Testing Spirotetramat as an Alternative Solution to ...

Journal of Economic Entomology Advance Access published July 29, 2015 INSECTICIDE RESISTANCE

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RESISTANCE MANAGEMENT

Testing Spirotetramat as an Alternative Solution to Abamectin for Cacopsylla pyri (Hemiptera: Psyllidae) Control: Laboratory and Field Tests STEFANO CIVOLANI,1,2,3 MAURO BOSELLI,4 ALDA BUTTURINI,4 MILVIA CHICCA,1 STEFANO CASSANELLI,5 MARIA GRAZIA TOMMASINI,6 VASSILIS ASCHONITIS,1 AND ELISA ANNA FANO1

J. Econ. Entomol. 1–6 (2015); DOI: 10.1093/jee/tov228

ABSTRACT Aim of the study was to investigate the performance of the new insecticide “spirotetramat” as an alternative solution of “abamectin” for the control of Cacopsylla pyri L. (Hemiptera: Psyllidae) in the context of an IPM program in European pear, Pyrus communis L.. Laboratory bioassays for the estimation of LC50 and LC90 of both insecticides were performed using four populations collected in Emilia-Romagna (Italy) orchards where different pest management strategies were used (organic, integrated, and conventional). The same populations were also analyzed for the main insecticide detoxifying activities in nymphs by spectrofluorimetric in vitro assays. The performance of the two insecticides was also tested on field on one population under integrated pest management conditions. The laboratory experiments showed that the LC90 of spirotetramat were lower than the highest field concentration allowed in Europe (172.80 mg AI liter 1) giving reassurance about the efficacy of the product. Concerning the abamectin, the laboratory bioassays did not show strong indications of resistance development of C. pyri populations of Emilia-Romagna. A similarity in enzyme detoxifying activity was observed in both insecticides indicating a general absence of a significant insecticide resistance. The field trial showed a high efficacy (>90 %) of spirotetramat on C. pyri already after 15 d from application, and it was significantly higher from abamectin. Overall, spirotetramat is one more choice for C. pyri control, as well as abamectin in order to minimize the risks of occurrence of insecticide resistance. KEY WORDS spirotetramat, Cacopsylla pyri, psylla control, insecticide resistance The pear psylla, Cacopsylla pyri L. (Hemiptera: Psyllidae), is a common pest of pear orchards (Pyrus communis L.) in Europe, USA, and Canada (Berrada et al. 1996). The C. pyri usually presents four to five generations per year in this region and damages pear trees mainly through honeydew excretion by nymphs, which harms leaves tissues and creates fruits russeting. Honeydew also acts as a growth medium for black sooty molds, whose presence on fruit drastically reduces their market value. In addition, C. pyri is a vector of “Candidatus Phytoplasma pyri” (Seemu¨ller and Schneider 2004) responsible of Pear Decline disease (PD) that reduces tree vigor and may be even lethal to trees. During the past two decades, C. pyri generally caused less damage to pear orchards of EmiliaRomagna (northern Italy), probably due to the success of correct control programs based on integrated pest

1 Department of Life Sciences and Biotechnology, University of Ferrara, Italy. 2 InnovaRicerca s.r.l. Monestirolo, Ferrara, Italy. 3 Corresponding author, e-mail: [email protected]. 4 Servizio Fitosanitario Regione Emilia-Romagna, Italy. 5 Department of Life Sciences, University of Modena and Reggio Emilia, Italy. 6 Centro Ricerche Produzioni Vegetali, Cesena, Italy.

management (IPM) (Galassi and Sattin 2014). These control programs included new selective chemical or microbiological agents (e.g., Granulovirus—CpGV) specifically targeted to another pear key pest, Cydia pomonella L. (Lepidoptera: Tortricidae). These selective insecticides do not reduce natural enemies of C. pyri, particularly Anthocoris nemoralis F. (Anthocoridae: Hemiptera), the main natural predator of pear psyllids (Shaltiel and Coll, 2004). In this way, it is also succeeded the biological-natural control of the pest due to Anthocoris nemoralis F. (Shaltiel and Coll, 2004). Until now, for control of sucking insect pests the available chemical insecticides from different chemical classes were few (Ishaaya 2001, Bru¨ck et al. 2009): in the case of C. pyri, the only one employed from 2005 to 2011 was abamectin (Civolani 2012). In the past, the repeated use of chemical active ingredients against C. pyri (e.g., organophosphorous, pyrethroids, and chitin-inibitors, and some others like amitraz which was banned in Italy from 2004) caused the development of resistance; thus, several insecticides employed to control the pear psylla showed a sharp decline in activity (Berrada et al. 1995, Civolani 2012). After a sudden outbreak of C. pyri populations in some pear orchards of Emilia-Romagna (Italy) in 2005, a survey of abamectin bioassays was performed from 2005 to 2008 on these orchards. Overall, the bioassays data indicated

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that C. pyri populations of Emilia-Romagna had not yet developed resistance to abamectin (Civolani et al. 2010), and similar findings were also derived during the same years in C. pyri populations of Spain (Miarnau et al. 2010). A new insecticide, spirotetramat, has been introduced in 2012 in Italy against C. pyri. Spirotetramat is a persistent foliar active ingredient belonging to the class of tetramic acid derivates (Cantoni et al. 2008, Nauen et al. 2008, Bru¨ck et al. 2009) with a novel mode-of-action (MoA) that interferes with lipid biosynthesis and act as inhibitors of acetyl-coenzyme A carboxylase (ACCase) (Demaeght et al. 2013, Lu¨mmen et al. 2014). Spirotetramat is listed in Group 23 of the Insecticide Resistance Action Committee (IRAC) together with spirodiclofen and spiromesifen. In field applications, this active ingredient causes death at immature stages not only in psyllids, but also in aphids, scales, mealybugs, whiteflies and thrips (Cantoni et al. 2008, Nauen et al. 2008). In baseline studies, spirotetramat showed excellent activity against nymphs of Myzus persicae, Aphis gossypii, Phorodon humuli, and Bemisia tabaci (Bru¨ck et al. 2009). Up to date, no laboratory data are available on spirotetramat effects on C. pyri, unlike field ones (Pasqualini and Civolani 2010; Bangels et al. 2010, 2011; Jaworska et al. 2012). For this reason, the susceptibility to spirotetramat and abamectin was evaluated both in field and laboratory conditions based on foliar spray applications. The values of LC50 and LC90 were estimated for C. pyri populations collected in Emilia-Romagna orchards where different pest management strategies were used (organic, integrated, and conventional treatments). The same populations were also analyzed for the main insecticide detoxifying activities in nymphs by spectrofluorimetric in vitro assays, whose results were compared to bioassays. Thus, aim of the study is to provide an up to date “baseline” data set for spirotetramat against C. pyri in Emilia-Romagna region as a reference for future comparisons, taking into account previous representative works which were conducted in Switzerland for amitraz (Schaub et al. 2001, 2002) and in Spain and Italy for abamectin (Civolani et al. 2010, Miarnau et al. 2010). Materials and Methods Insect Populations. C. pyri populations were collected by aspirator (pooter) sucking of adults falling onto a sheet after beating tray technique in a 50-ml plastic test tube in July of 2012 from four pear orchards of Emilia-Romagna region (Italy) (age of trees between 12–20 yr) where different pest management strategies were applied. The four orchards are located in the province of Ferrara, the most important province of pear production in the region, and labeled from FE1 to FE4. The FE1 population was collected from an isolated organic farm located in the municipality of Diamantina, and it was used as control since chemical insecticides are not applied in this farm. The control farm was chosen based on its isolation and long distance from other treated pear orchards in order to

avoid C. pyri immigration, as suggested for C. pyricola by Croft et al. (1989) and for C. pyri by Schaub et al. (1996). The FE2 population was collected from an IPM farm of the municipality of Montesanto, where all options to reduce pest populations with priority to nonchemical measures were applied according to IOBC (2008) (i.e., chemical treatments were not performed on a preventive basis, but only when required). In this farm, the practice against C. pyri was always a single treatment of abamectin per year (in May on the C.pyri second generation) and no significant economical damages in the orchard have ever been detected. On the contrary, the FE3 and FE4 populations were collected from the municipalities of Baura and Massafiscaglia, respectively, where IPM was not applied and several preventive and repeated insecticide treatments were performed. Before 2012, the C. pyri control in these two farms was carried out using two or more abamectin treatments per year. A high infestation by C. pyri occurred during 2011 in the FE4 farm with heavy economic damages. After 2012 the control strategy in both FE3 and FE4 changed to two treatments per year; one with abamectin and one with spirotetramat. Foliar Spray Bioassays in Laboratory Conditions. Foliar spray bioassays were performed in 2012 following a slightly modified procedure after Schaub et al. (1996). About 200 unsexed C. pyri adults from the respective populations of he four orchards were released in a two rearing cages (BugDorm-2120 Insect Rearing Tent 60 by 60 by 60 cm, MegaView Science Co., Ltd. Taichung, Taiwan) containing 18 micropropagated untreated and potted pear plants (cv. Bartlett) per cage. The adults were kept at 23 6 1 C, 65% RH, and a photoperiod of 16:8 (L:D) h in order to obtain eggs of the same age after 4 d from the release. The number of leaves per plant containing eggs ranged from 10 to 20. C. pyri eggs were counted on the infested pear plants until the cumulative number of eggs on the leafs, starting counting from the tip, reached at least 100 eggs by visual inspection with a magnifying lens. One–two days before hatching of eggs (final embryonic development—yellow stage eggs), spirotetramat IRAC Group 23 (Movento 48 SC, 48 g liter 1 suspension concentrate formulation, Bayer CropScience, Milan, Italy) and abamectin IRAC Group 6 as a chemical reference (Vertimec EC, 18 g liter 1 emulsifiable concentrate formulation, Syngenta Crop Protection, Milan, Italy) were applied by a hand sprayer until dripping. The serial concentrations which were tested were from 0.18 to 13.50 mg abamectin liter 1 and 0.48 to 120.00 mg spirotetramat liter 1 in distilled water covering a mortality range of 5 to 95%. These ranges of concentrations were selected after preliminary trials in order to get observations covering the full range of LC probit curves for optimizing their calibration. Three pear plants (three replicates) were treated for each serial insecticide concentration. A proper control batch of three plants sprayed only with distilled water was also prepared for each population. The surviving nymphs were scored 10 d after treatment. Nymphs were considered alive when they could move and produce honeydew. The dead nymphs were

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CIVOLANI ET AL.: ACTIVITY OF SPIROTETRAMAT ON Cacopsylla pyri

estimated by subtracting the number of surviving nymphs to the total number of eggs counted before insecticide applications. The experiments follow a complete randomized experimental design. Enzymatic Activity. For each population, levels of detoxication activities due to mixed-function oxidases (MFO), glutathione S-transferases (GST), and carboxylesterases (EST) were analyzed in whole homogenates of C. pyri nymphs, mass-reared on pear plants. For each population, three groups of about 80 nymphs were homogenized in 150 ml of ice-cold 0.1 M sodium phosphate buffer, pH 7.6, (plus 0.1% Triton-X for the carboxylesterase assay) in a glass Potter homogenizer. The homogenates were centrifuged at 10,000 g at 4 C for 15 min and aliquots of 30 ml of resulting supernatants were used to determine cytochrome P450dependent monooxygenase activity (MFO), while 5 ml only were used for glutathione S-transferase (GST) as well as carboxylesterase (EST) assessments. Spectrofluorimetric in vitro assays (FLUOstar OPTIMA, BMG LabTech, Offenburg, Germany) were carried out in 96well microplates (Corming Life Sciences, Lowell, MA) using different substrates: 7-ethoxy-coumarin (7-EC) for MFO, 1-chloro-2,4-dinitrobenzene (CDNB) for GST and 1-naphtyl acetate (1-NA) for EST tests as previously described (Stumpf and Nauen 2002, Rauch and Nauen 2002). Field Study. A field trial was carried out according to the Good Experimental Practice (GEP) following the EPPO/OEPP guidelines (https://archives.eppo.int/ EPPOStandards/efficacy.htm) based on the protocol number PP 1/44(2). The efficacy of spirotetramat (Movento 48 SC) in the control of C. pyri secondgeneration nymphs was evaluated in a commercial pear orchard (cv Kaiser Alexander of 15 years old) located in Malalbergo (Bologna, Italy) (BO1 population). The pest management treatments used during the previous years is similar with the ones used in FE2. Four adjacent parallel replicate blocks with five plots per block and three trees per plot (24 m2 per plot) were used based on a randomized blocks design (five plots per

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block, one for control, two treatments of spirotetramat and two of abemectin). The spirotetramat application was performed at two different doses (180.0 and 216.0 g AI ha 1) plus mineral oil (4500 g AI ha 1), (Oliocin 696 g liter 1 water oil emulsifiable formulation, Bayer CropScience, Milan, Italy) and it was compared with a chemical reference, abamectin (Vertimec EC) applied at two different doses (16.8–27.0 g AI ha 1) plus mineral oil (4500 g AI ha 1) (Oliocin 696 g liter 1 water oil emulsifiable formulation, Bayer CropScience, Milan, Italy) and with an untreated control. Mineral oil control was not used since it does not have insecticide activity when is used alone. It is a common practice to mix it with chemical insecticides because it increases adhesion and penetration of the insecticides in the plant tissues. All treatments were applied at an equivalent water volume of 1250 liter ha 1 using a spray mistblower (Stihl SR 420, Andreas Stihl S.p.A., Milan, Italy). All treatments were applied once on 9 May 2012 in order to control the second generation (standard practice when no outbreak of the pest is observed in the next generation). The nymphs on 10 previously marked shoots per plot were scored, respectively, at 8, 15, 23 and 29 days after application (DAA). Data Analysis. The estimates of LC50 and LC90 for nymph mortality and their 95% confidence limits (CL) were obtained using the PoloPlus Version 2.0 (LeOra Software Company, Petaluma, CA) based on Finney’s Probit analysis (1971) (Robertson et al. 2007). Significant differences were determined by non-overlapping of the 95% confidence limits of LC50 and LC90 and by the hypothesis of equality of slopes and intercepts between the Probit-log dose curves of FE2, FE3, and FE4 versus F1 for P < 0.05. Resistance factors (RF) were calculated by dividing the LC50 and LC90 values of the C. pyri FE2, FE3, and FE24 by the respective LC50 and LC90 values of the C. pyri of FE1. The enzymatic activities were normalized on protein content per each extract according to Bradford procedure and their values were subjected to an analysis of variance (ANOVA). Means were separated by a

Table 1. Probit-log dose response regression line of Cacopsylla pyri nymphs, treated with spirotetramat and abamectin at the nearhatching yellow egg stage Population Total number of individuals tested

Spirotetramat FE1 FE2 FE3 FE4 Abamectin FE1 FE2 FE3 FE4 a

Number and range of concentrations applied

Slope 6 SE

LC50 mg AI liter

1

RF50a

LC90 mg AI liter

(95% CL)

c

RF90

v2 (df)

P-valueb

Number

Range

1,734 2,585 1,430 3,584

7 7 7 7

0.48–120.0c 0.48–120.0 0.48–120.0 0.48–120.0

1.52 6 0.08 9.71 (4.57–14.92) 1.11 6 0.05 6.51 (1.93–12.09) 1.28 6 0.09 12.53 (0.71–26.36) 1.37 6 0.05 9.73 (3.33–16.96)

– 0.67 1.29 1

67.3 (42.45–158.62) 91.04 (52.44–244.79) 125.22 (64.5–1130.1) 83.5 (55.17–154.14)

– 1.35 1.86 1.24

208 (18) 273 (18) 295 (17) 363 (20)

– 90 %) of spirotetramat on C. pyri in the field trial already at 15 DAA, which was also confirmed at 23 DAA and 29 DAA. These values agree with those recently observed in other C. pyri field trials (Pasqualini and Civolani 2010; Bangels et al. 2010, 2011; Jaworska et al. 2012). Besides the high field mortality of spirotetramat observed in BO1 population, all the LC90 values obtained in the laboratory experiments on FE1, FE2, FE3, and FE4 populations were lower than the highest field concentration allowed in Europe (172.80 mg AI liter 1), giving reassurance about the efficacy of spirotetramat and providing a reference baseline for future comparisons of C. pyri population resistance to the active ingredient. Concerning the abamectin, as a reference insecticide, the laboratory bioassays did not show strong indications of resistance development of C. pyri populations of Emilia-Romagna, verifying the results of previous surveys in years 2007 and 2008 (Civolani et al. 2010). Moreover, the LC50 and LC90 values of the FE2, FE3, and FE4 in comparison to FE1 are within the natural variation, as reported by Schaub et al. (2002). Besides bioassays, biochemical profiles may be performed for monitoring insecticide resistance. In our study mixed-function oxidases (MFO) and glutathione S-transferases (GST) were comparable among C. pyri populations even some differences were detected for carboxylesterases (EST). However, the differences detected for EST based on our bioassay data apparently have no effect on spirotetramat and abamectin resistance in C. pyri. High levels of GST and MFO activities were also detected in abamectin-resistant strains of Bemisia tabaci, Tetranychus urticae and Plutella xilostella, in the last case associated to high levels of EST (Stumpf and Nauen 2002, Wang and Wu 2007, Qian 2008). The similarity in enzyme detoxifying activity observed in our study probably reflects the natural variation among C. pyri populations, and it is consistent with the general absence of a significant insecticide resistance. Overall, spirotetramat is considered to be safe to most beneficial insects (Bru¨ck et al. 2009) and for this reason can be considered a valuable alternative to abamectin, including rotation, in order to minimize the risks of occurrence of insecticide resistance.

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Acknowledgments This work was supported by the project “CRPV-Servizi di supporto per l’applicazione dei Disciplinari di Produzione Integrata e delle norme di Produzione Biologiche nell’ambito del PSR-Mis. 214, azioni 1 e 2” founded by Emilia-Romagna Region (PSR 2007\2013 Mis. 511).

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