Applied Crop Protection 2015 IX Insecticide resistance experiments Caroline Kaiser, Dorte H. Højland, Karl-Martin V. Jensen and Michael Kristensen Insecticide resistance is less of an issue than that of herbicide and fungicide resistance in Denmark. This can be largely attributed to insect pests generally being less of a problem than weeds and diseases, therefore the consumption of insecticides is significantly less than the consumption of herbicides and fungicides. Insecticide resistance in pests of outdoor crops has been known in Denmark since the 1980s when resistance to pyrethroids in the peach-potato aphid in sugar beets was found. In the 1990s, coating of sugar beet seeds with neonicotinoids eliminated this problem. Reports of failure of pyrethroids are widespread from Christmas tree to strawberry production. This may be related to resistance, but this has not been verified. In agricultural crops, the first resistant pollen beetle population was found in 1999 in France. Throughout the 2000s the resistance was systematically studied in a small number of populations all over Europe. The overall picture was a strong resistance to lambda-cyhalothrin, while there was less widespread resistance to tau-fluvalinate. In these studies, no resistance towards the neonicotinoid thiacloprid was observed (Slater et al. 2011). Pyrethroid resistance in the pollen beetle (Meligethes aeneus F.) In a two-year study the current level of resistance from Danish pollen beetle populations was carried out. Pollen beetle populations were collected from 49 randomly selected fields in collaboration with consultants and farmers in 2014 (Kristensen et al. 2015). In 2015, selected locations were sampled again as closely as possible to the previous collection sites. In total 14 populations were collected from winter oilseed rape fields. Further five populations from Germany were tested in 2015 (Figure 1). All bioassays were conducted regarding the established IRAC Adult-Vial-Test methods No. 11 for lambda-cyhalothrin and No. 21 for thiacloprid. In addition to the recommended concentrations three concentrations were added to the testing for lambda-cyhalothrin and two for thiacloprid. In 2015 the tested concentrations for lambda-cyhalothrin were 500%, 100% (0.075 μg cm -2 ), 20%, 4% and 0.8%. The effect of thiacloprid was assessed for 200% (1.44 μg cm -2 ), 100%, 20%, 4% and 0.8% of the field application rate. Each concentration for both active ingredients were tested with a minimum of 2 replicates which were compared with two control glasses. The beetles were assessed after 24 h exposure in the coated glass vials and were grouped into affected or alive. Both dead and moribund beetles were classified as affected. 115
Figure 1. Monitoring locations from the two-year study in 2014 and 2015. In this map the locations of the two-year monitoring are shown. Every circle or square symbolises one population, collected in an oilseed rape field. Results Investigating pyrethroid resistance In 2015 the majority of the pollen beetle populations tested with the adult vial glasses for lambda-cyhalothrin in Denmark were classified as moderately resistant according to the IRAC classification scheme (Zimmer and Nauen 2011). The pollen beetle populations from Germany were tested for comparison. Those populations were mainly categorised as moderately resistant (Figure 2). Figure 2. Results for the lambda-cyhalothrin monitoring according to the IRAC classification scheme. The different colours represent the different classes of the pyrethroid resistance. None of the tested populations in 2015 was declared as highly susceptible or highly resistant. 116
Neonicotinoid monitoring The majority of the tested Danish populations were classified as highly susceptible in 2015. Only one population was classified as susceptible. Only one of the tested German populations showed a slightly reduced susceptibility to thiacloprid (Figure 3). Figure 3. Results for the thiacloprid monitoring according to the IRAC classification scheme. The different colours represent the different classes of the thiacloprid resistance. Pyrethroid resistance in the cabbage stem flea beetle (Psylloides chrysocephala) The cabbage stem flea beetle is the most devastating pest of oilseed rape, after pollen beetles, and has previously been controlled by seed treatment with neonicotinoids and spraying with pyrethroids. The European Commission has suspended any use of neonicotinoid products in crops that attract bees for three years. The rape sown in autumn 2014 was therefore not treated with neonicotinoids, which led to a slight increase in the frequency of treatment with pyrethroids compared to previous years. In the fall of 2015 seeds were again treated due to a dispensation for treatment with neonicotinoids. It is not yet known whether this has affected pyrethroid use. In 2010 pyrethroid-resistant populations of cabbage stem flea beetles were found in Schleswig-Holstein (Zimmer et al. 2014). Monitoring of pyrethroid resistance in cabbage stem flea beetles in Denmark was initiated in 2014 when cabbage stem flea beetle collections from nine fields were done in collaboration with consultants and farmers. In 2015 an additional eight populations were collected post-harvest at seed storage facilities and one from a field in South Jutland and Western Zealand (Figure 4). Beetles were stored at least 24 h in climatic chambers at 4-6ºC with food and water supply before testing. For the bioassay only live and fit beetles were used for the assessment. Ten beetles were selected for each glass vial. Six concentrations of lambda-cyhalothrin equivalent to 0.16%, 0.8%, 4%, 20%, 50% and 100% of the recommended field rate were used. Populations were classified according to the IRAC guidelines. 117
Results Pyrethroid monitoring Collection sites 2015 Figure 4. Collection sites in Denmark 2015. The colour indicates the resistance level for the given population. Green: 100% mortality with 20% λ-cyhalothrin field dose, Orange: 90-99% mortality with 20% λ-cyhalothrin field dose, Red: < 90% mortality with 20% λ-cyhalothrin field dose or < 100% mortality with 50% λ-cyhalothrin field dose. IRAC guidelines divide cabbage stem flea beetles into three categories in terms of pyrethroid resistance: susceptible, decreased susceptibility and resistance suspected. For both sites in Western Zealand and four out of five in South Jutland there was full mortality at 20% of the field dose and in most cases also a high mortality rate (> 80%) at 4% of the field dose, placing these populations in the susceptible category. For a single sample in South Jutland the mortality was < 90% using 20% field dose, so this population was suspected of resistance according to IRAC guidelines. We tried to locate the possible resistance by contacting the farmers who had supplied the oilseed rape from which the resistant population was taken. However, further analysis of beetles from a field with possible control failure showed no resistance. The results of pyrethroid bioassays in 2015 are similar to those of 2014 in which a single population was found to have decreased susceptibility, while the remaining eight populations were susceptible to pyrethroids. Detection of the kdr mutation The knock-down resistance mutation (kdr) is a point mutation of the sodium channel believed to be involved in pyrethroid resistance. For each population 10 randomly selected cabbage stem flea beetles were tested for the presence of the kdr mutation. We found no occurrence of the mutation in Zealand, but found it in almost all (four of five) populations from Jutland in various degrees. The population in which resistance was suspected, the frequency of kdr was 89%. The beetles which survived 20% of the field dose were homozygous for the kdr mutation. Furthermore, some beetles were sent from the southern part of Sweden and tested, but no presence of kdr was detected. The kdr mutation was also found in the 2014 populations in susceptible beetles. 118
Conclusions The current resistance situation in pollen beetles to pyrethroids and neonicotinoids were investigated in 2014 and 2015. In the bioassays with pyrethroid (IRAC No. 11) moderately resistant populations dominated. The Danish populations were classified as highly susceptible towards neonicotinoids (IRAC No. 21). The experiments performed in 2015 correlate well with those from 2014. Resistance is not widespread in cabbage stem flea beetles, despite the kdr mutation being widely distributed in Jutland. The fact that we did not find it in populations from Zealand does not necessarily mean that it is not there. It is possible that it occurs rarely (we found it in a very low frequency in 2014). In order to exclude the presence we would have to test many more beetles. The presence of resistance mutations in several populations indicates incipient resistance development. So continued monitoring is a key to foreseeing possible control problems. Investigations of resistance in oilseed rape pests continue in the coming years in connection with a research project funded by the Innovation Fund and Bayer CropScience. References Kristensen, M., C. Kaiser and D. H. Højland (2015). Insecticide resistance experiments. In: L. N. Jørgensen, B. J. Nielsen, P. K. Jensen, P. Kudsk, S. K. Mathiassen, P. Hartvig & M. Kristensen (eds.) Applied Crop Protection 2014. Aarhus University DCA Danish Centre for Food and Agriculture, DCA Report No. 58: 141-143. Slater, R., S. Ellis, J. P. Genay, U. Heimbach, G. Huart, M. Sarazin, C. Longhurst, A. Muller, R. Nauen, J. L. Rison and F. Robin (2011). Pyrethroid resistance monitoring in European populations of pollen beetle (Meligethes spp.): a coordinated approach through the Insecticide Resistance Action Committee (IRAC). Pest Manag Sci 67: 633-638. Zimmer, C. T., A. Müller, U. Heimbach and R. Nauen (2014). Target-site resistance to pyrethroid insecticides in German populations of the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae). Pestic Biochem Physiol 108: 1-7. Zimmer, C. T. and R. Nauen (2011). Pyrethroid resistance and thiacloprid baseline susceptibility of European populations of Meligethes aeneus (Coleoptera: Nitidulidae) collected in winter oilseed rape. Pest Manag Sci 67: 599-608. 119