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 Gvozdenac1, Dragica Janković,3 Gordana Forgić,4 Gordana Mrdak4 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; 4SOE Agroinstitute, Sombor, e-mail: [email protected] 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 1-5 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 warm-blooded organisms) (Šovljanski et al. 2004, Inđić et al. 2006a, Klokočar-Šmit 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 (Mota-Sanchez 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 (24-72 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 (lc-p lines, LC50, LC95, LC99, 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 100% 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 1-5, 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 = 100%) 2 - sensitive (100> 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* 1.5l/ha – label rate chlorpyrifos (480 g/l) (organophosphate) Pyrinex 48-EC 7.5 l/ha – 5 x higher 15 l/ha – 10 x higher 0.3l/ha - label rate cypermethrin (200 g/l) (pyrethroid) Cipkord 20-EC 1.5 l/ha – 5 x higher 3 l/ha – 10 x higher 60 g/ha - label rate thiamethoxam (250 g/kg) (neonicotinoid) Actara 25-WG 70 g/ha - label rate 140 g/ha – 2 x higher 25 g/ha - label rate fipronil (800 g/kg) (pyrazole) Regent 800-WG 50 g/ha – 2 x higher 125 g/ha – 5 x higher * - Insecticides were applied in 400 l/ha; Results and discussion Biology of CPB is thoroughly studied in our agro-ecological 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