Screening test in determination of Colorado potato beetle (Leptinotarsa decemlineata Say.) sensitivity to insecticides Dušanka Inđić, 1 Slavica Vuković, 1 Petar Vukša, 2 Mila Grahovac, 1 Sonja Gvozdenac 1, Dragica Janković, 3 Gordana Forgić, 4 Gordana Mrdak 4 1 University of Novi Sad, Faculty of Agriculture, Novi Sad, Serbia, 2 University of Belgrade, Faculty of Agriculture, Belgrade, Zemun; 3 Provincial Center for plant protection FS of Vojvodina, Novi Sad; 4 SOE Agroinstitute, Sombor, email: vukovic@polj.uns.ac.rs Abstract Sensitivity of 15 field populations of Colorado potato beetle (Leptinotarsa decemlineata Say.) CPB (Apatin, Doroslovo, Karavukovo, Stanišić, Žarkovac, Sremska Mitrovica, Platičevo, Ležimir, Klenak, Stejanovci, Grocka, Korman, Skela, Vrelo and Vinča) was determined. Bioassay was performed using screening test that allows rapid assessment of sensitivity of overwintered adults to insecticides. Sensitivity to four insecticides most commonly used in CPB control in Serbia (chlorpyrifos, cypermethrin, thiamethoxam and fipronil) was assessed. Insecticides were applied at label rates, and two, five and 10 fold higher rates by soaking method. Insect mortality was assessed after 72 h. Sensitivity of CPB adults was assessed according to modified IRAC method (2009) using 15 scale. Out of 15 monitored populations of CPB, three were highly sensitive, six were sensitive, one slightly resistant and five resistant to chlorpyrifos label rate. One population was highly sensitive, seven sensitive, two slightly resistant and five were resistant to cypermethrin. To thiamethoxam label rate, ten populations were highly sensitive and five sensitive. Ten populations were highly sensitive and five sensitive to fipronil label rate. The application of insecticides at higher rates (two, five and 10 fold), that is justified only in bioassays, provided a rapid insight into sensitivity of field populations of CPB to insecticides. Key words: Leptinotarsa decemlineata, insecticides, monitoring, sensitivity Introduction Having in mind the share of potato in human nutrition, even among high risk groups (pregnant women, babies, and children in intensive growth) but also quantities and diversity of pesticides used in potato protection during vegetation and storage, the quality of this aliment is of great importance. Recently, this problem has been supported by alarming data on the chronic effect of pesticides ( The Pesticide Action Network UK ) since the residues were found in 57% of potato tuber samples and 50% of samples of potato chips (Anonymous, 2005). Professional community is familiar with possible consequences of chronic toxicity of pesticides (impact on growth and reproductive ability, endocrine and nervous systems, the occurrence of cancer and other alterations in warmblooded organisms) (Šovljanski et al. 2004, Inđić et al. 2006a, KlokočarŠmit et al., 2006). Negative effect of pesticide application, apart from the abovementioned, is also reflected in occurrence of insect resistance to certain insecticides. According to several authors (Stanković et al. 2004, Inđić et al., 2006b), CPB resistance to carbamates and organophosphates, as well as to pyrethroids (Inđić, 1997; Perić et al. 1997) was registered in a number of populations in Serbia. Literature data indicate that CPB resistance was noted for 42 insecticides, belonging to different chemical groups (organophosphates, carbamates, 115
Screening test in determination of Colorado potato beetle (Leptinotarsa decemlineata Say.) sensitivity to insecticides pyrethroids). This fact justifies CPB ranking among 10 species that most rapidly develop resistance (MotaSanchez et al., 2006; Whalon et al., 2008). Heim et al. (1990) stated that there were differences in CPB sensitivity to insecticides even between populations originating from the same area under potato production, but as quoted by Inđić (1997) also between life stages and generations. In practice, the problem of resistance in our region is resolved in accordance with principles of integrated pest management using different measures (manual collection, less frequent use of biocides and growth regulators) which reduce insecticide selection pressure. However, major producers mainly use chemical measures within the strategy of delaying the mentioned problem that involve application of insecticide mixtures containing active ingredient with different modes of action. Although producers are familiar with other measures, such as the use of predators and parasitoids, plant extracts, combined crops (Copping and Menn, 2000; Gokce et al., 2006), selection of potato varieties for resistance to CPB or spatial distance of potato fields, which directly or indirectly reduce insecticide selection pressure, they are not present in practice. Until 2008 there was no organized CPB resistance monitoring in Serbia. Only after the Ministry of Agriculture of Serbia recognized and accepted the screening test, sensitivity of 41 CPB populations to five insecticides (belonging to five chemical groups) was evaluated. The aim of the study was the creation of simple and rapid test for detection of sensitivity levels of CPB to insecticides and confirmation of resistance, as well as providing simplified presentation of results. The method is based on assessment of sensitivity of overwintered adults to insecticides. On one hand, it allows quick formation (2472 h) of information network related to CPB sensitivity in different production regions, and further establishment of strategy for rational use of insecticides in CPB control. On the other hand, the test provides initial inventory and insight in sensitivity of populations, so that further expensive, sophisticated (lcp lines, LC 50, LC 95, LC 99, CR, tests at the molecular level), time consuming tests are only employed for high risk populations. Material and methods The site selection For monitoring of CPB sensitivity to insecticides 15 sites on the territory of Serbia (Apatin, Doroslovo, Karavukovo, Stanišić, Žarkovac, Sremska Mitrovica, Platičevo, Ležimir, Klenak, Stejanovci, Grocka, Korman, Skela, Vrelo and Vinča) were selected based on the advice of experts from Agricultural extension service, and were marked with GPS coordinates. Applied insecticides Insecticides (Table 1) belonging to four chemical groups were used in this assay: organophosphates (chlorpyrifos), pyrethroids (cypermethrin), neonicotinoids (thiamethoxam) and pyrazoles (fipronil), which differ in history of application. Chlorpyrifos has been in use for nearly 40 years in our region, cypermethrin about 30 years, while thiamethoxam and fipronil for more than 10 years, considering that recently thiamethoxam has become the most frequently used in CPB control. Test insect This bioassay included overwintered adults of CPB field population, which were not in direct contact with insecticides prior to testing. Insects were kept in laboratory conditions, without additional feeding, at temperature of 23±2 C and normal photoperiod (16/8h). Toxicological experiment Bioassay was based on the assumption that CPB populations have reduced sensitivity to insecticides, and on evaluation of the response of overwintered adults to insecticide label rates (the rate determined in field experiments during the registration process and found to cause % mortality) and higher rates. Chlorpyrifos and cypermethrin, which have a long history of use, were applied at five and 10 fold higher quantities than the label rate, and thiamethoxam and fipronil at two and/or fivefold higher rates. Insecticides were applied by insects soaking for 5 sec. The experiment was set up in four replicates with 30 adults per replication (sex ratio 1:1). The sex was determined 116
according to Tribelj and Korol (2001) instructions. Assessment of insecticide efficacy consisted of counting the number of dead (with no signs of vitality), paralyzed (uncoordinated movements and inability to move) and alive insects (normal mobility and vitality). The effect was determined 24, 48 and 72 h after insecticide application. Results were corrected for mortality in the control (Schneider Orelli, 1947) and expressed as the efficacy (E%) achieved only after 72 h. Sensitivity was evaluated on the scale 15, which was created as a slight modification of IRAC method No. 011 (Anonymous, 2009) that refers to pollen beetles (Meligethes spp.). 1 highly sensitive populations (E = %) 2 sensitive (> E 95%) 3 slightly resistant (95> E 90%) 4 resistant (90> E 50%) 5 highly resistant (E <50%) Table 1. Insecticides and rates applied in tests Active ingredient (chemical group) Preparation Apllied rate* chlorpyrifos (480 g/l) (organophosphate) cypermethrin (200 g/l) (pyrethroid) thiamethoxam (250 g/kg) (neonicotinoid) fipronil (800 g/kg) (pyrazole) * Insecticides were applied in 400 l/ha; Pyrinex 48EC Cipkord 20EC Actara 25WG Regent 800WG 1.5l/ha label rate 7.5 l/ha 5 x higher 15 l/ha 10 x higher 0.3l/ha label rate 1.5 l/ha 5 x higher 3 l/ha 10 x higher 60 g/ha label rate 70 g/ha label rate 140 g/ha 2 x higher 25 g/ha label rate 50 g/ha 2 x higher 125 g/ha 5 x higher Results and discussion Biology of CPB is thoroughly studied in our agroecological conditions, and regardless of global climate change, according to present available data there are two generations per year. Overwintered adults and first generation of larvae damage potato plants from sprouting to flowering, while adults of first generation and second generation larvae damage plants from tuber formation until green leaves are present. For CPB control in Serbia there are 43 registered products, based on 20 active ingredients, out of which 18 are of chemical origin, divided into 10 groups (organophosphates, carbamates, pyrethroids, neonicotinoids, pyrazoles, benzoilfenilureas, benzoilureas, macrolides, semikarbazons, antaranil diamides) and two bioinsecticides. Analyzing the efficacy of recommended application (label) rates of insecticides, we tended to simulate conditions and effects that persist in field. The application of two, five or 10 fold higher rates 117
Screening test in determination of Colorado potato beetle (Leptinotarsa decemlineata Say.) sensitivity to insecticides aimed to verify that individuals from the same population survive higher rates of insecticides. The abovementioned indicate which part of CPB population is not sensitive, tolerates, or demonstrates reduced sensitivity to the label rates, or indicates that individuals have developed resistance. Given the fact that there was no correlation between increase in insecticide doses and mortality in the population, i.e. that survival rate was very high regardless of rates applied, but also that the population became resistant, sets the necessity for unconditional exclusion of these compounds from use in potato production, strategy compliance, and continuous mapping of these populations. The sensitivity of CPB populations to chlorpyrifos, cypermethrin, thiamethoxam and fipronil was classified (Table 2 and 3) based on the insecticide efficacy achieved in screening test (72 h of exposure) and slightly modified scale for classification of insecticide sensitivity (Anonymous, 2009). Overwintered adults were chosen as test insect in the assessment of sensitivity to insecticides for a number of reasons. First, it is familiar that they were not in direct contact with insecticides, and are easily maintained before inclusion in the test, they do not have to be fed, and the effects of insecticides (mortality, paralysis, vitality) are easily visible and measurable. Out of 15 surveyed CPB populations only two (S. Mitrovica and Klenak) were highly sensitive to label rate of chlorpyrifos, while six (Apatin, Doroslovo, Karavukovo, Stanišić, Žarkovac and Platičevo) were sensitive and all others were slightly to highly resistant. Namely, only one population from Stejanovci was slightly resistant, while five were resistant, to this insecticide. Five populations (Grocka, Korman, Skela, Vrelo and Vinča) of 15 that were classified as resistant remained in the same category even when treated with higher rates of this insecticide. Given indicates distinct heterogeneity in sensitivity of these populations to chlorpyrifos. According to Wegorek et al. (2011) sensitivity of CPB adults to chlorpyrifos, for three populations from the territory of Poland, monitored in three consecutive years (20082010), and classified according to resistance coefficient (RC), varied within the same population. Results of this study indicate that the same population (Krotoszyn) in 2008 and 2010, in respect to RC was not resistant to chlorpyrifos, while in 2009 it showed slight resistance or some variation in sensitivity. One (Karavukovo) of 15 CPB populations were highly sensitive to cypermethrin label rates. Seven populations (Apatin, Doroslovo, Stanišić, Žarkovac, S. Mitrovica, Platičevo and Klenak) were sensitive, while two populations originating from Ležimir and Stejanovci were slightly resistant, five populations (Grocka, Korman, Skela, Vrelo and Vinča) were resistant to cypermethrin. Three populations (Grocka, Skela and Vrelo) of seven that were classified as slightly resistant to resistant remained in the same category even after cypermethrin was applied at 5 fold higher rates. Populations from Ležimir, Stejanovci, Grocka, Korman, Vrelo and Vinča expressed slight increase in sensitivity when cypermethrin was applied at higher rates, therefore, regardless the applied rate, they were classified as resistant to highly resistant. A number of authors found the differences in sensitivity (LC 50 ) between different stages of CPB to insecticides (Zehnder and Gelernter, 1989, Zhao et al., 2000). According to Inđić (1994, 1997), the toxicity of chlorpyrifos and cypermethrin to overwintered adults, adults and larvae of first generation (stage III) originating from the same population (Zmajevo) differed. LC 50 values for chlorpyrifos were 761, 69 and 972 mg a.i./l respectively, and for cypermethrin 44, 55 and 2 mg a.i./l respectively, indicating heterogeneity in sensitivity among these life stages of CPB. Results of Zamojska et al. (2011) showed that the use of neonicotinoid compounds instead of pyrethroids had very good effect in terms of increased sensitivity to pyrethroids, which is a good example for antiresistance strategy. CPB sensitivity to thiamethoxam and fipronil is shown in Table 3. These two insecticides are, unlike the previous one in use for shorter period, over 10 years, and were introduced almost at the same time. Given the mentioned facts, we used thiamethoxam at label rates (0.06 and 0.07 kg/ha) and two fold higher (0.14 kg/ha), while the product based on fipronil was also applied at label rate (0.025 kg/ha), two and five fold higher (0.05 and 0.125 kg/ha), assuming that CPB sensitivity to these compounds has not yet been compromised. 118
Ten of 15 CPB populations demonstrated high sensitivity to recommended application (label) rate of thiamethoxam. Efficacy of thiamethoxam was 96.5% in five populations originating from Apatin, Doroslovo, S. Mitrovica, Platičevo and Ležimir and that were classified as sensitive. Increase in thiamethoxam application rate caused slightly increased sensitivity in one population (Doroslovo), yet in one population (Platičevo) increased application rate did not cause increase in sensitivity. Whalon and Ferro (1998) stated, which is also our opinion, that the introduction of neonicotinoid based insecticides in 1997, brought a relief in production areas where CPB had become resistant to other insecticides. However, first cases of resistance to neonicotinoid compounds (imidacloprid) were registered in commercial potato crops in several U.S. shortly after its introduction (MotaSanchez et al., 2006; Alyokhin et al., 2006). Alyokhin et al. (2007) noted a significant variability in neonicotinoid resistance between CPB populations. These authors recorded 37 fold higher resistance to imidacloprid, and 10 fold higher to thiamethoxam compared to sensitive, laboratory populations. In production of Serbia, efficacy of thiametoxam is so far satisfactory, although neonicotinoid based products are used for tubers treatment before planting (imidacloprid) and for foliar application, but are in accordance with IPM. When fipronil was applied at label rate, ten from 15 populations of CPB were highly sensitive, and regardless of the applied rates the efficacy was %. Five populations (Apatin, Stanišić, Ležimir, Stejanovci and Vrelo) were sensitive. In one population (Stanišić), the sensitivity was not increased with the increase of application rates of fipronil. Results of Inđić et al. (2012) showed that from 15 monitored populations of CPB in Serbia, two were sensitive to label rate of chlorpyrifos, one was slightly resistant, 11 were resistant and one population was highly resistant. Concerning cypermethrin, two populations were sensitive, two slightly resistant, five were resistant and six highly resistant. Highly sensitive to thiamethoxam label rate were 12 populations, while three were sensitive. In the case of fipronil applied at label rate, two populations were highly sensitive, six sensitive, one slightly resistant and six were resistant. The test presented in the paper can be performed in scant experimental conditions, with no special requirements for specific equipment. Handling procedures are very simple and the results achieved are evident and do not require particular statistical analysis, aside from precise classification on the scale from 1 to 5. The results obtained in screening test, based on the sensitivity of overwintered adults of CPB to insecticides are useful in rational selection of products to be used against CPB larvae which are about to occur in the vegetation, and for excluding insecticides that adults are less susceptible/ resistant to, and choosing the most effective one. Table 2. Sensitivity of overwintered CPB adults to chlorpyrifos and cypermethrin, scale 15 Sites Apatin Doroslovo Karavukovo Kg; l/ha 1.5 efficacy (%) of efficacy (%) of chlorpyrifos Kg; cypermethrin l/ha 1 2 3 4 5 1 2 3 4 5 96.4 7.5 1.5 15 3.0 1.5 96.4 7.5 1.5 15 3.0 0.3 0.3 1.5 0.3 97.1 7.5 1.5 99.0 15 3.0 96.5 96.5 119
Screening test in determination of Colorado potato beetle (Leptinotarsa decemlineata Say.) sensitivity to insecticides Stanišić 1.5 0.3 7.5 1.5 15 3,0 98.2 Žarkovac 1.5 96.3 7.5 1.5 15 3.0 0.3 98.1 97.2 Sremska Mitrovica 1.5 7.5 1.5 15 3.0 0.3 97.4 Platičevo 1.5 96.6 7.5 1.5 15 98.3 3.0 0.3 Ležimir 1.5 7.5 1.5 15 98.3 3.0 0.3 91.6 Klenak 1.5 7.5 1.5 15 3.0 0.3 97.4 Stejanovci 1.5 94.1 7.5 98.3 1.5 15 3.0 0.3 94.1 Grocka 1.5 57.1 7.5 70.0 1.5 15 85.7 3.0 0.3 96.1 72.7 85.7 Korman 1.5 70.1 7.5 75.3 1.5 15 85.7 3.0 0.3 98.7 92.2 62.3 Skela 1.5 85.0 7.5 91.3 1.5 15 92.5 3.0 0.3 92.5 73.8 85.0 Vrelo 1.5 76.3 7.5 96.3 1.5 15 98.8 3.0 0.3 68.8 82.5 Vinča 1.5 84.2 7.5 93.4 1.5 15 3.0 0.3 98.7 80.3 1 highly sensitive population (E= %); 2 sensitive ( > E 95%); 3 slightly resistant (95>E 90%); 4 resistant (90>E 50%); 5 highly resistant (E < 50%) 120
Table 3. Sensitivity of overwintered CPB adults to thiamethoxam and fipronil, scale 15 Sites Apatin Doroslovo Karavukovo Stanišić Žarkovac Sremska Mitrovica Platičevo Ležimir Klenak Stejanovci Grocka Korman Skela Vrelo Vinča efficacy (%) of efficacy (%) of Kg; l/ thiamethoxam Kg; l/ fipronil ha ha 1 2 3 4 5 1 2 3 4 5 0.06 0.025 0.07 0.05 0.14 0.125 0.06 97.4 0.025 0.07 96.5 0.05 0.14 0.125 0.06 0.025 0.07 0.05 0.14 0.125 0.06 0.025 98.2 0.07 97.3 0.05 98.2 0.14 0.125 98.2 0.06 0.025 0.07 0.05 0.14 0.125 0.06 0.025 0.07 0.05 0.14 0.125 0.06 0.025 0.07 0.05 0.14 0.125 0.06 0.025 0.07 0.05 0.14 0.125 0.06 0.025 0.07 0.05 0.14 0.125 0.06 0.025 98.3 0.07 0.05 0.14 0.125 0.06 0.025 0.07 0.05 0.14 0.125 0.06 0.025 0.07 0.05 0.14 0.125 0.06 0.025 0.07 0.05 0.14 0.125 0.06 0.025 98.8 0.07 0.05 0.14 0.125 0.06 0.025 0.07 0.05 0.14 0.125 1 highly sensitive population (E= %); 2 sensitive ( > E 95%); 3 slightly resistant (95>E 90%); 4 resistant (90>E 50%); 5 highly resistant (E < 50%) 121
Screening test in determination of Colorado potato beetle (Leptinotarsa decemlineata Say.) sensitivity to insecticides Conclusion Based on the results of screening test on sensitivity of 15 CPB populations from Serbia to insecticides (label rates) the following conclusions can be drawn: Three populations were highly sensitive, six were sensitive, one was slightly resistant and five were resistant to chlorpyrifos; One population was highly sensitive, seven were sensitive, two slightly resistant and five were resistant to cypermethrin; 10 populations were highly sensitive and four were sensitive to thiamethoxam; 10 populations were highly sensitive and five sensitive to fipronil. Acknowledgment The research was conducted within the project III 46008, funded by the Ministry of Education and Science of the Republic of Serbia. References Alyokhin A, Dively G, Patterson M, Rogers D, Mahoney Mand Wollam J (2006): Susceptibility of imidaclopridresistant Colorado potato beetles to nonneonicotinoid insecticides in the laboratory and field trials. Am. J. Potato Res. 83: 485 494. Alyokhin A, Dively G, Patterson M, Castaldo C, Rogers D, Mahoney M and Wollam J (2007): Resistance and crossresistance to imidacloprid and thiamethoxam in the Colorado potato beetle Leptinotarsa decemlineata. Pest Manag. Sci. 63: 32 41. Anonymous (2005): Pesticides in Your Food, 2005. www.panuk.org. Web page accessed: 10.05.2005. Anonymous (2009): IRAC Susceptibility Test Methods Series, Version 3, Method No. 011. Copping LG, and Menn JJ (2000): Biopesticides: a review of their action, applications and efficacy. Pest Manag. Sci. 56: 651676. Gokce A, Isaacs R and Whalon ME (2006): Behavioural response of Colorado potato beetle (Leptinotarsa decemlineata) larvae to selected plant extracts. Pest Manag.Sci. 62: 1052 1057. Heim DC, Kennedy GG, Van Duzn JW (1990): Survey of insecticide resistance among North Carolina Colorado potato beetle (Coleoptera: Chrysomelidae) populations. J. Econ. Entomol. 83: 1229 1235. Inđić D (1994): Efekti zajedničkog delovanja insekticida na krompirovu zlaticu Leptinotarsa decemlineata Say. Doktorska disertacija, Univerzitet u Beogradu, Poljoprivrdeni fakultet, 1107. Inđić D (1997): Effect of joint action of insecticides on colorado potato beetle (Leptinotarsa decemlineata Say). Review of Research Work at the Faculty of Agriculture, Belgrade 42 (1): 722. Inđic D, Klokočar Šmit Z i Vuković S (2006a): Pesticidi visokog rizika i njihova zamena u zaštiti krompira. Studija, Poljoprivredni fakultet i Pokrajinski sekretarijat za nauku i tehnološki razvoj APV, Novi Sad, pp.139. Inđić D, Vuković S KlokošarŠmit Z (2006b): Rapid detection of Colorado potato beetle (Leptinotarsa decemlineata Say.) sensitivity to insecticides. XVII Czech and Slovak Plant Protection Conference, Prague, Czech, pp. 459464. 122
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