45 ab AntagonistKarate Zeon 66.0 � 4.00 abc Additive Galgotrin 94.0 � 4.00 ab AntagonistMatch 52.0 � 9.17 bcd Synergistic Karate Zeon 94.0 � 2.45 ab AntagonistHostathion 50.0 � 6.32 bcd Additive Match 90.0 � 4.47 ab AntagonistIntrepid 48.0 � 4.90 bcd Additive Hostathion 88.0 � 3.74 ab AntagonistGalgotrin 48.0 � 9.80 bcd Additive Intrepid 88.0 � 3.74 ab AntagonistCertero 48.0 � 10.6 bcd Additive Certero 86.0 � 2.45 ab AntagonistTalcord 46.0 � 6.78 bcd Additive Talcord 86.0 � 2.45 ab AntagonistTracer 44.0 � 6.00 bcd Additive Tracer 86.0 � 2.45 ab AntagonistTurbo 40.0 � 7.07 bcd Additive Turbo 82.0 � 7.35 ab AntagonistValon 38.0 � 11.6 bcd Additive Valon 82.0 � 7.35 ab AntagonistDeltaphos 36.0 � 10.3 cd Antagonist Deltaphos 80.0 � 5.48 abc AntagonistDimilin 34.0 � 2.45 d Antagonist Dimilin 80.0 � 5.48 abc AntagonistStallion 32.0 � 3.74 d Additive Stallion 76.0 � 6.00 abc AntagonistDecis 30.0 � 5.48 d Antagonist Decis 68.0 � 5.83 bc AntagonistFolidol 30.0 � 4.47 d Antagonist Folidol 68.0 � 5.83 bc AntagonistFastac 30.0 � 3.16 d Antagonist Fastac 50.0 � 5.48 c AntagonistControla 28.0 � 3.74 d e Controla 50.0 � 5.48 c e

a Entomopathogenic nematode without insecticide.b Po: Percentage of observed mortality.c Interaction between treatments: antagonist (X2 > 3.84 e Po < Pe), additive (X2< 3.84), synergistic (X2 > 3.84 e Po > Pe), being 3,84 correspondent to 1 liberty degree at

P � 0.05.d Means followed by the same letter are not statistically different by the Tukey test at P � 0.05.

A.S. Negrisoli et al. / Crop Protection 29 (2010) 677e683680

insects observed, Le is the number of living insects expected, Do isthe number of dead insects observed and De is the number of deadinsects expected. In order to use a value of X2 ¼ 3.84, a degree offreedom was considered (n�1) and P ¼ 0.05, and the additiveinteraction was represented by X2 < 3.84, antagonism by X2 > 3.84and Po < Pe, and synergism by X2 > 3.84 and Po > Pe.

3. Results and discussion

3.1. Efficacy of insecticide and EPN mixture to control Spodopterafrugiperda in laboratory

Larval mortality of S. frugiperda was significantly different afterthe treatments with insecticides þ nematodes for two days

Table 5Effect of insecticides over mortality (mean � SE) of Spodoptera frugiperda larvae attwo and four days after exposure in laboratory (temperature 22 � 1 �C, relativehumidity 70 � 10% and photoperiod of 12 h).

Treatment 2 days after treatment Treatment 4 days after treatment

Folidol 42.0 � 3.74 a Folidol 98.0 � 2.00 aKarate Zeon 42.0 � 3.74 a Certero 96.0 � 2.45 abHostathion 32.0 � 4.90 a Decis 96.0 � 2.45 abStallion 32.0 � 4.90 a Lorsban 94.0 � 4.00 abcTalcord 32.0 � 3.74 a Fastac 94.0 � 2.45 abcDimilin 32.0 � 2.00 a Hostathion 92.0 � 2.00 abcDeltaphos 30.0 � 7.07 a Match 88.0 � 5.83 abcCertero 30.0 � 4.47 a Talcord 88.0 � 2.00 abcGalgotrin 30.0 � 4.47 a Turbo 86.0 � 6.78 abcLorsban 26.0 � 6.00 a Deltaphos 86.0 � 4.00 abcMatch 26.0 � 4.00 a Valon 80.0 � 17.6 abcFastac 26.0 � 2.45 a Galgotrin 80.0 � 3.16 abcdValon 24.0 � 5.10 a Stallion 80.0 � 6.32 abcdIntrepid 22.0 � 7.35 a Vexter 80.0 � 7.35 abcdTracer 22.0 � 6.63 a Intrepid 66.0 � 4.00 bcdDecis 22.0 � 2.00 a Tracer 66.0 � 10.3 bcdTurbo 20.0 � 3.16 a Karate Zeon 64.0 � 8.12 cdVexter 18.0 � 4.00 a Dimilin 52.0 � 4.90 dControla 6.0 � 4.00 b Controla 6.0 � 4.00 e

a Water without insecticide.

(F ¼ 5.54; df ¼ 53, P ¼ 0.001; Cv ¼ 22.9). The insect mortality wasstatistically equal after the insecticides have been applied withoutEPNs (P ¼ 0.19), differing only for the control (water) (Tables 2e5).In H. indica þ insecticides interaction, synergism was registeredonly with Lorsban� (86%), additive effect with Galgotrin�, Tracer�,Intrepid� and Deltaphos� (Table 2). In S. carpocapsaeþ insecticidesinteraction, Match�, Lorsban�, Galgotrin�, Intrepid�, KarateZeon�, Vexter� and control caused highermortality of S. frugiperda,with an additive effect observed in insect mortality with the firstthree insecticides (Table 3). In S. glaseri þ insecticides interaction,mortality in control treatment was of 28%, evidencing the lowpathogenicity of this EPN two days after inoculation. In this inter-action, synergismwith Vexter�, Lorsban�, Match, antagonist effectwith Deltaphos�, Dimilin�, Folidol� and Fastac� and an additiveeffect on pest mortality with the rest of the insecticides wasobserved (Table 4).

Larval mortality of S. frugiperdawas significantly different whenthese were submitted to treatments with insecticidesþ nematodesfor four days (F ¼ 5.54; P ¼ 0.001; df ¼ 53, Cv ¼ 22.9) (Tables 2e5).Additive interaction was observed only in combination of Vexter�

and S. glaseri, with the rest being considered as antagonist. InH. indicaþ insecticides interaction, Lorsban� and Stallion� resultedin higher insect mortality, with Decis� and Folidol� as the insec-ticides resulting in the lowest mortality of S. frugiperda (Table 2). InS. carpocapsaeþ insecticides interaction, Fastac�, Stallion� resultedin the highest mortalities when applied together with the ento-mopathogen (Table 3). In contrast, among all combinations ofS. glaseri þ insecticide, Decis�, Folidol� and Fastac� resulted in thelowest mortality, close to control (Table 4). In treatments with onlyinsecticide application, only Intrepid�, Tracer�, Karate Zeon� andDimilin� resulted in mortality lower than 80%, which is consideredthe minimum accepted for insecticides at full dose (Table 5).

In general, an increase in S. frugiperda larval mortality submittedto treatments (nematodes þ insecticides) was observed over time,with the difference between evaluation periods being higher ininsecticides treatments (Tables 2e4). These results suggest thatnematodes increase S. frugiperda mortality two days after

A.S. Negrisoli et al. / Crop Protection 29 (2010) 677e683 681

treatment application if compared to treatments without nema-todes. In particular, insect mortality in treatments with Certero�,Tracer�, Intrepid�, Galgotrin� and Lorsban� in combination withH. indica was equal in both evaluation periods, with the sameresults observed using the insecticides treatments with Hos-tathion�, Intrepid�, Galgotrin�, Karate Zeon� and Match� whenmixed with S. carpocapse and the product Karate Zeon� with S.glaseri.

Concerning the significant increase in S. frugiperda mortalitywith time, in treatments with application of EPNs associated toinsecticides, Epsky and Capinera (1993) verified that S. frugiperdamortality was correlated positively (exponential response) withtime exposure to S. capocapsae in laboratory, in agree with thisstudy. In addition, mortality increase of Popillia japonica Newman,1841 and Exomala orientalis (Waterhouse, 1875) (Coleoptera: Scar-abaeidae) was observed associated with time exposure in labora-tory and in the field with the combination of neo-nicotinicinsecticides (imidaclopride and tiametoxam) and Heterorhabditisbacteriophora (Koppenhöfer et al., 2002, 2003).

The species S. glaseri caused the lowest mortality in bothexposure periods to the pest if compared with H. indica and S.carpocapsae. Despite the difficulty in comparing these studies,Garcia et al. (2008) registered lower mean lethal dose (LD50) ofSteinernema sp. compared to H. indica (same strain as in presentwork) to control third instar larvae of S. frugiperda. Besides, thereare evidence that H. indica is less virulent than S. carpocapsae andSteinernema sp. Analogous. Thus, Fuxa et al. (1988) verified thatSteinernema feltiae showed low LD50 (10.8 IJs/mL) over fifth instarlarvae of S. frugiperda in laboratory.

There are few studies evaluating efficacy of mixtures of insec-ticides and entomopathogenic nematodes aiming to control Lepi-doptera, with the average being tested on Scarabaeidae species.Baweja and Sehgal (1997), evaluating the interaction ofH. bacteriophora and malathion to control Spodoptera litura inlaboratory, observed that the nematode's parasitism rate decreasedwith the increase in insecticide dose. On the other hand, Zhanget al. (1994), while determining the effect of 14 organic-phospho-rates, seven carbamates, four pirethroids, cartap and imidacloprideon S. carpocapsae infectivity on S. litura, observed that only cartap(10 mg/mL) and profenofos (100 mg/mL), after product rinsing,reduced nematode pathogenicity.

The synergistic and additive effect for most treatments withassociation of insecticides and S. glaseri and with two days' expo-sure, showed a relatively low performance of S. frugiperdamortalityif compared with other species (Table 4). This is in agreement withthe studies on Scarabaeidae species that have shown low suscep-tibility to some EPNs. In this regard, Koppenhöfer and Kaya (1998)and Koppenhöfer et al., (2002) demonstrated that combining EPNs(H. bacteriophora and S. glaseri) and neonicotinoids (imicloprideand tiametoxam) has a synergistic effect to control P. japonica,Cyclocephala hirta LeConte, 1861, Cyclocephala pasadenae Casey,1915 (Coleoptera: Scarabaeidae) and E. orientalis in laboratory andgreen-house conditions.

Concerning the interaction of insecticides with other entomo-pathogenic organisms aiming to control pests from the genusSpodoptera, Jayanthi and Padmavathamma (2001) evaluated theinteraction among Bacillus thuringiensis var. kurstaki (Berliner,1915)(Dipel�), nuclear polyhedrosis virus (SplVPN) and Beauveria bassi-ana (Bals.), with half of the recommended dose of fenvalerate andmonocrotofos (unknown commercial product). They observedhigher control of S. litura with the mixture of chemical and bio-logical insecticides, with higher pest mortality observed in treat-ment using Dipel plus fenvalerate. In a similar way, Khattab (2007)observed an increase in Spodoptera littoralis mortality after expo-sure to a mixture of espinosine (Spinosad�) plus the nuclear

polyhedrosis virus (SpliVPN), with reduction of the entomopatho-gen's lethal dose on the pest in association with the insecticide.

3.2. Efficacy of EPNs and insecticide mixture to control Spodopterafrugiperda in the field

Generally, the mortality of third instar larvae of S. frugiperdawasdifferent among treatments in the first experiment in the field(2007/2008) (F ¼ 89.9; df ¼ 8; P ¼ 0.001; Cv ¼ 9.36). A significantincrease in insect mortality was observed when EPNs were appliedin association with the insecticides in comparison with only EPNs,with additive interaction in these treatments, although the incre-ment was not significant if compared to the insecticide Lorsban�

alone and associated with EPNs. Third instar larval mortality of S.frugiperda was different between treatments during the secondexperiment in the field (2008/2009) (F ¼ 46.9; df ¼ 8; P ¼ 0.001;Cv¼13.88). A significant increment in insectmortalitywasobservedwhen EPNs were applied in association with the insecticides incomparison with only EPNs, with additive interaction in thesetreatments, although the increment was not significant if comparedto the insecticides Lorsban� and Match� alone and associated withEPNs. Among all treatments, association of H. indica and Match�

resulted in thehighestmortality of S. frugiperda, with 62.5% (assay1)and 57.5% mortality (assay 2), despite not being significantlydifferent from the other treatments with the nematodeeinsecticidecombination (Figs. 1 and 2).

Low mortality produced by the products when isolated may berelated to the product concentration, since they were applied athalf of the recommended dose. These results evidence a highermortality rate during the first experimental period (2007/2008), ifcompared to the second one (2008/2009). This fact may be relatedto a difference in methodology used in both periods. During thefirst period, plant apexes were covered with thin cloth bags withthe objective of preventing larvae from escaping and, as a conse-quence, a micro-climatic condition was established inside the bagsthat may have favored EPNs. However, as this is an artificial situa-tion in the field, during the second period experiment plants werenot covered, so abiotic factors such as temperature, UV radiationand desiccation may be responsible for the lower performance ofEPNs, as reported by Glazer (2002).

Results revealed that insecticides were the most importantfactor in larval mortality, in detriment of EPNs, which showed lowefficacy in field conditions with no more than 22.5% mortality intreatment with S. carpocapsae, a rate higher than that observed byGarcia et al. (2008) whenmortality equal to zerowas observed afterapplication of H. indica IBCB-n5 and Steinernema sp. applied overS. frugiperda in the corn husk under controlled conditions. Theseauthors evaluated different application technologies and surfactanteffect on EPNs, observing that IJs of both species were compatiblewith all application techniques and adjuvants, so these factors arenot responsible for EPN inefficacy in laboratory.

Some hypotheses were proposed by Garcia (2006) regardinginefficacy of EPNs to control S. frugiperda when directed to cornhusk using high volume of mixture (800 L/ha): the insertion angleof the corn leaf of up to 30� and narrow channel shaped, mayprevent the formation of a water layer essential for nematodemovement towards larvae, as well as for inoculum concentration.Besides this fact, the author emphasizes that application in highvolumes of mixture are far over the needs for chemical control ofthis insect, and may make control economically unfeasible as wellas forming a water film so thick that it may hinder EPN movement.

In agreement with observations recorded with EPNs andinsecticides in the present work, Mendez et al. (2002) verified thatthe mixture of espinosine at 3ppm (Tracer�) with nucleopolyhe-drovirus of S. frugiperda (SfMNPV) resulted in higher mortality of

Fig. 1. Mortality (% � SE) of Spodoptera frugiperda larvae four days after exposure to association between insecticides and entomopathogenic nematodes Steinernema carpocapsaeand Heterorhabditis indica in the field. Letters above the boxes show differences between each insecticide alone and associated to each nematode (Tukey, P � 0,05). The signal “þ”

shows additive interaction between EPN and insecticide (Capão do Leão, RS, 2007/2008).

A.S. Negrisoli et al. / Crop Protection 29 (2010) 677e683682

this pest (90%) in the field, compared with the entomopathogenapplied without insecticide (12e37%).

Research is needed in order to determine the economicallyoptimal concentration of EPNs in the field for application directed tocorn husk, associated with selected insecticides (Lorsban� andMatch�). Besides that, pre-pupal and pupal stages of the insect,which are associated with soil, may be susceptible to EPNs in fieldconditions, considering that Molina-Ochoa et al. (2003) observednatural occurrence of Steinernematidae and Heterorhabditidae insoil samples collected in 64 localities (10.9%) associated with pop-ulations of S. frugiperda in corn, sorghum and pastures in Mexico.

In this regard, Cabanillas and Raulston (1996) evaluated appli-cation of Steinernema riobravis and S. carpocapse on Helicoverpa zeapupae and pre-pupae in corn crops in Texas, USA; they observedmortality of up to 97% caused by S. riobravis in the field, for thehighest concentration (20 � 109/ha), applied through irrigationrows, with persistence of up to 36 days after the treatment andwith80% of pest control when compared with 14% pest control (natural)in the control treatment. S. carpocapsae was not efficient for pestcontrol due to high soil temperatures (approximately 31 �C at20 cm depth). Thus, it is important to emphasize that S. riobraviswas isolated in 1990 in Texas, USA, being a native strain andtherefore adapted to local conditions, unlike S. carpocapsae (strainAll), which originated from the state of Georgia, USA, with a rela-tively more moderate climate.

Fig. 2. Mortality (% � SE) of Spodoptera frugiperda larvae four days after exposure to assoccapsae and Heterorhabditis indica in the field. Letters above the boxes show differences betwe“þ” shows additive interaction between EPN and insecticide (Capão do Leão, RS, 2008/200

In the same way, studies of indigenous EPNs isolates must beperformed in corn producing regions of Rio Grande do Sul, Brazil, inorder to identify and select more efficient isolates to control cornpests, especially those having a soil stage, such as S. frugiperda, H.zea and Diabrotica speciosa. Consequently, the high cost of EPNapplication may be minimized through the control of more thanone pest in this crop, thus reducing selection pressure. This isa potential strategy to be used against the problem of pest resis-tance caused by chemical insecticides.

The present work is the first to record the interaction of EPNswith chemical insecticides aiming to control S. frugiperda. Thecontrol by association, using an insecticide in sub-lethal dosecompatible with an entomopathogen, represents a possibility forcontrolling S. frugiperda in applications directed to the corn husk(foliar application), through spraying or even through irrigationtechnologies, depending on the grower's technological availability.

Unfortunately, the results showed low efficiency of mixing EPNswith insecticides to control the pest. Hence, new studies must beperformed to determine optimal EPNs and insecticide concentra-tions that may make technologies economically feasible, as well asevaluations of susceptibility of pre-pupal and pupal stages of S.frugiperda in the soil, with the possibility of a multiple controlaimed at other corn pests and in this way, improving economicalfeasibility of such technologies, reducing environmental costs ofproduction.

iation between insecticides and the entomopathogenic nematodes Steinernema carpo-en each insecticide alone and associated to each nematode (Tukey, P � 0,05). The signal9).

A.S. Negrisoli et al. / Crop Protection 29 (2010) 677e683 683

With regard to future prospects, and considering that in a shorttime the Brazilian market will have bio-insecticides based on EPNsto control pests in sugar-cane in the state of São Paulo (L.G. Leite, IB/CEIB, personal communication), the management of S. frugiperdaresistance to insecticides through theuse of suchproducts should beinvestigated. As pointed out by Omoto (2000), this strategy couldtake place bymeans of multiple attacks, with amixture of biologicaland chemical insecticides and by moderation, using reduceddosages of chemical insecticides associated with bio-insecticides.

Acknowledgements

The authors thank Dr. Claudia Dolinski from UniversidadeEstadual Norte Fluminense (UENF/RJ), Brazil, for reading themanuscript. Aldomario SantoNegrisoli Júnior received a scholarshipfrom the Conselho Nacional de Pesquisa e Desenvolvimento (CNPq).

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