Article pubs.acs.org/JAFC
Nitenpyram, Dinotefuran, and Thiamethoxam Used as Seed Treatments Act as Efficient Controls against Aphis gossypii via High Residues in Cotton Leaves Zhengqun Zhang,‡,∥ Xuefeng Zhang,†,∥ Yao Wang,† Yunhe Zhao,† Jin Lin,† Feng Liu,† and Wei Mu*,† †
College of Plant Protection and ‡College of Horticulture Science and Engineering, Shandong Agricultural University, 61 Daizong Street, Tai’an 271018, China S Supporting Information *
ABSTRACT: The effects of eight neonicotinoid seed treatments against the cotton aphid Aphis gossypii and its natural enemies in Bt cotton fields were evaluated, and the concentrations of these neonicotinoids in cotton leaves and soil were also investigated. The results showed that all neonicotinoid seed treatments efficiently reduced A. gossypii populations throughout the cotton seedling stage. The percentages of curly leaf plants in all of the neonicotinoid seed treatments were below the threshold for economic loss. Among the eight tested neonicotinoid seed treatments, nitenpyram, dinotefuran, and thiamethoxam showed high control efficiency against A. gossypii. Residues of the three neonicotinoids were higher than those of other neonicotinoids in cotton leaves. Moreover, residues of dinotefuran and nitenpyram remained at low levels in the soil. However, the abundance of natural enemies in the cotton field was to some extent influenced by neonicotinoid seed treatments. Therefore, neonicotinoids nitenpyram, dinotefuran, and thiamethoxam used as seed treatment can provide effective protection that should play an important role in the management of early-season A. gossypii in Bt cotton fields; however, the risks of neonicotinoids to the environment should also be considered. KEYWORDS: neonicotinoid seed treatment, A. gossypii (Glover), natural enemies, leave residue, soil residue
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the undesirable use of pesticides and labor costs.11 Choosing the most appropriate time for foliar spraying is critical but difficult, particularly for the small farmers involved in Chinese agriculture.12 Delaying insecticide applications too long leads to greater crop damage and yield losses, whereas frequently applied insecticides kill natural enemies in cotton fields and weaken their biocontrol services.13 Presently, seed treatments represent an important measure in integrated pest management systems because they are relatively safe, easy to handle, cause less pollution, and allow more precise targeting of the active insecticide ingredient to the pest organism.14 Seed treatments with systemic insecticides could provide longer term protection and have fewer side effects on natural enemies than sprayed applications of insecticides.15 Neonicotinoid insecticides are agonists of nicotinic acetylcholine receptors16 and have good plant systemicity (via roots) and excellent biological activity against sucking insect pests and certain chewing species and are suitable for use as seed treatments for wheat, cotton, potato, and other crops to control piercing−sucking herbivores.17−19 Furthermore, neonicotinoids used as seed treatments are effective against various aphid species, including wheat aphids,15 soybean aphids,20 and cotton aphids.21 Neonicotinoid seed treatments also provide earlyseason seedling protection against a range of sucking pests such as leafhoppers, whiteflies, and thrips in cotton fields.14,22,23
INTRODUCTION Bt cotton was approved for commercial planting in China in 1997 and has since been planted over large areas to prevent losses from the cotton bollworm Helicoverpa armigera.1 Bt cotton can effectively control Lepidoptera larvae, but it has no significant protective effect against piercing−sucking cotton plant herbivores such as the cotton aphid, Aphis gossypii.2 At present, A. gossypii is a commonly occurring and economically important cotton pest that causes serious damage in Bt cotton fields. A. gossypii infests cotton plants by sucking phloem sap, causing damaged leaves to roll up, resulting in retarded growth or even growth cessation and death.3 Moreover, A. gossypii may cause indirect damage because it can transmit several debilitating plant viruses.4 An infestation of A. gossypii can result in a considerable yield loss if not controlled effectively. Chemical control is currently the main control measure for A. gossypii in cotton fields in China.5 Other nonchemical control measures including cultural control (e.g., intercropping with other crops6) and physical control (e.g., yellow sticky traps7) are used as supplemental control measures. The common practice to manage A. gossypii is foliar spraying of insecticides due to their rapid action and high efficacy. Chemical foliar sprays of insecticides such as organophosphates, carbamates, pyrethroids, and neonicotinoids have been widely used to control A. gossypii in cotton fields in China. However, due to the high fecundity of A. gossypii,8 the short residual effects of sprayed insecticides,9 and insect development of resistance to commonly used insecticides,10 cotton farmers need to spray foliar insecticides approximately three times to manage A. gossypii at the seedling stage, which increases both © XXXX American Chemical Society
Received: Revised: Accepted: Published: A
August 23, 2016 November 4, 2016 November 22, 2016 November 22, 2016 DOI: 10.1021/acs.jafc.6b03430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Weather conditions and number of Aphis gossypii in untreated control plots in 2013 (a), 2014 (b), and 2015 (c).
management of A. gossypii and their impacts on natural enemy populations in cotton fields. Additionally, the concentrations of neonicotinoids in cotton leaves and soil were determined to investigate differences in the control effects of various neonicotinoids on A. gossypii. The data obtained here can be
However, neonicotinoid seed treatments still have some negative effects on animals beneficial to agroecosystems such as arthropods, bees, and birds.24 The objective of this research was to evaluate the efficacy of eight neonicotinoid insecticides used as seed treatments for the B
DOI: 10.1021/acs.jafc.6b03430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
90 min at 200 rpm using a mechanical shaker. The mixture was then transferred into a 100 mL centrifuge tube, and 5 g of NaCl and 8 g of anhydrous MgSO4 were added, followed by vigorous manual shaking for 1 min. Then, the mixture was centrifuged at 4000 rpm for 5 min. Then, 25 mL of the supernatant was evaporated until nearly dry using a rotary vacuum evaporator at 40 °C and blown dry using an aurilave. The residue was reconstituted with 2 mL of acetonitrile. A dual-layer GCB/PSA SPE cartridge (500 mg of graphitized carbon black (GCB) in the upper layer and 500 mg of primary−secondary amine (PSA) in the lower layer; Anpel, Shanghai, China) was used for cleanup. The SPE cartridge was preconditioned with 5 mL of acetonitrile. The concentrated extract was transferred to the SPE cartridge and rinsed three times with 6 mL of acetonitrile. The eluents were collected and evaporated to dryness under a nitrogen stream at 40 °C. The residue was dissolved in 2 mL of acetonitrile and passed through a 0.22 μm membrane filter, ready for analysis. The soil samples were air-dried and strained through a 10-mesh sieve prior to extraction. The soil (20 g) was placed into a 250 mL triangular flask and 50 mL of acetonitrile was added. This mixture was shaken for 1 h and then ultrasonically extracted for 30 min. Then, 3 g of NaCl was added, followed by vigorous manual shaking for 1 min. After a 30 min settling period, 25 mL of the supernatant was transferred to a 100 mL flat-bottomed flask and then evaporated to near dryness with a rotary evaporator at 40 °C. Finally, the remainder was blown dry by an aurilave. The residue was then reconstituted with 2 mL of acetonitrile and transferred into a 2 mL microcentrifuge tube containing 50 mg of PSA and 150 mg of anhydrous magnesium sulfate. The tube was vortex-mixed for 1 min and centrifuged at 12,000 rpm for 3 min. The supernatant was passed through a 0.22 μm membrane filter, ready for analysis. The extracts were analyzed by an Agilent 1290 Infinity UHPLC system coupled to an Agilent 6460 MS/MS system. Chromatographic separation was performed using an Agilent SB-C18 column (100 mm × 2.1 mm i.d., 1.8 μm particle size) at 30 °C. The injection volume was 2 μL, and the flow rate was 0.2 mL min−1. The mobile phase was acetonitrile (A) and 0.1% formic acid in water (B). The mobile phase gradient programs were as follows: imidacloprid, acetamiprid, sulfoxaflor, and thiacloprid, 0−2.5 min, 70% A; 2.5−4 min, 100% A; 4−5 min, 70% A; thiamethoxam, nitenpyram, and dinotefuran, 0−2.5 min, 30% A; 2.5−4 min, 100% A; 4−5 min, 30% A; clothianidin, 0−2.5 min, 60% A; 2.5−4 min, 100% A; 4−5 min, 60% A. The retention times of pesticides in the respective mobile phase were 1.34 min (imidacloprid), 2.09 min (thiamethoxam), 1.40 min (clothianidin), 1.72 min (nitenpyram), 1.50 min (dinotefuran), 1.36 min (acetamiprid), 1.38 min (sulfoxaflor), and 1.41 min (thiacloprid). The tandem mass spectrometry was performed in positive electrospray ionization (ESI+) mode, and the source parameters were as follows: gas temperature, 325 °C; gas flow, 8 L min−1; nebulizer gas, 40 psi; and capillary voltage, 4000 V. Nitrogen gas served as the nebulizer, and argon gas was used as the collision gas. The multiple reaction monitoring (MRM) mode was selected, and the MRM parameters of tested insecticides are presented in Table S2, including the precursor ion, production, fragmentation voltage, and collision energies. The analytical method was validated. The linear standard curves were obtained from the matrix-matched working standard solutions, at concentrations ranging from 0.002 to 2.0 mg L−1. The recovery experiments were carried out at the three spike levels of 0.01, 0.1, and 1 mg kg−1 from the cotton leaves and soil samples and replicated three times for each level. These samples were processed as described above. The matrix-matched calibration curves had good linearity; the correlation coefficients (R2) were >0.99 for the eight neonicotinoids (Table S3). Mean recovery values for neonicotinoids were 76.53− 97.23% in cotton leaves and soil samples, which were in the acceptable range (70−120%) specified by the SANCO guidelines.26 The limit of quantitation (LOQ) and limit of detection (LOD) for neonicotinoids in cotton leaves and soils were 0.002 and 0.001 mg kg−1, respectively. Data Analyses. All statistical analyses were carried out using SPSS statistical software (version 18.0, SPSS Inc., Chicago, IL, USA). Statistically significant mean values were compared using one-way ANOVA tests followed by Tukey’s HSD method (P < 0.05). The
used to select the most efficient neonicotinoids when used as seed treatments and most suitable for strengthening the control measures for A. gossypii in Chinese cotton fields.
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MATERIALS AND METHODS
Cotton Seeds and Insecticides. The transgenic Bt cotton seeds, variety Xinqiu-1, were supplied by Shandong Xinqiu Agricultural Science and Technology Co., Ltd. The seeds were delinted and selected before seed treatment with the insecticides. Eight neonicotinoid insecticides were used in this study: imidacloprid (Gaucho 600 g L−1 FS; Bayer CropScience (China) Co., Ltd., Hangzhou, China), thiamethoxam (Cruiser 70% WS, Syngenta Crop Protection (Suzhou) Co., Ltd., Suzhou, China), clothianidin (Poncho 600 g L−1 FS, Bayer CropScience LP, Monheim, Germany), nitenpyram (50% SG, Jiangshan Agrochemical & Chemical Co., Ltd., Nantong, China), dinotefuran (20% SG, Mitsui Chemicals, Inc., Bangkok, Thailand), acetamiprid (20% SG, Shandong United Pesticide Industry Co., Ltd., Tai’an, China), sulfoxaflor (22% SC, Dow AgroSciences LLC, Shanghai, China), and thiacloprid (48% SC Noposion Agrochemicals Co., Ltd., Shenzhen, China). These insecticide formulations were diluted to a uniform slurry with water before seed treatment. Field Experiments. The experiments were conducted in 2013, 2014, and 2015 at the cotton breeding center of Shandong Xin Qiu Agricultural Science and Technology Co., Ltd., in Xiajin, Shandong, China (site: 36.93° N, 115.95° E). The soil type used was silty loam (clay 12.15%, silt 61.88%, and sand 25.97%) with a pH of 7.53 and 1.41% organic content. Data concerning the weather during the trials are shown in Figure 1. The region has a long history of cotton cultivation, and A. gossypii populations are abundant. Farmers usually sow cotton seeds in late April, covering the ground with translucent plastic film to keep the seedlings warm. The harvest occurs in midSeptember; the area produces only one crop per year. Ten treatments were arranged in a randomized complete block design with four replications. These treatments consisted of eight neonicotinoid (imidacloprid, thiamethoxam, clothianidin, nitenpyram, dinotefuran, acetamiprid, sulfoxaflor, and thiacloprid) seed treatments at the rate of 4 g of active ingredient (AI) kg−1 seed, an untreated treatment, and a foliar spray treatment performed in accordance with the pest management program of the cotton breeding center (details of the spraying treatment are listed in Table S1). The cotton seeds were sown on April 25, 2013, April 23, 2014, and April 27, 2015, in furrows made by a mechanical furrow opener (80 cm apart and 5 cm deep) via manual dibbling: three seeds per hole and 25 cm apart. Approximately 20 kg of seeds per hectare (ha) was used. The resulting plant densities were approximately 45,000 per hectare. Each plot consisted of 10 7-m-long rows. Plots were separated by 1.6 m of bare cultivated ground. Pendimethalin was applied after sowing at the rate of 800 g of AI ha−1. Then, every row was covered with translucent plastic film. The number of A. gossypii was counted every 5 days on 30 randomly selected plants from each plot, beginning on May 12, 2013, May 10, 2014, and May 13, 2015, and continuing until mid-June, when the cotton aphid numbers decreased to a very low density. When the cotton aphids were counted, natural enemies (including ladybirds, lacewings, syrphids, predatory bugs, and spiders) were also monitored. The number of curly leaf plants caused by A. gossypii was assessed from 200 randomly selected plants per plot. Residual Determination of Neonicotinoids in Cotton Leaves and Soil. Ten cotton plants were randomly selected from each plot. Their leaves were collected, and the soil around the plants was sampled using a soil auger (8 cm inner diameter, 15 cm depth) every 5 days from May 13 through June 14 of 2015. The soil samples from each plot were pooled and divided, and approximately 500 g of soil was stored. All samples were stored at −20 °C until analysis. The extraction and cleanup of samples followed the method of Zhang et al.25 The leaf samples were finely chopped using a blender, and 10 g of the sample was transferred into a 250 mL triangular flask. Then, 50 mL of acetonitrile was added, and the mixture was shaken for C
DOI: 10.1021/acs.jafc.6b03430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 2. Population dynamics (a, c, e) and mean numbers (b, d, f) of A. gossypii per 30 plants in each plot in 2013, 2014, and 2015. Different letters indicate significant differences among treatments (Tukey’s HSD test, P < 0.05). DAS, days after sowing. percentages of curly leaf plants were arcsine square root transformed prior to analysis, but untransformed data were presented.
development of A. gossypii populations. Intense rainfall can decimate A. gossypii populations.31 A rainstorm of 18.3 mm on June 8, 2015, resulted in a huge reduction of the cotton aphid population in the untreated control group: from 3628.0 per 30 plants on June 7 to just 599.8 per 30 plants on June 9. The 22.6 mm rainfall on June 10, 2013, and the 17.6 mm rainfall on June 6, 2014, also reduced the numbers of A. gossypii. The three study years showed large populations of A. gossypii; the highest population density occurred in the untreated control plots on June 9, 2013 (45 days after sowing (DAS)), reaching 7825.3 per 30 plants. The population densities of A. gossypii in the neonicotinoid seed treatments were much lower than those in the untreated control plots throughout the seedling stage (except in the last sampling, when only very low densities were present) (Figure 2). However, there were no significant differences in the A. gossypii population densities among the eight neonicotinoid seed treatments during the early
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RESULTS AND DISCUSSION Effect of Neonicotinoid Seed Treatments on A. gossypii Population. A. gossypii is a major recurrent pest in the cotton seedling stage.27 Cotton aphid outbreaks occurred from mid-May until mid-June at the study site. The population densities of A. gossypii peaked on June 9, 2013, June 4, 2014, and June 7, 2015 (Figure 1), and their population dynamics were affected by climatic conditions, including temperature and rainfall.28 The temperature significantly influenced the intrinsic growth rate and survival rate of A. gossypii; 25−30 °C was the optimal temperature range for population development.29 Sudden high temperatures affected the mortality and fecundity of A. gossypii.30 For example, weather data showed that high air temperatures in late May of 2014 resulted in slower D
DOI: 10.1021/acs.jafc.6b03430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
treatments were significantly less than the untreated control plots and below the economic threshold (Figure 3). The results of these field trials over three years confirmed that neonicotinoid seed treatments can control A. gossypii effectively in cotton at the seedling stage. All eight neonicotinoid seed treatments (at a rate of 4 g of AI kg−1) reduced A. gossypii damage, keeping it below the economic threshold throughout the cotton seedling stage. Nitenpyram, dinotefuran, and thiamethoxam exhibited greater control efficiency for A. gossypii than the other tested neonicotinoids, whetrsd thiacloprid and sulfoxaflor were less efficient in these field experiments. The differences in control efficacy of neonicotinoids against A. gossypii were related to the neonicotinoid residues in the cotton leaves. The residues of dinotefuran, thiamethoxam, and nitenpyram in cotton leaves were higher than the residues from the other neonicotinoids. The higher residues corresponded to better control effects on cotton aphids. In contrast, thiacloprid and sulfoxaflor had low residue levels in cotton leaves, which resulted in relatively poor control efficacy against A. gossypii. The control effect differences among the neonicotinoids were also related to the toxicity of the insecticides to this pest. Zhang et al. found that the toxicities of thiamethoxam and nitenpyram to A. gossypii from this cotton-planting region were higher than those of other tested neonicotinoids.10 However, Shi et al. showed that dinotefuran and thiamethoxam are the most effective insecticides for use against A. gossypii compared to other neonicotinoids.33 In this study, in the later sampling periods even with a very low concentration in cotton leavesall of the neonicotinoid seed treatments provided excellent control efficiency against A. gossypii. One possible explanation for this phenomenon is that the insecticides effectively suppressed the aphid population to a very low level during the earlier study period; consequently, the insects had insufficient time to develop again. Additionally, the sublethal concentrations of neonicotinoids affected the growth and fertility of A. gossypii.33 Finally, some studies have shown that some A. gossypii populations have developed resistance to neonicotinoids after exposure,10,34 necessitating resistance monitoring for this pest to insecticides. Effect of Neonicotinoid Seed Treatments on Natural Enemies. In cotton fields, ladybeetles (Harmonia axyridis, Propylaea japonica, and Coccinella septempunctata), lacewing (Chrysopa phyllochroma and C. sinica), syrphid (Epistrophe balteata, Syrphus nitens, and S. corollae), predatory bugs (Orius sauteri, O. minutes, and Geocoris pallidipennis), and spiders (Lycosidae, Gnaphosidae, and Titanoecidae) may all be found on cotton plants or in the surrounding areas. Natural enemies (except spiders) were more abundant in the untreated plots than in the treated plots, and no significant differences in the numbers of natural enemies were observed among the eight neonicotinoid seed treatments. The predators in plots with spray treatments tended to have lower abundance than in those with seed treatments, but the effects were not statistically significant (Figure 4). Our results showed that the neonicotinoid seed treatments reduced the abundance of natural enemies in cotton fields. This result may be explained by the following two factors: (1) the low prey density in the seed treatment plots failed to attract natural enemies;27 and (2) cotton leaves and extrafloral nectarcontaining insecticides are ingested directly by predators as supplemental diet.35 First, the abundance of prey is the main factor that determines the distribution of natural enemies.36
sampling period (17−30 DAS in 2013, 17−32 DAS in 2014, and 16−26 DAS in 2015). In 2013, among the different neonicotinoid-treated plots, low aphid densities occurred in those treated with nitenpyram and dinotefuran; however, these values were not significantly different compared with spray treatments at 40 and 45 DAS (P < 0.001) (Figure 2a). In 2014, aphid densities in the plots treated with nitenpyram, dinotefuran, and thiamethoxam were significantly higher than densities in the plots treated with thiacloprid at 37 DAS (F9,39 = 223.8, P < 0.001). At 42 DAS, aphid density in the nitenpyramtreated plots was significantly lower than in the thiaclopridtreated plots, but not significantly different from the other seven neonicotinoid treatments (F9,39 = 210.8, P < 0.001) (Figure 2c). In 2015, at 31 DAS, significantly lower aphid density occurred in plots treated with thiamethoxam than in plots treated with thiacloprid (F9,39 = 84.8, P < 0.001). Aphid densities under the dinotefuran and thiamethoxam treatments were significantly lower than those under the thiacloprid and sulfoxaflor treatments at 36 DAS (F9,39 = 111.2, P < 0.001), but no significant differences were observed among the neonicotinoid seed treatments at 41 DAS (Figure 2e). The population densities of A. gossypii in all treatments decreased drastically on June 9 (43 DAS) due to the intense rainfall (18.3 mm) on June 7, but aphid densities with neonicotinoid seed treatments were still significantly lower than untreated control densities (F9,39 = 21.2, P < 0.001). The densities of A. gossypii in plots with spray treatment remained at low levels throughout the seedling stages during experiments (Figure 2). The population densities of A. gossypii in plots treated with neonicotinoids and in plots with the spray treatment were not significantly different, and they were all significantly lower than those in the control plots (in 2013, F9,70 = 3.743, P < 0.001; in 2014, F9,70 = 4.283, P < 0.001; in 2015, F9,70 = 4.634, P < 0.001) (Figure 2b,d,f). The interaction between year, neonicotinoid seed treatment, and sampling date had a significant effect on the numbers of A. gossypii in cotton fields (F = 63.54, P < 0.001) (Table 1). The percentage of curly leaf Table 1. Repeated Measures MANOVA Parameters for Effects of Year, Neonicotinoid Seed Treatment, Sampling Date, and Interactions on the Numbers of A. gossypii in Cotton Fields source
df
F
P values
year neonicotinoid seed treatment sampling date year × neonicotinoid seed treatment year × sampling date neonicotinoid seed treatment × sampling date year × neonicotinoid seed treatment × sampling date
2 9 7 18 14 63 126
505.7 442.3 750.5 289.6 342.6 67.30 63.54
