Crop Protection 91 (2017) 82e88
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Determination of resistance and resistance mechanisms to thiacloprid in Cydia pomonella L. (Lepidoptera: Tortricidae) populations collected from apple orchards in Isparta Province, Turkey _ ¸ ci a, Recep Ay b, * Mesut Is a b
Fruit Research Institute, Department of Plant Protection, Isparta, Turkey Suleyman Demirel University, Faculty of Agriculture, Department of Plant Protection, 32260, Isparta, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 June 2016 Received in revised form 21 September 2016 Accepted 23 September 2016
The codling moth, Cydia pomenella is considered as the most important pest of apple worldwide and it causes significant economic losses yearly in orchards where it is not controlled effectively. The purpose of this study was to determine the resistance ratios to thiacloprid and the detoxification enzymes of Cydia pomonella from apple orchards in Isparta, Turkey. Populations of codling moth were collected from six orchards in the region and the diapausing larvae were treated with thiacloprid and chlorpyrifos by topical application. The LD50 values of field and a susceptible population were used to determine the resistance ratios to thiacloprid and chlorpyrifos. The corresponding LD50 values of C. pomonella populations showed a low (5.5e6.7 fold) or medium resistance (11.2e16.5 fold) against thiacloprid but were susceptible to chlorpyrifos. In studies conducted with synergists, piperonyl butoxide (PBO) and S,S,S, tributyl phosphorotrithioate (DEF) had a significant synergistic effect on two populations (from Gelendost and Senirkent) that medium resistance to thiacloprid. The levels of detoxifying enzymes [esterase, glutathion eSe transferase (GST) and cytochrome P450 monooxygenase (P450)] were investigated using biochemical methods and differed depending on the population. Based on the results of the enzyme analyses, the P450 and esterase enzymes may play a role in the resistance in codling moths to thiacloprid. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Cydia pomonella Resistance Thiacloprid Chlorpyrifos Detoxifying enzymes Negative cross-resistance
1. Introduction The codling moth (Cydia pomonella L.) is an important pest of apples, pears, quince and walnuts that is found in all countries, with the exception of Japan, Taiwan, Korea, parts of eastern China and Western Australia and possibly Brazil. Larvae feeding in the fruits cause two types of damage. First, the larvae create a deep entrance hole as they bore toward the pip of the fruit and the second type occurs from shallow holes in the fruit (Barnes, 1991; Beers et al., 1993, 2003; Pedigo and Rice, 2009). Turkey is the third largest apple producer behind China and the USA (Anonymous, 2015a). In Isparta, apple production has reached 634,000 tons produced on 21,761 ha which represents 22% of the countrywide production and it contributes annually to the economy of Isparta by approximately
* Corresponding author. E-mail address:
[email protected] (R. Ay). http://dx.doi.org/10.1016/j.cropro.2016.09.015 0261-2194/© 2016 Elsevier Ltd. All rights reserved.
231 million dollars (Anonymous, 2015b). In the region of Isparta that is located at south western of Turkey, the codling moth has two generations per year. Producers prefer to use chemicals for the control of the pest. When pesticides fail, the producer increases the dose or the frequency of insecticide application. Unfortunately, the use of high doses to manage pests causes the occurrence of resistance (FAO, 2012). The resistance to insecticides in the codling moth first developed in two different populations to lead arsenate (Hough, 1928). The insecticide DDT began to replace arsenate at the end of the 1940s and the first resistance to DDT was registered in 1951, with widespread resistance to DDT emerging within the next 10 years. Because of the resistance to DDT, the widespread use of organophosphorus insecticides began in the mid - 1950s, providing effective protection against the codling moth (Dunley and Welter, 2000). The resistance to organophosphorus insecticides has since been reported in different countries by many researchers, including the USA (Welter et al., 1991; Bush et al., 1993; Knight et al., 1994),
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North Africa (Blomefield, 1994) and France (Sauphanor et al., 1998). For the insecticide growth regulator diflubenzuron, resistance was identified for the first time in Europe (Waldner, 1993), in Italy (Riedl and Zelger, 1994) and in the southwestern France (Sauphanor et al., 1994). In the following years, the organophosphate group of insecticides was examined for the development of resistance by Sauphanor et al. (2000); Knight et al. (2001), Reyes et al. (2007), Stara and Kocourek (2007) and Voudouris et al. (2011). Reyes et al. (2007), Voudouris et al. (2011), Cichon et al. (2013) _ ¸ ci and Ay (2013, 2014) studied the development of resisand Is tance to the neonicotinoid group of insecticides, including thiacloprid. The resistance of the codling moth to other groups of insecticides has also been examined (Knight et al., 2001; Reyes et al., 2007; Sauphanor et al., 1997; Ioriatti et al., 2007; Sanchez et al., 2008). Thiacloprid is a neonicotinoid insecticide and is in the N-cyanoamidines group, the classification of which is based on the molecular properties of the group (Elbert et al., 2008). For the neonicotinoid insecticides, the mode of action is connected to the nicotinic ACh receptors, with the insecticide behaving as ACh (acetylcholine) affecting the insect's central nervous system of the insect (Elbert et al., 2008; Millar and Denholm, 2007; Jeschke and Nauen, 2008). Thiacloprid is a broad-spectrum, systemic and traslaminar neonicotinoid insecticide. Thiacloprid has been registered since 2006 and used in Turkey against Cydia pomonella and Aphis pomi Deg. attacking apple (Anonymous, 2016). Chlorpyrifos is abroad-spectrum and contact-acting organophosphate insecticide and acaricide. Since 1981, it is has been registered and used in Turkey on agricultural crops against many pests including C. pomonella (Anonymous, 2016). In this study, populations of codling moth collected from apple orchards in Isparta were investigated for the ratios and the mechanisms of resistance to thiacloprid and chlorpyrifos.
2. Materials and methods 2.1. Insects The INRA (AVIGNON)/French National Institute for Agricultural Research provided the susceptible population (Sv) that was used as the reference strain in this study. It was collected from orchards in the southwest regions of France and has been maintained in the laboratory since 1995 (Bouvier et al., 2001; Berling et al., 2009). Field populations were collected from the city and the districts of Isparta. The properties of these populations are represented in Table 1. Samples were collected from a few neighbourhood apple orchards at each site. Diapausing larvae of field populations were collected from apple orchards by using corrugated cardboard tree bands. All populations were maintained with a 16:8 h L:D photoperiod at 25 ± 1 C with 60± 5% relative humidity: the production of codling moths under the same conditions was conducted with
Table 1 Collection site and date of Cydia pomonella populations tested for their response to thiacloprid and their detoxification enzyme. Collection site
Collection date
INRA (France) (Susceptible Sv) Isparta Central Senirkent Gelendost irdir Tepeli-Eg irdir Serpil-Eg Yalvaç
e 13.07.2011 12.06.2012 11.06.2012 30.06.2013 02.07.2013 05.07.2013
83
artificial feed (Codling Moth Diet; Soutland Products Inc.) (Reuveny and Cohen, 2007; Stara and Kocourek, 2007). Bioassays were carried out on fifth-stage larvae from the first to third generation laboratory population. 2.2. Insecticide and synergist bioassays 2.2.1. Insecticide bioassays The tests were based on the method described by Sauphanor et al. (1997) and a commercial formulation of the insecticide thiacloprid (Calypso OD 240 g/l; BayerCropscience) and chlorpyrifos (Dursban 4 EC 480 g/l; Dow Agrosciences) was used in this study. Accordingly, the LD50 values of the C. pomonella populations to thiacloprid were determined by topical application. First, the insecticide was diluted with distilled water to prepare the different treatment concentrations. The test treatments were 1 control þ6 concentrations, with 5 replications of each. Each replication included five fifth-stage larvae. The insecticide treatments of 1.0 ml, were applied to the middle of the thorax on the dorsaline of the fifth-stage larvae with a micropipette. Distilled water was applied as the control. Following the application of thiacloprid, the larvae were transferred to an artificial diet in Petri dishes in groups of five, and after 72 h, the live and dead larvae were counted. Before each experiment, mortality tests were performed to determine the concentration range for approximately 10e90% mortality. Experiments in which control mortality exceeded 10% were repeated. The pooled data were analyzed using Probit analysis (POLO PC) computer program (LeOra Software, 1994) to estimate the LD50 and LD90 values with 95% CLs. The resistance ratio was calculated according to the formula: RR ¼ LD50 value of the orchard population/LD50 value of the susceptible population. The resistance ratios were classified according to Koh et al. (2009): low resistance RR 10, medium resistance 10 < RR 40, high resistance 40 < RR 160 and very-high resistance RR > 160. 2.2.2. Synergist bioassays Three synergists, piperonyl butoxide (PBO), S,S,S, tributyl phosphorotrithioate (DEF) and diethyl maleate (DEM), were used to determine whether enzymes played a role in the metabolism of resistance to the insecticide. The monooxygenase enzyme inhibitor PBO (2000 mg/l), the GST enzyme inhibitor DEM (1000 mg/l) and the esterase enzyme inhibitor DEF (1000 mg/l) were prepared for use (Van Leeuwen et al., 2004; Wang et al., 2009; Van Leeuwen and Tirry, 2007). All synergist were dissolved in acetone: distillated water (1:1; v: v) The synergist solutions were applied to the thorax dorsaline of the fifth instars larvae as 2.0 ml of PBO, 1.0 ml of DEF, and 1.0 ml of DEM with a micropipette one hour before the insecticide applications. The synergists were dissolved in acetone. One hour after the application of the synergists, the insecticide concentrations were applied to the larvae as described previously. The treatments for insecticide tests were 1 control þ6 concentrations, with 5 replications. Only the synergist was applied to the control. The live and dead larvae were counted after 72 h. Experiments where control mortality exceeded 10% were repeated. The synergistic ratio (SR) was calculated using the following formula: SR ¼ LD50 of thiacloprid without a synergist/LD50 of thiacloprid with a synergist (Kim et al., 2004). 2.3. Biochemical studies 2.3.1. Enzyme extract-preparation Some of the larvae used for bioassay studies were stored at 80 C for biochemical studies in a deep freeze. Ten fifth instar codling moth larvae were homogenized in 2.0 ml of homogenization buffer (0.1 M sodium phosphate buffer, pH: 7.6, and 1 mM
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Editic acid (EDTA), 1 mM 1,4-dithiothreitol (DTT), 1 mM phenylmethyl sulfonylfluoride (PMSF) and 1 mM phenylthiourea (PTU)). The homogenate was centrifuged for 20 min at 15,000 g and then for 20 min at 20,000 g at þ4 C. Then, the clear supernatant was collected and used as the enzyme resources for analysis of activity of photometric esterase (EST), GST and P450 and the fluorometric of P450 (Qian et al., 2008). 2.3.2. Photometric reading of total esterase, GST and P450 enzyme activity The detoxification enzymes of codling moth were determined using the method adapted from Qian et al. (2008) using a microplate reader. To determine the esterase enzyme activity, 90 ml of 0.1 M sodium phosphate buffer (pH: 7.6) and 200 ml of a solution with 10 mM 1-naphthyl acetate and 4 mM fast blue RR salt were added to each microplate well. The reaction was started with the addition of 10 ml of the enzyme source from a stock solution. The control was read without the homogenate. The esterase activity was measured continuously for 10 min at 450 nm and 27 C in a VersaMax kinetic microplate reader (Molecular Devices) and the data were analyzed using Softmax PRO software to develop the kinetics plots with linear regression. The tests were conducted with 3 or 4 replications. For the determination of GST activity, 1-chloro-2,4dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were both used as substrate. For the activity with CNDB as the substrate, 10 ml of the enzyme source from a stock solution, 90 ml of Tris HCL buffer (0.05 M, pH: 7.5), 100 ml of 1.2 mM CDNB and 100 ml of 6 mM glutathione (GSH) were added to each cell of the microplate. For the activity with DCNB as the substrate, 100 ml of the enzyme source from a stock solution, 100 ml of 1.2 mM DCNB and 100 ml of 6 mM GSH were placed in the wells of a microplate (VersaMax, Molecular Devices). The GST activity was read at 27 C and 340 nm for 10 min in a microplate reader. The control was read without the homogenate. The tests were conducted with 3 or 4 replications. For the determination of cytochrome P450 monooxygenase enzyme activity, p-nitroanisole (PNOD) was used as the substrate. The method to determine the activity of this enzyme was adapted from Qian et al. (2008). For the PNOD (p-nitroanisole) test, a mixture with 90 ml of the enzyme source þ 100 ml of 2 mM pnitroanisole was added to each well of a microplate and incubated at 27 C for 5 min. The reaction was started with the addition of 10 ml of 9.6 mM NADPH. The control cells were read without the homogenate. The enzyme activity was read at 405 nm for 10 min in 25 s intervals (VersaMax, Molecular Devices). The P450 enzyme readings were performed in at least triplicate. 2.3.3. Photometric identification of acetylcholinesterase (AChE) enzyme activity in C. pomonella The activity of acetylcholinesterase (AChE) was determined using a method modified from Stumpf et al. (2001). Ten fifth instar larvae of C. pomonella were homogenized with a plastic pestle in 0.1 M phosphate buffer (2 ml, pH: 7.5) with 0.1% Triton X-100 in an Eppendorf tube. The tissues were dissolved for 25 min on ice and the homogenate was centrifuged at 4 C at 11,000 rpm for 25 min; the supernatant was used as the source of the enzyme. To measure AChE activity, 50 ml of the enzyme solution in 100 ml of Acetylcholine iodide (ATChI), 100 ml of 5.5-dithiobis (2-nitrobenzoic acid) (DTNB) and 50 ml of phosphate buffer were put into the microplate cells. The final concentration of ATChI and DTNB was 0.5 mM in the 300 ml. The AChE activity was read for 20 min at 23 C and 405 nm with the microplate reader (VersaMax, Molecular Devices). The control cells were read without the homogenate. For the AChE readings, at least 3 replications were used. The Bradford (1976),
method was used to determine the total protein amount. 2.3.4. Fluorometric identification of GST enzyme activity in C. pomonella For the fluorometric determination of GST enzyme activity, the method of Ioriatti et al. (2007) was adapted. Five fifth instars larvae were placed in an Eppendorf tube, homogenized with a plastic pestle in 1 ml of Hepes Buffer (50 mM, pH: 7), and centrifuged at 4 C at 15,000 g for 15 min. After centrifuging, the supernatant was used as the source of the enzyme. To measure the GST enzyme activity, 30 ml of supernatant, 168 ml of 100 mM GSH (in Hepes Buffer), and 2 ml of 30 mM MCNB (dissolved in methanol) were added to black microplate cells. After incubation at 22 C for 20 min, the activity was read as excitation at 380 nm and emission at 450 nm (SpectraMax Gemini XS model; Molecular Devices). 2.3.5. Fluorometric identification of P450 enzyme in C. pomonella For the fluorometric determination of P450 enzyme activity, the method of Rauch and Nauen (2003) was adapted. The codling moth larvae were homogenized as described in the chapter “Enzyme extract-preparation.” Fifty microliters of homogenate, 80 ml of 0.5 mM 7-ethoxycoumarin and 10 ml of 9.6 mM NADPH were added to the black wells of a 96-well microtiter plate. The plate was incubated by shaking at 44 g at 30 C in a centrifuge for 30 min. To remove the NADP, the plate cells received 10 ml of 100 mM oxidised glutathion and 13 ml of glutathion reductase (1.3 U) and were incubated for 10 min. To stop the reaction, 125 ml of 50% acetonitrile (in Trizma base buffer (0.05 M, pH: 10)) was added. The activity was read as excitation at 390 nm and emission at 465 nm as the endpoint (SpectraMax Gemini XS model; Molecular Devices). The Bradford (1976), method was used to determine the total protein amount for all enzymes. The activity of the enzymes (mOD/ min/mg protein or RFU/30 min/mg protein) was analyzed with Softmax PRO software as values of mg of protein. The data were analyzed using the General Linear Model (GLM) procedure of SAS (1999) by using population in the model and PDIFF statement was used to compare enzyme activity means by population. As for significance level (P < 0.05) was accepted as statistically significant (SAS, 1999). 3. Results 3.1. Bioassays The LD values and the resistance ratios to thiacloprid and chlorpyrifos in the populations of C. pomonella collected from the apple orchards of Isparta, Gelendost, Senirkent, Tepeli, Serpil, and Yalvaç in the city of Isparta are presented in Table 2. The resistance ratios represented by LD50 values of orchard populations of the codling moth relative those of the Sv population against thiacloprid ranged between 5.5 and 16.5fold. Thus, all orchard populations showed low or medium resistance to thiacloprid (Table 2). Using the LD90 values, the resistance ratios ranged between 2.5 and 14.9fold. Resistance against chlorpyrifos in codling moth populations has not been determined (Table 2). Resistance ratios of orchards populations to chlorpyrifos did not differ significantly compared to the Sv population at confidence limits of LD50. 3.2. Synergist-insecticide application To determine the effects of synergists on the efficacy of thiacloprid, the synergists DEF, PBO, and DEM were examined, with the results provided in Table 3. The effects of the three synergists were significant in the populations (Gelendost and Senirkent) with medium resistance to thiacloprid, based on the resistance ratios
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Table 2 Concentration-response data for Cydia pomonella to thiacloprid and chlorpyrifos including a laboratory susceptible strain (Sv) and various populations collected from apple orchars fields. Insecticide
Population
n*
Slope ± SE
X2
h
LD50 (ml/l) (%95 CL**)
Thiacloprid
Sv
175
1.25 ± 0.22
3.43
0.86
Isparta
200
2.13 ± 0.28
2.69
0.54
Gelendost
200
1.11 ± 0.28
4.95
0.99
Senirkent
225
1.46 ± 0.19
10.25
1.71
Tepeli
200
1.42 ± 0.25
3.12
0.62
Serpil
225
1.20 ± 0.16
1.94
0.32
Yalvaç
225
1.32 ± 0.17
3.11
0.52
Sv
175
2.13 ± 0.20
6.80
1.70
Isparta
200
1.48 ± 0.20
0.36
0.07
Gelendost
200
1.45 ± 0.22
1.76
0.35
Senirkent
175
1.90 ± 0.27
1.82
0.45
Tepeli
200
1.64 ± 0.24
1.93
0.39
Serpil
200
1.52 ± 0.24
2.21
0.44
Yalvaç
200
1.46 ± 0.20
1.18
0.24
2.62 1.74e4.13 17.47 13.52e23.05 29.32 19.17e51.50 43.16 25.31e88.09 16.32 9.00e25.78 15.97 10.78e23.27 14.52 10.09e20.53 1.76 0.71e3.05 1.93 1.36e2.71 1.18 0.72e1.74 1.04 0.75e1.39 1.18 0.75e1.69 1.68 1.04e2.48 1.33 0.90e1.87
Chlorpyrifos
LD50 RR***
6.66 11.19 16.47 6.22 6.09 5.54
1.10
