Crop Protection 29 (2010) 677e683
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Efficacy of entomopathogenic nematodes (Nematoda: Rhabditida) and insecticide mixtures 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 Silva Laboratório 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
a b s t r a c t
Article history: Received 20 August 2009 Received in revised form 11 January 2010 Accepted 4 February 2010
The main insect pest in Brazilian corn is fall armyworm, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae). Entomopathogenic nematodes (EPNs) can be used to control this pest, and can be applied together with various insecticides. Thus, the objective of this work was to evaluate the efficacy of mixtures of EPNs and insecticides to control S. frugiperda in corn crops. In laboratory bioassays three species 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 between insecticides and EPNs on S. frugiperda larvae was evaluated against the insect's third instar, 2 and 4 days after applications in laboratory. Experiments in the field were performed in two consecutive years, with located application of H. indica and S. carpocapsae (250 IJs/cm2) mixed with chlorpyrifos (0.3 L/ha) and lufenuron (0.15 L/ha) on the corn husk. In laboratory, after two days exposure the interaction between chlorpyrifos and H. indica was synergistic, while interaction with cypermethrin, spinosad, methoxyfenozide and deltamethrin þ triazofos was additive, as was interaction between lufenuron, chlorpyrifos and cypermethrin with S. carpocapsae. In contrast, the interaction between chlorpyrifos (VexterÔ and LorsbanÔ) and lufenuron with S. glaseri was synergistic. In the field, the best treatment was the mixture of H. indica with lufenuron (0.15 L/ha), with 62.5% and 57.5% larval mortality in the two evaluation years in the field, respectively. Ó 2010 Elsevier Ltd. All rights reserved.
Keywords: Heterorhabditis indica Steinernema carpocapsae S. glaseri Fall armyworm Pesticides Biological control
1. Introduction Among the major insect pests in corn crops in Brazil, fall armyworm, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is considered the most important one. Insect infestations occur from plant emergence until ear formation, can result in yield reductions of up to 34%infestation (Cruz et al., 1995, 1997). Natural occurrence of Steinernematidae and Heterorhabditidae in soil samples collected in 64 localities (corresponding to 10.9%) associated with populations of S. frugiperda in corn and sorghum crops and pastures in Mexico, have demonstrated these entomopathogens' potential to control this pest in such agro-ecosystems (Molina-Ochoa et al., 2003). Various foliage application technologies to control S. frugiperda were tested using Heterorhabditis indica Poinar, Karunakar and David, 1992 and Steinernema sp and the
* Corresponding author. Tel.: þ55 53 3275 9031. E-mail address:
[email protected] (A.S. Negrisoli). 0261-2194/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2010.02.002
compatibility of these EPNs with tensioactives, resulting in low efficacy in the pest control. (Garcia et al., 2008). In chemical control of S. frugiperda, the use of high concentrations of the same active ingredient allied with to the absence of refuge areas leads to high selective pressure, resulting in development of resistance to these chemicals. On the other hand, the combination of a stressor and a natural enemy may increase or decrease insect adaptability to the agrochemicals, depending on the intrinsic characteristic of the system (Gould et al., 1991; Johnson et al., 1997; Koppenhöfer and Kaya, 1998). Thus, the objective of this work was to evaluate the efficacy of control of S. frugiperda in the laboratory and field, by associating insecticides and EPNs. 2. Materials and methods Insect rearing, EPN multiplication and bioassays were performed at the Laboratório de Biologia de Insetos e Controle Biológico of the Departmento de Fitossanidade at the Faculdade de Agronomia “Eliseu Maciel” of the Universidade Federal de Pelotas/UFPel, Brazil.
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Table 1 Characteristics 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 ha1)b
Spray volume (L ha1)c
0.050 0.025 0.300 0.300 0.100 0.175
200e300 150 100e300 100e300 10e20d 300
0.050 0.012 0.015
200e400 300 250
Technical
Commercial
Beta cyfluthrin Cypermethrin Chlorpyrifos Chlorpyrifos Deltamethrin Deltamethrin þ Triazofos Diflubenzuron Gamacyhalothrin Lambdacyhalothrin
Turbo Galgotrin Lorsban 480 BR Vexter Decis 25 EC Deltaphos EC
ECe EC EC EC EC EC
II I II II III I
SCM SCM CI CI SCM SCM þ CI
Dimilin Stallion 150 CS Karate Zeon 250 CS Match Intrepid 240 SC Folidole 600 Talcord 250 CE Valon 384 EC Tracer Hostathion 400 BR Certero Fastac
WP CS CS
IV III III
Csi SCM SCM
Pyrethroid Pyrethroid Organophosphate Organophosphate Pyrethroid Pyrethroid þ Organophosphate Benzoylurea Pyrethroid Pyrethroid
EC SC EC EC EC SC EC
IV IV II I II III II
Csi Mmh CI SCM SCM NS CI
Benzoylurea Diacylhydrazine Organophosphate Pyrethroid Pyrethroid Espinosines Organophosphate
0.150 0.075 0.337 0.050 0.0325 0.050 0.250
150e400 200e400 100 e e 100e300 100e400
SC SC
IV III
Csi SCM
Benzoylurea Pyrethroid
0.025 0.025
200e300 150e300
Lufenuron Methoxyfenozide Methil parathion Permethrin Permethrin Espinosad Triazofos Triflumuron Alpha cypermethrin a b c d e
T.C. ¼ Toxicological class of the a.i. 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. Corresponding to terrestrial application. Corresponding to aereal application. Recommended for S. frugiperda in corn when the study was performed.
Larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae) were reared in a controlled room at 28 2 C, 60 10% relative humidity (RH) and 12 h photoperiod (Parra, 1998). Fall armyworm were reared in a controlled room at 25 2 C, 70 10% relative humidity (RH) and 12 h photoperiod, according to the methodology proposed by Greene et al. (1976). Entomopathogenic nematodes (EPNs) H. indica Poinar, 1976 strain IBCB-n5 (isolated in Itapetininga, São Paulo, Brazil), Steinernema carpocapsae (Weiser, 1955) strain Sta Rosa and S. glaseri (Steiner, 1929) strain Sta Rosa (isolated in Santa Rosa do Viterbo, Sao Paulo, Brazil), were multiplied on last instar G. mellonella, according to methodology described by Kaya and Stock (1997). Every day IJs were collected and stored in (zip-lock type) plastic bags containing polyurethane sponge blocks. In order to reduce contaminant proliferation EPNs were maintained in an environmentally controlled chamber at 12 C and 24 h darkness, using distilled water with 0.1 % sodium hypochlorite solution during the whole process.
2.1. Efficacy of insecticide and EPN mixture to control Spodoptera frugiperda in laboratory Third instar S. frugiperda were submitted to the treatments in plastic boxes (150 mL) containing wet sand (10%, w/w) as substrate and 5g of artificial diet, and inoculated with the following treatments: 1- insecticide at the half recommended dose; 2- entomopathogenic nematodes; 3- a combination of each insecticide (half recommended dose); 4- control (water) and each nematode, considering ten larvae as one replicate, with a total of ten replicates for each treatment. Localized application of the treatments was made with a micro-pipette (100 mL capacity), corresponding to a 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 was adjusted to 100 IJs per container (2.6 IJs/cm2). Each container received one larva and mortality was registered 2 and 4 days after application. The experimental statistical design was factorial, mortality values were submitted to analysis of variance and
differences between treatment means were estimated by Tukey's HDS test at P < 0.05 probability. 2.2. Efficacy of insecticide and EPN mixture, to control Spodoptera frugiperda in the field The experiments were performed in the county of Capão do Leão, near the Federal University of Pelotas, Rio Grande do Sul, Brazil, during 2007/2008 (first assay) and 2008/2009 (second assay). The efficacy of control of S. frugiperda in corn plantations in the field by the combination of two nematode species (H. indica and Steinernema carpocapse, 250 IJs/cm2) and two insecticides (MatchÔ 0.15 L/ha and LorsbanÔ 0.3 L/ha) previously selected in the laboratory, was evaluated. The experimental area was prepared by incorporating organic manure in the soil (87.5 m3/ha), and weeds were controlled mechanically. Corn (cv. AS 32 AgroesteÔ) planting was performed with the aid of a manual planter (two seeds per hollow) and chemical fertilizer (250 kg/ha of 5e20e20 formulation), with a distance of 0.5 m between rows and plants. After emergence, plots (4 4 m) were delimited with wood sticks, with a total of 64 plants per plot, considering two lines on each side of the area as border lines. One month after plant emergence soil-cover fertilization was done with urea (70 Kg/ha). After plants gained an average of eight leaves, one third instar S. furgiperda larva was placed in each plant. After 24 h, plants were manually sprayed with the aid of a pressurized sprayer (PCP-1P GuaranyÔ) containing spraying mixture (close to soaking point) prepared with the following treatments: 1- insecticide at half the recommended dose; 2- entomopathogenic nematodes and 3- combination of each insecticide (half doses); 4- control (water) and each nematode. Treatments were applied between 5:00 and 6:30 p.m. During the first experimental period (2007/2008), the apex of each plant was covered with a fine cloth bag after spraying and tied with a fine rope to prevent larvae escaping. During the second experimental period (2008/2009), plant apexes were not covered. Treatments were applied in 10 and 30 plants selected randomly inside each plot in
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Table 2 Interaction between insecticides and entomopathogenic nematodes Heterorhabditis indica 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
Lorsban Galgotrin Tracer Intrepid Deltaphos Karate Zeon Controla Certero Dimilin Stallion Match Fastac Talcord Vexter Hostathion Turbo Valon Decis Folidol
86.0 72.0 70.0 66.0 64.0 64.0 60.0 54.0 52.0 52.0 48.0 46.0 44.0 42.0 42.0 40.0 42.0 32.0 24.0
6.00 3.74 7.75 9.80 4.00 6.00 5.48 7.48 8.60 3.74 15.3 10.7 5.10 9.70 7.35 4.47 8.00 10.6 6.78
ad ab abc abc abc abc abcd bcde bcde bcde bcde bcde bcde bcde bcde cde cde de e
Ic
Treatments
Pob
Synergistic Additive Additive Additive Additive Antagonist e Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist
Lorsban Stallion Galgotrin Match Fastac Controla Vexter Dimilin Karate Zeon Intrepid Turbo Valon Tracer Certero Talcord Hostathion Deltaphos Decis Folidol
90.0 90.0 88.0 88.0 88.0 88.0 86.0 84.0 82.0 78.0 76.0 76.0 74.0 68.0 68.0 68.0 66.0 58.0 58.0
Ic
4.47 7.75 4.90 4.90 2.00 2.00 6.78 2.45 5.83 3.74 5.10 5.10 7.48 9.17 9.17 8.00 4.00 3.74 3.74
ae a ab ab ab ab ab ab ab ab ab ab ab ab ab ab ab b b
Antagonist Antagonist Antagonist Antagonist Antagonist e Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist
a
Entomopathogenic nematode without insecticide. 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. b
experiment 1 and 2 respectively, considering each plot as one replicate. Each treatment had five replicates. The experimental statistical design was completely randomized and larval mortality was evaluated four days after treatments. 2.3. Data analysis S. frugiperda mortality data was submitted to analysis of variance and differences between treatment means were estimated by Tukey's HDS test at P < 0.05 probability. In order to evaluate the effect of the interaction between insecticides and nematodes
a binomial test was used; the observed and expected mortality percentages of Robertson and Preslier (1992) were compared, modified by Nishimatsu and Jackson (1998). Expected mortality percentage was obtained through the formula: Pe [ Po D (1LPo) (P1) D (1LPo)(1LP1)(P2), where Pe represents the expected mortality for the combination of EPNs and insecticides; Po represents mortality in control treatment (insect's natural mortality); P1 represents mortality after treatment only with insecticide, and P2 represents mortality after treatment with nematodes only. The value 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
Table 3 Interaction between insecticides and entomopathogenic nematodes Steinernema carpocapsae 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 Treatments Match Lorsban Galgotrin Controla Karate Zeon Intrepid Vexter Dimilin Fastac Stallion Decis Hostathion Tracer Deltaphos Certero Valon Folidol Talcord Turbo a
4 days after treatment Po
b
90.0 90.0 82.0 78.0 70.0 62.0 60.0 56.0 46.0 40.0 38.0 34.0 34.0 32.0 26.0 26.0 22.0 22.0 22.0
I
4.47 3.16 3.74 5.83 3.16 5.48 8.37 10.9 2.45 7.07 9.70 7.48 5.10 5.83 6.78 5.10 7.35 3.74 3.74
e
a a a a abc abcd abcd abcd bcde cde de de de de e e e e e
c
Additive Additive Additive e Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist
Treatments Lorsban Stallion Galgotrin Match Fastac Controla Vexter Dimilin Karate Zeon Intrepid Turbo Valon Tracer Certero Talcord Hostathion Deltaphos Decis Folidol
Ic
Po b 98.0 98.0 96.0 94.0 94.0 92.0 86.0 86.0 84.0 78.0 76.0 74.0 74.0 72.0 72.0 64.0 64.0 56.0 36.0
2.00 2.00 1.87 2.45 2.45 4.06 5.10 5.10 6.78 4.90 8.60 2.45 2.45 8.60 8.60 2.45 2.45 9.80 10.8
d
a a ab ab ab ab abc abc abc abc abc abc abc abc abc bcd bcd cd d
Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist
Entomopathogenic nematode without insecticide. 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. b
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Table 4 Interaction 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
Vexter Lorsban Karate Zeon Match Hostathion Intrepid Galgotrin Certero Talcord Tracer Turbo Valon Deltaphos Dimilin Stallion Decis Folidol Fastac Controla
88.0 68.0 66.0 52.0 50.0 48.0 48.0 48.0 46.0 44.0 40.0 38.0 36.0 34.0 32.0 30.0 30.0 30.0 28.0
5.83 17.1 4.00 9.17 6.32 4.90 9.80 10.6 6.78 6.00 7.07 11.6 10.3 2.45 3.74 5.48 4.47 3.16 3.74
ad ab abc bcd bcd bcd bcd bcd bcd bcd bcd bcd cd d d d d d d
Ic
Treatments
Pob
Ic
Synergistic Synergistic Additive Synergistic Additive Additive Additive Additive Additive Additive Additive Additive Antagonist Antagonist Additive Antagonist Antagonist Antagonist e
Vexter Lorsban Galgotrin Karate Zeon Match Hostathion Intrepid Certero Talcord Tracer Turbo Valon Deltaphos Dimilin Stallion Decis Folidol Fastac Controla
100 0.0 ae 96.0 2.45 ab 94.0 4.00 ab 94.0 2.45 ab 90.0 4.47 ab 88.0 3.74 ab 88.0 3.74 ab 86.0 2.45 ab 86.0 2.45 ab 86.0 2.45 ab 82.0 7.35 ab 82.0 7.35 ab 80.0 5.48 abc 80.0 5.48 abc 76.0 6.00 abc 68.0 5.83 bc 68.0 5.83 bc 50.0 5.48 c 50.0 5.48 c
Additive Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist Antagonist e
a
Entomopathogenic nematode without insecticide. 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. b
insects observed, Le is the number of living insects expected, Do is the number of dead insects observed and De is the number of dead insects expected. In order to use a value of X2 ¼ 3.84, a degree of freedom was considered (n1) and P ¼ 0.05, and the additive interaction was represented by X2 < 3.84, antagonism by X2 > 3.84 and 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 Spodoptera frugiperda in laboratory Larval mortality of S. frugiperda was significantly different after the treatments with insecticides þ nematodes for two days Table 5 Effect of insecticides over mortality (mean SE) of Spodoptera frugiperda larvae at two and four days after exposure in laboratory (temperature 22 1 C, relative humidity 70 10% and photoperiod of 12 h). Treatment
2 days after treatment
Treatment
4 days after treatment
Folidol Karate Zeon Hostathion Stallion Talcord Dimilin Deltaphos Certero Galgotrin Lorsban Match Fastac Valon Intrepid Tracer Decis Turbo Vexter Controla
42.0 3.74 a 42.0 3.74 a 32.0 4.90 a 32.0 4.90 a 32.0 3.74 a 32.0 2.00 a 30.0 7.07 a 30.0 4.47 a 30.0 4.47 a 26.0 6.00 a 26.0 4.00 a 26.0 2.45 a 24.0 5.10 a 22.0 7.35 a 22.0 6.63 a 22.0 2.00 a 20.0 3.16 a 18.0 4.00 a 6.0 4.00 b
Folidol Certero Decis Lorsban Fastac Hostathion Match Talcord Turbo Deltaphos Valon Galgotrin Stallion Vexter Intrepid Tracer Karate Zeon Dimilin Controla
98.0 2.00 a 96.0 2.45 ab 96.0 2.45 ab 94.0 4.00 abc 94.0 2.45 abc 92.0 2.00 abc 88.0 5.83 abc 88.0 2.00 abc 86.0 6.78 abc 86.0 4.00 abc 80.0 17.6 abc 80.0 3.16 abcd 80.0 6.32 abcd 80.0 7.35 abcd 66.0 4.00 bcd 66.0 10.3 bcd 64.0 8.12 cd 52.0 4.90 d 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 was statistically equal after the insecticides have been applied without EPNs (P ¼ 0.19), differing only for the control (water) (Tables 2e5). In H. indica þ insecticides interaction, synergism was registered only with LorsbanÔ (86%), additive effect with GalgotrinÔ, TracerÔ, IntrepidÔ and DeltaphosÔ (Table 2). In S. carpocapsae þ insecticides interaction, MatchÔ, LorsbanÔ, GalgotrinÔ, IntrepidÔ, Karate ZeonÔ, VexterÔ and control caused higher mortality of S. frugiperda, with an additive effect observed in insect mortality with the first three insecticides (Table 3). In S. glaseri þ insecticides interaction, mortality in control treatment was of 28%, evidencing the low pathogenicity of this EPN two days after inoculation. In this interaction, synergism with VexterÔ, LorsbanÔ, Match, antagonist effect with DeltaphosÔ, DimilinÔ, FolidolÔ and FastacÔ and an additive effect on pest mortality with the rest of the insecticides was observed (Table 4). Larval mortality of S. frugiperda was significantly different when these were submitted to treatments with insecticides þ nematodes for 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 VexterÔ and S. glaseri, with the rest being considered as antagonist. In H. indica þ insecticides interaction, LorsbanÔ and StallionÔ resulted in higher insect mortality, with DecisÔ and FolidolÔ as the insecticides resulting in the lowest mortality of S. frugiperda (Table 2). In S. carpocapsae þ insecticides interaction, FastacÔ, StallionÔ resulted in the highest mortalities when applied together with the entomopathogen (Table 3). In contrast, among all combinations of S. glaseri þ insecticide, DecisÔ, FolidolÔ and FastacÔ resulted in the lowest mortality, close to control (Table 4). In treatments with only insecticide application, only IntrepidÔ, TracerÔ, Karate ZeonÔ and DimilinÔ resulted in mortality lower than 80%, which is considered the minimum accepted for insecticides at full dose (Table 5). In general, an increase in S. frugiperda larval mortality submitted to treatments (nematodes þ insecticides) was observed over time, with the difference between evaluation periods being higher in insecticides treatments (Tables 2e4). These results suggest that nematodes increase S. frugiperda mortality two days after
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treatment application if compared to treatments without nematodes. In particular, insect mortality in treatments with CerteroÔ, TracerÔ, IntrepidÔ, GalgotrinÔ and LorsbanÔ in combination with H. indica was equal in both evaluation periods, with the same results observed using the insecticides treatments with HostathionÔ, IntrepidÔ, GalgotrinÔ, Karate ZeonÔ and MatchÔ when mixed with S. carpocapse and the product Karate ZeonÔ with S. glaseri. Concerning the significant increase in S. frugiperda mortality with time, in treatments with application of EPNs associated to insecticides, Epsky and Capinera (1993) verified that S. frugiperda mortality was correlated positively (exponential response) with time exposure to S. capocapsae in laboratory, in agree with this study. In addition, mortality increase of Popillia japonica Newman, 1841 and Exomala orientalis (Waterhouse, 1875) (Coleoptera: Scarabaeidae) was observed associated with time exposure in laboratory and in the field with the combination of neo-nicotinic insecticides (imidaclopride and tiametoxam) and Heterorhabditis bacteriophora (Koppenhöfer et al., 2002, 2003). The species S. glaseri caused the lowest mortality in both exposure 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) of Steinernema sp. compared to H. indica (same strain as in present work) to control third instar larvae of S. frugiperda. Besides, there are evidence that H. indica is less virulent than S. carpocapsae and Steinernema sp. Analogous. Thus, Fuxa et al. (1988) verified that Steinernema feltiae showed low LD50 (10.8 IJs/mL) over fifth instar larvae of S. frugiperda in laboratory. There are few studies evaluating efficacy of mixtures of insecticides and entomopathogenic nematodes aiming to control Lepidoptera, with the average being tested on Scarabaeidae species. Baweja and Sehgal (1997), evaluating the interaction of H. bacteriophora and malathion to control Spodoptera litura in laboratory, observed that the nematode's parasitism rate decreased with the increase in insecticide dose. On the other hand, Zhang et al. (1994), while determining the effect of 14 organic-phosphorates, seven carbamates, four pirethroids, cartap and imidaclopride on 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 with association of insecticides and S. glaseri and with two days' exposure, showed a relatively low performance of S. frugiperda mortality if compared with other species (Table 4). This is in agreement with the studies on Scarabaeidae species that have shown low susceptibility 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 (imiclopride and tiametoxam) has a synergistic effect to control P. japonica, Cyclocephala hirta LeConte, 1861, Cyclocephala pasadenae Casey, 1915 (Coleoptera: Scarabaeidae) and E. orientalis in laboratory and green-house conditions. Concerning the interaction of insecticides with other entomopathogenic organisms aiming to control pests from the genus Spodoptera, Jayanthi and Padmavathamma (2001) evaluated the interaction among Bacillus thuringiensis var. kurstaki (Berliner, 1915) (DipelÔ), nuclear polyhedrosis virus (SplVPN) and Beauveria bassiana (Bals.), with half of the recommended dose of fenvalerate and monocrotofos (unknown commercial product). They observed higher control of S. litura with the mixture of chemical and biological insecticides, with higher pest mortality observed in treatment using Dipel plus fenvalerate. In a similar way, Khattab (2007) observed an increase in Spodoptera littoralis mortality after exposure to a mixture of espinosine (SpinosadÔ) plus the nuclear
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polyhedrosis virus (SpliVPN), with reduction of the entomopathogen's lethal dose on the pest in association with the insecticide. 3.2. Efficacy of EPNs and insecticide mixture to control Spodoptera frugiperda in the field Generally, the mortality of third instar larvae of S. frugiperda was different 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 significant increase in insect mortality was observed when EPNs were applied in association with the insecticides in comparison with only EPNs, with additive interaction in these treatments, although the increment was not significant if compared to the insecticide LorsbanÔ alone and associated with EPNs. Third instar larval mortality of S. frugiperda was different between treatments during the second experiment in the field (2008/2009) (F ¼ 46.9; df ¼ 8; P ¼ 0.001; Cv ¼ 13.88). A significant increment in insect mortality was observed when EPNs were applied in association with the insecticides in comparison with only EPNs, with additive interaction in these treatments, although the increment was not significant if compared to the insecticides LorsbanÔ and MatchÔ alone and associated with EPNs. Among all treatments, association of H. indica and MatchÔ resulted in the highest mortality of S. frugiperda, with 62.5% (assay 1) and 57.5% mortality (assay 2), despite not being significantly different from the other treatments with the nematodeeinsecticide combination (Figs. 1 and 2). Low mortality produced by the products when isolated may be related to the product concentration, since they were applied at half of the recommended dose. These results evidence a higher mortality rate during the first experimental period (2007/2008), if compared to the second one (2008/2009). This fact may be related to a difference in methodology used in both periods. During the first period, plant apexes were covered with thin cloth bags with the objective of preventing larvae from escaping and, as a consequence, a micro-climatic condition was established inside the bags that may have favored EPNs. However, as this is an artificial situation in the field, during the second period experiment plants were not covered, so abiotic factors such as temperature, UV radiation and desiccation may be responsible for the lower performance of EPNs, as reported by Glazer (2002). Results revealed that insecticides were the most important factor in larval mortality, in detriment of EPNs, which showed low efficacy in field conditions with no more than 22.5% mortality in treatment with S. carpocapsae, a rate higher than that observed by Garcia et al. (2008) when mortality equal to zero was observed after application of H. indica IBCB-n5 and Steinernema sp. applied over S. frugiperda in the corn husk under controlled conditions. These authors evaluated different application technologies and surfactant effect on EPNs, observing that IJs of both species were compatible with all application techniques and adjuvants, so these factors are not responsible for EPN inefficacy in laboratory. Some hypotheses were proposed by Garcia (2006) regarding inefficacy of EPNs to control S. frugiperda when directed to corn husk using high volume of mixture (800 L/ha): the insertion angle of the corn leaf of up to 30 and narrow channel shaped, may prevent the formation of a water layer essential for nematode movement towards larvae, as well as for inoculum concentration. Besides this fact, the author emphasizes that application in high volumes of mixture are far over the needs for chemical control of this insect, and may make control economically unfeasible as well as forming a water film so thick that it may hinder EPN movement. In agreement with observations recorded with EPNs and insecticides in the present work, Mendez et al. (2002) verified that the mixture of espinosine at 3ppm (TracerÔ) with nucleopolyhedrovirus of S. frugiperda (SfMNPV) resulted in higher mortality of
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Fig. 1. Mortality (% SE) of Spodoptera frugiperda larvae four days after exposure to association between insecticides and entomopathogenic nematodes Steinernema carpocapsae and 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).
this pest (90%) in the field, compared with the entomopathogen applied without insecticide (12e37%). Research is needed in order to determine the economically optimal concentration of EPNs in the field for application directed to corn husk, associated with selected insecticides (LorsbanÔ and MatchÔ). Besides that, pre-pupal and pupal stages of the insect, which are associated with soil, may be susceptible to EPNs in field conditions, considering that Molina-Ochoa et al. (2003) observed natural occurrence of Steinernematidae and Heterorhabditidae in soil samples collected in 64 localities (10.9%) associated with populations of S. frugiperda in corn, sorghum and pastures in Mexico. In this regard, Cabanillas and Raulston (1996) evaluated application of Steinernema riobravis and S. carpocapse on Helicoverpa zea pupae and pre-pupae in corn crops in Texas, USA; they observed mortality of up to 97% caused by S. riobravis in the field, for the highest concentration (20 109/ha), applied through irrigation rows, with persistence of up to 36 days after the treatment and with 80% of pest control when compared with 14% pest control (natural) in the control treatment. S. carpocapsae was not efficient for pest control due to high soil temperatures (approximately 31 C at 20 cm depth). Thus, it is important to emphasize that S. riobravis was isolated in 1990 in Texas, USA, being a native strain and therefore adapted to local conditions, unlike S. carpocapsae (strain All), which originated from the state of Georgia, USA, with a relatively more moderate climate.
In the same way, studies of indigenous EPNs isolates must be performed in corn producing regions of Rio Grande do Sul, Brazil, in order to identify and select more efficient isolates to control corn pests, especially those having a soil stage, such as S. frugiperda, H. zea and Diabrotica speciosa. Consequently, the high cost of EPN application may be minimized through the control of more than one pest in this crop, thus reducing selection pressure. This is a potential strategy to be used against the problem of pest resistance caused by chemical insecticides. The present work is the first to record the interaction of EPNs with chemical insecticides aiming to control S. frugiperda. The control by association, using an insecticide in sub-lethal dose compatible with an entomopathogen, represents a possibility for controlling S. frugiperda in applications directed to the corn husk (foliar application), through spraying or even through irrigation technologies, depending on the grower's technological availability. Unfortunately, the results showed low efficiency of mixing EPNs with insecticides to control the pest. Hence, new studies must be performed to determine optimal EPNs and insecticide concentrations that may make technologies economically feasible, as well as evaluations of susceptibility of pre-pupal and pupal stages of S. frugiperda in the soil, with the possibility of a multiple control aimed at other corn pests and in this way, improving economical feasibility of such technologies, reducing environmental costs of production.
Fig. 2. Mortality (% SE) of Spodoptera frugiperda larvae four days after exposure to association between insecticides and the entomopathogenic nematodes Steinernema carpocapsae and 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, 2008/2009).
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