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This article was downloaded by: [UPM], [ELISA VIÑUELA]On: 06 March 2012, At: 08:58Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
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Effect of emamectin benzoate undersemi-field and field conditions on keypredatory biological control agentsused in vegetable greenhousesFermin Amor a , Pilar Medina a , Paloma Bengochea a , MónicaCánovas b , Pedro Vega b , Rui Correia b , Federico García c ,Manuel Gómez c , Flor Budia a , Elisa Viñuela a & Juan AntonioLópez ba Unidad de Protección de Cultivos, Escuela Técnica Superior deIngenieros Agrónomos, Universidad Politécnica de Madrid, CiudadUniversitaria s/n, Madrid, Spainb Syngenta Agro S.A, Ribera del Loira, 8-10, 3°, Madrid, Spainc Syngenta Agro S.A. – Bioline, Jazminero, 1, Edificio Guay, Ofic.3, Aguadulce, 04720, Almería, Spain
Available online: 20 Dec 2011
To cite this article: Fermin Amor, Pilar Medina, Paloma Bengochea, Mónica Cánovas, Pedro Vega,Rui Correia, Federico García, Manuel Gómez, Flor Budia, Elisa Viñuela & Juan Antonio López (2012):Effect of emamectin benzoate under semi-field and field conditions on key predatory biologicalcontrol agents used in vegetable greenhouses, Biocontrol Science and Technology, 22:2, 219-232
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RESEARCH ARTICLE
Effect of emamectin benzoate under semi-field and field conditions onkey predatory biological control agents used in vegetable greenhouses
Fermin Amora, Pilar Medinaa*, Paloma Bengocheaa, Monica Canovasb,
Pedro Vegab, Rui Correiab, Federico Garcıac, Manuel Gomezc, Flor Budiaa,
Elisa Vinuelaa and Juan Antonio Lopezb
aUnidad de Proteccion de Cultivos, Escuela Tecnica Superior de Ingenieros Agronomos,Universidad Politecnica de Madrid, Ciudad Universitaria s/n, Madrid, Spain; bSyngenta AgroS.A., Ribera del Loira, 8-10, 38, Madrid, Spain; cSyngenta Agro S.A. � Bioline, Jazminero, 1,
Edificio Guay, Ofic. 3, 04720 Aguadulce, Almerıa, Spain
(Received 11 October 2011; final version received 13 December 2011)
Predatory arthropods are commonly used as biological control agents (BCAs).They are released in commercial vegetable greenhouses as primary elements ofintegrated pest management programmes for some of the most devastating pestson pepper and tomato in southeastern Spain. Emamectin benzoate, a macro-cyclic lactone insecticide derived from the avermectin family of natural products,is being developed for the control of Lepidoptera pests on a variety of crops inEurope including vegetables. The compatibility of emamectin benzoate with thepredatory BCAs Amblyseius swirskii Athias-Henriot and Orius laevigatus (Fieber)in field trials (direct spray and aged residues) and Macrolophus pygmaeus(Rambur) and Chrysoperla carnea (Stephens) in semi-field studies was studied.Emamectin benzoate at the highest recommended concentration (14.25 mg L�1)was compatible with A. swirskii and O. laevigatus when applied 3 days beforethe introduction of the arthropods, but it was toxic when directly sprayed.M. pygmaeus and C. carnea adults survived to direct spray applications.
Keywords: emamectin benzoate; Amblyseius swirskii; Orius laevigatus;Macrolophus pygmaeus; Chrysoperla carnea; greenhouse crops
Introduction
Spain is the most important producer and exporter of fresh greenhouse vegetables in
Europe with approximately 59,000 ha dedicated to these crops. The main production
area is located in the southeastern (SE) regions of Almeria and Murcia, where
peppers (Capsicum annuum L.) and tomatoes (Solanum lycopersicum L.) under
greenhouses are the major crops [Ministerio de Medio Ambiente, Rural y Marino
(MARM) 2010a]. Currently, the most devastating pests affecting these two crops are
the western flower thrips Frankliniella occidentalis (Pergande) (Thysanoptera:
Thripidae), the tobacco whitefly Bemisia tabaci (Gennadius) (Homoptera: Aleyr-
odidae) and the tomato borer Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae),
which were recently introduced (Urbaneja, Mouton, and Molla 2008). These pests
are commonly managed through integrated pest management (IPM) programmes
primarily based on the release of commercial arthropods as biological control agents
*Corresponding author. Email: [email protected]
Biocontrol Science and Technology,
Vol. 22, No. 2, February 2012, 219�232
ISSN 0958-3157 print/ISSN 1360-0478 online
# 2012 Taylor & Francis
http://dx.doi.org/10.1080/09583157.2011.650152
http://www.tandfonline.com
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(BCAs). These include several major predatory organisms, such as the phytoseiid
Amblyseius swirskii Athias-Henriot and the hemipterans Orius laevigatus (Fieber),
Macrolophus pygmaeus (Rambur) [formerly cited and commercialised as
Macrolophus caliginosus (Wagner) (�Macrolophus melanotoma (Costa)) according
to Martınez-Cascales, Cenis, Cassis, and Sanchez (2006)] and Nesidiocoris tenuis
(Reuter). The phytoseiid mite is particularly useful against the whitefly B. tabaci and
can also feed on first instar thrips. O. laevigatus controls F. occidentalis and the othertrue bugs are being studied to regulate T. absoluta populations (Gabarra, Zapata,
Castane, Riudavets, and Arno 2006; Gabarra, Arno, and Riudavets 2008; Van der
Blom 2008; Van der Blom, Robledo, Torres, Sanchez, and Contreras 2008). The
green lacewing Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) is also
considered a key generalist BCA in vegetables greenhouses (Gabarra et al. 2008; Van
der Blom 2008). However, because effective BCAs are not available for controlling all
pests, such as the noctuid caterpillars Helicoverpa armigera (Hubner) and Spodoptera
exigua (Hubner) and because they are not always able to maintain populations of
foliar piercing-sucking arthropods under economic thresholds, pesticides remain an
important management tool in greenhouse IPM to control key pests.
Emamectin benzoate (4ƒ-deoxy-4 ƒ-methylamino-4 ƒ-epiavermectin B1 benzoate)
is a new macro-cyclic lactone insecticide derived from the avermectin family of
natural products. This product has been developed for the control of Lepidoptera
pests on a variety of vegetable crops worldwide (Liguori, Cestari, Serrati, and
Fusarini 2008), such as T. absoluta, H. armigera and S. exigua (Liguori et al. 2010;Lopez et al. 2010). The compound is not systemic, but it exhibits translaminar
activity (Willis and McDowell 1989). Its main physiological mode of action is to
stimulate the release of the neurotransmitter g-aminobutyric acid (GABA), thus
causing a continuous flow of chloride ions into muscle cells resulting in a suppression
of contraction and paralysis (Ishaaya, Kontsedalov, and Horowitz 2002). Emamectin
benzoate acts via ingestion and, to a lesser extent, by contact (Dybas and Babu
1988). It is rapidly taken up by plants and metabolised on the surface by photo-
oxidation to non-toxic levels which favours its selectivity for biological control
organisms (Ishaaya et al. 2002). This insecticide is currently being evaluated for
inclusion in Annex I of the European directive 91/414/EEC which regulates the
Registration of Plant Protection Products and was recently approved for its use on
vegetables in Spain (MARM 2010b).
Knowledge of pesticide selectivity to BCAs is necessary for a successful
implementation of biological and chemical control methods in IPM programmes.
Fresh residues of emamectin benzoate have been shown to be harmful to a related
species of Orius, Orius insidiosus, in laboratory and extended laboratory tests(Studebaker and Kring 2003a,Studebaker and Kring 2003b). Under field conditions,
aged residues were found to be harmless to other phytoseiids, Euseius alatus DeLeon
and Euseius citrifolius Denmark and Muma in citrus (Reis, Neto, Franco, and
Teodoro 2004). Thus, for A. swirskii and O. laevigatus, field trials have been
implemented because they are the most important BCAs used widely in commercial
pepper greenhouses in SE Spain. Given the preliminary information on these two
BCAs, the effects of direct spray on established populations and of aged residues on
inoculative introductions have been studied to determine whether the product is
compatible with existing BCA populations. Alternatively, in case of negative effects
of direct spraying on BCAs, the optimal re-entry interval must be determined. For
220 F. Amor et al.
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C. carnea and M. pygmaeus, less used in SE Spain in comparison with the former
BCAs, semi-field assays have been performed to evaluate the level of compatibility of
fresh residues of the insecticide.
Therefore, the objective of this study is to determine the level of selectivity of the
insecticide emamectin benzoate for predatory BCAs commonly used in greenhouse
vegetable production in SE Spain.
Materials and methods
Field trials
In field tests, the compounds were tested under typical conditions in a commercial
crop. European Plant Protection Organization (EPPO) guideline PP1/151(2), ‘Side-
effects on Phytoseiulus persimilis’ (EPPO 2004) was considered for the general
aspects in the establishment of these trials.
To evaluate BCA population dynamics under commercial field conditions, four
trials were conducted during 2007 in Torre Pacheco, Murcia, Spain, under multiple
tunnel greenhouses of three extruded layer Indasol† (Solplast, Murcia) plastic at ex
CIFACITA S.L. facilities. Greenhouses were automatically acclimatised with Aco†
Vision Clima 727 equipment (Hortimax Growing Solutions S.L., Almeria, Spain).
Two trials on A. swirskii (direct spray and aged residues) and two trials on
O. laevigatus (direct spray and aged residues) were carried out. In all the trials, a fully
randomised design with 3 replicates and 160 m2 (272 plants) plots were used. To
avoid arthropod cross-contamination and spray drifts, plots were isolated both
laterally and on the top, with an anti-thrips net 10�14 thread cm�2 (Botanica
equipment S.L., Alcantarilla, Murcia) sealed between the sides (anchored to the
ground) and the top, resulting in cages of 8 m width, 20 m length and 3.5 m height,
separated and surrounded by 1 m corridors. The entrance to the plots consisted of
overlaps in the lateral nets (1.5 m overlap) closed with several wire loops.
Biological control arthropods were supplied by Syngenta Agro S.A. Bioline
(Aguadulce, Almeria) and released following the methodology provided by the
supplier. In the aged residue trials, the following use rates were employed: A. swirskii
(Swirskiiline† as in twin Gemini† sachets) at 250 mobiles per plant and O. laevigatus
(Oriline† activ) at 3.6 mobiles per plant. These release rates are considerably higher
than those commercially recommended (see below for the direct spray trials) to
ensure a faster development of the arthropod populations. In these trials, the
inoculative BCA introductions were performed 3, 7 and 14 days after insecticide
application (aged residues of 3, 7 and 14 days). On each plant, A. swirskii mites were
introduced by hanging a twin sachet (containing 250 mobiles) on a leaf petiole
approximately 20�25 cm from the ground forming an inverted V with the sides
containing the emergence holes inwards. For the inoculation of O. laevigatus, 2
bottles of 500 mobiles each were uniformly distributed over each plot, by tapping the
bottle and releasing about the same amount of the predators on the growing area of
every two plants. In the direct spray tests, populations of both BCAs, A. swirskii and
O. laevigatus, were already well established at the time of insecticide application
(Table 1) and came from inoculative releases performed on 13 April 2007 (31 days
before application) at commercially recommended rates, A. swirskii (Swirskiiline†
as) at 100 mobiles m�2 and O. laevigatus (Oriline† activ) at 3 mobiles m�2 and
Biocontrol Science and Technology 221
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Table 1. Average population density of Amblyseius swirskii and Orius laevigatus (number of mobile forms/plant) in the direct spray treatment of
emamectin benzoate over the different sampling dates. Field studies on greenhouse pepper.
Mobile forms per plant (9SE)
Treatment
Concentration
(mg a.i. L�1)
0 DBAA,C
(14 May 2007)
3 DAAA,B
(17 May 2007)
7 DAAA,C
(21 May 2007)
14 DAAA,B
(28 May 07)
21 DAAA,B
(4 June 2007)
Amblyseius swirskii
Control 12.83 (91.30)a 11.00 (91.80)a 9.17 (91.86)a 14.00 (91.00)a 14.33 (91.92)ab
Emamectin
benzoate
14.25 11.50 (91.61)a 2.83 (90.88)b 4.67 (90.73)a 1.50 (90.29)b 5.67 (91.69)a
Thiamethoxam 100.00 14.00 (93.25)a 4.67 (90.93)b 11.50 (92.29)a 11.00 (91.76)a 23.17 (95.43)b
Orius laevigatus
Control 2.02 (90.34)a 2.20 (90.29)a 1.22 (90.21)a 1.92 (90.17)a 2.30 (90.48)a
Emamectin
benzoate
14.25 1.93 (90.42)a 0.10 (90.03)b 0.08 (90.08)b 0b 0.05 (90.03)b
Thiamethoxam 100.00 2.23 (90.14)a 0.02 (90.02)b 0.07 (90.07)b 0b 0b
Note: DBA, days before application; DAA, days after application. Three replicates of 20 plants per replicate.AMeans within columns followed by the same letter are not significantly different (ANOVA, LSD; a�0.05) for A. swirskii.BMeans within columns followed by the same letter are not significantly different (KW, notched boxplot; a�0.05) for O. laevigatus.CMeans within columns followed by the same letter are not significantly different (ANOVA, LSD; a�0.05) for O. laevigatus.
22
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following the methodology described earlier for the aged residue trials. In both cases,
the establishment and viability of the BCAs was ensured by maintaining an adequate
presence of their prey F. occidentalis from a natural infestation, although these
polyphagous predators can subsist feeding on other pests as well as on plant pollen.
Trials were conducted on pepper (C. annuum L.) var. Coyote in the direct spray
trials and var. Bilbo in the aged residue trials. Crops were grown on artificial media(coconut fibre Hidrosac†, Poliexmur, S.A., San Gines, Murcia) and arrayed at 1.5 m
row distance�0.4 m plant distance (1.7 plants m�2). Dates (planting and last
evaluation) and environmental conditions (Ta daily averages and relative humidity
[RH]) of the trials were as follows: direct spray trials (16 January 2007�14 June 2007):
14�25 8C, 67�89% RH; and aged residue trials (2 June 2007�24 October 2007): 19�30 8C, 70�81% RH.
The formulated insecticide emamectin benzoate 95 g kg�1 SG (Affirm†,
Syngenta Agro S.A., Madrid) was tested as an experimental product at the maximum
field-recommended concentration of the active ingredient, 14.25 mg L�1. Two other
insecticides were included in the study as references: thiamethoxam 400 g kg�1 WG
(Actara†, Syngenta Agro S.A., Madrid) in A. swirskii and O. laevigatus direct sprays
and lambda-cyhalothrin (100 g L�1 SC, Karate† Zeon†, Syngenta Agro S.A.,
Madrid) in aged residue trials. Thiamethoxam was chosen because neonicotinoids
have been shown to be harmful to many BCAs, in particular, to O. laevigatus
(Delbeke, Vercruysse, Tirry, De Clercq, and Degheele 1997; Van de Veire et al. 2002;Sterk, Heuts, Merck, and Bock 2003) as has imidacloprid (Angeli, Baldessari,
Maines, and Duso 2005). The use of separate references in the two trials is because
thiamethoxam was harmless to A. swirskiii in the direct spray trial, while pyrethroids
are generally known to be harmful to predatory mites (Aliniazee and Cranham 1980;
Bostanian and Belanger 1985; Bostanian and Racette 1997). Reference products were
applied at the recommended concentrations of 100 mg L�1 and 20 mg L�1 of active
ingredient, respectively (thiamethoxam and lambda-cyhalothrin). Insecticides were
applied as foliar spray until run off with a motorised knapsack sprayer (MS068,
Maruyama) and double cone nozzle gun (1.5 mm diameter), using a spray volume of
600 L ha�1 in the direct spray assay and 1000 L ha�1 in the aged residues assay. The
differences in volume are justified by the different growth stages, 61 and 75 BBCH,
respectively (Meier 2001), and size of the plants at application (50�60 and 80�90 cm
high, respectively). Output pressure was always 8 atm. The control plots were
sprayed with tap water under the same earlier conditions. In the direct spray trials,
application was performed on 14 May 2007, once the level of leaf colonisation by the
arthropods reached more than 75% (according to an evaluation made at 7 days
before application, on the 10 upper leaves, flowers included, of 20 plants per plotselected at random in the middle of the plots). In the aged residue trials, the products
were sprayed on 11 September, 7 September and 31 August 2007, which correspond
to 3, 7 and 14 days before the introduction of the arthropods (14 September 2007).
In all the trials, assessments were made on the 10 upper leaves, flowers included,
of 20 plants per plot selected at random in the middle of the plots. Leaves were
collected in plastic bags, kept in an ice-chest and transported to the laboratory.
Arthropods were identified and counted under a stereoscopic microscope such that
leaves and flowers were evaluated individually and the remaining arthropods in the
plastic bags were collected with a brush into a Petri dish, identified and reported as
number of mobile forms (nymphs and adults) per plant. Sampling in the direct spray
Biocontrol Science and Technology 223
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trials was performed at 0 days before application, and 3, 7, 14 and 21 days after
application. The first evaluation after treatment was made at 3 days after application
taking into consideration that, according to previous studies, several days are needed
for the insecticide to kill target insects (Ishaaya et al. 2002; Ishaaya, Barazani,Kontsedalov, and Horowitz 2007), so differences among earlier treatments would not
be expected. The rest of the evaluations were made at 7-day intervals after
application. In the aged residue trials, samplings were taken at 0 Days Before
BCA Introduction (DBI), and at 7, 17, 22, 29, 36 and 43 Days After Introduction
(DAI). The first evaluation after the introduction of the BCAs was made at 7 DAI to
allow populations to settle down uniformly across the untreated plots. Evaluations
were made following at approximately 7-day intervals after introduction.
Semi-field tests
The methodology used in these tests was based on the semi-field test described for
O. laevigatus (Van de Veire et al. 2002), and modified to the different species and
conditions of the plants and facilities.
To evaluate the acute toxic effects of the direct spray application of emamectin
benzoate on adults of M. pygmaeus and C. carnea, two semi-field trials were
conducted on tomato plants in an experimental greenhouse equipped with coolingand heating systems under controlled environmental conditions (2595 8C; 4095%
RH) during May�July 2009 at the facilities of Escuela Tecnica Superior de
Ingenieros Agronomos (ETSIA), Universidad Politecnica de Madrid (UPM),
Madrid, Spain. A completely randomised design with 10 replicates (1 plant per
plot) was used.
Macrolophus pygmaeus (Macroline†) was supplied by Syngenta Agro S.A.
Bioline (Aguadulce, Almeria). C. carnea came from the colony reared at the Unidad
de Proteccion de Cultivos, ETSIA, UPM, Madrid. To ensure adequate quality, onthe day of the reception, M. pygmaeus adults were selected from the commercial
package and kept in groups of 30 (in plastic ventilated round cages with Ephestia
kuehniella Zeller and Artemia salina Leach eggs as ad libitum food) for 24 h, and
unhealthy stocks were discarded. C. carnea adults were selected 24 h after emergence.
In each of the replicates (1 plant), 15 individuals were used. In the M. pygmaeus
trials, a mix of E. kuehniella and A. salina eggs was supplied after the insecticide
application as ad libitum food in a small container hung on each plant. C. carnea was
provided with an artificial diet (Vogt et al. 2000).Bioassays were conducted on tomato (S. lycopersicum L.) var. Montfavet† 63/5
HF1 (Vilmorin Iberica S.A., Alicante), seeded in tray cells with sterilised peat
substrate (pH 6.5) and transplanted into individual pots (22 cm diameter) 6 weeks
later to obtain homogeneous pesticide-free plants. Each plant (ca. 20 cm height) was
surrounded by a plastic cylinder (21 cm diameter, 30 cm height) sealed to the surface
of the pot with foam and covered with a fine cloth mesh in the upper part to allow
ventilation. Cylinder cages had two lateral windows (15�15 cm2) covered with the
mesh.The formulated insecticide emamectin benzoate 95 g kg�1 SG (Affirm†,
Syngenta Agro S.A., Madrid) was tested as experimental product at the highest
recommended concentration of the active ingredient, 14.25 mg L�1. Imidacloprid at
200 g L�1 SC (Confidor†, Bayer CropSciences S.A., Valencia) was used, as a
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reference product, at the recommended concentration of 150 mg L�1 of active
ingredient, based on its harmfulness to the BCAs, M. pygmaeus (Tedeschi, Tirry, Van
de Veire, and de Clercq 2002; Van de Veire and Tirry 2003) and C. carnea (Huerta,
Medina, Smagghe, Castanera, and Vinuela 2003; Nasreen, Mustafa, and Ashafaq
2005).
Applications were made 1 h after the insect introduction on the tomato plants
until run off with a 1 L hand sprayer. Control plants were treated with distilled water
as described earlier.
Insect mortality (%) was assessed at 24, 48 and 72 h after treatment. Mortality in
the control treatments was 0% for C. carnea and 21% for M. pygmaeus, below the
25% recommended by the standards of the IOBC (Hassan 1985).
Data analysis
Data were subjected to an analysis of variance using Statgraphics† Plus v. 5.0 (STSC
1987), and differences among the means were determined with a LSD test performed
at a significance level of a�0.05. A non-parametric Kruskal�Wallis (KW) test and a
median discrimination by the notched boxplot were conducted at a�0.05 when the
normal distribution (Kolmogorov test) and/or homoscedasticity (Bartlett test)
hypothesis were not confirmed. Corrected data were calculated (Abbott 1925) and
according to the IOBC scheme (Sterk et al. 1999), the insecticides were classified into
one of the following categories: 1, harmless (B25%); 2, slightly harmful (25�50%); 3,
moderately harmful (51�75%) or 4, harmful (�75%).
Results
Amblyseius swirskii
The analysis of the number of mobile forms per plant in the direct spray trial at 0
days before application revealed that there were no significant differences (F�0.32;
df �2,6; P�0.741) in populations among treatments. Compared with the control
treatment, the reference product thiamethoxam caused a significant reduction in the
population only at 3 days after application (moderately harmful) (F�10.35;
df �2,6; P�0.031) whereas emamectin benzoate caused significant reduction at 3
days after application (moderately harmful), and at 14 days after application
(harmful) (F�30.66; df �2,6; PB0.001). No statistical differences were shown at 7
days after application (F�3.92; df �2,6; P�0.081), and neither differed from the
control plots at 21 days after application; however, statistical differences were shown
between both compounds (F�1.75; df �2,6; PB0.001), as emamectin benzoate was
significantly more toxic than thiamethoxam in this evaluation (Table 1).
In the aged residue trial, no significant effect was seen among treatments 7 days
after the arthropod introduction (F�2.87; df �4,10; P�0.080). Lambda-cyhalo-
thrin 3 days residue caused significant population reduction from 17 DAI (harmful)
(F�5.22; df �4,10; P�0.016) until the last evaluation, 43 DAI (F�6.59; df �4,10;
P�0.006). None of the emamectin benzoate residues (3, 7 and 14) differed
significantly from the control plots in any of the assessments throughout the
experiment (7, 17, 22, 29, 36 and 43 DAI) (Table 2).
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Table 2. Average population density of Amblyseius swirskii and Orius laevigatus (number of mobile forms/plant) over the different sampling dates when
the natural enemies were exposed to aged residues (3, 7 and 14 days old) of emamectin benzoate. Field studies on greenhouse pepper.
Mobile forms per plant (9SE)
TreatmentConcentration(mg a.i. L�1)
0 DBI (14September
2007)
7 DAIA,C (21September
2007)17 DAIA,B (1October 2007)
22 DAIA,C (6October 2007)
29 DAIA,C (13October 07)
36 DAIA,C (20October 2007)
43 DAIA,C (27October 2007)
Amblyseius swirskii
Control 0 4.98 (91.98)a 28.67 (99.69)a 26.33 (912.87)ab 28.67 (98.38)a 42.33 (98.35)a 18.67 (93.84)aEmamectin benzoate 3 days (AD:
11 September 2007)14.25 0 4.25 (91.03)a 26.00 (92.56)a 17.25 (91.98)b 28.83 (95.87)a 28.17 (92.09)a 12.42 (90.74)a
Emamectin benzoate 7 days(AD:07 September 2007)
14.25 0 3.10 (90.73)a 28.00 (94.39)a 43.50 (93.97)a 13.67 (92.67)ab 31.92 (96.42)a 14.70 (92.06)a
Emamectin benzoate 14 days (AD:31 August 2007)
14.25 0 2.28 (91.03)a 36.33 (95.02)a 22.50 (95.01)ab 22.83 (91.59)a 29.17 (94.06)a 11.83 (91.01)a
Lambda-cyhalothrin 3 days (AD:11/09/07)
20.00 0 0.37 (90.07)a 3.00 (91.26)b 2.92 (90.58)c 5.67 (90.73)b 7.17 (90.93)b 4.17 (90.88)b
Orius laevigatus
Control 0 0.08 (90.08)a 2.17 (90.17)a 2.92 (90.65)ab 4.00 (90.52)a 2.92 (90.30)b 2.58 (90.51)bEmamectin benzoate 3 days (AD:
11 September 07)14.25 0 0.08 (90.08)a 1.83 (90.08)a 3.33 (90.60)a 2.50 (90.38)a 2.33 (90.27)b 1.92 (90.17)bc
Emamectin benzoate 7 days(AD:07 September 2007)
14.25 0 0.08 (90.08)a 2.17 (90.17)a 1.58 (90.36)b 3.25 (90.63)a 4.42 (90.30)a 3.08 (90.22)a
Emamectin benzoate 14 days (AD:31 August 2007)
14.25 0 0.33 (90.08)a 1.92 (90.16)a 3.17 (90.44)a 3.42 (90.58)a 3.92 (90.22)a 3.00 (90.25)a
Lambda-cyhalothrin 3 days (AD:11 September 07)
20.00 0 0.02 (90.02)a 0.08 (90.02)b 0.08 (90.08)c 0.09 (90.08)b 0.05 (90.05)c 0.13 (90.06)c
Note: AD, application date; DBI, dates before insects introduction; DAI, days after insects introduction. Three replicates of 20 plants per replicate.AMeans within columns followed by the same letter are not significantly different (ANOVA, LSD; a�0.05) for A. swirskii.BMeans within columns followed by the same letter are not significantly different (KW, notched boxplot; a�0.05) for O. laevigatus.CMeans within columns followed by the same letter are not significantly different (ANOVA, LSD; a�0.05) for O. laevigatus.
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Orius laevigatus
In the direct spray trial, there were no significant differences (F�0.23; df �2,6;
P�0.798) in populations among treatments at 0 days before application. Compared
with the control treatment, both emamectin benzoate and the reference product,
thiamethoxam, caused significant reduction of the population at 3 days after
application (KW �6.94; P�0.031), 7 days after application (F�23.70; df �2,6;
P�0.001), 14 days after application (KW �6.72; P�0.035) and 21 days afterapplication (KW �6.76; P�0.034), and both were classified both as harmful in all
the cases (Table 1).
In the aged residue trial, no significant effect was seen among treatments at 7
DAI (F�2.68; df �4,10; P�0.094). Lambda-cyhalothrin 3 days residue caused
significant population reduction (harmful) from 17 DAI (KW �9.78; P�0.044)
until the last evaluation, 43 DAI (F�18.47; df �4,10; PB0.001). None of the
emamectin benzoate residues (3, 7 and 14) differed significantly from the control
plots in assessments 7, 17, 22, 29 DAI. Numbers of O. laevigatus found were evensignificantly higher in comparison with the control in the evaluations 36 and 43 DAI
(Table 2).
Macrolophus pygmaeus
A direct spray application of emamectin benzoate at the highest recommended rate
(14.25 mg L�1) in the semi-field direct spray trial in tomato plants was notsignificantly different from the control at 24 and 48 h but showed significantly higher
mortality at 72 h (F�77.8; df �2,27; PB0.001) that was however classified as
harmless (16% corrected mortality). The reference product, the neonicotinoid
insecticide imidacloprid (150 mg L�1), produced significantly higher mortality at
24 h (F�336.79; df �2,27; PB0.001), 48 h (F�192.58; df �2,27; PB0.001) and
72 h (F�77.8; df �2,27; PB0.001) after application and was classified as harmful
in all cases (Table 3).
Chrysoperla carnea
The effect of emamectin benzoate application in tomato plants on C. carnea at the
highest recommended rate (14.25 mg L�1) in the semi-field direct spray trial was not
significantly different from the control at 24, 48 or 72 h after application. In contrast,
the reference insecticide, imidacloprid (150 mg L�1), produced a significantly highermortality at 24 h (58%) (KW �26.11; PB0.001), 48 h (67.33%) (KW �26.08;
PB0.001) and 72 h (69.33%) (KW �26.07; PB0.001) after application and was
classified as moderately harmful in all cases (Table 3).
Discussion
Emamectin benzoate is less toxic to a variety of beneficial organisms than it is to its
main targets, according to our results.
Amblyseius swirskii is a relatively new BCA and few data exist on its susceptibility
to pesticides, and no studies assessing the effects of emamectin benzoate were found.
In our study, conducted in a commercial greenhouse, emamectin benzoate was
Biocontrol Science and Technology 227
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harmful to A. swirskii on peppers up to 14 days after application when sprayed
directly on an established population. Previous studies on the predatory mite Euseius
victoriensis Womersley (Acari: Phytoseiidae) using a ‘worst case scenario’ direct
overspray assays showed that emamectin benzoate is highly toxic after a single-use
application at the highest recommended rate, 1000 L ha�1 (Bernard et al. 2010),
suggesting that it may not be completely safe for phytoseiids as demonstrated in our
test field. Abamectin, the first avermectin of commercial interest, is known for its
unprecedented potency against a broad spectrum of phytophagous mites, members
of Tetranychidae, Eriophyidae and Tarsonemidae. It is markedly less toxic to other
phytophagous mites, such as Panonychus ulmi (Koch), and its toxicity to insects is
more variable (Jansson and Dybas 1998). In contrast, emamectin benzoate is highly
potent against a broad spectrum of Lepidoptera, but not comparable to abamectin as
acaricide. The toxicity of emamectin benzoate on mites depends more on the species,
and field studies in citrus carried out showed no effect of fresh emamectin benzoate
residues (20 g ha�1) on E. alatus and E. citrifolius (Reis et al. 2004).
In aged residue trials, 3, 7 and 14 day residues of the insecticide were harmless to
the predators as it has been previously demonstrated for foliar residues of emamectin
benzoate. Within 1 day of application, and often within a few hours after application,
the residues were only slightly toxic (B20% mortality) to most beneficial insects,
including the honey bee, Apis mellifera L., and several predators and parasitoids
(Jansson and Dybas 1998). In the related insecticide, abamectin, 1 day residues (0.108
mg L�1) were harmless to A. swirskii in a field test on greenhouse cucumber showing
effects similar to the aged emamectin benzoate residues in our trial (Gradish, Scott-
Dupree, Shipp, Harrisa, and Ferguson 2011). On O. laevigatus, emamectin benzoate
is harmful up to 21 days after application in direct spray applications but 3, 7 and 14
days residue exposure is harmless. For another Orius species, O. insidiosus, it was
found in laboratory and extended laboratory tests that fresh residues of emamectin
benzoate, as well as abamectin, were moderately harmful, to harmful for nymphs and
Table 3. Average mortality of Macrolophus pygmaeus and Chrysoperla carnea adults after
direct spray of emamectin benzoate at 24, 48 and 72 h after application. Semi-field studies on
tomato plants.
Average mortality (%) (9SE)
Treatment
Concentration
(mg a.i. L�1) 24 h 48 h 72 h
Macrolophus pygmaeus
Control 8.0091.33a 16.6692.85a 21.3393.11a
Emamectin benzoate 14.25 10.6692.47a 18.6692.59a 34.0095.20b
Imidacloprid 150.00 86.6693.14b 86.6693.14b 86.6693.14c
Chrysoperla carnea
Control 0a 0a 0a
Emamectin benzoate 14.25 0.6790.67a 0.6790.67a 0.6790.67a
Imidacloprid 150.00 58.0093.72b 67.3393.77b 69.3394.12b
Note: Means within columns followed by the same letter are not significantly different (ANOVA, LSD;a�0.05 for M. pygmaeus and KW, notched boxplot, a�0.05 for C. carnea).
228 F. Amor et al.
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adults of the predator (Studebaker and Kring 2003a,Studebaker and Kring 2003b).
These results confirm our data from the direct spray trial and may be in accordance
with our aged residue results, considering that, unlike in a laboratory tests,
emamectin benzoate trial may be degraded on the leaf surface enough to prevent
any toxic effects after 3 days due to its fast degradation under field conditions by
photodegradation (Willis and McDowell 1989; Prabhu, Wehner, Egan, and Tway
1991; Wrzesinski et al. 1996).
Collectively, our field data demonstrated that emamectin benzoate provides, if
not physiological selectivity because of its direct toxicity, at least, ecological
selectivity for A. swirskii and O. laevigatus. This ecological selectivity is related to
the short half-life of the compound on foliage, and within an IPM programme, this
pesticide can be used 3 days before inoculative release of the natural enemies tested in
this study.
Under semi-field conditions, emamectin benzoate is harmless at the highest
recommended rate of 14.25 mg L�1 when sprayed directly onto M. pygmaeus adults
in tomato plants. Similarly, it was found under laboratory test conditions that
exposure to fresh residues of emamectin benzoate is harmless to nymphs of this
hemipteran predator (Tedeschi et al. 2002; Van de Veire and Tirry 2003). Previous
results obtained by the authors in residue bioassays under semi-field conditions
confirmed also the harmless effect on M. pygmaeus adults when exposed to fresh and
1 day residues of the insecticide (Lopez et al. 2011). Other naturally derived products,
abamectin and spinosad, are also harmless to nymphs after fresh residue exposure
(Van de Veire and Tirry 2003). On C. carnea, emamectin benzoate (14.25 mg L�1)
has been shown to be harmless in direct spray tests under semi-field conditions in
tomato plants up to 72 h. The harmless effects of two direct sprays of emamectin
benzoate at 13.5 g ha�1 on C. carnea larvae were observed in cotton under field
conditions (Sechser, Ayoub, and Monuir 2003). In the related species
C. externa (Hagen), no negative effects of topical or direct spray applications with
emamectin benzoate were found in laboratory or extended laboratory tests on L1, L2
and L3 larvae (Ferreira, Carvalho, Botton, and Lasmar 2006). Similarly, another
avermectin, abamectin, showed also no negative effects on adults when exposed to
fresh residues and was slightly harmful to L1 larvae (Giolo, Medina, Gruetzmacher,
and Vinuela 2009).
In conclusion, emamectin benzoate is compatible with A. swirskii and
O. laevigatus when applied 3 days before predator introduction, and with
M. pygmaeus and C. carnea in direct spray application. Taking into account these
considerations would complement biological control of the BCAs in pepper
greenhouses.
Acknowledgements
This work was partially supported by the Spanish Ministry of Education and Culture (projectAGL 2007-66399-C03-01 and AGL2010-22196-C02-02 to Elisa Vinuela). F. Amor andP. Bengochea acknowledge the Ministry of Education and Culture and UniversidadPolitecnica de Madrid (UPM), respectively, for doctoral fellowships.
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