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Synergistic effect of entomopathogenic nematodes and thiamethoxam in controlling Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae)
Biological Control 111 (2017) 53–60
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Synergistic effect of entomopathogenic nematodes and thiamethoxam in controlling Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae)
MARK
⁎
Haibin Wua,b, Qingtao Gongb, Kun Fanb, Ruihong Sunb, Yongyu Xua, , Kunpeng Zhangb a b
College of Plant Protection, Shandong Agricultural University, Tai’an, Shandong 271018, PR China Shandong Institute of Pomology, Tai’an, Shandong 271000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Entomopathogenic nematode Thiamethoxam Bradysia odoriphaga Synergistic effect Integrated pest management
Six entomopathogenic nematode species (EPNs) (Heterorhabditis bacteriophora H06 (H06), Steinernema carpocapsae NC116 (NC116), Steinernema carpocapsae All (All), Steinernema longicaudum X-7 (X-7), Steinernema feltiae SF-SN (Sf) and Heterorhabditis indica LN2 (LN2) were tested for virulence against 3rd instar Bradysia odoriphaga, and their interactions with thiamethoxam against 3rd instar B. odoriphaga under laboratory and greenhouse conditions were also evaluated. S. feltiae SF-SN, which is the most virulent, was selected to evaluate the synergism effects with thiamethoxam at different concentrations under laboratory conditions. S. feltiae SF-SN and thiamethoxam were either applied alone or in combination, and the combined application manner was evaluated in the greenhouse. Under laboratory conditions, a synergistic effect was found between six EPNs and thiamethoxam. The combination effects of Sf, All and LN2 species caused significantly higher mortality than the other three treatments (X-7, H06 and NC116). Furthermore, the combination of Sf and All species led to a significantly higher control effect than LN2 in greenhouse tests. In further greenhouse tests, the combination effect of Sf (0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ha) applied twice (28 day intervals) was significantly higher than a single treatment of Sf + thiamethoxam at twice the dosage. Compared to the single thiamethoxam application, the combination effects of Sf and thiamethoxam had the highest value at over 90% of the control effects for up to 6 weeks. Therefore, the integrated technique of the synergism of S. feltiae SF-SN species and thiamethoxam to control B. odoriphaga could be useful for integrated pest management in the future.
1. Introduction The Chinese chive (Allium tuberosum Rottl. ex Spreng.) is a perennial herbaceous vegetable with high economic value in eastern Asia and is grown over a vast geographical area from Asia through the Middle East to Europe and North America (Imahori et al., 2004; Misawa and Kuninaga, 2013). The chive maggot, Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae), is the major pest that restricts Chinese chive production (Li et al., 2015). This insect feeds on seven plant families and more than thirty plants species, including Chinese chives, garlic (Allium sativum L.), onion (A. cepa L.), and cucumber (Cucumis sativus L.) (Yang and Zhang, 1985; Feng and Zheng, 1987; Li et al., 2007), and it also causes production losses in mushroom sheds (Shi et al., 2001). Its larvae feed on the roots and stems of chives and cause 30–80% production losses in the absence of insecticide treatment. In severe cases, new Chinese chives need to be planted again (Ma et al., 2013). Currently, the predominant strategy for the management of this pest is to apply conventional insecticides, such as organophosphates, carbamates, and neonicotinoids (Zhang et al., 2014b). Thiamethoxam ⁎
is a second-generation neonicotinoid that has stomach and contact activity, and it operates by interfering with the nicotinic acetylcholine receptors in the insect’s nervous system (Maiensfisch et al., 2001; Torres et al., 2003). Cloyd and Dickinson (2006) reported that thiamethoxam was highly effective against second and third instar larvae of the fungus gnat Bradysia sp nr. coprophila L. (Diptera: Sciaridae). In addition, thiamethoxam has been reported to control B. odoriphaga at 6.0 kg a.i./ha and may help farmers more effectively manage B. odoriphaga (Zhang et al., 2015b). However, chemigation was a traditional method to control this pest and needed a mass amount of thiamethoxam (12.0 kg a.i./ha in some places) because of the dilution effects of water and soil on pesticides (Zhang et al., 2015b). The control efficacy was unsatisfactory due to the overlapping generations of the insect, and most of its life stages occur in sheltered circumstances (Wang et al., 2011). This challenge led to the excessive use of chemical insecticides, which caused pollution in the environment and left a high amount of residue on Chinese chives that were sold on the market (Zhang et al., 2016). Entomopathogenic nematodes (EPNs) have an indirect role in
Corresponding author at: College of Plant Protection, Shandong Agricultural University, 61 Daizong Street, Tai’an, Shandong 271018, PR China. E-mail address: [email protected] (Y. Xu).
http://dx.doi.org/10.1016/j.biocontrol.2017.05.006 Received 27 December 2016; Received in revised form 29 March 2017; Accepted 12 May 2017 Available online 12 May 2017 1049-9644/ © 2017 Published by Elsevier Inc.
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around an apple tree at the Shandong Institute of Pomology (SIP), Tai’an (35°48′ N, 117°28′ E), Shandong Province, China (Sun and Li, 2007). Five other isolates, including Steinernema carpocapsae NC116 (NC116), S. carpocapsae All (All), S. longicaudum X-7 (X-7), S. feltiae SFSN (Sf) and H. indica LN2 (LN2), were provided free-living in a foam substrate by Tai’an Lvnong Biological Co. Ltd. (Tai’an, China). All EPN species and isolates were cultured in the last-instar of the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae) at room temperature for 7–10 days (Kaya and Stock, 1997). Nematode infective juveniles (IJs) emerged from insect cadavers into White traps (White, 1927). The harvested IJs were washed 3 times by sedimentation in tap water, and were used immediately (laboratory experiment) or were stored in tap water before use (field experiment). Storage was carried out at a density of 1000 IJs/mL, in a 90 mm diameter plastic food tubes (each containing 50 mL of nematode suspension) with snap-on lids. The batches of collected IJs were stored at 10–12 °C for no longer than 2 weeks, after which fresh batches were obtained using the same methods as described above to ensure maximal efficacy (Langford et al., 2014; Ma et al., 2013). Only fresh batches of IJs were used in the experiment (Demarta et al., 2014). IJs were acclimated at room temperature for 1 h, and an Olympus SZ2-ILST microscope (×100) and a microscope counting plate were used to calibrate the solutions to the required concentrations and to check nematode activity prior to application (Langford et al., 2014; Ma et al., 2013). The nematode concentrations required were prepared by volumetric dilutions in distilled water. Colonies of B. odoriphaga were originally obtained from a Chinese chive field at Tai′an in Shandong, China (36°18′ N, 117°13′ E) during September 2013 and were maintained at a constant temperature of 25 ± 1 °C and Relative Humidity (R.H.) of 65–75% with a photoperiod of 12 h:12 h L:D in the insectary of SIP, Tai’an. The thiamethoxam formulation used was Actara (Swiss Syngenta Crop Protection Co. Ltd., China), which is a wettable granule (WG) and contains 250 g/kg of active ingredient. Chinese chive seeds (Dugenhong) were purchased from Shouguang Dafeng seedling Co. Ltd. (Shandong, China) and were sown in April 2013, grown to the seedling stage and then transplanted to a greenhouse at Tai′an in Shandong, China (36°38′ N, 117°43′ E), in 0.20 m rows at a rate of approximately 3,000,000 plants/ha in March 2013. In order to ensure the consistency of the test conditions (soil texture, soil structure, soil organic content and so on), the greenhouse (6 W × 80 L m) is evenly divided into two parts (part A and part B). Two field trials were conducted in part A and part B, respectively.
protecting plants through parasitizing a wide range of insects in natural and agricultural systems (Duncan and McCoy, 1996; Lacey and Unruh, 1998; Shapiro and McCoy, 2000). The third-stage infective juvenile (IJ) is the only stage in the life cycle of the nematodes that lives in the soil. EPNs of the families Steinernematidae and Heterorhabditidae may be particularly useful for the control of pests that occur in cryptic habitats (Ehlers, 1996). A number of studies have reported on the application of EPNs as biological control agents of pests in the genus Bradysia (Harris et al., 1995; Sun and Li, 2007; Wu et al., 2014a). However, EPN field efficacy is often inconsistent and unsatisfactory (Georgis and Gaugler, 1991; Klein, 1993) due to various biotic (Kaya, 2002; Kaya and Koppenhöfer, 1996) and abiotic factors (Smits, 1996; Glazer, 2002). Additionally, the choice of application equipment, as well as the manner in which the nematodes are applied, can have a substantial impact on pest control efficacy (David et al., 2006). In the search for a suitable host, IJs interact synergistically with some insecticides (Thurston et al., 1994; Nishimatsu and Jackson, 1998; Koppenhöfer et al., 2000). Neonicotinoids, for instance, were shown to be a synergist of EPNs against scarab beetle larvae (Koppenhöfer et al., 2000, 2003). The strategic combination of biological controls with reduced application rates and dosages of chemical insecticide synergists may, therefore, represent a valuable approach for the suppression of soil insect pests. When applied together, they may act independently and cause an additive effect, or interact with each other in a synergistic or antagonistic way (Jacques and Morris, 1981). Additive or synergistic interactions have been reported in the combined application of Steinernema glaseri and imidacloprid against 3rd instar of the masked chafers Cyclocephala hirta LeConte and C. pasadenae Casey (Coleoptera: Scarabaeidae) (Koppenhöfer et al., 2000). Mortality of Agrotis ypsilon Rottemberg increased after mixed application of matrine and S. carpocapsae NC116, which showed a synergistic interaction (Wu et al., 2015a). To increase the effectiveness of controlling B. odoriphaga with EPNs, produce high-quality Chinese chives with less residual pesticide, save money from using EPNs and pesticides and protect the environment, a combined application of nematodes and insecticides has been developed recently. Previous study also showed that neonicotinoid insecticides, such as imidacloprid and thiamethoxam, had no effect on the survival of nematodes and may be used as a synergist for nematodes (Yan et al., 2012). Additive or synergistic interactions have been reported for the combined application of three EPNs (Heterorhabditis bacteriophora H06, S. carpocapsae All and S. feltiae SF-SN) and three insecticides (imidacloprid, chlorpyrifos and beta-cypermethrin) against B. odoriphaga under laboratory conditions. (Wu et al., 2014a). However, previous study found that these three insecticides had high toxicities to Eisenia foetida Savigny (Opisthopora: Lumbricidea), and only thiamethoxam had a low selective toxicity to E. foetida in the pot experiments (Zhang et al., 2014a). Therefore, combined application of nematodes and thiamethoxam may be an important alternative for controlling B. odoriphaga and needs further study under both laboratory and greenhouse conditions. The objectives of this study were to (1) select a virulent EPN species, (2) evaluate its interaction with thiamethoxam against B. odoriphaga, (3) select the best synergism effect of the most virulent species and thiamethoxam using appropriate concentrations under laboratory conditions, and (4) optimize the combined application methods and respective concentrations of nematodes species and thiamethoxam against B. odoriphaga under greenhouse conditions. It was anticipated that additive or synergistic interactions would be achieved by the combined use of the two types of control agents and thus improve the overall pest control efficacy under greenhouse conditions.
2.2. Effects of the nematode concentrations on susceptibility of 3rd instar B. odoriphaga Six EPN species (H06, NC116, All, X-7, Sf and LN2) were tested to investigate the effect of IJs concentrations on the pathogenicity of the nematodes to 3rd instar B. odoriphaga. Zhang et al. (2016) reported B. odoriphaga was primarily observed in spring and autumn, with the highest level reaching 54.67 larvae per quadrat (20 cm × 20 cm; soil surface area: 0.04 m2) in the field. Based on our preliminary investigation of population dynamics, the occurrences of B. odoriphaga could reach an average of 60 larvae per quadrat (20 cm × 20 cm) in the greenhouse (15,000,000 larvae/ha). Following Wu et al. (2014b) with few modification, the method of calculating the amount of IJs applied was established according to the population densities of B. odoriphaga larvae in the greenhouse (30 IJs/larva, equal to 0.45 billion IJs/ha). Four rates (30, 60, 100 and 200 IJs/larva, equal to 0.45, 0.9, 1.5 and 3.0 billion IJs/ha, respectively) were used for each species. The test concentrations used in our study were based on previous studies (Sun and Li, 2007; Wu et al., 2014a, 2015). One Petri dish was treated as a single replication. Fresh Chinese chive pseudo stems (5.0 mm) were cut and put in a 90 mm diameter Petri dish containing a 90 mm diameter filter paper that was moistened with 1.0 mL of test solution. After
2. Materials and methods 2.1. Nematodes, insects, insecticide, plant material Heterorhabditis bacteriophora H06 (H06) was isolated from the soil 54
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instars) was almost the 3rd instar larvae. Following Zhang et al. (2015a) and Ma et al. (2013) with few modification, the numbers of B. odoriphaga larvae at the pseudo stems/bulbs and roots was determined after chipping away the soil without harming the plants for each plot (Z-shaped sampling) by taking five square soil plugs (20 cm × 20 cm quadrat) randomly with a shovel, at a depth of 15 cm. The number of B. odoriphaga larvae in each treatment were counted at 0, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 days after the application of the agents. After investigation, the larvae and soil were put back.
treatment, the larvae were maintained at a constant temperature of 25 ± 1 °C and R.H. of 65–75% with a photoperiod of 12 h:12 h L:D. Controls were identical to the treatments except that no IJs were added. Each treatment was replicated four times (approximately 30 larvae per replication) over a short period and under the same condition (Ma et al., 2013; Wu et al., 2014a,b, 2015b). Mortality was assessed after 72 h. Larvae were considered dead if they were unable to move in a coordinated manner when disturbed with the point of a writing brush. 2.3. Preliminary combination screening of EPNs and thiamethoxam against B. odoriphaga
2.5. Best synergism effects of EPN species and thiamethoxam against B. odoriphaga
The interaction between six EPN species and thiamethoxam against 3rd instar B. odoriphaga larvae was tested under laboratory conditions. In order to maximize combination screening of EPNs and thiamethoxam, and reduce the cost of using EPN, nematode (100 IJs/larva, equal to 1.5 billion IJs/ha) applied alone or added to the thiamethoxam were based on previous studies (Ma et al., 2013; Sun et al., 2004; Sun and Li, 2007). Thiamethoxam applied alone or in combination with a nematode were based on the LC50 (12 mg a.i./L) concentration from previous studies (Zhang et al., 2014a). In addition, an untreated water control was included. Other experimental conditions used were the same as the conditions in Section 2.2.
To optimize the combination of nematodes and thiamethoxam against B. odoriphaga, the most virulent EPN species from the experiments in Sections 2.2–2.4 was used. The S. feltiae SF-SN species with three rates (30, 60 and 100 IJs/larva, equal to 0.45, 0.9 and 1.5 billion IJs/ha, respectively) and thiamethoxam with four concentrations (8, 12, 15 and 20 mg a.i./L) were used, in addition to an untreated water control. All treatments were replicated four times (approximately 30 larvae per replication). The experimental conditions were the same as the conditions in Section 2.2. 2.6. Optimized control methods of nematodes S. feltiae SF-SN species and thiamethoxam against B. odoriphaga in the greenhouse
2.4. Combined effects of EPN isolates and thiamethoxam against B. odoriphaga in the greenhouse
To evaluate the efficacy of nematodes and thiamethoxam against B. odoriphaga, the most virulent Sf species and thiamethoxam concentrations from the Section 2.5 experiment was used. The experiments were conducted in the part B of the greenhouses, which were divided into 20 plots on 11 September 2015. The absence of natural populations of EPNs in the greenhouse was confirmed through sampling. During the experiment, the average temperature, R.H. and soil moisture were 21.5 ± 2.4 °C, 52.1 ± 2.8% and 23.7 ± 2.6% VWC (volumetric water content), respectively. The experiment included 5 treatments: a water control, thiamethoxam (6.0 kg a.i./ha), Sf (3.0 billion IJs/ha), Sf (1.50 billion IJs/ha) + thiamethoxam (2.0 kg a.i./ha), and Sf (0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ha). The thiamethoxam was applied alone at 6.0 kg a.i./ha or in combination with Sf at 2.0 or 1.0 kg a.i./ha. Sf species was applied alone at 3.0 billion IJs/ha or in combination with the thiamethoxam at 1.50 or 0.75 billion IJs/ha. All treatments were applied at September 10. But, Sf (0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ha) were applied in two dressings (September 10 and October 8). Four blocks and a randomized complete block design were used. Before trial treatments, the developmental stage of B. odoriphaga (94% 3rd instars, 6% 4th instars) was almost the 3rd instar larvae. The numbers of B. odoriphaga were counted at 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70 and 77 days after treatments. Other experimental procedures, methods and the soils used were the same as the ones in Section 2.4.
The combined effectiveness of different nematode species and thiamethoxam against B. odoriphaga were conducted in part A of the greenhouses, which were divided into 32 plots on 31 March 2015. Each plot (0.8 W × 5 L m) was separated by 0.3 m of bare cultivated ground. To prevent oviposition interference in adult B. odoriphaga, each plot was covered with 80 screen mesh (0.8 W × 5 L × 1.8 H m). The absence of natural populations of EPNs in the greenhouse was confirmed through sampling. The soil was a loamy sand texture comprised of 45.7% sand, 37.5% silt, 16.8% clay, 2.8% organic matter with a pH of 6.4. During the experiment, the average temperature, R.H. and soil moisture were 20.8 ± 1.9 °C, 50.6 ± 3.5% and 21.8 ± 2.1% VWC (volumetric water content), respectively. Temperature, R.H. and soil moisture were collected with a ZigWSN®C-A date logger throughout the experiment. The experiment included 8 treatments: a water control, thiamethoxam, All, Sf, LN2, All + thiamethoxam, Sf + thiamethoxam, and LN2 + thiamethoxam. Following Zhang et al. (2015a) and Ma et al. (2013) with few modification, all agents were applied using the directional spray-washing method by a 5.0 L hand-held pump sprayer. Chinese chives plants were sprayed with each agent at the roots according to good agricultural practice in each plot. Each plot received each agent with a volume of water equivalent to 1.0 L of water per m2. IJs suspended in the same volume of water was prepared and applied alone at 3.0 billion IJs/ha (equal to 300,000 IJs/m2) or in combination with the thiamethoxam at 1.5 billion IJs/ha (equal to 150,000 IJs/m2). Thiamethoxam was also prepared in the same volume of water and applied alone at one thousand times dilutions (6.0 kg a.i./ha) or in combination with a nematode at three thousand times dilutions (2.0 kg a.i./ha). Each agent was gently mixed in a plastic bucket before application was transferred to the spray tanks. To prevent sedimentation of the IJs in the tank, the tanks were well shaken before and during application. All treatments were done at the same time in the evening to minimize the risk of desiccation of IJs. Four blocks and a randomized complete block design were used. An auto-watering system was set up in the greenhouse. All experimental treatments were watered every two days with a total amount of 5.0 mm per watering during the experiment. Before applying the agents, the initial density of B. odoriphaga was investigated, and its developmental stage (95% 3rd instars, 5% 4th
2.7. Statistical analysis A chi-square (X2) test was used to test the interaction of EPNs and thiamethoxam. Before analysis, all mortality data were corrected for control mortality (Abbott, 1925). The method for determining the type of interaction (synergistic, additive, or antagonistic) was first described by Finney (1964) and then modified by McVay et al. (1977). The expected additive proportional mortality ME for the EPN/thiamethoxam combinations was calculated by ME = MN + MT (1 − MN), where MN and MT are the observed proportional mortalities relatively caused by EPNs and thiamethoxam alone. A chi-square test was then carried out using the formula X2 = (MNT − ME)2/ME, where MNT represents the observed mortality for the EPN/thiamethoxam combination. The 55
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Fig. 1. Mortality of 3rd instar B. odoriphaga when exposed to different IJ concentrations of (A) S. carpocapsae All, (B) H. indica LN2, (C) S. feltiae SF-SN, (D) S. longicaudum X-7, (E) H. bacteriophora H06, (F) S. carpocapsae NC116. Error bars represent standard errors. Different letters of Tukey’s HSD test are indicative of significant differences (P < 0.05) between means.
calculated value from the chi-square test was then compared to the chisquare table value for one degree of freedom. If calculated values are greater than the table value (X21, 0.05 = 3.84), non-additive effects, e.g., synergistic or antagonistic, could be suspected between the two agents (Finney, 1964). If the differences MNT–ME had a positive value, the interaction was considered synergistic, and if the difference was negative, the interaction was considered antagonistic. All data were transformed using an arcsin formula. Analysis of variance (ANOVA) with SPSS software (SAS Institute, 2012) was used to test the significant differences among treatments in both laboratory and greenhouse experiments. The results are reported as significant in Tukey’s Honestly Significant Difference (Tukey HSD) Test (p < 0.05).
Table 1 Interaction of various EPN species (100 IJs/larva) and thiamethoxam (12 mg a.i./L) on 3rd instar B. odoriphaga.
3. Results 3.1. EPN density effects on B. odoriphaga mortality There were significant differences among various EPN species in mortality (F = 25.08, DF = 5; P < 0.001) used One-way ANOVA (Fig. 1). Two-way ANOVA was used to test the differences between treatments with different EPN species and IJ concentrations. Significant differences were found among EPN species (F = 29.86, DF = 5; P < 0.001) and IJ concentrations (F = 88.63, DF = 4; P < 0.001). Significant interactions were detected between EPN species and IJ concentrations (F = 5.26, DF = 20; P < 0.001). As IJ concentrations increased, so did B. odoriphaga mortality with higher mortality at 200 IJs/larva and lower mortality at 30 IJs/larva. However, there was no difference in larval mortality among IJ concentrations (60, 100 and 200 IJs/larva), except LN2 species (Fig. 1B). Therefore, 100 IJs/larva was used in the following laboratory studies.
Treatment
Observed mortality (%)a
Expected mortality (%)b
X2
Type of interaction
S. feltiae SF-SN S. carpocapsae All S. longicaudum X-7 H. indica LN2 H. bacteriophora H06 S. carpocapsae NC116 Thiamethoxam S. feltiae SF-SN + thiamethoxam S. carpocapsae All + thiamethoxam S. longicaudum X7 + thiamethoxam H. indica LN2 + thiamethoxam H. bacteriophora H06 + thiamethoxam S. carpocapsae NC116 + thiamethoxam
16.81 ± 3.46 ab* 13.45 ± 1.61 ab 14.29 ± 0.97 ab 23.53 ± 1.61 b 7.56 ± 1.68 a 9.24 ± 1.37 a 42.50 ± 2.47 c 94.96 ± 3.22 e
– – – – – – – 53.16
– – – – – – – 39.44
– – – – – – – Synergistic
89.92 ± 3.63 e
51.27
34.96
Synergistic
65.55 ± 2.87 d
51.74
4.42
Synergistic
96.64 ± 1.37 e
56.95
33.20
Synergistic
66.39 ± 2.74 d
47.96
8.50
Synergistic
70.59 ± 4.20 d
48.90
11.54
Synergistic
a
Observed mortality was corrected for control mortality (0.83% at 3 days). Expected mortality ME = MN + MT (1 − MN), where MN and MT are the observed proportional mortalities relatively caused by EPN and thiamethoxam alone. * Different letters of Tukey’s HSD test indicate significant differences (P < 0.05) between means. Data are presented as means ± standard error. b
3.3. Combinatorial effects of EPNs and thiamethoxam against B. odoriphaga under greenhouse conditions
3.2. Combination effects of EPNs and thiamethoxam against B. odoriphaga
The B. odoriphaga larvae were sampled after application of differed treatments from March 27 to June 5, 2015 (Fig. 2). During the experimental months, the main occurrences of B. odoriphaga were from April 17 to May 15 with the highest level reaching 77.00 larvae/ quadrat in untreated plots on day 49 after applying different treatments. Compared to the untreated control, B. odoriphaga larvae were dramatically reduced after application of the treatments. The combination treatments caused significantly lower population densities compared to each of the respective single Sf, All and LN2 species from day 7 to day 42 (Fig. 2A). After 7 d, the control effects of treatments with a combination of low concentration thiamethoxam (2.0 kg a.i./ha) with
The combination treatments caused significantly higher mortality than each of the respective single agents (F = 175.08, DF = 12; P < 0.001). Synergistic effects were found between EPN species and thiamethoxam. However, there was significantly different in the observed mortality of B. odoriphaga among the combination treatments of various EPN species and thiamethoxam. The combination effects of Sf, All and LN2 species caused significantly higher mortality than X-7, H06 and NC116, respectively (F = 22.00, DF = 5; P < 0.001). Among these treatments, LN2 and thiamethoxam achieved the strongest mortality for the combined application (Table 1). 56
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Fig. 2. Population densities and control efficiency of B. odoriphaga at different times after the application of different treatments. Fig. 2A represent the number of larvae per quadrat and Fig. 2B represent the control effects relative to the water control to B. odoriphaga at different times, respectively. Error bars represent standard errors. Different letters of Tukey’s HSD test are indicative of significant differences (P < 0.05) between means.
detected in the interaction between Sf species and lower concentrations thiamethoxam (8, 12 and 15 mg a.i./L), and an additive effect was found between the nematode and higher concentration of thiamethoxam (20 mg a.i./L). Among them, the combination effect of Sf species (30, 60 and 100 IJs/larva) and thiamethoxam (12, 15 and 20 mg a.i./L) caused no significant differences (F = 0.68, DF = 8; P = 0.709) (Table 2).
Sf, All and LN2 species were higher compared to treatments with any one EPN species alone. The combination of Sf and All species caused significantly higher control effects than that of LN2 from day 7 to day 28, respectively. However, the combination effects of Sf and All species were gradually reduced from day 28 to day 70. For the single thiamethoxam (6.0 kg a.i./ha) treatment, the control effects were the highest from day 21 to day 56 and could last up to months after application at over 80% of the control effects (Fig. 2B).
3.5. Optimized control effects of S. feltiae SF-SN species and thiamethoxam against B. odoriphaga under greenhouse conditions
3.4. Synergism effect of S. feltiae SF-SN species and thiamethoxam against B. odoriphaga
B. odoriphaga larvae were sampled after different treatments were applied from September 10 to November 26, 2015 (Fig. 3). During the experiment, the main occurrences of B. odoriphaga were from September 10 to October 29 with the highest level reaching 79.25 larvae/ quadrat in untreated plots on day 35 after different treatments were applied. Compared to the untreated controls, B. odoriphaga larvae were
When the Sf species (30, 60 and 100 IJs/larva) and higher concentrations thiamethoxam (12, 15 and 20 mg a.i./L) were applied simultaneously, the combination treatments caused significantly higher mortality than each of the respective single agents, except thiamethoxam (20 mg a.i./L) (F = 134.00, DF = 18; P < 0.001). Synergism was 57
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Table 2 Interaction of S. feltiae SF-SN species and thiamethoxam on 3rd instar B. odoriphaga under laboratory conditions. Treatment
S. feltiae SF-SN (IJs/larva)
Thiamethoxm (mg a.i./L)
Observed mortality (%)a
Expected mortality (%)b
X2
Type of interaction
S. feltiae SF-SN
30 60 100
– – –
4.20 ± 1.68 a* 10.08 ± 0.84 a 13.45 ± 1.61 a
– – –
– – –
– – –
Thiamethoxam
– – – –
8 12 15 20
15.13 43.70 55.46 82.35
± ± ± ±
0.84 a 2.87 bc 3.46 cd 2.11 e
– – – –
– – – –
– – – –
S. feltiae SF-SN + thiamethoxam
30 30 30 30 60 60 60 60 100 100 100 100
8 12 15 20 8 12 15 20 8 12 15 20
35.00 90.84 95.84 92.51 50.01 93.34 95.84 94.18 62.51 91.67 95.01 94.17
± ± ± ± ± ± ± ± ± ± ± ±
6.29 2.15 2.36 1.36 5.51 3.44 1.92 0.96 4.91 1.60 2.50 2.15
22.43 55.28 68.80 99.71 28.42 59.25 71.94 100.96 31.85 61.52 73.74 101.67
17.08 52.23 31.04 1.28 35.10 46.98 25.78 1.44 58.50 38.22 21.99 1.26
Synergistic Synergistic Synergistic Additive Synergistic Synergistic Synergistic Additive Synergistic Synergistic Synergistic Additive
b e e e bcd e e e d e e e
a
Observed mortality was corrected for control mortality (1.60% at 3 days). Expected mortality ME = MN + MT (1 − MN), where MN and MT are the observed proportional mortalities relatively caused by S. feltiae SF-SN species and thiamethoxam alone. * Different letters of Tukey’s HSD test indicate significant differences (P < 0.05) between means. Data are presented as means ± standard error. b
+ thiamethoxam (1.0 kg a.i./ha))twice caused significantly lower population densities compared to that of Sf species alone (Fig. 3A). After 7 d, the control effects of treatments with a combination of low concentrations thiamethoxam (1.0, 2.0 kg a.i./ha) with Sf species was higher
dramatically reduced after the applications of single thiamethoxam (6.0 kg a.i./ha) treatment, Sf (1.5 billion IJs/ha) + thiamethoxam (2.0 kg a.i./ha), Sf ((0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ ha))twice. The combination treatments of Sf ((0.75 billion IJs/ha)
Fig. 3. Population densities and control efficiency of B. odoriphaga at different times after the application of different treatments. Fig. 3A represent the number of larvae per quadrat, and Fig. 3B represents the control effects relative to the water control to B. odoriphaga at different times, respectively. Error bars represent standard errors. Different letters of Tukey’s HSD test indicate significant differences (P < 0.05) between means. twice represents treatment applied twice (September 10 and October 8).
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determine whether any synergistic effects occur between Sf species and thiamethoxam at the different concentrations against B. odoriphaga. In our study, the combination effects of Sf species (30, 60 and 100 IJs/ larva) and thiamethoxam (12, 15 and 20 mg a.i./L) caused no significant differences, but with a dramatic increase synergistic effects from a concentrations of 8–12 mg a.i./L thiamethoxam. This result indicates that thiamethoxam was mainly responsible for the higher B. odoriphaga larvae mortality, and an increase in concentrations from 30 to 100 IJs/larva did not enhance the impact of S. feltiae in the interaction (Table 2). However, the underlying mechanism for the interaction between S. feltiae and thiamethoxam against B. odoriphaga remains unknown. No matter how well-suited an entomopathogenic nematode is to a targeted pest, the application will fail if the technology is not available for application of nematodes, including the application equipment and methods (Shapiro and McCoy, 2000). In the greenhouse experiments, the combination treatments of Sf (1.5 billion IJs/ha) and thiamethoxam (2.0 kg a.i./ha) caused significantly lower population densities and significantly higher control effects than each of the respective single Sf, All and LN2 species from day 7 to day 42 (Fig. 2). However, the combination effects of Sf and thiamethoxam were gradually reduced with the increase of B. odoriphaga larvae densities from day 28. Therefore, the combination effects of Sf (0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ha) were applied twice on days 1 and 28, and the combination effects were gradually increased from day 28 and were significantly higher compared to treatment with Sf (1.5 billion IJs/ha) + thiamethoxam (2.0 kg a.i./ha) once (Fig. 3B). Compared to a single thiamethoxam (6.0 kg a.i./ha) treatment, the combination effects did not decline with time and persisted well in the greenhouse experiments with over 90% of the control effects up to 6 weeks after application. The good persistence of synergistic interactions is important for obtaining sustainable control, which may eventually lead to lower costs (Susurluk and Ehlers, 2008). Persisting populations of B. odoriphaga larvae likely aided the establishment and propagation of the Sf species from March to early June and from September to November. There is a greater scope for using the combination of Sf species and thiamethoxam for controlling B. odoriphaga larvae at peak damage, which often occurs in late spring and autumn. However, Chinese chives have a very low B. odoriphaga population density from late June to August every year. These results were consistent with the results that Zhang et al. (2015a) reported. This period was in the summer in China with temperatures higher than 30 °C, which could suppress the individual development and population establishment of B. odoriphaga (Li et al., 2015; Zhang et al., 2015a). Therefore, the combination of thiamethoxam and Sf applied at lower rates would significantly reduce the amount of both control agents when applied singly, leading not only to a persistence of control effects but also to lower risks to humans and the environment. This is considered as a promising alternative strategy for the integrated management of B. odoriphaga, because of its superior control effect, low rates of application, long-term controls and low cost (Ma et al., 2013). In addition, our previous study also found that sticky cards with black color could efficiently trap the adults (Wu et al., 2015b). The integrated technique of synergism of EPNs, thiamethoxam and sticky cards to control B. odoriphaga should be studied further. Overall, synergistic interactions were mostly detected between S. feltiae SF-SN and thiamethoxam against 3rd instar B. odoriphaga. The combination of the nematodes and insecticides may achieve an effect comparable or superior to thiamethoxam for the curative control of this pest. Therefore, the integrated techniques of the synergism of S. feltiae SF-SN species and thiamethoxam to control B. odoriphaga could be useful in future integrated pest management.
compared to Sf species alone, except at 63 and 70 days. Furthermore, the combination effect of Sf ((0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ha))twice were gradually increased to a significantly higher level compared to a single treatment with Sf (1.5 billion IJs/ha) + thiamethoxam (2.0 kg a.i./ha) from day 35 to day 70. Compared to a single thiamethoxam (6.0 kg a.i./ha) treatment, the combination effects attained their highest values at over 90% of the control effects from day 35 to day 77 (Fig. 3B). 4. Discussion Previous studies have reported that the older dipterous larvae were more susceptible to EPNs compared to the younger stages (Sirjani et al., 2009). Also, other studies have shown that nematode virulence was overall superior against the 3rd instar B. odoriphaga larvae (Sun and Li, 2007; Wu et al., 2014a). As the 3rd instar B. odoriphaga larvae were larger (approximately 3–4 mm length) and may have larger natural openings for nematode invasion, the application of selected EPN against the 3rd instar larval populations may therefore generate better control results (Ma et al., 2013). Our study clearly showed that 3rd instar B. odoriphaga larvae were susceptible to infection with the six EPNs. However, there were significant differences in virulence among species and isolates (Fig. 1). In the concentration response assay, the mortality of B. odoriphaga larvae tended to increase with an increase of an EPN concentration up to 100 IJs/larva, after which the mortality remained stable, except for H. indica LN2. In contrast to the other five EPNs, we found a concentration response in the application of H. indica LN2, for which a higher density was required to cause a high mortality in B. odoriphaga. The larger effective concentration range were very similar to previously found by Langford et al. (2014) who tested the H. bacteriophora infectivity in Bactrocera tryoni Froggatt (Diptera: Tephritidae) at three concentrations (50, 100 and 200 IJs/cm2). This effect could be due to a number of factors, including varying pathogenicity in target hosts (Dowds and Peters, 2002), different foraging behaviors of EPN species and isolates (Gaugler and Bilgrami, 2004). Therefore, the six EPN species were considered further in the subsequent experiments of the present study. The tandem application of biological control agents and chemical insecticides to achieve a greater total effect than the sum of their individual effects may be a promising approach for insect pest management in different agricultural systems (Rodriguez and Peck, 2009). The preliminary screening experiments showed there was a synergistic interaction between thiamethoxam and six EPN species to the 3rd instar B. odoriphaga under laboratory conditions. The strongest interactions were observed in thiamethoxam + LN2 combinations. Thiamethoxam + All combinations and thiamethoxam + Sf combinations also indicated a significant synergistic interaction (Table 1). In the greenhouse experiments, however, the combination of Sf and All species caused significantly higher control effects than that of LN2 from day 7 to day 28, respectively (Fig. 2B). Unfavorable environmental conditions, soil type, habitats for pests and other variable factors can substantially influence the survival and performance of EPNs (Koppenhöfer et al., 2000; Susurluk and Ehlers, 2008). B. odoriphaga is a cold-adapted pest, and it can cause damage when the soil temperature is as low as 10–15 °C. The adults cannot propagate at temperatures exceeding 30 °C. However, the warm-adapted species H. indica plays an important role in causing high mortality of pests at 25–30 °C (Sun et al., 2004; Jagdale et al., 2007). In contrast, the coldadapted species S. feltiae yielded significantly better B. coprophila control than H. indica at 22 °C (Jagdale et al., 2004). On the other hand, the S. feltiae species is sold commercially to control dipteran larvae, such as sciarid flies, and it appeared that these larvae were probably an important natural host (Peters, 1996). However, present applications of EPNs in China are usually costlier than chemical pesticides (Ma et al., 2013). In order to lower the dose of both Sf species and thiamethoxam, further laboratory tests are needed to
Acknowledgments We thank Tai’an Lvnong Biological Co. Ltd. (Tai’an, China) for providing nematode resources. This project was supported by the 59
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nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) against the chive gnat, Bradysia odoriphaga. J. Pest Sci. 86, 551–561. Maiensfisch, P., Huerlimann, H., Rindlisbacher, A., Gsell, L., Dettwiler, H., Haettenschwile, J., Sieger, E., Walti, M., 2001. The discovery of thiamethoxam: a second-generation neonicotinoid. Pest Manage. Sci. 57, 165–176. McVay, J.R., Gudauskas, R.T., Harper, J.D., 1977. Effect of Bacillus thuringiensis and chemical insecticides on Spodoptera littoralis (Lepidoptera: Noctuidae). J. Econ. Entomol. 77, 590–885. Misawa, T., Kuninaga, S., 2013. First report of white leaf rot on Chinese chives caused by Rhizoctonia solani AG-2-1. J. Gen. Plant Pathol. 79, 280–283. Nishimatsu, T., Jackson, J.J., 1998. Interaction of insecticides, entomopathogenic nematodes, and larvae of the western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 91, 410–418. Peters, A., 1996. The natural host range of Steinernema and Heterorhabditis spp. and their impact on insect populations. Biocontrol Sci. Technol. 6, 389–402. Rodriguez, A.M., Peck, D.C., 2009. Synergies between biological and neonicotinoid insecticides for the curative control of the white grubs Amphimallon majale and Popillia japonica. Biol. Control 51, 169–180. SAS Institute, 2012. JMP Users Guide. Version 10.0. SAS Institute Inc., Cary, NC. Shapiro, D.I., McCoy, W.W., 2000. Virulence of entomopathogenic nematodes to Diaprepes abbreviatus (Coleoptera: Curculionidae) in the laboratory. J. Econ. Entomol. 93, 1090–1095. Shi, Y.C., Zheng, J.Q., Zhang, Y., 2001. Mushroom major pest survey briefing in suburbs. Plant Pro. Technol. Exten. 21, 18–19. Sirjani, F.O., Lewis, E.E., Kaya, H.K., 2009. Evaluation of entomopathogenic nematodes against the olive fruit fly, Bactrocera oleae (Diptera: Tephritidae). Biol. Control 48, 274–280. Smits, P.H., 1996. Post-application persistence of entomopathogenic nematodes. Biocontrol Sci. Technol. 6, 379–387. Sun, R.H., Li, A.H., 2007. Studies on combination effect of entomopathogenic nematode H06 and insecticide against Bradysia odoriphaga. Chin. J. Pesticide Sci. 9, 66–70. Sun, R.H., Li, A.H., Han, R.C., Cao, L., Liu, X.L., 2004. Factors affecting the control of Bradysia odoriphaga with entomopathogenic nematode Heterorhabditis indica LN2. Nat. Enem. Insects 26, 150–155. Susurluk, A., Ehlers, R.U., 2008. Field persistence of the entomopathogenic nematode Heterorhabditis bacteriophora in different crops. Biol. Control 53, 627–641. Thurston, G.S., Kaya, H.K., Gaugler, R., 1994. Characterizing the enhanced susceptibility of milky disease-infected scarabaeid grubs to entomopathogenic nematodes. Biol. Control 4, 67–73. Torres, J.B., Silva-Torres, C.S.A., Barros, R., 2003. Relative effects of the insecticide thiamethoxam on the predator Podisus nigrispinus and the tobacco whitefly Bemisia tabaci in nectaried and nectariless cotton. Pest Manage. Sci. 59, 315–323. Wang, W.J., Zhang, T., Chen, J.M., Chen, Z.D., 2011. Present situation and control technology of pesticide residue in Chinese chives. Shandong Agric. Sci. 10, 82–84. White, G.F., 1927. A method for obtaining infective nematode larvae from cultures. Science 66, 302–303. Wu, H.B., Xin, L., Gong, Q.T., Zhang, K.P., Cao, G.P., Sun, R.H., 2014a. Evaluation of the effects of infection by different entomopathogenic nematodes and chemical pesticides on Bradysia odoriphaga. Chin. J. Appl. Entomol. 51, 1060–1068. Wu, S.H., Youngman, R.R., Kok, L.T., Laub, C.A., Pfeiffer, D.G., 2014b. Interaction between entomopathogenic nematodes and entomopathogenic fungi applied to third instar southern masked chafer white grubs, Cyclocephala lurida, (Coleoptera: Scarabaeidae), under laboratory and greenhouse conditions. Biol. Control 76, 65–73. Wu, H.B., Fan, K., Xin, L., Cao, G.P., Sun, R.H., 2015a. Virulence and control effect of entomopathogenic nematodes against Agrotis ypsilon Rottemberg. J. Plant Prot. 42, 244–250. Wu, H.B., Gong, Q.T., Zhang, K.P., Zhang, X.P., Sun, R.H., 2015b. The efficacy of entomopathogenic nematodes and black sticky card to Bradysia odoriphaga. J. Plant Prot. 42, 632–638. Yan, X., Moens, M., Han, R., Chen, S., De Clercq, P., 2012. Effects of selected insecticides on osmotically treated entomopathogenic nematodes. J. Plant Dis. Protect 119, 152–158. Yang, J.K., Zhang, X.M., 1985. Notes on the fragrant onion gnats with descriptions of two new species of Bradysia odoriphaga (Diptera: Sciaridae). Acta Agri. Univ. Pekinen. 11, 153–156. Zhang, P., Chen, C.Y., Li, H., Liu, F., Mu, W., 2014a. Selective toxicity of seven neonicotinoid insecticides to Bradysia odoriphaga and Eisenia foetida. J. Plant Prot. 41, 79–86. Zhang, P., Liu, F., Mu, W., Wang, Q.H., Li, H., Chen, C.Y., 2014b. Life table study of the effects of sublethal concentrations of thiamethoxam on Bradysia odoriphaga Yang and Zhang. Pestic. Biochem. Physiol. 111, 31–37. Zhang, P., He, M., Zhao, Y.H., Ren, Y.P., Wei, Y., Mu, W., Liu, F., 2015a. Dissipation dynamics of clothianidin and its control efficacy against Bradysia odoriphaga Yang and Zhang in Chinese chive ecosystems. Pest Manage. Sci. 32, 617–622. Zhang, P., Zhao, Y.H., Han, J.K., Zhai, Y.B., Mu, W., Liu, F., 2015b. Control effects of thiamethoxam and clothianidin against Bradysia odoriphaga with different application methods. J. Plant Prot. 42, 645–650. Zhang, P., He, M., Wei, Y., Zhao, Y.H., Ren, Y.P., Mu, W., Liu, F., 2016. Comparative soil distribution and dissipation of phoxim and thiamethoxam and their efficacy in controlling Bradysia odoriphaga Yang and Zhang in Chinese chive ecosystems. Crop Prot. 9, 1–8.
Special Fund for Agro-scientific Research in the Public Interest (201303027) and the Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences (2015YQN30). References Abbott, W.S., 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Cloyd, R.A., Dickinson, A., 2006. Effect of Bacillus thuringiensis subsp. israelensis and neonicotinoid insecticides on the fungus gnat Bradysia sp nr. coprophila (Lintner) (Diptera: Sciaridae). Pest Manage. Sci. 62, 171–177. David, I.S., Dawn, H.G., Simon, J.P., Jane, P.F., 2006. Application technology and environmental considerations for use of entomopathogenic nematodes in biological control. Biol. Control 5, 124–133. Demarta, L., Hibbard, B.E., Bohn, M.O., Hiltpold, I., 2014. The role of root architecture in foraging behavior of entomopathogenic nematodes. J. Invertebr. Pathol. 122, 32–39. Dowds, B.C.A., Peters, A., 2002. Virulence mechanisms. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, pp. 79–98. Duncan, L.W., McCoy, C.W., 1996. Vertical distribution in soil, persistence, and efficacy against citrus root weevil (Coleoptera: Curculionidae) of two species of entomogenous nematodes (Rhabditida: Steinernematidae; Heterorhabditidae). Environ. Entomol. 25, 174–178. Ehlers, R.U., 1996. Current and future use of nematodes in biocontrol: practice and commercial aspects in regard to regulatory policies. Biocontrol Sci. Technol. 6, 303–316. Feng, H.Q., Zheng, F.Q., 1987. Studies of the occurrence and control of Bradysia odoriphaga Yang et Zhang. J. Shandong Agric. Univ. 18, 71–80. Finney, D.J., 1964. Probit Analysis. Cambridge University Press, London. Gaugler, R., Bilgrami, A.L. (Eds.), 2004. Nematode Behaviour. CABI Publishing, Cambridge. Georgis, R., Gaugler, R., 1991. Predictability in biological control using entomopathogenic nematodes. J. Econ. Entomol. 84, 713–720. Glazer, I., 2002. Survival biology. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Wallingford, UK, pp. 169–187. Harris, M.A., Oetting, R.D., Gardner, W.A., 1995. Use of entomopathogenic nematodes and a new monitoring technique for control of fungus gnats, Bradysia coprophila (Diptera: Sciaridae), in floriculture. Biol. Control 5, 412–418. Imahori, Y., Suzuki, Y., Uemura, K., Kishioka, I., Fujiwara, H., Ueda, Y., Chachin, K., 2004. Physiological and quality responses of Chinese chive leaves to low oxygen atmospheres. Postharvest Biol. Technol. 31, 295–303. Jacques, R.P., Morris, O.N., 1981. Compatibility of pathogens with other methods of pest control and crops protection. In: Burges, H.D. (Ed.), Microbial Control of Pests and Plant Disease 1970–1980. Academic Press, London, pp. 695–715. Jagdale, G.B., Casey, M.L., Grewal, P.S., Lindquist, P.K., 2004. Application rate and timing, potting medium and host plant effects on the efficacy of Steinernema feltiae against the fungus gnat, Bradysia coprophila, in floriculture. Biol. Control 29, 296–305. Jagdale, G.B., Casey, M.L., Cañas, L., Grewal, P.S., 2007. Effect of entomopathogenic nematodes species, split application and potting medium on the control of the fungus gnat, Bradysia difformis (Diptera: Sciaridae), in the greenhouse at alternation cold and warm temperatures. Biol. Control 43, 23–30. Kaya, H.K., 2002. Natural enemies and other antagonists. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Wallingford, UK, pp. 189–203. Kaya, H.K., Koppenhöfer, A.M., 1996. Effects of microbial and other antagonistic organism and competition on entomopathogenic nematodes. Biocontrol Sci. Technol. 6, 357–371. Kaya, H.K., Stock, S.P., 1997. Techniques in insect nematology. In: Lacey, L.A. (Ed.), Manual Techniques in Insect Pathology. Academic Press, San Diego, CA. Klein, M.G., 1993. Biological control of scarabs with entomopathogenic nematodes. In: Bedding, R., Akhurst, R., Kaya, H. (Eds.), Nematodes and the Biological Control of Insect Pests. CSIRO, East Melbourne, Australia, pp. 49–58. Koppenhöfer, A.M., Brown, I.M., Gaugler, R., Grewal, P.S., Kaya, H.K., Klein, M.G., 2000. Synergism of entomopathogenic nematodes and imidacloprid against white grubs: greenhouse and field evaluation. Biol. Control 19, 245–251. Koppenhöfer, A.M., Cowles, R.S., Cowles, E.A., Fuzy, E.M., Kaya, H.K., 2003. Effect of neonicotinoid synergists on entomopathogenic nematode fitness. Entomol. Exp. Appl. 106, 7–18. Lacey, L.A., Unruh, T.R., 1998. Entomopathogenic nematodes for control of coding moth: effect of nematode species, dosage, temperature and humidity under laboratory conditions. Biol. Control 13, 190–197. Langford, E.A., Nielsen, U.N., Johnson, S.N., Riegler, M., 2014. Susceptibility of queensland fruit fly, Bactrocera tryoni, (Froggatt) (Diptera: Tephritidae), to entomopathogenic nematodes. Biol. Control 69, 34–39. Li, H., Zhu, F., Zhou, X.M., Li, N., 2007. Bionomics and control of the root maggot, Bradysia odoriphaga, infested on the watermelon. Entomol. Knowl. 44, 834–836. Li, W.X., Yang, Y.T., Xie, W., Wu, Q.J., Xu, B.Y., Wang, S.L., Zhu, X., Wang, S.J., Zhang, Y.J., 2015. Effects of temperature on the age-stage, two-sex life table of Bradysia odoriphaga (Diptera: Sciaridae). J. Econ. Entomol. 108, 126–134. Ma, J., Chen, S.L., Moens, M., Han, R., De Clercq, P., 2013. Efficacy of entomopathogenic
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Synergistic effect of entomopathogenic nematodes and thiamethoxam in controlling Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae)
MARK
⁎
Haibin Wua,b, Qingtao Gongb, Kun Fanb, Ruihong Sunb, Yongyu Xua, , Kunpeng Zhangb a b
College of Plant Protection, Shandong Agricultural University, Tai’an, Shandong 271018, PR China Shandong Institute of Pomology, Tai’an, Shandong 271000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Entomopathogenic nematode Thiamethoxam Bradysia odoriphaga Synergistic effect Integrated pest management
Six entomopathogenic nematode species (EPNs) (Heterorhabditis bacteriophora H06 (H06), Steinernema carpocapsae NC116 (NC116), Steinernema carpocapsae All (All), Steinernema longicaudum X-7 (X-7), Steinernema feltiae SF-SN (Sf) and Heterorhabditis indica LN2 (LN2) were tested for virulence against 3rd instar Bradysia odoriphaga, and their interactions with thiamethoxam against 3rd instar B. odoriphaga under laboratory and greenhouse conditions were also evaluated. S. feltiae SF-SN, which is the most virulent, was selected to evaluate the synergism effects with thiamethoxam at different concentrations under laboratory conditions. S. feltiae SF-SN and thiamethoxam were either applied alone or in combination, and the combined application manner was evaluated in the greenhouse. Under laboratory conditions, a synergistic effect was found between six EPNs and thiamethoxam. The combination effects of Sf, All and LN2 species caused significantly higher mortality than the other three treatments (X-7, H06 and NC116). Furthermore, the combination of Sf and All species led to a significantly higher control effect than LN2 in greenhouse tests. In further greenhouse tests, the combination effect of Sf (0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ha) applied twice (28 day intervals) was significantly higher than a single treatment of Sf + thiamethoxam at twice the dosage. Compared to the single thiamethoxam application, the combination effects of Sf and thiamethoxam had the highest value at over 90% of the control effects for up to 6 weeks. Therefore, the integrated technique of the synergism of S. feltiae SF-SN species and thiamethoxam to control B. odoriphaga could be useful for integrated pest management in the future.
1. Introduction The Chinese chive (Allium tuberosum Rottl. ex Spreng.) is a perennial herbaceous vegetable with high economic value in eastern Asia and is grown over a vast geographical area from Asia through the Middle East to Europe and North America (Imahori et al., 2004; Misawa and Kuninaga, 2013). The chive maggot, Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae), is the major pest that restricts Chinese chive production (Li et al., 2015). This insect feeds on seven plant families and more than thirty plants species, including Chinese chives, garlic (Allium sativum L.), onion (A. cepa L.), and cucumber (Cucumis sativus L.) (Yang and Zhang, 1985; Feng and Zheng, 1987; Li et al., 2007), and it also causes production losses in mushroom sheds (Shi et al., 2001). Its larvae feed on the roots and stems of chives and cause 30–80% production losses in the absence of insecticide treatment. In severe cases, new Chinese chives need to be planted again (Ma et al., 2013). Currently, the predominant strategy for the management of this pest is to apply conventional insecticides, such as organophosphates, carbamates, and neonicotinoids (Zhang et al., 2014b). Thiamethoxam ⁎
is a second-generation neonicotinoid that has stomach and contact activity, and it operates by interfering with the nicotinic acetylcholine receptors in the insect’s nervous system (Maiensfisch et al., 2001; Torres et al., 2003). Cloyd and Dickinson (2006) reported that thiamethoxam was highly effective against second and third instar larvae of the fungus gnat Bradysia sp nr. coprophila L. (Diptera: Sciaridae). In addition, thiamethoxam has been reported to control B. odoriphaga at 6.0 kg a.i./ha and may help farmers more effectively manage B. odoriphaga (Zhang et al., 2015b). However, chemigation was a traditional method to control this pest and needed a mass amount of thiamethoxam (12.0 kg a.i./ha in some places) because of the dilution effects of water and soil on pesticides (Zhang et al., 2015b). The control efficacy was unsatisfactory due to the overlapping generations of the insect, and most of its life stages occur in sheltered circumstances (Wang et al., 2011). This challenge led to the excessive use of chemical insecticides, which caused pollution in the environment and left a high amount of residue on Chinese chives that were sold on the market (Zhang et al., 2016). Entomopathogenic nematodes (EPNs) have an indirect role in
Corresponding author at: College of Plant Protection, Shandong Agricultural University, 61 Daizong Street, Tai’an, Shandong 271018, PR China. E-mail address: [email protected] (Y. Xu).
http://dx.doi.org/10.1016/j.biocontrol.2017.05.006 Received 27 December 2016; Received in revised form 29 March 2017; Accepted 12 May 2017 Available online 12 May 2017 1049-9644/ © 2017 Published by Elsevier Inc.
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around an apple tree at the Shandong Institute of Pomology (SIP), Tai’an (35°48′ N, 117°28′ E), Shandong Province, China (Sun and Li, 2007). Five other isolates, including Steinernema carpocapsae NC116 (NC116), S. carpocapsae All (All), S. longicaudum X-7 (X-7), S. feltiae SFSN (Sf) and H. indica LN2 (LN2), were provided free-living in a foam substrate by Tai’an Lvnong Biological Co. Ltd. (Tai’an, China). All EPN species and isolates were cultured in the last-instar of the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae) at room temperature for 7–10 days (Kaya and Stock, 1997). Nematode infective juveniles (IJs) emerged from insect cadavers into White traps (White, 1927). The harvested IJs were washed 3 times by sedimentation in tap water, and were used immediately (laboratory experiment) or were stored in tap water before use (field experiment). Storage was carried out at a density of 1000 IJs/mL, in a 90 mm diameter plastic food tubes (each containing 50 mL of nematode suspension) with snap-on lids. The batches of collected IJs were stored at 10–12 °C for no longer than 2 weeks, after which fresh batches were obtained using the same methods as described above to ensure maximal efficacy (Langford et al., 2014; Ma et al., 2013). Only fresh batches of IJs were used in the experiment (Demarta et al., 2014). IJs were acclimated at room temperature for 1 h, and an Olympus SZ2-ILST microscope (×100) and a microscope counting plate were used to calibrate the solutions to the required concentrations and to check nematode activity prior to application (Langford et al., 2014; Ma et al., 2013). The nematode concentrations required were prepared by volumetric dilutions in distilled water. Colonies of B. odoriphaga were originally obtained from a Chinese chive field at Tai′an in Shandong, China (36°18′ N, 117°13′ E) during September 2013 and were maintained at a constant temperature of 25 ± 1 °C and Relative Humidity (R.H.) of 65–75% with a photoperiod of 12 h:12 h L:D in the insectary of SIP, Tai’an. The thiamethoxam formulation used was Actara (Swiss Syngenta Crop Protection Co. Ltd., China), which is a wettable granule (WG) and contains 250 g/kg of active ingredient. Chinese chive seeds (Dugenhong) were purchased from Shouguang Dafeng seedling Co. Ltd. (Shandong, China) and were sown in April 2013, grown to the seedling stage and then transplanted to a greenhouse at Tai′an in Shandong, China (36°38′ N, 117°43′ E), in 0.20 m rows at a rate of approximately 3,000,000 plants/ha in March 2013. In order to ensure the consistency of the test conditions (soil texture, soil structure, soil organic content and so on), the greenhouse (6 W × 80 L m) is evenly divided into two parts (part A and part B). Two field trials were conducted in part A and part B, respectively.
protecting plants through parasitizing a wide range of insects in natural and agricultural systems (Duncan and McCoy, 1996; Lacey and Unruh, 1998; Shapiro and McCoy, 2000). The third-stage infective juvenile (IJ) is the only stage in the life cycle of the nematodes that lives in the soil. EPNs of the families Steinernematidae and Heterorhabditidae may be particularly useful for the control of pests that occur in cryptic habitats (Ehlers, 1996). A number of studies have reported on the application of EPNs as biological control agents of pests in the genus Bradysia (Harris et al., 1995; Sun and Li, 2007; Wu et al., 2014a). However, EPN field efficacy is often inconsistent and unsatisfactory (Georgis and Gaugler, 1991; Klein, 1993) due to various biotic (Kaya, 2002; Kaya and Koppenhöfer, 1996) and abiotic factors (Smits, 1996; Glazer, 2002). Additionally, the choice of application equipment, as well as the manner in which the nematodes are applied, can have a substantial impact on pest control efficacy (David et al., 2006). In the search for a suitable host, IJs interact synergistically with some insecticides (Thurston et al., 1994; Nishimatsu and Jackson, 1998; Koppenhöfer et al., 2000). Neonicotinoids, for instance, were shown to be a synergist of EPNs against scarab beetle larvae (Koppenhöfer et al., 2000, 2003). The strategic combination of biological controls with reduced application rates and dosages of chemical insecticide synergists may, therefore, represent a valuable approach for the suppression of soil insect pests. When applied together, they may act independently and cause an additive effect, or interact with each other in a synergistic or antagonistic way (Jacques and Morris, 1981). Additive or synergistic interactions have been reported in the combined application of Steinernema glaseri and imidacloprid against 3rd instar of the masked chafers Cyclocephala hirta LeConte and C. pasadenae Casey (Coleoptera: Scarabaeidae) (Koppenhöfer et al., 2000). Mortality of Agrotis ypsilon Rottemberg increased after mixed application of matrine and S. carpocapsae NC116, which showed a synergistic interaction (Wu et al., 2015a). To increase the effectiveness of controlling B. odoriphaga with EPNs, produce high-quality Chinese chives with less residual pesticide, save money from using EPNs and pesticides and protect the environment, a combined application of nematodes and insecticides has been developed recently. Previous study also showed that neonicotinoid insecticides, such as imidacloprid and thiamethoxam, had no effect on the survival of nematodes and may be used as a synergist for nematodes (Yan et al., 2012). Additive or synergistic interactions have been reported for the combined application of three EPNs (Heterorhabditis bacteriophora H06, S. carpocapsae All and S. feltiae SF-SN) and three insecticides (imidacloprid, chlorpyrifos and beta-cypermethrin) against B. odoriphaga under laboratory conditions. (Wu et al., 2014a). However, previous study found that these three insecticides had high toxicities to Eisenia foetida Savigny (Opisthopora: Lumbricidea), and only thiamethoxam had a low selective toxicity to E. foetida in the pot experiments (Zhang et al., 2014a). Therefore, combined application of nematodes and thiamethoxam may be an important alternative for controlling B. odoriphaga and needs further study under both laboratory and greenhouse conditions. The objectives of this study were to (1) select a virulent EPN species, (2) evaluate its interaction with thiamethoxam against B. odoriphaga, (3) select the best synergism effect of the most virulent species and thiamethoxam using appropriate concentrations under laboratory conditions, and (4) optimize the combined application methods and respective concentrations of nematodes species and thiamethoxam against B. odoriphaga under greenhouse conditions. It was anticipated that additive or synergistic interactions would be achieved by the combined use of the two types of control agents and thus improve the overall pest control efficacy under greenhouse conditions.
2.2. Effects of the nematode concentrations on susceptibility of 3rd instar B. odoriphaga Six EPN species (H06, NC116, All, X-7, Sf and LN2) were tested to investigate the effect of IJs concentrations on the pathogenicity of the nematodes to 3rd instar B. odoriphaga. Zhang et al. (2016) reported B. odoriphaga was primarily observed in spring and autumn, with the highest level reaching 54.67 larvae per quadrat (20 cm × 20 cm; soil surface area: 0.04 m2) in the field. Based on our preliminary investigation of population dynamics, the occurrences of B. odoriphaga could reach an average of 60 larvae per quadrat (20 cm × 20 cm) in the greenhouse (15,000,000 larvae/ha). Following Wu et al. (2014b) with few modification, the method of calculating the amount of IJs applied was established according to the population densities of B. odoriphaga larvae in the greenhouse (30 IJs/larva, equal to 0.45 billion IJs/ha). Four rates (30, 60, 100 and 200 IJs/larva, equal to 0.45, 0.9, 1.5 and 3.0 billion IJs/ha, respectively) were used for each species. The test concentrations used in our study were based on previous studies (Sun and Li, 2007; Wu et al., 2014a, 2015). One Petri dish was treated as a single replication. Fresh Chinese chive pseudo stems (5.0 mm) were cut and put in a 90 mm diameter Petri dish containing a 90 mm diameter filter paper that was moistened with 1.0 mL of test solution. After
2. Materials and methods 2.1. Nematodes, insects, insecticide, plant material Heterorhabditis bacteriophora H06 (H06) was isolated from the soil 54
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instars) was almost the 3rd instar larvae. Following Zhang et al. (2015a) and Ma et al. (2013) with few modification, the numbers of B. odoriphaga larvae at the pseudo stems/bulbs and roots was determined after chipping away the soil without harming the plants for each plot (Z-shaped sampling) by taking five square soil plugs (20 cm × 20 cm quadrat) randomly with a shovel, at a depth of 15 cm. The number of B. odoriphaga larvae in each treatment were counted at 0, 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70 days after the application of the agents. After investigation, the larvae and soil were put back.
treatment, the larvae were maintained at a constant temperature of 25 ± 1 °C and R.H. of 65–75% with a photoperiod of 12 h:12 h L:D. Controls were identical to the treatments except that no IJs were added. Each treatment was replicated four times (approximately 30 larvae per replication) over a short period and under the same condition (Ma et al., 2013; Wu et al., 2014a,b, 2015b). Mortality was assessed after 72 h. Larvae were considered dead if they were unable to move in a coordinated manner when disturbed with the point of a writing brush. 2.3. Preliminary combination screening of EPNs and thiamethoxam against B. odoriphaga
2.5. Best synergism effects of EPN species and thiamethoxam against B. odoriphaga
The interaction between six EPN species and thiamethoxam against 3rd instar B. odoriphaga larvae was tested under laboratory conditions. In order to maximize combination screening of EPNs and thiamethoxam, and reduce the cost of using EPN, nematode (100 IJs/larva, equal to 1.5 billion IJs/ha) applied alone or added to the thiamethoxam were based on previous studies (Ma et al., 2013; Sun et al., 2004; Sun and Li, 2007). Thiamethoxam applied alone or in combination with a nematode were based on the LC50 (12 mg a.i./L) concentration from previous studies (Zhang et al., 2014a). In addition, an untreated water control was included. Other experimental conditions used were the same as the conditions in Section 2.2.
To optimize the combination of nematodes and thiamethoxam against B. odoriphaga, the most virulent EPN species from the experiments in Sections 2.2–2.4 was used. The S. feltiae SF-SN species with three rates (30, 60 and 100 IJs/larva, equal to 0.45, 0.9 and 1.5 billion IJs/ha, respectively) and thiamethoxam with four concentrations (8, 12, 15 and 20 mg a.i./L) were used, in addition to an untreated water control. All treatments were replicated four times (approximately 30 larvae per replication). The experimental conditions were the same as the conditions in Section 2.2. 2.6. Optimized control methods of nematodes S. feltiae SF-SN species and thiamethoxam against B. odoriphaga in the greenhouse
2.4. Combined effects of EPN isolates and thiamethoxam against B. odoriphaga in the greenhouse
To evaluate the efficacy of nematodes and thiamethoxam against B. odoriphaga, the most virulent Sf species and thiamethoxam concentrations from the Section 2.5 experiment was used. The experiments were conducted in the part B of the greenhouses, which were divided into 20 plots on 11 September 2015. The absence of natural populations of EPNs in the greenhouse was confirmed through sampling. During the experiment, the average temperature, R.H. and soil moisture were 21.5 ± 2.4 °C, 52.1 ± 2.8% and 23.7 ± 2.6% VWC (volumetric water content), respectively. The experiment included 5 treatments: a water control, thiamethoxam (6.0 kg a.i./ha), Sf (3.0 billion IJs/ha), Sf (1.50 billion IJs/ha) + thiamethoxam (2.0 kg a.i./ha), and Sf (0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ha). The thiamethoxam was applied alone at 6.0 kg a.i./ha or in combination with Sf at 2.0 or 1.0 kg a.i./ha. Sf species was applied alone at 3.0 billion IJs/ha or in combination with the thiamethoxam at 1.50 or 0.75 billion IJs/ha. All treatments were applied at September 10. But, Sf (0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ha) were applied in two dressings (September 10 and October 8). Four blocks and a randomized complete block design were used. Before trial treatments, the developmental stage of B. odoriphaga (94% 3rd instars, 6% 4th instars) was almost the 3rd instar larvae. The numbers of B. odoriphaga were counted at 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70 and 77 days after treatments. Other experimental procedures, methods and the soils used were the same as the ones in Section 2.4.
The combined effectiveness of different nematode species and thiamethoxam against B. odoriphaga were conducted in part A of the greenhouses, which were divided into 32 plots on 31 March 2015. Each plot (0.8 W × 5 L m) was separated by 0.3 m of bare cultivated ground. To prevent oviposition interference in adult B. odoriphaga, each plot was covered with 80 screen mesh (0.8 W × 5 L × 1.8 H m). The absence of natural populations of EPNs in the greenhouse was confirmed through sampling. The soil was a loamy sand texture comprised of 45.7% sand, 37.5% silt, 16.8% clay, 2.8% organic matter with a pH of 6.4. During the experiment, the average temperature, R.H. and soil moisture were 20.8 ± 1.9 °C, 50.6 ± 3.5% and 21.8 ± 2.1% VWC (volumetric water content), respectively. Temperature, R.H. and soil moisture were collected with a ZigWSN®C-A date logger throughout the experiment. The experiment included 8 treatments: a water control, thiamethoxam, All, Sf, LN2, All + thiamethoxam, Sf + thiamethoxam, and LN2 + thiamethoxam. Following Zhang et al. (2015a) and Ma et al. (2013) with few modification, all agents were applied using the directional spray-washing method by a 5.0 L hand-held pump sprayer. Chinese chives plants were sprayed with each agent at the roots according to good agricultural practice in each plot. Each plot received each agent with a volume of water equivalent to 1.0 L of water per m2. IJs suspended in the same volume of water was prepared and applied alone at 3.0 billion IJs/ha (equal to 300,000 IJs/m2) or in combination with the thiamethoxam at 1.5 billion IJs/ha (equal to 150,000 IJs/m2). Thiamethoxam was also prepared in the same volume of water and applied alone at one thousand times dilutions (6.0 kg a.i./ha) or in combination with a nematode at three thousand times dilutions (2.0 kg a.i./ha). Each agent was gently mixed in a plastic bucket before application was transferred to the spray tanks. To prevent sedimentation of the IJs in the tank, the tanks were well shaken before and during application. All treatments were done at the same time in the evening to minimize the risk of desiccation of IJs. Four blocks and a randomized complete block design were used. An auto-watering system was set up in the greenhouse. All experimental treatments were watered every two days with a total amount of 5.0 mm per watering during the experiment. Before applying the agents, the initial density of B. odoriphaga was investigated, and its developmental stage (95% 3rd instars, 5% 4th
2.7. Statistical analysis A chi-square (X2) test was used to test the interaction of EPNs and thiamethoxam. Before analysis, all mortality data were corrected for control mortality (Abbott, 1925). The method for determining the type of interaction (synergistic, additive, or antagonistic) was first described by Finney (1964) and then modified by McVay et al. (1977). The expected additive proportional mortality ME for the EPN/thiamethoxam combinations was calculated by ME = MN + MT (1 − MN), where MN and MT are the observed proportional mortalities relatively caused by EPNs and thiamethoxam alone. A chi-square test was then carried out using the formula X2 = (MNT − ME)2/ME, where MNT represents the observed mortality for the EPN/thiamethoxam combination. The 55
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Fig. 1. Mortality of 3rd instar B. odoriphaga when exposed to different IJ concentrations of (A) S. carpocapsae All, (B) H. indica LN2, (C) S. feltiae SF-SN, (D) S. longicaudum X-7, (E) H. bacteriophora H06, (F) S. carpocapsae NC116. Error bars represent standard errors. Different letters of Tukey’s HSD test are indicative of significant differences (P < 0.05) between means.
calculated value from the chi-square test was then compared to the chisquare table value for one degree of freedom. If calculated values are greater than the table value (X21, 0.05 = 3.84), non-additive effects, e.g., synergistic or antagonistic, could be suspected between the two agents (Finney, 1964). If the differences MNT–ME had a positive value, the interaction was considered synergistic, and if the difference was negative, the interaction was considered antagonistic. All data were transformed using an arcsin formula. Analysis of variance (ANOVA) with SPSS software (SAS Institute, 2012) was used to test the significant differences among treatments in both laboratory and greenhouse experiments. The results are reported as significant in Tukey’s Honestly Significant Difference (Tukey HSD) Test (p < 0.05).
Table 1 Interaction of various EPN species (100 IJs/larva) and thiamethoxam (12 mg a.i./L) on 3rd instar B. odoriphaga.
3. Results 3.1. EPN density effects on B. odoriphaga mortality There were significant differences among various EPN species in mortality (F = 25.08, DF = 5; P < 0.001) used One-way ANOVA (Fig. 1). Two-way ANOVA was used to test the differences between treatments with different EPN species and IJ concentrations. Significant differences were found among EPN species (F = 29.86, DF = 5; P < 0.001) and IJ concentrations (F = 88.63, DF = 4; P < 0.001). Significant interactions were detected between EPN species and IJ concentrations (F = 5.26, DF = 20; P < 0.001). As IJ concentrations increased, so did B. odoriphaga mortality with higher mortality at 200 IJs/larva and lower mortality at 30 IJs/larva. However, there was no difference in larval mortality among IJ concentrations (60, 100 and 200 IJs/larva), except LN2 species (Fig. 1B). Therefore, 100 IJs/larva was used in the following laboratory studies.
Treatment
Observed mortality (%)a
Expected mortality (%)b
X2
Type of interaction
S. feltiae SF-SN S. carpocapsae All S. longicaudum X-7 H. indica LN2 H. bacteriophora H06 S. carpocapsae NC116 Thiamethoxam S. feltiae SF-SN + thiamethoxam S. carpocapsae All + thiamethoxam S. longicaudum X7 + thiamethoxam H. indica LN2 + thiamethoxam H. bacteriophora H06 + thiamethoxam S. carpocapsae NC116 + thiamethoxam
16.81 ± 3.46 ab* 13.45 ± 1.61 ab 14.29 ± 0.97 ab 23.53 ± 1.61 b 7.56 ± 1.68 a 9.24 ± 1.37 a 42.50 ± 2.47 c 94.96 ± 3.22 e
– – – – – – – 53.16
– – – – – – – 39.44
– – – – – – – Synergistic
89.92 ± 3.63 e
51.27
34.96
Synergistic
65.55 ± 2.87 d
51.74
4.42
Synergistic
96.64 ± 1.37 e
56.95
33.20
Synergistic
66.39 ± 2.74 d
47.96
8.50
Synergistic
70.59 ± 4.20 d
48.90
11.54
Synergistic
a
Observed mortality was corrected for control mortality (0.83% at 3 days). Expected mortality ME = MN + MT (1 − MN), where MN and MT are the observed proportional mortalities relatively caused by EPN and thiamethoxam alone. * Different letters of Tukey’s HSD test indicate significant differences (P < 0.05) between means. Data are presented as means ± standard error. b
3.3. Combinatorial effects of EPNs and thiamethoxam against B. odoriphaga under greenhouse conditions
3.2. Combination effects of EPNs and thiamethoxam against B. odoriphaga
The B. odoriphaga larvae were sampled after application of differed treatments from March 27 to June 5, 2015 (Fig. 2). During the experimental months, the main occurrences of B. odoriphaga were from April 17 to May 15 with the highest level reaching 77.00 larvae/ quadrat in untreated plots on day 49 after applying different treatments. Compared to the untreated control, B. odoriphaga larvae were dramatically reduced after application of the treatments. The combination treatments caused significantly lower population densities compared to each of the respective single Sf, All and LN2 species from day 7 to day 42 (Fig. 2A). After 7 d, the control effects of treatments with a combination of low concentration thiamethoxam (2.0 kg a.i./ha) with
The combination treatments caused significantly higher mortality than each of the respective single agents (F = 175.08, DF = 12; P < 0.001). Synergistic effects were found between EPN species and thiamethoxam. However, there was significantly different in the observed mortality of B. odoriphaga among the combination treatments of various EPN species and thiamethoxam. The combination effects of Sf, All and LN2 species caused significantly higher mortality than X-7, H06 and NC116, respectively (F = 22.00, DF = 5; P < 0.001). Among these treatments, LN2 and thiamethoxam achieved the strongest mortality for the combined application (Table 1). 56
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Fig. 2. Population densities and control efficiency of B. odoriphaga at different times after the application of different treatments. Fig. 2A represent the number of larvae per quadrat and Fig. 2B represent the control effects relative to the water control to B. odoriphaga at different times, respectively. Error bars represent standard errors. Different letters of Tukey’s HSD test are indicative of significant differences (P < 0.05) between means.
detected in the interaction between Sf species and lower concentrations thiamethoxam (8, 12 and 15 mg a.i./L), and an additive effect was found between the nematode and higher concentration of thiamethoxam (20 mg a.i./L). Among them, the combination effect of Sf species (30, 60 and 100 IJs/larva) and thiamethoxam (12, 15 and 20 mg a.i./L) caused no significant differences (F = 0.68, DF = 8; P = 0.709) (Table 2).
Sf, All and LN2 species were higher compared to treatments with any one EPN species alone. The combination of Sf and All species caused significantly higher control effects than that of LN2 from day 7 to day 28, respectively. However, the combination effects of Sf and All species were gradually reduced from day 28 to day 70. For the single thiamethoxam (6.0 kg a.i./ha) treatment, the control effects were the highest from day 21 to day 56 and could last up to months after application at over 80% of the control effects (Fig. 2B).
3.5. Optimized control effects of S. feltiae SF-SN species and thiamethoxam against B. odoriphaga under greenhouse conditions
3.4. Synergism effect of S. feltiae SF-SN species and thiamethoxam against B. odoriphaga
B. odoriphaga larvae were sampled after different treatments were applied from September 10 to November 26, 2015 (Fig. 3). During the experiment, the main occurrences of B. odoriphaga were from September 10 to October 29 with the highest level reaching 79.25 larvae/ quadrat in untreated plots on day 35 after different treatments were applied. Compared to the untreated controls, B. odoriphaga larvae were
When the Sf species (30, 60 and 100 IJs/larva) and higher concentrations thiamethoxam (12, 15 and 20 mg a.i./L) were applied simultaneously, the combination treatments caused significantly higher mortality than each of the respective single agents, except thiamethoxam (20 mg a.i./L) (F = 134.00, DF = 18; P < 0.001). Synergism was 57
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Table 2 Interaction of S. feltiae SF-SN species and thiamethoxam on 3rd instar B. odoriphaga under laboratory conditions. Treatment
S. feltiae SF-SN (IJs/larva)
Thiamethoxm (mg a.i./L)
Observed mortality (%)a
Expected mortality (%)b
X2
Type of interaction
S. feltiae SF-SN
30 60 100
– – –
4.20 ± 1.68 a* 10.08 ± 0.84 a 13.45 ± 1.61 a
– – –
– – –
– – –
Thiamethoxam
– – – –
8 12 15 20
15.13 43.70 55.46 82.35
± ± ± ±
0.84 a 2.87 bc 3.46 cd 2.11 e
– – – –
– – – –
– – – –
S. feltiae SF-SN + thiamethoxam
30 30 30 30 60 60 60 60 100 100 100 100
8 12 15 20 8 12 15 20 8 12 15 20
35.00 90.84 95.84 92.51 50.01 93.34 95.84 94.18 62.51 91.67 95.01 94.17
± ± ± ± ± ± ± ± ± ± ± ±
6.29 2.15 2.36 1.36 5.51 3.44 1.92 0.96 4.91 1.60 2.50 2.15
22.43 55.28 68.80 99.71 28.42 59.25 71.94 100.96 31.85 61.52 73.74 101.67
17.08 52.23 31.04 1.28 35.10 46.98 25.78 1.44 58.50 38.22 21.99 1.26
Synergistic Synergistic Synergistic Additive Synergistic Synergistic Synergistic Additive Synergistic Synergistic Synergistic Additive
b e e e bcd e e e d e e e
a
Observed mortality was corrected for control mortality (1.60% at 3 days). Expected mortality ME = MN + MT (1 − MN), where MN and MT are the observed proportional mortalities relatively caused by S. feltiae SF-SN species and thiamethoxam alone. * Different letters of Tukey’s HSD test indicate significant differences (P < 0.05) between means. Data are presented as means ± standard error. b
+ thiamethoxam (1.0 kg a.i./ha))twice caused significantly lower population densities compared to that of Sf species alone (Fig. 3A). After 7 d, the control effects of treatments with a combination of low concentrations thiamethoxam (1.0, 2.0 kg a.i./ha) with Sf species was higher
dramatically reduced after the applications of single thiamethoxam (6.0 kg a.i./ha) treatment, Sf (1.5 billion IJs/ha) + thiamethoxam (2.0 kg a.i./ha), Sf ((0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ ha))twice. The combination treatments of Sf ((0.75 billion IJs/ha)
Fig. 3. Population densities and control efficiency of B. odoriphaga at different times after the application of different treatments. Fig. 3A represent the number of larvae per quadrat, and Fig. 3B represents the control effects relative to the water control to B. odoriphaga at different times, respectively. Error bars represent standard errors. Different letters of Tukey’s HSD test indicate significant differences (P < 0.05) between means. twice represents treatment applied twice (September 10 and October 8).
58
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determine whether any synergistic effects occur between Sf species and thiamethoxam at the different concentrations against B. odoriphaga. In our study, the combination effects of Sf species (30, 60 and 100 IJs/ larva) and thiamethoxam (12, 15 and 20 mg a.i./L) caused no significant differences, but with a dramatic increase synergistic effects from a concentrations of 8–12 mg a.i./L thiamethoxam. This result indicates that thiamethoxam was mainly responsible for the higher B. odoriphaga larvae mortality, and an increase in concentrations from 30 to 100 IJs/larva did not enhance the impact of S. feltiae in the interaction (Table 2). However, the underlying mechanism for the interaction between S. feltiae and thiamethoxam against B. odoriphaga remains unknown. No matter how well-suited an entomopathogenic nematode is to a targeted pest, the application will fail if the technology is not available for application of nematodes, including the application equipment and methods (Shapiro and McCoy, 2000). In the greenhouse experiments, the combination treatments of Sf (1.5 billion IJs/ha) and thiamethoxam (2.0 kg a.i./ha) caused significantly lower population densities and significantly higher control effects than each of the respective single Sf, All and LN2 species from day 7 to day 42 (Fig. 2). However, the combination effects of Sf and thiamethoxam were gradually reduced with the increase of B. odoriphaga larvae densities from day 28. Therefore, the combination effects of Sf (0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ha) were applied twice on days 1 and 28, and the combination effects were gradually increased from day 28 and were significantly higher compared to treatment with Sf (1.5 billion IJs/ha) + thiamethoxam (2.0 kg a.i./ha) once (Fig. 3B). Compared to a single thiamethoxam (6.0 kg a.i./ha) treatment, the combination effects did not decline with time and persisted well in the greenhouse experiments with over 90% of the control effects up to 6 weeks after application. The good persistence of synergistic interactions is important for obtaining sustainable control, which may eventually lead to lower costs (Susurluk and Ehlers, 2008). Persisting populations of B. odoriphaga larvae likely aided the establishment and propagation of the Sf species from March to early June and from September to November. There is a greater scope for using the combination of Sf species and thiamethoxam for controlling B. odoriphaga larvae at peak damage, which often occurs in late spring and autumn. However, Chinese chives have a very low B. odoriphaga population density from late June to August every year. These results were consistent with the results that Zhang et al. (2015a) reported. This period was in the summer in China with temperatures higher than 30 °C, which could suppress the individual development and population establishment of B. odoriphaga (Li et al., 2015; Zhang et al., 2015a). Therefore, the combination of thiamethoxam and Sf applied at lower rates would significantly reduce the amount of both control agents when applied singly, leading not only to a persistence of control effects but also to lower risks to humans and the environment. This is considered as a promising alternative strategy for the integrated management of B. odoriphaga, because of its superior control effect, low rates of application, long-term controls and low cost (Ma et al., 2013). In addition, our previous study also found that sticky cards with black color could efficiently trap the adults (Wu et al., 2015b). The integrated technique of synergism of EPNs, thiamethoxam and sticky cards to control B. odoriphaga should be studied further. Overall, synergistic interactions were mostly detected between S. feltiae SF-SN and thiamethoxam against 3rd instar B. odoriphaga. The combination of the nematodes and insecticides may achieve an effect comparable or superior to thiamethoxam for the curative control of this pest. Therefore, the integrated techniques of the synergism of S. feltiae SF-SN species and thiamethoxam to control B. odoriphaga could be useful in future integrated pest management.
compared to Sf species alone, except at 63 and 70 days. Furthermore, the combination effect of Sf ((0.75 billion IJs/ha) + thiamethoxam (1.0 kg a.i./ha))twice were gradually increased to a significantly higher level compared to a single treatment with Sf (1.5 billion IJs/ha) + thiamethoxam (2.0 kg a.i./ha) from day 35 to day 70. Compared to a single thiamethoxam (6.0 kg a.i./ha) treatment, the combination effects attained their highest values at over 90% of the control effects from day 35 to day 77 (Fig. 3B). 4. Discussion Previous studies have reported that the older dipterous larvae were more susceptible to EPNs compared to the younger stages (Sirjani et al., 2009). Also, other studies have shown that nematode virulence was overall superior against the 3rd instar B. odoriphaga larvae (Sun and Li, 2007; Wu et al., 2014a). As the 3rd instar B. odoriphaga larvae were larger (approximately 3–4 mm length) and may have larger natural openings for nematode invasion, the application of selected EPN against the 3rd instar larval populations may therefore generate better control results (Ma et al., 2013). Our study clearly showed that 3rd instar B. odoriphaga larvae were susceptible to infection with the six EPNs. However, there were significant differences in virulence among species and isolates (Fig. 1). In the concentration response assay, the mortality of B. odoriphaga larvae tended to increase with an increase of an EPN concentration up to 100 IJs/larva, after which the mortality remained stable, except for H. indica LN2. In contrast to the other five EPNs, we found a concentration response in the application of H. indica LN2, for which a higher density was required to cause a high mortality in B. odoriphaga. The larger effective concentration range were very similar to previously found by Langford et al. (2014) who tested the H. bacteriophora infectivity in Bactrocera tryoni Froggatt (Diptera: Tephritidae) at three concentrations (50, 100 and 200 IJs/cm2). This effect could be due to a number of factors, including varying pathogenicity in target hosts (Dowds and Peters, 2002), different foraging behaviors of EPN species and isolates (Gaugler and Bilgrami, 2004). Therefore, the six EPN species were considered further in the subsequent experiments of the present study. The tandem application of biological control agents and chemical insecticides to achieve a greater total effect than the sum of their individual effects may be a promising approach for insect pest management in different agricultural systems (Rodriguez and Peck, 2009). The preliminary screening experiments showed there was a synergistic interaction between thiamethoxam and six EPN species to the 3rd instar B. odoriphaga under laboratory conditions. The strongest interactions were observed in thiamethoxam + LN2 combinations. Thiamethoxam + All combinations and thiamethoxam + Sf combinations also indicated a significant synergistic interaction (Table 1). In the greenhouse experiments, however, the combination of Sf and All species caused significantly higher control effects than that of LN2 from day 7 to day 28, respectively (Fig. 2B). Unfavorable environmental conditions, soil type, habitats for pests and other variable factors can substantially influence the survival and performance of EPNs (Koppenhöfer et al., 2000; Susurluk and Ehlers, 2008). B. odoriphaga is a cold-adapted pest, and it can cause damage when the soil temperature is as low as 10–15 °C. The adults cannot propagate at temperatures exceeding 30 °C. However, the warm-adapted species H. indica plays an important role in causing high mortality of pests at 25–30 °C (Sun et al., 2004; Jagdale et al., 2007). In contrast, the coldadapted species S. feltiae yielded significantly better B. coprophila control than H. indica at 22 °C (Jagdale et al., 2004). On the other hand, the S. feltiae species is sold commercially to control dipteran larvae, such as sciarid flies, and it appeared that these larvae were probably an important natural host (Peters, 1996). However, present applications of EPNs in China are usually costlier than chemical pesticides (Ma et al., 2013). In order to lower the dose of both Sf species and thiamethoxam, further laboratory tests are needed to
Acknowledgments We thank Tai’an Lvnong Biological Co. Ltd. (Tai’an, China) for providing nematode resources. This project was supported by the 59
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nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) against the chive gnat, Bradysia odoriphaga. J. Pest Sci. 86, 551–561. Maiensfisch, P., Huerlimann, H., Rindlisbacher, A., Gsell, L., Dettwiler, H., Haettenschwile, J., Sieger, E., Walti, M., 2001. The discovery of thiamethoxam: a second-generation neonicotinoid. Pest Manage. Sci. 57, 165–176. McVay, J.R., Gudauskas, R.T., Harper, J.D., 1977. Effect of Bacillus thuringiensis and chemical insecticides on Spodoptera littoralis (Lepidoptera: Noctuidae). J. Econ. Entomol. 77, 590–885. Misawa, T., Kuninaga, S., 2013. First report of white leaf rot on Chinese chives caused by Rhizoctonia solani AG-2-1. J. Gen. Plant Pathol. 79, 280–283. Nishimatsu, T., Jackson, J.J., 1998. Interaction of insecticides, entomopathogenic nematodes, and larvae of the western corn rootworm (Coleoptera: Chrysomelidae). J. Econ. Entomol. 91, 410–418. Peters, A., 1996. The natural host range of Steinernema and Heterorhabditis spp. and their impact on insect populations. Biocontrol Sci. Technol. 6, 389–402. Rodriguez, A.M., Peck, D.C., 2009. Synergies between biological and neonicotinoid insecticides for the curative control of the white grubs Amphimallon majale and Popillia japonica. Biol. Control 51, 169–180. SAS Institute, 2012. JMP Users Guide. Version 10.0. SAS Institute Inc., Cary, NC. Shapiro, D.I., McCoy, W.W., 2000. Virulence of entomopathogenic nematodes to Diaprepes abbreviatus (Coleoptera: Curculionidae) in the laboratory. J. Econ. Entomol. 93, 1090–1095. Shi, Y.C., Zheng, J.Q., Zhang, Y., 2001. Mushroom major pest survey briefing in suburbs. Plant Pro. Technol. Exten. 21, 18–19. Sirjani, F.O., Lewis, E.E., Kaya, H.K., 2009. Evaluation of entomopathogenic nematodes against the olive fruit fly, Bactrocera oleae (Diptera: Tephritidae). Biol. Control 48, 274–280. Smits, P.H., 1996. Post-application persistence of entomopathogenic nematodes. Biocontrol Sci. Technol. 6, 379–387. Sun, R.H., Li, A.H., 2007. Studies on combination effect of entomopathogenic nematode H06 and insecticide against Bradysia odoriphaga. Chin. J. Pesticide Sci. 9, 66–70. Sun, R.H., Li, A.H., Han, R.C., Cao, L., Liu, X.L., 2004. Factors affecting the control of Bradysia odoriphaga with entomopathogenic nematode Heterorhabditis indica LN2. Nat. Enem. Insects 26, 150–155. Susurluk, A., Ehlers, R.U., 2008. Field persistence of the entomopathogenic nematode Heterorhabditis bacteriophora in different crops. Biol. Control 53, 627–641. Thurston, G.S., Kaya, H.K., Gaugler, R., 1994. Characterizing the enhanced susceptibility of milky disease-infected scarabaeid grubs to entomopathogenic nematodes. Biol. Control 4, 67–73. Torres, J.B., Silva-Torres, C.S.A., Barros, R., 2003. Relative effects of the insecticide thiamethoxam on the predator Podisus nigrispinus and the tobacco whitefly Bemisia tabaci in nectaried and nectariless cotton. Pest Manage. Sci. 59, 315–323. Wang, W.J., Zhang, T., Chen, J.M., Chen, Z.D., 2011. Present situation and control technology of pesticide residue in Chinese chives. Shandong Agric. Sci. 10, 82–84. White, G.F., 1927. A method for obtaining infective nematode larvae from cultures. Science 66, 302–303. Wu, H.B., Xin, L., Gong, Q.T., Zhang, K.P., Cao, G.P., Sun, R.H., 2014a. Evaluation of the effects of infection by different entomopathogenic nematodes and chemical pesticides on Bradysia odoriphaga. Chin. J. Appl. Entomol. 51, 1060–1068. Wu, S.H., Youngman, R.R., Kok, L.T., Laub, C.A., Pfeiffer, D.G., 2014b. Interaction between entomopathogenic nematodes and entomopathogenic fungi applied to third instar southern masked chafer white grubs, Cyclocephala lurida, (Coleoptera: Scarabaeidae), under laboratory and greenhouse conditions. Biol. Control 76, 65–73. Wu, H.B., Fan, K., Xin, L., Cao, G.P., Sun, R.H., 2015a. Virulence and control effect of entomopathogenic nematodes against Agrotis ypsilon Rottemberg. J. Plant Prot. 42, 244–250. Wu, H.B., Gong, Q.T., Zhang, K.P., Zhang, X.P., Sun, R.H., 2015b. The efficacy of entomopathogenic nematodes and black sticky card to Bradysia odoriphaga. J. Plant Prot. 42, 632–638. Yan, X., Moens, M., Han, R., Chen, S., De Clercq, P., 2012. Effects of selected insecticides on osmotically treated entomopathogenic nematodes. J. Plant Dis. Protect 119, 152–158. Yang, J.K., Zhang, X.M., 1985. Notes on the fragrant onion gnats with descriptions of two new species of Bradysia odoriphaga (Diptera: Sciaridae). Acta Agri. Univ. Pekinen. 11, 153–156. Zhang, P., Chen, C.Y., Li, H., Liu, F., Mu, W., 2014a. Selective toxicity of seven neonicotinoid insecticides to Bradysia odoriphaga and Eisenia foetida. J. Plant Prot. 41, 79–86. Zhang, P., Liu, F., Mu, W., Wang, Q.H., Li, H., Chen, C.Y., 2014b. Life table study of the effects of sublethal concentrations of thiamethoxam on Bradysia odoriphaga Yang and Zhang. Pestic. Biochem. Physiol. 111, 31–37. Zhang, P., He, M., Zhao, Y.H., Ren, Y.P., Wei, Y., Mu, W., Liu, F., 2015a. Dissipation dynamics of clothianidin and its control efficacy against Bradysia odoriphaga Yang and Zhang in Chinese chive ecosystems. Pest Manage. Sci. 32, 617–622. Zhang, P., Zhao, Y.H., Han, J.K., Zhai, Y.B., Mu, W., Liu, F., 2015b. Control effects of thiamethoxam and clothianidin against Bradysia odoriphaga with different application methods. J. Plant Prot. 42, 645–650. Zhang, P., He, M., Wei, Y., Zhao, Y.H., Ren, Y.P., Mu, W., Liu, F., 2016. Comparative soil distribution and dissipation of phoxim and thiamethoxam and their efficacy in controlling Bradysia odoriphaga Yang and Zhang in Chinese chive ecosystems. Crop Prot. 9, 1–8.
Special Fund for Agro-scientific Research in the Public Interest (201303027) and the Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences (2015YQN30). References Abbott, W.S., 1925. A method for computing the effectiveness of an insecticide. J. Econ. Entomol. 18, 265–267. Cloyd, R.A., Dickinson, A., 2006. Effect of Bacillus thuringiensis subsp. israelensis and neonicotinoid insecticides on the fungus gnat Bradysia sp nr. coprophila (Lintner) (Diptera: Sciaridae). Pest Manage. Sci. 62, 171–177. David, I.S., Dawn, H.G., Simon, J.P., Jane, P.F., 2006. Application technology and environmental considerations for use of entomopathogenic nematodes in biological control. Biol. Control 5, 124–133. Demarta, L., Hibbard, B.E., Bohn, M.O., Hiltpold, I., 2014. The role of root architecture in foraging behavior of entomopathogenic nematodes. J. Invertebr. Pathol. 122, 32–39. Dowds, B.C.A., Peters, A., 2002. Virulence mechanisms. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Oxon, pp. 79–98. Duncan, L.W., McCoy, C.W., 1996. Vertical distribution in soil, persistence, and efficacy against citrus root weevil (Coleoptera: Curculionidae) of two species of entomogenous nematodes (Rhabditida: Steinernematidae; Heterorhabditidae). Environ. Entomol. 25, 174–178. Ehlers, R.U., 1996. Current and future use of nematodes in biocontrol: practice and commercial aspects in regard to regulatory policies. Biocontrol Sci. Technol. 6, 303–316. Feng, H.Q., Zheng, F.Q., 1987. Studies of the occurrence and control of Bradysia odoriphaga Yang et Zhang. J. Shandong Agric. Univ. 18, 71–80. Finney, D.J., 1964. Probit Analysis. Cambridge University Press, London. Gaugler, R., Bilgrami, A.L. (Eds.), 2004. Nematode Behaviour. CABI Publishing, Cambridge. Georgis, R., Gaugler, R., 1991. Predictability in biological control using entomopathogenic nematodes. J. Econ. Entomol. 84, 713–720. Glazer, I., 2002. Survival biology. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Wallingford, UK, pp. 169–187. Harris, M.A., Oetting, R.D., Gardner, W.A., 1995. Use of entomopathogenic nematodes and a new monitoring technique for control of fungus gnats, Bradysia coprophila (Diptera: Sciaridae), in floriculture. Biol. Control 5, 412–418. Imahori, Y., Suzuki, Y., Uemura, K., Kishioka, I., Fujiwara, H., Ueda, Y., Chachin, K., 2004. Physiological and quality responses of Chinese chive leaves to low oxygen atmospheres. Postharvest Biol. Technol. 31, 295–303. Jacques, R.P., Morris, O.N., 1981. Compatibility of pathogens with other methods of pest control and crops protection. In: Burges, H.D. (Ed.), Microbial Control of Pests and Plant Disease 1970–1980. Academic Press, London, pp. 695–715. Jagdale, G.B., Casey, M.L., Grewal, P.S., Lindquist, P.K., 2004. Application rate and timing, potting medium and host plant effects on the efficacy of Steinernema feltiae against the fungus gnat, Bradysia coprophila, in floriculture. Biol. Control 29, 296–305. Jagdale, G.B., Casey, M.L., Cañas, L., Grewal, P.S., 2007. Effect of entomopathogenic nematodes species, split application and potting medium on the control of the fungus gnat, Bradysia difformis (Diptera: Sciaridae), in the greenhouse at alternation cold and warm temperatures. Biol. Control 43, 23–30. Kaya, H.K., 2002. Natural enemies and other antagonists. In: Gaugler, R. (Ed.), Entomopathogenic Nematology. CABI Publishing, Wallingford, UK, pp. 189–203. Kaya, H.K., Koppenhöfer, A.M., 1996. Effects of microbial and other antagonistic organism and competition on entomopathogenic nematodes. Biocontrol Sci. Technol. 6, 357–371. Kaya, H.K., Stock, S.P., 1997. Techniques in insect nematology. In: Lacey, L.A. (Ed.), Manual Techniques in Insect Pathology. Academic Press, San Diego, CA. Klein, M.G., 1993. Biological control of scarabs with entomopathogenic nematodes. In: Bedding, R., Akhurst, R., Kaya, H. (Eds.), Nematodes and the Biological Control of Insect Pests. CSIRO, East Melbourne, Australia, pp. 49–58. Koppenhöfer, A.M., Brown, I.M., Gaugler, R., Grewal, P.S., Kaya, H.K., Klein, M.G., 2000. Synergism of entomopathogenic nematodes and imidacloprid against white grubs: greenhouse and field evaluation. Biol. Control 19, 245–251. Koppenhöfer, A.M., Cowles, R.S., Cowles, E.A., Fuzy, E.M., Kaya, H.K., 2003. Effect of neonicotinoid synergists on entomopathogenic nematode fitness. Entomol. Exp. Appl. 106, 7–18. Lacey, L.A., Unruh, T.R., 1998. Entomopathogenic nematodes for control of coding moth: effect of nematode species, dosage, temperature and humidity under laboratory conditions. Biol. Control 13, 190–197. Langford, E.A., Nielsen, U.N., Johnson, S.N., Riegler, M., 2014. Susceptibility of queensland fruit fly, Bactrocera tryoni, (Froggatt) (Diptera: Tephritidae), to entomopathogenic nematodes. Biol. Control 69, 34–39. Li, H., Zhu, F., Zhou, X.M., Li, N., 2007. Bionomics and control of the root maggot, Bradysia odoriphaga, infested on the watermelon. Entomol. Knowl. 44, 834–836. Li, W.X., Yang, Y.T., Xie, W., Wu, Q.J., Xu, B.Y., Wang, S.L., Zhu, X., Wang, S.J., Zhang, Y.J., 2015. Effects of temperature on the age-stage, two-sex life table of Bradysia odoriphaga (Diptera: Sciaridae). J. Econ. Entomol. 108, 126–134. Ma, J., Chen, S.L., Moens, M., Han, R., De Clercq, P., 2013. Efficacy of entomopathogenic
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