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Can resistance in Bemisia tabaci (Homoptera: Aleyrodidae) be overcome with mixtures of neonicotinoids and insect growth regulators?
Crop Protection 44 (2013) 135e141
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Can resistance in Bemisia tabaci (Homoptera: Aleyrodidae) be overcome with mixtures of neonicotinoids and insect growth regulators? Muhammad Basit a, *, Shafqat Saeed a, Mushtaq Ahmad Saleem a, Ali H. Sayyed b a b
Department of Entomology, University College of Agriculture, Bahauddin Zakariya University Multan, Pakistan Institute of Biotehnology, Bahauddin Zakariya University Multan, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 March 2012 Received in revised form 17 October 2012 Accepted 20 October 2012
Tobacco whitefly, Bemisia tabaci is an important polyphagous insect pest which has developed resistance to various insecticides worldwide. Mixtures of insecticides with different modes of action may delay the onset of resistance. Bioassays were performed to investigate the effects of various mixtures of neonicotinoid and insect growth regulator (IGR) insecticides against a susceptible and a resistant strain. The results of the study showed that potentiation ratio (PR) of all neonicotinoids þ buprofezin or pyriproxyfen mixtures at 1:1, 10:1 and 20:1 ratios was greater than 1 suggesting synergistic interactions between insecticides. Maximum potentiation occurred at the 1:1 ratio (PR ¼ 1.69e7.56). The PRs for mixture of acetamiprid, thiamethoxam, thiacloprid or nitenpyram with buprofezin or pyriproxyfen at 1:10 and 1:20 ratios were less than 1 indicating antagonistic interactions. Addition of synergists, S, S, S, tri-butyl phosphorotrithioate (DEF) or piperonyl butoxide (PBO) in the insecticide solutions largely overcame the resistance to all tested neonicotinoids, indicating that the resistance was associated with esterases or mono-oxygenases, respectively. Likewise, addition of both DEF and PBO in mixture with neonicotinoids and IGRs also suggested a similar mechanism of resistance in B. tabaci to the tested insecticide groups. The mechanism of synergism between neonicotinoids and IGRs is unclear. Implications of using mixtures to counteract pesticide resistance are discussed. Mixtures of neonicotinoids with buprofezin or pyriproxyfen at a 1:1 ratio could be used to restore the efficacy of these neonicotinoids against B. tabaci. Ó 2012 Published by Elsevier Ltd.
Keywords: Bemisia tabaci Neonicotinoids IGRs Mixtures DEF PBO
1. Introduction The tobacco whitefly, Bemisia tabaci (Gennadius) is an economic agricultural pest world wide (Byrne et al., 1990). It is a highly adaptable and polyphagous insect pest that feeds on more than 700 plant species from 86 botanical families (Greathead, 1986). It directly causes injuries to crop plants by sucking phloem sap and indirectly by transmitting more than 100 plant viruses (Horowitz et al., 2003; Mugiira et al., 2008). The estimated loss caused by the whitefly is 3e4 millions of U.S. dollars each year in British Columbia, Canada (Moreau and Isman, 2012). In Pakistan, it caused the economic loss of US$5 billion from 1992 to 1997 due to transmission of cotton leaf curl virus disease (Briddon and Markham, 2000; Briddon, 2003; Basit et al., 2012). It has attained a major pest status because of its capabilities to replace existing biotypes, invading new geographical ranges and rapidly developing resistance to new pesticides including neonicotinoids and insect growth * Corresponding author. Tel.: þ92 0345 4602058; fax: þ92 619210068. E-mail address: [email protected] (M. Basit). 0261-2194/$ e see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.cropro.2012.10.021
regulators (IGRs) (Byrne and Bellows, 1991; Horowitz and Ishaaya, 1996; Perring, 2001). The usual response to the insecticide resistance problem is to increase the dosage and/or application frequencies and in some cases to substitute with new products. However, this has become expensive for both growers and industry. In the United States, cost of pesticide resistance has been estimated to be approximately $ 1.5 billion annually (Pimentel, 2005). Furthermore, availability of new products is limited because of the rising standards of environmental and toxicological safety. The most effective way to counteract the resistance problem is to reduce the selection pressure before resistance (Georghiou and Taylor, 1976; Ware, 2000) and to use resistance management strategies, which include use of insecticides in sequences, rotations and mixtures of two or more insecticides having different modes of action (Georghiou et al., 1983). Sequential use of insecticides has been discouraged due to the earlier development of resistance but the use of insecticides in rotations and mixtures merits consideration. Two insecticides with different modes of action in a mixture may synergize each other and increase the efficacy consequently
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reducing the input cost (Martin et al., 2003; Attique et al., 2006). Synergism theory is based on the ability of one molecule to interfere with the metabolic detoxification of another molecule (Corbett, 1974). Synergism between organophosphates or carbamates and pyrethroids has already been reported in a number of insect pests (Martin et al., 2003; Corbel et al., 2006; Attique et al., 2006). However, there is no documented report of mixing neonicotinoids with IGRs, which are being widely used in Pakistan and elsewhere for the control of B. tabaci. Neonicotinoids act agonistically on postsynaptic nicotinic acetylcholine receptors (Elbert et al., 2007) and have been shown to have no cross-resistance to IGRs (Basit et al., 2012). The aim of the present study was to determine the effect of mixing a neonicotinoid (imidacloprid, acetamiprid, thiamethoxam, thiacloprid or nitenpyram) with an IGR (buprofezin or pyriproxyfen) against the laboratory susceptible (Lab-PK) and acetamiprid selected population (Aceta-SEL) of B. tabaci. The AcetaSEL has been shown to have high level of resistance to neonicotinoids and cross-resistance to pyrethroid (bifenthrin) and endosulfan (Basit et al., 2011). To evaluate the mixtures of neonicotinoid insecticides and IGRs, a standard bioassay procedure was used to assess whether the mixture could yield additive, antagonistic or synergistic effects in the presence or absence of cytochrome P450 monooxigenase and an esterase inhibitor, pipronyl butoxide (PBO), and S, S, S, tri-butyl phosphorotrithioate (DEF), an esterase-specific inhibitor. Such studies could provide important information for devising resistance management strategies and so delay the development of resistance in B. tabaci. 2. Materials and methods 2.1. Insects The resistant strain of B. tabaci was developed in the laboratory of Bahaudin Zakarya University (BZU), Multan, Pakistan from a field collected population by exposing to different selection pressures of the neonicotinoid insecticide, acetamiprid. The susceptible population was developed in 2009 by single pair crosses and was held in the laboratory for more than two years before the start of this study. Both populations were reared on cotton plants (Gossypium hirsutum L. var CIM-496) in the entomological laboratory of BZU, under photoperiod of 16 h at 26 2 C. 2.2. Insecticides and inhibitors
fifteen whiteflies were briefly sedated with CO2 before placing them on the lower side of cotton leaves in each clip cage (replicate) and thus a total of 30e40 adults were tested per concentration. The bioassays were kept at a temperature of 26 2 C, 65% relative humidity and 16:8 (light: dark) photoperiod. Mortality was assessed after 48 h. 2.4. Effect of inhibitors on insecticide toxicity To evaluate the effect of inhibitors on the toxicity of insecticides alone and in mixture, PBO and DEF were used. Stock solution (1000 ppm) of PBO and DEF were prepared in acetone (analytical reagent grade, Fisher Scientific, Loughborogh, UK). To determine the dose at which mortality of B. tabaci was zero, a series of concentrations of DEF and PBO were tested. To test the effect of PBO and DEF on the toxicity of insecticides alone and in mixture, 100 ppm (zero % mortality) was added in each concentration tested. Mortality data were recorded after 48 h. The synergistic ratio was calculated by dividing the LC50 of the population treated with the insecticide alone by the LC50 of the population treated with insecticide plus synergist. 2.5. Evaluation of mixture Each insecticide mixture could give greater (synergism) or less (antagonism) than the expected additive effect (summation). To determine which of these possibilities resulted, mixture of two insecticides were tested at ratios of 1:1, 1:10, 10:1, 1:20 and 20:1 using serial dilutions. Synergism or antagonism can be assessed using various methods and we used the method described by Hoel (1987). Potency ratios were calculated by dividing the estimated lethal concentration (LC) values of the mixtures, calculated for joint similar action by the experimental LC values observed in the bioassay. If PR ¼ 1, the mixture was regarded ashaving additive action; if PR was <1, it showed an antagonistic action and if PR was >1, it exhibited a potentiating action. The estimated LC value of a mixture of A and B was computed as follows:
Estimated LCðA þ BÞ ¼
1 LCðAÞ þ mB=LCðBÞ
where mA and mB represent the proportion of A and B in the mixture; mA þ mB ¼ 1.
Commercial formulations used in the bioassays were: buprofezin 250 g a.i. kg1(FMC, Pakistan), pyriproxyfen 108 g a.i. kg1(Kanzo AG, Pakistan), acetamiprid 200 g a.i. l1 (AcelanÒ; FMC, Pakistan), imidacloprid 200 g a.i. l1 (ConfidorÒ, Bayer Crop Sciences, France), thiamethoxam 250 g a.i. kg1 (ActaraÒ, Syngenta, Berkshire UK), thiacloprid 480 g a.i. l1 (TalentÒ, Kanzo AG, Pakistan), nitenpyram 100 g a.i. l1 (PyramidÒ, Kanzo AG, Pakistan), Piperonyl butoxide (PBO, Sigma Ltd, UK), S, S, S, tri-butylphosphorotrithioate (DEF, Sigma, Ltd).
2.6. Data analysis
2.3. Bioassays
3. Results
The bioassays were carried out on whole cotton plants at two true leaf stages (20e22 days old) as described previously (Basit et al., 2011). Both leaves were washed, dried and dipped in the freshly prepared solutions for five to 10 s with slight agitation. Excessive liquid was allowed to drain off and the leaves were air dried for 1 h before confining the adults’ B. tabaci in clip cages. There was one clip cage on each cotton plant and thus each plant was used as a single replicate. Each treatment (concentration) was replicated 3e4 times, including distilled water controls. Ten to
3.1. Toxicity of tested insecticides alone and in combination to the Lab-PK and Aceta-SEL population
Mortality data were analyzed and LC50 values and their 95% fiducial limits were estimated using the POLO-PC computer based software (POLO, LeOra software, Menlo Park, California). Because of the inherent variability of bioassays, pairwise comparisons of LC50 values were done at the 5% significance level (where individual 95% FLs for two treatments do not overlap) (Litchfield and Wilcoxon, 1949).
The toxicities of acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid were similar to one another and to the Lab-PK (p < 0.05, overlapping 95% fiducial limits). Similarly, LC50 values of buprofezin and pyriproxyfen were also similar to each other for the Lab-PK (Table 1). However, mixtures of pyriproxyfen or buprofezin with acetamiprid, imidacloprid, thiamethoxam,
M. Basit et al. / Crop Protection 44 (2013) 135e141
137
Table 1 Toxicity of acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid alone and in combination with buprofezin, pyriproxyfen, DEF and PBO to the Lab-PK population of Bemisia tabaci. Ratio
LC50 (95% FL) (mg AI ml1)
LC50 (95% FL) (mg AI ml1) þDEF
LC50 (95% FL) (mg AI ml1) þPBO
PR
1:00 1:00 1:1 1:10 10:1 1:20 20:1
0.70 24.1 4.63 22.3 5.97 30.1 7.01
(0.30e1.22) (19.5e31.1) (2.58e6.45) (13.3e49.3) (3.01e9.99) (25.2e36.1) (3.59e10.7)
0.40 e 0.97 1.12 1.07 1.11 1.08
(0.35e0.63)
(0.12e0.45)
(0.60e1.45) (0.61e1.81) (0.62e1.65) (0.58e1.84) (0.56e1.75)
0.29 e 0.65 0.70 0.61 0.75 0.66
(0.28e1.12) (0.32e1.18) (0.25e1.07) (0.36e1.23) (0.29e1.12)
e e 0.29 0.26 0.12 0.30 0.10
Acetamiprid Acetamiprid þ buprofezin
1:00 1:1 1:10 10:1 1:20 20:1
2.00 5.12 24.7 7.46 25.5 8.26
(0.83e3.20) (2.00e8.25) (20.6e31.3) (3.92e11.4) (20.9e32.2) (4.62e12.5)
1.58 1.48 1.65 1.54 1.60 1.56
(0.98e2.45) (0.87e2.31) (0.99e2.53) (0.91e2.39) (0.94e2.50) (0.88e2.47)
0.76 0.73 0.87 0.78 0.89 0.69
(0.44e1.14) (0.34e1.23) (0.45e1.40) (0.38e1.29) (0.45e1.46) (0.29e1.18)
e 0.72 0.48 0.29 0.62 0.25
Thiamethoxam Thiamethoxam þ buprofezin
1:00 1:1 1:10 10:1 1:20 20:1
1.45 (0.90e2.18) 4.52 (1.66e7.36) 20.0 (15.5e27.0) 8.26 (4.62e12.5) 21.6 (17.1e28.4) 8.35 (4.12e12.9)
1.29 (0.74e2.07) 1.37 (0.83e1.09) 1.47 (0.90e2.21) 1.41 (0.88e2.12) 1.48 (0.88e2.29) 1.36 (0.85e2.04)
0.87 0.87 0.91 0.84 0.88 0.83
(0.33e1.63) (0.45e1.40) (0.50e1.45) (0.47e1.29) (0.51e1.35) (0.46e1.30)
e 0.60 0.50 0.19 0.54 0.18
Nitenpyram Nitenpyram þ buprofezin
1:00 1:1 1:10 10:1 1:20 20:1
1.17 4.27 20.7 7.01 22.6 10.4
1.10 1.01 1.11 1.09 1.13 1.05
0.88 0.82 0.82 0.83 0.88 0.88
(0.56e1.30) (0.41e1.35) (0.41e1.35) (0.39e1.41) (0.44e1.46) (0.43e1.47)
e 0.52 0.41 0.18 0.55 0.11
Thiacloprid Thiacloprid þ buprofezin
1:00 1:1 1:10 10:1 1:20 20:1
0.94 (0.54e1.47) 3.92 (1.12e6.75) 19.1 (14.5e26.6) 5.90 (2.36e9.58) 20.7 (16.1e28.1) 7.61 (4.21e11.4)
0.81 (0.41e1.33) 1.03 (0.56e1.65) 1.08 (0.60e1.70) 1.03 (0.57e1.65) 1.11 (0.61e1.78) 0.99 (0.53e1.58)
0.68 (0.32e1.15) 0.73 (0.34e1.23) 0.84 (0.41e1.41) 0.75 (0.33e1.29) 0.77 (0.39e1.24) 0.80 (0.38e1.35)
e 0.46 0.39 0.17 0.53 0.12
Pyriproxyfen Imidacloprid þ pyriproxyfen
1:00 1:1 1:10 10:1 1:20 20:1
20.0 3.79 20.0 6.21 23.8 7.61
e 1.26 1.47 1.39 1.42 1.34
e 0.50 0.61 0.64 0.69 0.61
(0.10e1.07) (0.26e1.03) (0.31e1.04) (0.35e1.09) (0.29e1.00)
e 0.35 0.28 0.12 0.36 0.09
Acetamiprid þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
4.93 (2.23e7.61) 25.5 (20.9e32.2) 8.47 (5.33e12.1) 26.3 (21.7e33.1) 9.51 (6.29e13.4)
1.55 (1.05e2.31) 2.75 (1.20e2.58) 1.68 (1.16e2.43) 2.62 (1.12e2.35) 1.50 (1.01e2.32)
0.68 (0.37e1.06) 0.78 (0.41e1.24) 0.73 (0.41e1.11) 0.77 (0.43e1.19) 0.81 (0.50e1.20)
0.73 0.43 0.25 0.53 0.22
Thiamethoxam þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
4.89 24.1 7.15 24.9 8.04
(1.67e8.13) (19.5e31.1) (4.47e10.0) (20.2e32.1) (5.27e11.1)
1.39 1.50 1.28 3.61 1.34
(0.95e2.03) (1.03e2.17) (0.86e1.88) (1.14e2.29) (0.91e1.93)
0.69 0.87 0.74 0.94 0.77
(0.36e1.09) (0.52e1.30) (0.41e1.15) (0.59e1.40) (0.43e1.19)
0.55 0.38 0.22 0.50 0.18
Nitenpyram þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
4.09 21.1 7.98 23.5 8.70
(1.68e6.48) (16.5e28.4) (4.67e11.7) (18.8e30.9) (5.51e12.4)
1.04 1.11 1.08 1.10 1.07
(0.64e1.58) (0.67e1.73) (0.67e1.64) (0.72e1.61) (0.64e1.64)
0.77 0.84 0.82 0.88 0.79
(0.43e1.19) (0.48e1.29) (0.48e1.24) (0.51e1.35) (0.45e1.20)
0.55 0.38 0.16 0.48 0.14
Thiacloprid þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
3.89 (1.42e6.35) 22.9 (18.5e29.7) 6.20 (3.21e9.30) 24.1 (19.5e31.2) 8.89 (5.59e12.8)
0.81 (0.46e1.24) 0.84 (0.49e1.29) 0.78 (0.44e1.19) 0.94 (0.58e1.40) 0.79 (0.44e1.23)
0.78 0.30 0.16 0.42 0.11
Insecticides tested Lab-PK Imidacloprid Buprofezin Imidacloprid þ buprofezin
(0.73e1.76) (1.45e7.09) (16.1e28.1) (3.50e10.7) (18.1e29.5) (7.18e14.5)
(15.5e27.0) (1.06e6.57) (15.5e27.0) (2.66e9.93) (19.2e31.0) (4.21e11.4)
nitenpyram or thiacloprid were more toxic to the Lab-PK at the 1:1, 1:10 and 1:20 ratios than the mixtures at 10:1 and 20:1 ratios (Table 1). The mixture of imidacloprid þ buprofezin exhibited significantly higher toxicity to the selected strain at the 1:1 ratio than the same mixture at the 1:10, 10:1, 1:20 and 20:1 ratios (p < 0.05,
(0.84e1.01) (0.63e1.50) (0.63e1.77) (0.71e1.59) (0.73e1.66) (0.66e1.55)
(0.50e2.12) (0.68e2.35) (0.62e2.26) (0.66e2.27) (0.56e2.21)
0.96 (0.58e1.45) 1.10 (0.72e1.62) 1.06 (0.68e1.57) 1.14 (0.75e1.68) 1.06 (0.68e1.55)
overlapping 95% fiducial limits) (Table 1). Likewise the same mixture gave higher toxicity at 10:1 and 201 ratios compared with 1:10 and 1:20 ratios. Mixture of acetamiprid, thiamethoxam, thiacloprid or nitenpyram with buprofezin also displayed similar results as that of the imidacloprid with buprofezin (Table 2). Similarly, mixing of imidacloprid with pyriproxyfen at a 1:1 ratio
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M. Basit et al. / Crop Protection 44 (2013) 135e141
Table 2 Toxicity of acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid alone and in combination with buprofezin, pyriproxyfen, DEF and PBO against Aceta-SEL population of Bemisia tabaci. Ratio
LC50 (95% FL) (mg AI ml1)
LC50 (95% FL) (mg AI ml1) þDEF
LC50 (95% FL) (mg AI ml1) þPBO
PR
1:00 1:00 1:1 1:10 10:1 1:20
41.3 220 14.8 158 40.3 191
(14.8e69.4) (169e301) (6.27e20.1) (90.7e155) (36.7e82.7) (111e252)
21.2 e 4.15 16.8 9.93 17.3
(11.1e31.3)
(1.49e6.79)
(2.80e5.87) (13.0e22.6) (6.40e14.4) (13.7e22.6)
4.18 e 1.16 6.10 3.30 7.63
(0.21e2.19) (4.65e8.22) (1.85e5.01) (5.97e10.5)
e e 4.67 0.99 1.11 0.95
Acetamiprid Acetamiprid þ buprofezin
20:1 1:00 1:1 1:10 10:1
41.8 237 135 680 230
(31.1e87.4) (150e506) (80.2e145) (550e1085) (180e403)
9.90 28.1 12.1 49.7 20.3
(6.29e14.5) (11.4e45.7) (5.94e18.6) (39.3e66.3) (13.2e29.5)
3.70 4.66 3.86 10.4 5.67
(2.25e5.49) (1.91e7.34) (1.56e6.21) (7.14e15.5) (2.66e9.21)
1.04 e 1.69 0.30 1.04
Thiamethoxam Thiamethoxam þ buprofezin
1:20 20:1 1:00 1:1 1:10 10:1
704 (673e1179) 235 (290e506) 375 (235e938) 100 (63.4e142) 650 (469e1085) 348 (290e465)
52.5 (41.3e71.3) 22.6 (15.6e32.0) 39.0 (24.9e51.5) 4.39 (1.76e7.15) 40.7 (30.9e57.1) 10.5 (3.75e17.5)
11.2 6.07 5.05 2.10 8.95 5.03
(7.72e16.7) (3.13e9.49) (1.85e8.32) (0.17e1.66) (6.64e12.8) (3.29e7.85)
0.34 1.03 e 2.77 0.35 1.01
Nitenpyram Nitenpyram þ buprofezin
1:20 20:1 1:00 1:1 1:10
748 355 163 53.7 236
(553e1093) (246e530) (121e209) (43.4e71.6) (192e304)
48.7 10.1 27.4 13.1 31.5
(38.9e63.8) (3.53e16.8) (18.1e37.4) (5.54e21.4) (23.4e44.0)
9.45 5.25 4.49 1.01 5.59
(7.06e13.5) (3.55e7.85) (2.75e6.10) (0.35e1.92) (3.65e8.68)
0.26 1.02 e 3.53 0.90
Thiacloprid Thiacloprid þ buprofezin
10:1 1:20 20:1 1:00 1:1
100 270 107 165 57.5
(72.8e163) (226e384) (73.2e190) (130e211) (46.8e75.7)
16.6 43.4 16.9 26.6 10.2
(10.2e24.0) (33.0e60.9) (10.7e24.0) (16.9e37.0) (7.99e14.0)
2.54 7.39 2.60 4.11 1.88
(1.52e3.91) (5.32e10.4) (1.52e4.07) (2.31e5.68) (0.81e3.35)
1.67 0.80 1.54 e 3.30
Pyriproxyfen Imidacloprid þ pyriproxyfen
1:10 10:1 1:20 20:1 1:00
233 90.4 300 96.8 200
(190e422) (77.2e157) (200e459) (79.4e180) (155e277)
30.0 14.6 34.1 15.2 e
(19.7e48.8) (8.18e21.6) (25.5e47.5) (8.86e22.4)
5.58 2.83 7.25 2.24 e
(4.02e7.60) (1.36e5.16) (5.13e10.6) (0.94e4.15)
0.91 1.87 0.72 1.72 e
Acetamiprid þ pyriproxyfen
1:1 1:10 10:1 1:20
9.07 150 39.2 180
(3.8e14.4) (90.4e165) (23.7e85.1) (100e187)
2.48 13.1 8.66 18.2
(1.06e3.97) (9.72e17.8) (5.09e12.9) (14.6e23.4)
0.89 8.18 5.11 12.1
(0.43e1.46) (5.27e15.0) (3.62e7.28) (8.69e17.0)
7.56 0.97 1.13 0.93
Thiamethoxam þ pyriproxyfen
20:1 1:1 1:10 10:1 1:20 20:1 1:1 1:10 10:1 1:20 20:1
43.0 (24.6e86.1) 107 (81.9e145) 495 (392e790) 221 (170e380) 640 (469e1068) 228 (169e385) 93.2 (62.0e138) 513 (381e636) 340 (200e350) 753 (400e60) 380 (227e380)
9.04 (5.55e13.2) 14.1 (8.00e20.1) 69.0 (53.6e94.8) 35.4 (28.0e46.2) 104 (84.0e143) 47.6 (37.7e61.4) 9.46 (6.58e13.4) 51.8 (40.8e67.8) 37.1 (26.6e51.7) 74.7 (60.46.5) 40.8 (29.7e57.2)
5.25 2.23 10.7 6.19 16.3 7.65 1.53 6.90 5.22 10.0 5.56
(3.77e7.37) (0.87e4.17) (7.17e13.8) (3.51e8.91) (12.3e22.4) (5.20e10.3) (0.44e3.15) (4.52e9.52) (2.99e7.56) (7.20e13.8) (3.30e7.98)
1.01 2.04 0.40 1.05 0.31 1.04 2.85 0.40 1.02 0.27 1.04
Nitenpyram þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
51.2 217 91.6 249 102
(41.6e62.4) (165 303) (65.2e134) (157e379) (70.3e161)
12.4 36.0 15.7 40.2 17.1
(6.73e18.2) (27.3e50.2) (9.53e22.8) (31.7e53.2) (11.1e24.1)
2.06 6.11 2.54 6.88 2.86
(1.06e3.05) (4.43e8.74) (1.45e3.67) (5.42e9.46) (1.79e4.01)
3.54 0.90 1.81 0.79 1.61
Thiacloprid þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
44.6 200 88.7 217 94.9
(35.9e59.8) (129.3e308) (61.1e142) (141e315) (60.9e153)
11.7 (4.95e18.3) 31.8 (20.2e47.7) 14.8 (6.68e23.0) 33.1 (22.1e48.0) 14.7 (6.14e23.3)
1.76 4.62 2.14 5.87 2.33
(0.80e2.70) (3.23e6.51) (0.81e3.48) (4.40e8.02) (0.76e4.94)
4.08 0.98 1.89 0.91 1.75
Insecticides tested Aceta-SEL Imidacloprid Buprofezin Imidacloprid þ buprofezin
significantly increased the effectiveness of the mixture compared with the mixture of these insecticides at 1:10, 10:1, 1:20 and 20:1 ratios. But the toxicity of that mixture at 1:10, 10:1, 1:20 and 20:1 was not significantly higher than the toxicity of imidacloprid alone (Table 2). However, the toxicity of that mixture at 10:1 and 20:1 ratios was at par with the toxicity of imidacloprid alone. Likewise,
mixture of acetamiprid, thiamethoxam, nitepyram or thiacloprid with pyriproxyfen at a 1:1 ratio displayed higher toxicity than the toxicity of these insecticides alone (Table 2). However, the efficacy of the mixture of acetamiprid, thiamethoxam, nitenpyram and thiacloprid with pyriproxyfen at 1:10, 10:1, 1:20 and 20:1 ratios was not significantly higher than the efficacy of these insecticides alone.
M. Basit et al. / Crop Protection 44 (2013) 135e141
When synergists (DEF and PBO) were added in the mixture, the LC50 values of buprofezin þ imidacloprid, acetamiprid, thiamethoxam, thiacloprid or nitepyram at all ratios were significantly reduced (Table 2). Likewise, mixture of pyriproxyfen with neonicotinoids þ PBO or DEF displayed similar results to those of the mixture of buprofezin with neonicotinoids at 1:1, 1:10, 10:1, 1:20 and 20:1 ratios (Table 2). To assess the synergism or antagonism between two insecticides, the potentiation ratio was calculated for each mixture. The PRs for neonicotinoids þ buprofezin at 1:1, 10:1 and 20:1 ratios against the selected strain were significantly greater than 1, indicating the synergistic interactions between the insecticides. Maximum synergism between neonicotinoids and buprofezin occurred at 1:1 ratios (Table 2). Nevertheless, PRs for neonicotinoids þ buprofezin were less than 1, indicating antagonistic interactions between the insecticides (Table 2). Similarly, the PR values for the mixture of neonicotinoids (imidacloprid, acetamiprid nitenpyram and thiacloprid) and pyriproxyfen at 1:1, 10:1 and 20:1 ratios were greater than 1, indicating synergistic interactions between the insecticides (Table 2). Synergism at 10:1 and 20:1 ratios was small compared with the synergism at 1:1 ratios. In contrast, the PR values for the mixture of neonicotinoids and pyriproxyfen at 1:10 and 1:20 ratios were less than 1, indicating antagonistic interactions between insecticides. 3.2. Effect of synergists on the tested insecticides The Aceta-SEL population was 119-, 59-, 452-, 139- and 175-fold resistant to acetmiprid, imidaloprid, thiamethoxam, nitenpyram and thiacloprid, respectively compared with Lab-PK (Table 3). The PBO largely overcame the resistance to acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid exhibiting 6-,
139
14-, 6-, 5-, and 6-fold resistance ratios, with synergistic ratios of 51, 10, 74, 36, and 40 for acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid, respectively. Similarly, DEF also synergized the tested insecticides with synergic ratios of 8, 2 10, 6 and 6 for acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid, respectively (Table 2). Likewise, PBO and DEF also synergized the mixtures of neonicotinoids and IGRs (Table 2). 4. Discussion Due to the development of resistance, safe and cost effective insecticides are being depleted rapidly; control of the resistant populations and strategies to slow down insecticide resistance evolution is based on the optimum use of existing compounds. In addition to sequence and rotation, use of mixtures of various compounds each acting on different sites has been adopted (Martin et al., 2000) Theoretically, under certain conditions, insecticide mixtures can delay the onset of resistance development more effectively than rotation of insecticides if resistance to each compound is independent and rare (Curtis, 1985). In West Africa, for example, mixture of organophosphate and pyrethroid delayed the development of resistance in cotton bollworm, Helicoverpa armigera (Hubner) for more than 20 years (Martin et al., 2003). In the present study, evaluation of insecticidal activity of neonicotinoids (imidacloprid, acetamiprid, thiamethoxam, nitenpyram and thiacloprid) with IGRs (buprofezin and pyriproxyfen) at different ratios was carried out. Bioassay results showed that the mixtures of neonicotinoids with IGRs (buprofezin and pyriproxifen) were more effective against the Aceta-SEL at 1:1 ratios than mixtures of these insecticides at 1:10, 10:1, 1:20 and 20:1 ratios (Table 2). But mixtures of these insecticides did not exhibit potentiation against Lab-PK. Synergism and antagonism were
Table 3 Toxicity of imidacloprid, acetamiprid, thiamethoxam, nitenpyram and thiacloprid with and without PBO and DEF to Lab-PK and Aceta-SEL strain of Bemisia tabaci. Strain
Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL a b
Treatment
Acetamiprid Imidacloprid Thiamethoxam Nitenpyram Thiacloprid Acetamiprid þ DEF Imidacloprid þ DEF Thiamethoxam þ DEF Nitenpyram þ DEF Thiacloprid þ DEF Acetamiprid þ PBO Imidacloprid þ PBO Thiamethoxam þ PBO Nitenpyram þ PBO Thiacloprid þ PBO Acetamiprid Imidacloprid Thiamethoxam Nitenpyram Thiacloprid Acetamiprid þ DEF Imidacloprid þ DEF Thiamethoxam þ DEF Nitenpyram þ DEF Thiacloprid þ DEF Acetamiprid þ PBO Imidacloprid þ PBO Thiamethoxam þ PBO Nitenpyram þ PBO Thiacloprid þ PBO
LC50 (95% FL) (mg a.i. ml1) 2.00(0.83e3.20) 0.70 (0.30e1.22) 1.45 (0.90e2.18) 1.17 (0.73e1.76) 0.94 (0.54e1.47) 1.58 (0.98e2.45) 0.40 (0.35e0.63) 1.29 (0.74e2.07) 1.10 (0.84e1.01) 0.81 (0.41e1.33) 0.76 (0.44e1.14) 0.29 (0.12e0.45) 0.87 (0.33e1.63) 0.88 (0.56e1.30) 0.68 (0.32e1.15) 237.0 (129e506) 41.3 (14.0e69.4) 375.2 (235e938) 162.6 (110e209) 164.8 (130e211) 28.2 (11.4e45.7) 21.2 (11.1e31.3) 39.0 (24.9e51.5) 27.4 (18.1e37.4) 26.6 (16.9e37.0) 4.66 (1.91e7.34) 4.18 (1.49e6.79) 5.05 (1.85e8.32) 4.49 (2.75e6.10) 4.11 (2.31e5.68)
Fit of Probit analysis Slope
SE
c
d.f
P
1.71 0.90 2.17 1.85 1.59 1.19 4.55 1.07 1.38 1.06 1.33 1.63 0.78 1.44 1.03 1.09 1.02 1.11 2.63 1.90 1.34 1.36 2.33 1.64 1.54 1.76 1.77 2.43 2.45 2.73
0.40 0.18 0.39 0.32 0.28 0.19 0.92 0.19 0.21 0.19 0.22 0.34 0.17 0.22 0.19 0.29 0.23 0.27 0.50 0.25 0.29 0.28 0.44 0.25 0.24 0.41 0.45 0.66 0.53 0.65
2.08 0.53 1.39 2.79 1.58 0.83 1.40 1.54 2.85 0.54 2.34 1.42 0.26 1.31 2.21 1.05 2.07 2.64 2.44 1.35 1.24 0.49 2.47 2.00 1.79 1.43 0.82 0.56 1.80 0.85
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 3 3 4 4 3 3 3 3 3
0.55 0.91 0.70 0.66 0.42 0.84 0.70 0.67 0.41 0.91 0.50 0.70 0.96 0.72 0.52 0.78 0.55 0.45 0.48 0.71 0.87 0.92 0.48 0.73 0.77 0.69 0.84 0.90 0.61 0.83
RR ¼ resistance ratio, calculated as (LC50 of field population)/(LC50 of Lab-PK). SR ¼ synergism ratio, calculated as (LC50 of selected population)/(LC50 of insecticide þ PBO or DEF).
2
RRa
SRb
e e e e e e e e e e e e e e e 119 59 452 139 175 18 53 30 21 33 6 14 6 5 6
e e e e e 1 1 2 1 1 <1 <1 1 1 1 e e e e e 8 2 10 6 6 51 10 74 36 40
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previously detected between permethrin and propoxur against two mosquito strains both having an identical enzymatic background but differing only by an insensitive acetylcholinasterase (Corbel et al., 2006). Synergism between pyrethroids and organophosphates has also been reported in several other insect pests like H. armigera (Gunning et al., 1999; Phokela et al., 1999), Spodoptera littoralis (Boisdual) (Ascher et al., 1986; Ahmad et al., 2009), Pectiniphora gossypiella (Saunders) (Keddis et al., 1986) and B. tabaci (Byrne and Devonshire, 1991). According to Corbel et al. (2006), there are two hypothesis of synergistic actions between insecticides. Synergistic actions may, between the molecules of different mode of actions, result from competitive substrate inhibition between insecticides in combination or disruption of the different physiological systems in insects. One molecule of the toxicant interferes with the metobolic detoxification of another, thereby increasing the toxicity of both (Corbett, 1974; Wilkinson, 1976). The OP prevents the degradation of pyrethroids by interfering with enzyme substrates thereby increasing the toxicity of pyrethroids. The present finding indicated the synergistic actions of neonicotinoids with IGRs (buprofezin or pyriproxyfen) at a 1:1 ratio against Aceta-SEL but displayed the antagonistic actions against Lab-PK (Table 2). This suggests that insecticides may be counteracting the mechanisms conferring insecticide resistance in the resistant population. Esterases and P450 mono-oxygenases are involved in the detoxification of neonicotinoids in resistant populations of B. tabaci (Karunker et al., 2009; Wang et al., 2009). Likewise, Esterases and mono-oxygenases are also responsible in the breakdown of pyriproxyfen in resistant B. tabaci (Hammock, 1985; Ishaaya and Horowitz, 1992). The increase of mixedfunction oxidase activity is also the major mechanism of resistance to buprofezin in Nilaparvata lugens (Stal) (Liu et al., 1998). The present synergistic studies with DEF, an esterase-specific inhibitor, and PBO, a mixed-function oxidase inhibitor, also suggest that resistance to neonicotinoid insecticides is MFO or esterase based (Table 2). The use of synergists in combination with mixtures also indicates a similar mechanism of resistance to neonicotinoids and IGRs in the selected population (Table 2). The physiological mechanism by which IGRs synergized the neonicotinoids at specific ratios is unclear and it could be due to the disruption of general physiological mechanisms. Further electrophysiological and biochemical investigations should be carried out to identify the synergistic mechanism in insects. Such an approach would be very helpful for better understanding the modes of action of insecticides and may contribute to improving pest control in the field. Mixtures should be used very carefully to counter the development of resistance in insects. They may give rise to multiple resistance mechanisms that extend to other chemical classes which may be difficult to manage as has occurred in the Plutella xyllostella in South East Asia (Sayyed et al., 2004). The use of synergistic mixtures as a resistance management strategy is a good option in the short run but they are not the best tool as a long-term management strategy. Potentiating mixtures can be used to reduce the cost of crop protection by use of lesser amounts of active ingredients, and labor costs by reducing spray applications. Antagonistic mixtures should not be used as they can exacerbate the problem by increasing insecticide application rates, accelerating the development of multiple resistance and adverse effects to the environment. Before recommending mixtures, study should be carried out that a particular mixture at the specific ratio displays the maximum synergism and is safe to natural enemies and the environment. Alternate strategies such as mosaics, rotations and non-chemical control measures should be adopted to avoid the development of multiple resistances in insect pests.
Acknowledgments We thank Dr Muhammad Afzal, University of Sargodha for providing a helping hand during the study. We are also indebted to Dr Muhammad Islam for technical help during experimentation and analysis of data. References Ahmad, M., Saleem, M.A., Sayyed, A.H., 2009. Efficacy of insecticide mixtures against pyrethroids- organophosphate resistant population of Spodoptera litura (Lepidoptera:Noctuidae). Pest Manag. Sci. 65, 266e274. Ascher, K.R.S., Eliyahu, M., Ishaaya, I., Zur, M., Benmoshe, E., 1986. Synergism of pyrethroideorganophosphorus insecticide mixtures in insects and their toxicity against Spodoptera littoralis larvae. Phytoparasitica 14, 101e110. Attique, M.N.R., Khaliq, A., Sayyed, A.H., 2006. Could resistance to insecticides in Plutella xylostella (Lep., Plutellidae) be overcome by insecticide mixtures? J. Appl. Entomol. 130, 122e127. Basit, M., Saleem, M.A., Saeed, S., Sayyad, A.H., 2012. 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Byrne, S.N., Cohen, A.C., Draeger, E.A., 1990. Water uptake from plant tissue by the eggs pedicel of the green house whitefly, Trialeurodes vaporariorum (Homoptera: Aleyrodidae). Can. J. Zool. 68, 1193e1195. Corbel, V., Stankiewicz, M., Bonnet, J., Grolleau, F., Hougard, J.M., Lapied, B., 2006. Synergism between insecticides permethrin and propoxur occurs through activation of pre-synaptic muscarinic negative feedback of acetylcholine release in the insect central nervous system. Neurotoxicology 27, 508e519. Corbett, J.R., 1974. The Biochemical Mode of Action of Pesticides. Academic Press Inc, New York. Curtis, C.F., 1985. Theoretical models of the use of insecticide mixtures for management of resistance. Bull. Entomol. Res. 75, 259e265. Elbert, A., Haas, M., Thielert, W., Nauen, R., 2007. Applied aspects of neonicotinoid uses. In: Proc XVI Internat Plant Prot. Cong. Glasgow, UK, vol. 3, pp. 620e621. Georghiou, G.P., Lab’lmes, T.A., Baker, J.D., 1983. 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Can resistance in Bemisia tabaci (Homoptera: Aleyrodidae) be overcome with mixtures of neonicotinoids and insect growth regulators? Muhammad Basit a, *, Shafqat Saeed a, Mushtaq Ahmad Saleem a, Ali H. Sayyed b a b
Department of Entomology, University College of Agriculture, Bahauddin Zakariya University Multan, Pakistan Institute of Biotehnology, Bahauddin Zakariya University Multan, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 March 2012 Received in revised form 17 October 2012 Accepted 20 October 2012
Tobacco whitefly, Bemisia tabaci is an important polyphagous insect pest which has developed resistance to various insecticides worldwide. Mixtures of insecticides with different modes of action may delay the onset of resistance. Bioassays were performed to investigate the effects of various mixtures of neonicotinoid and insect growth regulator (IGR) insecticides against a susceptible and a resistant strain. The results of the study showed that potentiation ratio (PR) of all neonicotinoids þ buprofezin or pyriproxyfen mixtures at 1:1, 10:1 and 20:1 ratios was greater than 1 suggesting synergistic interactions between insecticides. Maximum potentiation occurred at the 1:1 ratio (PR ¼ 1.69e7.56). The PRs for mixture of acetamiprid, thiamethoxam, thiacloprid or nitenpyram with buprofezin or pyriproxyfen at 1:10 and 1:20 ratios were less than 1 indicating antagonistic interactions. Addition of synergists, S, S, S, tri-butyl phosphorotrithioate (DEF) or piperonyl butoxide (PBO) in the insecticide solutions largely overcame the resistance to all tested neonicotinoids, indicating that the resistance was associated with esterases or mono-oxygenases, respectively. Likewise, addition of both DEF and PBO in mixture with neonicotinoids and IGRs also suggested a similar mechanism of resistance in B. tabaci to the tested insecticide groups. The mechanism of synergism between neonicotinoids and IGRs is unclear. Implications of using mixtures to counteract pesticide resistance are discussed. Mixtures of neonicotinoids with buprofezin or pyriproxyfen at a 1:1 ratio could be used to restore the efficacy of these neonicotinoids against B. tabaci. Ó 2012 Published by Elsevier Ltd.
Keywords: Bemisia tabaci Neonicotinoids IGRs Mixtures DEF PBO
1. Introduction The tobacco whitefly, Bemisia tabaci (Gennadius) is an economic agricultural pest world wide (Byrne et al., 1990). It is a highly adaptable and polyphagous insect pest that feeds on more than 700 plant species from 86 botanical families (Greathead, 1986). It directly causes injuries to crop plants by sucking phloem sap and indirectly by transmitting more than 100 plant viruses (Horowitz et al., 2003; Mugiira et al., 2008). The estimated loss caused by the whitefly is 3e4 millions of U.S. dollars each year in British Columbia, Canada (Moreau and Isman, 2012). In Pakistan, it caused the economic loss of US$5 billion from 1992 to 1997 due to transmission of cotton leaf curl virus disease (Briddon and Markham, 2000; Briddon, 2003; Basit et al., 2012). It has attained a major pest status because of its capabilities to replace existing biotypes, invading new geographical ranges and rapidly developing resistance to new pesticides including neonicotinoids and insect growth * Corresponding author. Tel.: þ92 0345 4602058; fax: þ92 619210068. E-mail address: [email protected] (M. Basit). 0261-2194/$ e see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.cropro.2012.10.021
regulators (IGRs) (Byrne and Bellows, 1991; Horowitz and Ishaaya, 1996; Perring, 2001). The usual response to the insecticide resistance problem is to increase the dosage and/or application frequencies and in some cases to substitute with new products. However, this has become expensive for both growers and industry. In the United States, cost of pesticide resistance has been estimated to be approximately $ 1.5 billion annually (Pimentel, 2005). Furthermore, availability of new products is limited because of the rising standards of environmental and toxicological safety. The most effective way to counteract the resistance problem is to reduce the selection pressure before resistance (Georghiou and Taylor, 1976; Ware, 2000) and to use resistance management strategies, which include use of insecticides in sequences, rotations and mixtures of two or more insecticides having different modes of action (Georghiou et al., 1983). Sequential use of insecticides has been discouraged due to the earlier development of resistance but the use of insecticides in rotations and mixtures merits consideration. Two insecticides with different modes of action in a mixture may synergize each other and increase the efficacy consequently
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reducing the input cost (Martin et al., 2003; Attique et al., 2006). Synergism theory is based on the ability of one molecule to interfere with the metabolic detoxification of another molecule (Corbett, 1974). Synergism between organophosphates or carbamates and pyrethroids has already been reported in a number of insect pests (Martin et al., 2003; Corbel et al., 2006; Attique et al., 2006). However, there is no documented report of mixing neonicotinoids with IGRs, which are being widely used in Pakistan and elsewhere for the control of B. tabaci. Neonicotinoids act agonistically on postsynaptic nicotinic acetylcholine receptors (Elbert et al., 2007) and have been shown to have no cross-resistance to IGRs (Basit et al., 2012). The aim of the present study was to determine the effect of mixing a neonicotinoid (imidacloprid, acetamiprid, thiamethoxam, thiacloprid or nitenpyram) with an IGR (buprofezin or pyriproxyfen) against the laboratory susceptible (Lab-PK) and acetamiprid selected population (Aceta-SEL) of B. tabaci. The AcetaSEL has been shown to have high level of resistance to neonicotinoids and cross-resistance to pyrethroid (bifenthrin) and endosulfan (Basit et al., 2011). To evaluate the mixtures of neonicotinoid insecticides and IGRs, a standard bioassay procedure was used to assess whether the mixture could yield additive, antagonistic or synergistic effects in the presence or absence of cytochrome P450 monooxigenase and an esterase inhibitor, pipronyl butoxide (PBO), and S, S, S, tri-butyl phosphorotrithioate (DEF), an esterase-specific inhibitor. Such studies could provide important information for devising resistance management strategies and so delay the development of resistance in B. tabaci. 2. Materials and methods 2.1. Insects The resistant strain of B. tabaci was developed in the laboratory of Bahaudin Zakarya University (BZU), Multan, Pakistan from a field collected population by exposing to different selection pressures of the neonicotinoid insecticide, acetamiprid. The susceptible population was developed in 2009 by single pair crosses and was held in the laboratory for more than two years before the start of this study. Both populations were reared on cotton plants (Gossypium hirsutum L. var CIM-496) in the entomological laboratory of BZU, under photoperiod of 16 h at 26 2 C. 2.2. Insecticides and inhibitors
fifteen whiteflies were briefly sedated with CO2 before placing them on the lower side of cotton leaves in each clip cage (replicate) and thus a total of 30e40 adults were tested per concentration. The bioassays were kept at a temperature of 26 2 C, 65% relative humidity and 16:8 (light: dark) photoperiod. Mortality was assessed after 48 h. 2.4. Effect of inhibitors on insecticide toxicity To evaluate the effect of inhibitors on the toxicity of insecticides alone and in mixture, PBO and DEF were used. Stock solution (1000 ppm) of PBO and DEF were prepared in acetone (analytical reagent grade, Fisher Scientific, Loughborogh, UK). To determine the dose at which mortality of B. tabaci was zero, a series of concentrations of DEF and PBO were tested. To test the effect of PBO and DEF on the toxicity of insecticides alone and in mixture, 100 ppm (zero % mortality) was added in each concentration tested. Mortality data were recorded after 48 h. The synergistic ratio was calculated by dividing the LC50 of the population treated with the insecticide alone by the LC50 of the population treated with insecticide plus synergist. 2.5. Evaluation of mixture Each insecticide mixture could give greater (synergism) or less (antagonism) than the expected additive effect (summation). To determine which of these possibilities resulted, mixture of two insecticides were tested at ratios of 1:1, 1:10, 10:1, 1:20 and 20:1 using serial dilutions. Synergism or antagonism can be assessed using various methods and we used the method described by Hoel (1987). Potency ratios were calculated by dividing the estimated lethal concentration (LC) values of the mixtures, calculated for joint similar action by the experimental LC values observed in the bioassay. If PR ¼ 1, the mixture was regarded ashaving additive action; if PR was <1, it showed an antagonistic action and if PR was >1, it exhibited a potentiating action. The estimated LC value of a mixture of A and B was computed as follows:
Estimated LCðA þ BÞ ¼
1 LCðAÞ þ mB=LCðBÞ
where mA and mB represent the proportion of A and B in the mixture; mA þ mB ¼ 1.
Commercial formulations used in the bioassays were: buprofezin 250 g a.i. kg1(FMC, Pakistan), pyriproxyfen 108 g a.i. kg1(Kanzo AG, Pakistan), acetamiprid 200 g a.i. l1 (AcelanÒ; FMC, Pakistan), imidacloprid 200 g a.i. l1 (ConfidorÒ, Bayer Crop Sciences, France), thiamethoxam 250 g a.i. kg1 (ActaraÒ, Syngenta, Berkshire UK), thiacloprid 480 g a.i. l1 (TalentÒ, Kanzo AG, Pakistan), nitenpyram 100 g a.i. l1 (PyramidÒ, Kanzo AG, Pakistan), Piperonyl butoxide (PBO, Sigma Ltd, UK), S, S, S, tri-butylphosphorotrithioate (DEF, Sigma, Ltd).
2.6. Data analysis
2.3. Bioassays
3. Results
The bioassays were carried out on whole cotton plants at two true leaf stages (20e22 days old) as described previously (Basit et al., 2011). Both leaves were washed, dried and dipped in the freshly prepared solutions for five to 10 s with slight agitation. Excessive liquid was allowed to drain off and the leaves were air dried for 1 h before confining the adults’ B. tabaci in clip cages. There was one clip cage on each cotton plant and thus each plant was used as a single replicate. Each treatment (concentration) was replicated 3e4 times, including distilled water controls. Ten to
3.1. Toxicity of tested insecticides alone and in combination to the Lab-PK and Aceta-SEL population
Mortality data were analyzed and LC50 values and their 95% fiducial limits were estimated using the POLO-PC computer based software (POLO, LeOra software, Menlo Park, California). Because of the inherent variability of bioassays, pairwise comparisons of LC50 values were done at the 5% significance level (where individual 95% FLs for two treatments do not overlap) (Litchfield and Wilcoxon, 1949).
The toxicities of acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid were similar to one another and to the Lab-PK (p < 0.05, overlapping 95% fiducial limits). Similarly, LC50 values of buprofezin and pyriproxyfen were also similar to each other for the Lab-PK (Table 1). However, mixtures of pyriproxyfen or buprofezin with acetamiprid, imidacloprid, thiamethoxam,
M. Basit et al. / Crop Protection 44 (2013) 135e141
137
Table 1 Toxicity of acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid alone and in combination with buprofezin, pyriproxyfen, DEF and PBO to the Lab-PK population of Bemisia tabaci. Ratio
LC50 (95% FL) (mg AI ml1)
LC50 (95% FL) (mg AI ml1) þDEF
LC50 (95% FL) (mg AI ml1) þPBO
PR
1:00 1:00 1:1 1:10 10:1 1:20 20:1
0.70 24.1 4.63 22.3 5.97 30.1 7.01
(0.30e1.22) (19.5e31.1) (2.58e6.45) (13.3e49.3) (3.01e9.99) (25.2e36.1) (3.59e10.7)
0.40 e 0.97 1.12 1.07 1.11 1.08
(0.35e0.63)
(0.12e0.45)
(0.60e1.45) (0.61e1.81) (0.62e1.65) (0.58e1.84) (0.56e1.75)
0.29 e 0.65 0.70 0.61 0.75 0.66
(0.28e1.12) (0.32e1.18) (0.25e1.07) (0.36e1.23) (0.29e1.12)
e e 0.29 0.26 0.12 0.30 0.10
Acetamiprid Acetamiprid þ buprofezin
1:00 1:1 1:10 10:1 1:20 20:1
2.00 5.12 24.7 7.46 25.5 8.26
(0.83e3.20) (2.00e8.25) (20.6e31.3) (3.92e11.4) (20.9e32.2) (4.62e12.5)
1.58 1.48 1.65 1.54 1.60 1.56
(0.98e2.45) (0.87e2.31) (0.99e2.53) (0.91e2.39) (0.94e2.50) (0.88e2.47)
0.76 0.73 0.87 0.78 0.89 0.69
(0.44e1.14) (0.34e1.23) (0.45e1.40) (0.38e1.29) (0.45e1.46) (0.29e1.18)
e 0.72 0.48 0.29 0.62 0.25
Thiamethoxam Thiamethoxam þ buprofezin
1:00 1:1 1:10 10:1 1:20 20:1
1.45 (0.90e2.18) 4.52 (1.66e7.36) 20.0 (15.5e27.0) 8.26 (4.62e12.5) 21.6 (17.1e28.4) 8.35 (4.12e12.9)
1.29 (0.74e2.07) 1.37 (0.83e1.09) 1.47 (0.90e2.21) 1.41 (0.88e2.12) 1.48 (0.88e2.29) 1.36 (0.85e2.04)
0.87 0.87 0.91 0.84 0.88 0.83
(0.33e1.63) (0.45e1.40) (0.50e1.45) (0.47e1.29) (0.51e1.35) (0.46e1.30)
e 0.60 0.50 0.19 0.54 0.18
Nitenpyram Nitenpyram þ buprofezin
1:00 1:1 1:10 10:1 1:20 20:1
1.17 4.27 20.7 7.01 22.6 10.4
1.10 1.01 1.11 1.09 1.13 1.05
0.88 0.82 0.82 0.83 0.88 0.88
(0.56e1.30) (0.41e1.35) (0.41e1.35) (0.39e1.41) (0.44e1.46) (0.43e1.47)
e 0.52 0.41 0.18 0.55 0.11
Thiacloprid Thiacloprid þ buprofezin
1:00 1:1 1:10 10:1 1:20 20:1
0.94 (0.54e1.47) 3.92 (1.12e6.75) 19.1 (14.5e26.6) 5.90 (2.36e9.58) 20.7 (16.1e28.1) 7.61 (4.21e11.4)
0.81 (0.41e1.33) 1.03 (0.56e1.65) 1.08 (0.60e1.70) 1.03 (0.57e1.65) 1.11 (0.61e1.78) 0.99 (0.53e1.58)
0.68 (0.32e1.15) 0.73 (0.34e1.23) 0.84 (0.41e1.41) 0.75 (0.33e1.29) 0.77 (0.39e1.24) 0.80 (0.38e1.35)
e 0.46 0.39 0.17 0.53 0.12
Pyriproxyfen Imidacloprid þ pyriproxyfen
1:00 1:1 1:10 10:1 1:20 20:1
20.0 3.79 20.0 6.21 23.8 7.61
e 1.26 1.47 1.39 1.42 1.34
e 0.50 0.61 0.64 0.69 0.61
(0.10e1.07) (0.26e1.03) (0.31e1.04) (0.35e1.09) (0.29e1.00)
e 0.35 0.28 0.12 0.36 0.09
Acetamiprid þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
4.93 (2.23e7.61) 25.5 (20.9e32.2) 8.47 (5.33e12.1) 26.3 (21.7e33.1) 9.51 (6.29e13.4)
1.55 (1.05e2.31) 2.75 (1.20e2.58) 1.68 (1.16e2.43) 2.62 (1.12e2.35) 1.50 (1.01e2.32)
0.68 (0.37e1.06) 0.78 (0.41e1.24) 0.73 (0.41e1.11) 0.77 (0.43e1.19) 0.81 (0.50e1.20)
0.73 0.43 0.25 0.53 0.22
Thiamethoxam þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
4.89 24.1 7.15 24.9 8.04
(1.67e8.13) (19.5e31.1) (4.47e10.0) (20.2e32.1) (5.27e11.1)
1.39 1.50 1.28 3.61 1.34
(0.95e2.03) (1.03e2.17) (0.86e1.88) (1.14e2.29) (0.91e1.93)
0.69 0.87 0.74 0.94 0.77
(0.36e1.09) (0.52e1.30) (0.41e1.15) (0.59e1.40) (0.43e1.19)
0.55 0.38 0.22 0.50 0.18
Nitenpyram þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
4.09 21.1 7.98 23.5 8.70
(1.68e6.48) (16.5e28.4) (4.67e11.7) (18.8e30.9) (5.51e12.4)
1.04 1.11 1.08 1.10 1.07
(0.64e1.58) (0.67e1.73) (0.67e1.64) (0.72e1.61) (0.64e1.64)
0.77 0.84 0.82 0.88 0.79
(0.43e1.19) (0.48e1.29) (0.48e1.24) (0.51e1.35) (0.45e1.20)
0.55 0.38 0.16 0.48 0.14
Thiacloprid þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
3.89 (1.42e6.35) 22.9 (18.5e29.7) 6.20 (3.21e9.30) 24.1 (19.5e31.2) 8.89 (5.59e12.8)
0.81 (0.46e1.24) 0.84 (0.49e1.29) 0.78 (0.44e1.19) 0.94 (0.58e1.40) 0.79 (0.44e1.23)
0.78 0.30 0.16 0.42 0.11
Insecticides tested Lab-PK Imidacloprid Buprofezin Imidacloprid þ buprofezin
(0.73e1.76) (1.45e7.09) (16.1e28.1) (3.50e10.7) (18.1e29.5) (7.18e14.5)
(15.5e27.0) (1.06e6.57) (15.5e27.0) (2.66e9.93) (19.2e31.0) (4.21e11.4)
nitenpyram or thiacloprid were more toxic to the Lab-PK at the 1:1, 1:10 and 1:20 ratios than the mixtures at 10:1 and 20:1 ratios (Table 1). The mixture of imidacloprid þ buprofezin exhibited significantly higher toxicity to the selected strain at the 1:1 ratio than the same mixture at the 1:10, 10:1, 1:20 and 20:1 ratios (p < 0.05,
(0.84e1.01) (0.63e1.50) (0.63e1.77) (0.71e1.59) (0.73e1.66) (0.66e1.55)
(0.50e2.12) (0.68e2.35) (0.62e2.26) (0.66e2.27) (0.56e2.21)
0.96 (0.58e1.45) 1.10 (0.72e1.62) 1.06 (0.68e1.57) 1.14 (0.75e1.68) 1.06 (0.68e1.55)
overlapping 95% fiducial limits) (Table 1). Likewise the same mixture gave higher toxicity at 10:1 and 201 ratios compared with 1:10 and 1:20 ratios. Mixture of acetamiprid, thiamethoxam, thiacloprid or nitenpyram with buprofezin also displayed similar results as that of the imidacloprid with buprofezin (Table 2). Similarly, mixing of imidacloprid with pyriproxyfen at a 1:1 ratio
138
M. Basit et al. / Crop Protection 44 (2013) 135e141
Table 2 Toxicity of acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid alone and in combination with buprofezin, pyriproxyfen, DEF and PBO against Aceta-SEL population of Bemisia tabaci. Ratio
LC50 (95% FL) (mg AI ml1)
LC50 (95% FL) (mg AI ml1) þDEF
LC50 (95% FL) (mg AI ml1) þPBO
PR
1:00 1:00 1:1 1:10 10:1 1:20
41.3 220 14.8 158 40.3 191
(14.8e69.4) (169e301) (6.27e20.1) (90.7e155) (36.7e82.7) (111e252)
21.2 e 4.15 16.8 9.93 17.3
(11.1e31.3)
(1.49e6.79)
(2.80e5.87) (13.0e22.6) (6.40e14.4) (13.7e22.6)
4.18 e 1.16 6.10 3.30 7.63
(0.21e2.19) (4.65e8.22) (1.85e5.01) (5.97e10.5)
e e 4.67 0.99 1.11 0.95
Acetamiprid Acetamiprid þ buprofezin
20:1 1:00 1:1 1:10 10:1
41.8 237 135 680 230
(31.1e87.4) (150e506) (80.2e145) (550e1085) (180e403)
9.90 28.1 12.1 49.7 20.3
(6.29e14.5) (11.4e45.7) (5.94e18.6) (39.3e66.3) (13.2e29.5)
3.70 4.66 3.86 10.4 5.67
(2.25e5.49) (1.91e7.34) (1.56e6.21) (7.14e15.5) (2.66e9.21)
1.04 e 1.69 0.30 1.04
Thiamethoxam Thiamethoxam þ buprofezin
1:20 20:1 1:00 1:1 1:10 10:1
704 (673e1179) 235 (290e506) 375 (235e938) 100 (63.4e142) 650 (469e1085) 348 (290e465)
52.5 (41.3e71.3) 22.6 (15.6e32.0) 39.0 (24.9e51.5) 4.39 (1.76e7.15) 40.7 (30.9e57.1) 10.5 (3.75e17.5)
11.2 6.07 5.05 2.10 8.95 5.03
(7.72e16.7) (3.13e9.49) (1.85e8.32) (0.17e1.66) (6.64e12.8) (3.29e7.85)
0.34 1.03 e 2.77 0.35 1.01
Nitenpyram Nitenpyram þ buprofezin
1:20 20:1 1:00 1:1 1:10
748 355 163 53.7 236
(553e1093) (246e530) (121e209) (43.4e71.6) (192e304)
48.7 10.1 27.4 13.1 31.5
(38.9e63.8) (3.53e16.8) (18.1e37.4) (5.54e21.4) (23.4e44.0)
9.45 5.25 4.49 1.01 5.59
(7.06e13.5) (3.55e7.85) (2.75e6.10) (0.35e1.92) (3.65e8.68)
0.26 1.02 e 3.53 0.90
Thiacloprid Thiacloprid þ buprofezin
10:1 1:20 20:1 1:00 1:1
100 270 107 165 57.5
(72.8e163) (226e384) (73.2e190) (130e211) (46.8e75.7)
16.6 43.4 16.9 26.6 10.2
(10.2e24.0) (33.0e60.9) (10.7e24.0) (16.9e37.0) (7.99e14.0)
2.54 7.39 2.60 4.11 1.88
(1.52e3.91) (5.32e10.4) (1.52e4.07) (2.31e5.68) (0.81e3.35)
1.67 0.80 1.54 e 3.30
Pyriproxyfen Imidacloprid þ pyriproxyfen
1:10 10:1 1:20 20:1 1:00
233 90.4 300 96.8 200
(190e422) (77.2e157) (200e459) (79.4e180) (155e277)
30.0 14.6 34.1 15.2 e
(19.7e48.8) (8.18e21.6) (25.5e47.5) (8.86e22.4)
5.58 2.83 7.25 2.24 e
(4.02e7.60) (1.36e5.16) (5.13e10.6) (0.94e4.15)
0.91 1.87 0.72 1.72 e
Acetamiprid þ pyriproxyfen
1:1 1:10 10:1 1:20
9.07 150 39.2 180
(3.8e14.4) (90.4e165) (23.7e85.1) (100e187)
2.48 13.1 8.66 18.2
(1.06e3.97) (9.72e17.8) (5.09e12.9) (14.6e23.4)
0.89 8.18 5.11 12.1
(0.43e1.46) (5.27e15.0) (3.62e7.28) (8.69e17.0)
7.56 0.97 1.13 0.93
Thiamethoxam þ pyriproxyfen
20:1 1:1 1:10 10:1 1:20 20:1 1:1 1:10 10:1 1:20 20:1
43.0 (24.6e86.1) 107 (81.9e145) 495 (392e790) 221 (170e380) 640 (469e1068) 228 (169e385) 93.2 (62.0e138) 513 (381e636) 340 (200e350) 753 (400e60) 380 (227e380)
9.04 (5.55e13.2) 14.1 (8.00e20.1) 69.0 (53.6e94.8) 35.4 (28.0e46.2) 104 (84.0e143) 47.6 (37.7e61.4) 9.46 (6.58e13.4) 51.8 (40.8e67.8) 37.1 (26.6e51.7) 74.7 (60.46.5) 40.8 (29.7e57.2)
5.25 2.23 10.7 6.19 16.3 7.65 1.53 6.90 5.22 10.0 5.56
(3.77e7.37) (0.87e4.17) (7.17e13.8) (3.51e8.91) (12.3e22.4) (5.20e10.3) (0.44e3.15) (4.52e9.52) (2.99e7.56) (7.20e13.8) (3.30e7.98)
1.01 2.04 0.40 1.05 0.31 1.04 2.85 0.40 1.02 0.27 1.04
Nitenpyram þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
51.2 217 91.6 249 102
(41.6e62.4) (165 303) (65.2e134) (157e379) (70.3e161)
12.4 36.0 15.7 40.2 17.1
(6.73e18.2) (27.3e50.2) (9.53e22.8) (31.7e53.2) (11.1e24.1)
2.06 6.11 2.54 6.88 2.86
(1.06e3.05) (4.43e8.74) (1.45e3.67) (5.42e9.46) (1.79e4.01)
3.54 0.90 1.81 0.79 1.61
Thiacloprid þ pyriproxyfen
1:1 1:10 10:1 1:20 20:1
44.6 200 88.7 217 94.9
(35.9e59.8) (129.3e308) (61.1e142) (141e315) (60.9e153)
11.7 (4.95e18.3) 31.8 (20.2e47.7) 14.8 (6.68e23.0) 33.1 (22.1e48.0) 14.7 (6.14e23.3)
1.76 4.62 2.14 5.87 2.33
(0.80e2.70) (3.23e6.51) (0.81e3.48) (4.40e8.02) (0.76e4.94)
4.08 0.98 1.89 0.91 1.75
Insecticides tested Aceta-SEL Imidacloprid Buprofezin Imidacloprid þ buprofezin
significantly increased the effectiveness of the mixture compared with the mixture of these insecticides at 1:10, 10:1, 1:20 and 20:1 ratios. But the toxicity of that mixture at 1:10, 10:1, 1:20 and 20:1 was not significantly higher than the toxicity of imidacloprid alone (Table 2). However, the toxicity of that mixture at 10:1 and 20:1 ratios was at par with the toxicity of imidacloprid alone. Likewise,
mixture of acetamiprid, thiamethoxam, nitepyram or thiacloprid with pyriproxyfen at a 1:1 ratio displayed higher toxicity than the toxicity of these insecticides alone (Table 2). However, the efficacy of the mixture of acetamiprid, thiamethoxam, nitenpyram and thiacloprid with pyriproxyfen at 1:10, 10:1, 1:20 and 20:1 ratios was not significantly higher than the efficacy of these insecticides alone.
M. Basit et al. / Crop Protection 44 (2013) 135e141
When synergists (DEF and PBO) were added in the mixture, the LC50 values of buprofezin þ imidacloprid, acetamiprid, thiamethoxam, thiacloprid or nitepyram at all ratios were significantly reduced (Table 2). Likewise, mixture of pyriproxyfen with neonicotinoids þ PBO or DEF displayed similar results to those of the mixture of buprofezin with neonicotinoids at 1:1, 1:10, 10:1, 1:20 and 20:1 ratios (Table 2). To assess the synergism or antagonism between two insecticides, the potentiation ratio was calculated for each mixture. The PRs for neonicotinoids þ buprofezin at 1:1, 10:1 and 20:1 ratios against the selected strain were significantly greater than 1, indicating the synergistic interactions between the insecticides. Maximum synergism between neonicotinoids and buprofezin occurred at 1:1 ratios (Table 2). Nevertheless, PRs for neonicotinoids þ buprofezin were less than 1, indicating antagonistic interactions between the insecticides (Table 2). Similarly, the PR values for the mixture of neonicotinoids (imidacloprid, acetamiprid nitenpyram and thiacloprid) and pyriproxyfen at 1:1, 10:1 and 20:1 ratios were greater than 1, indicating synergistic interactions between the insecticides (Table 2). Synergism at 10:1 and 20:1 ratios was small compared with the synergism at 1:1 ratios. In contrast, the PR values for the mixture of neonicotinoids and pyriproxyfen at 1:10 and 1:20 ratios were less than 1, indicating antagonistic interactions between insecticides. 3.2. Effect of synergists on the tested insecticides The Aceta-SEL population was 119-, 59-, 452-, 139- and 175-fold resistant to acetmiprid, imidaloprid, thiamethoxam, nitenpyram and thiacloprid, respectively compared with Lab-PK (Table 3). The PBO largely overcame the resistance to acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid exhibiting 6-,
139
14-, 6-, 5-, and 6-fold resistance ratios, with synergistic ratios of 51, 10, 74, 36, and 40 for acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid, respectively. Similarly, DEF also synergized the tested insecticides with synergic ratios of 8, 2 10, 6 and 6 for acetamiprid, imidacloprid, thiamethoxam, nitenpyram and thiacloprid, respectively (Table 2). Likewise, PBO and DEF also synergized the mixtures of neonicotinoids and IGRs (Table 2). 4. Discussion Due to the development of resistance, safe and cost effective insecticides are being depleted rapidly; control of the resistant populations and strategies to slow down insecticide resistance evolution is based on the optimum use of existing compounds. In addition to sequence and rotation, use of mixtures of various compounds each acting on different sites has been adopted (Martin et al., 2000) Theoretically, under certain conditions, insecticide mixtures can delay the onset of resistance development more effectively than rotation of insecticides if resistance to each compound is independent and rare (Curtis, 1985). In West Africa, for example, mixture of organophosphate and pyrethroid delayed the development of resistance in cotton bollworm, Helicoverpa armigera (Hubner) for more than 20 years (Martin et al., 2003). In the present study, evaluation of insecticidal activity of neonicotinoids (imidacloprid, acetamiprid, thiamethoxam, nitenpyram and thiacloprid) with IGRs (buprofezin and pyriproxyfen) at different ratios was carried out. Bioassay results showed that the mixtures of neonicotinoids with IGRs (buprofezin and pyriproxifen) were more effective against the Aceta-SEL at 1:1 ratios than mixtures of these insecticides at 1:10, 10:1, 1:20 and 20:1 ratios (Table 2). But mixtures of these insecticides did not exhibit potentiation against Lab-PK. Synergism and antagonism were
Table 3 Toxicity of imidacloprid, acetamiprid, thiamethoxam, nitenpyram and thiacloprid with and without PBO and DEF to Lab-PK and Aceta-SEL strain of Bemisia tabaci. Strain
Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Lab-PK Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL Aceta-SEL a b
Treatment
Acetamiprid Imidacloprid Thiamethoxam Nitenpyram Thiacloprid Acetamiprid þ DEF Imidacloprid þ DEF Thiamethoxam þ DEF Nitenpyram þ DEF Thiacloprid þ DEF Acetamiprid þ PBO Imidacloprid þ PBO Thiamethoxam þ PBO Nitenpyram þ PBO Thiacloprid þ PBO Acetamiprid Imidacloprid Thiamethoxam Nitenpyram Thiacloprid Acetamiprid þ DEF Imidacloprid þ DEF Thiamethoxam þ DEF Nitenpyram þ DEF Thiacloprid þ DEF Acetamiprid þ PBO Imidacloprid þ PBO Thiamethoxam þ PBO Nitenpyram þ PBO Thiacloprid þ PBO
LC50 (95% FL) (mg a.i. ml1) 2.00(0.83e3.20) 0.70 (0.30e1.22) 1.45 (0.90e2.18) 1.17 (0.73e1.76) 0.94 (0.54e1.47) 1.58 (0.98e2.45) 0.40 (0.35e0.63) 1.29 (0.74e2.07) 1.10 (0.84e1.01) 0.81 (0.41e1.33) 0.76 (0.44e1.14) 0.29 (0.12e0.45) 0.87 (0.33e1.63) 0.88 (0.56e1.30) 0.68 (0.32e1.15) 237.0 (129e506) 41.3 (14.0e69.4) 375.2 (235e938) 162.6 (110e209) 164.8 (130e211) 28.2 (11.4e45.7) 21.2 (11.1e31.3) 39.0 (24.9e51.5) 27.4 (18.1e37.4) 26.6 (16.9e37.0) 4.66 (1.91e7.34) 4.18 (1.49e6.79) 5.05 (1.85e8.32) 4.49 (2.75e6.10) 4.11 (2.31e5.68)
Fit of Probit analysis Slope
SE
c
d.f
P
1.71 0.90 2.17 1.85 1.59 1.19 4.55 1.07 1.38 1.06 1.33 1.63 0.78 1.44 1.03 1.09 1.02 1.11 2.63 1.90 1.34 1.36 2.33 1.64 1.54 1.76 1.77 2.43 2.45 2.73
0.40 0.18 0.39 0.32 0.28 0.19 0.92 0.19 0.21 0.19 0.22 0.34 0.17 0.22 0.19 0.29 0.23 0.27 0.50 0.25 0.29 0.28 0.44 0.25 0.24 0.41 0.45 0.66 0.53 0.65
2.08 0.53 1.39 2.79 1.58 0.83 1.40 1.54 2.85 0.54 2.34 1.42 0.26 1.31 2.21 1.05 2.07 2.64 2.44 1.35 1.24 0.49 2.47 2.00 1.79 1.43 0.82 0.56 1.80 0.85
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 3 3 4 4 3 3 3 3 3
0.55 0.91 0.70 0.66 0.42 0.84 0.70 0.67 0.41 0.91 0.50 0.70 0.96 0.72 0.52 0.78 0.55 0.45 0.48 0.71 0.87 0.92 0.48 0.73 0.77 0.69 0.84 0.90 0.61 0.83
RR ¼ resistance ratio, calculated as (LC50 of field population)/(LC50 of Lab-PK). SR ¼ synergism ratio, calculated as (LC50 of selected population)/(LC50 of insecticide þ PBO or DEF).
2
RRa
SRb
e e e e e e e e e e e e e e e 119 59 452 139 175 18 53 30 21 33 6 14 6 5 6
e e e e e 1 1 2 1 1 <1 <1 1 1 1 e e e e e 8 2 10 6 6 51 10 74 36 40
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M. Basit et al. / Crop Protection 44 (2013) 135e141
previously detected between permethrin and propoxur against two mosquito strains both having an identical enzymatic background but differing only by an insensitive acetylcholinasterase (Corbel et al., 2006). Synergism between pyrethroids and organophosphates has also been reported in several other insect pests like H. armigera (Gunning et al., 1999; Phokela et al., 1999), Spodoptera littoralis (Boisdual) (Ascher et al., 1986; Ahmad et al., 2009), Pectiniphora gossypiella (Saunders) (Keddis et al., 1986) and B. tabaci (Byrne and Devonshire, 1991). According to Corbel et al. (2006), there are two hypothesis of synergistic actions between insecticides. Synergistic actions may, between the molecules of different mode of actions, result from competitive substrate inhibition between insecticides in combination or disruption of the different physiological systems in insects. One molecule of the toxicant interferes with the metobolic detoxification of another, thereby increasing the toxicity of both (Corbett, 1974; Wilkinson, 1976). The OP prevents the degradation of pyrethroids by interfering with enzyme substrates thereby increasing the toxicity of pyrethroids. The present finding indicated the synergistic actions of neonicotinoids with IGRs (buprofezin or pyriproxyfen) at a 1:1 ratio against Aceta-SEL but displayed the antagonistic actions against Lab-PK (Table 2). This suggests that insecticides may be counteracting the mechanisms conferring insecticide resistance in the resistant population. Esterases and P450 mono-oxygenases are involved in the detoxification of neonicotinoids in resistant populations of B. tabaci (Karunker et al., 2009; Wang et al., 2009). Likewise, Esterases and mono-oxygenases are also responsible in the breakdown of pyriproxyfen in resistant B. tabaci (Hammock, 1985; Ishaaya and Horowitz, 1992). The increase of mixedfunction oxidase activity is also the major mechanism of resistance to buprofezin in Nilaparvata lugens (Stal) (Liu et al., 1998). The present synergistic studies with DEF, an esterase-specific inhibitor, and PBO, a mixed-function oxidase inhibitor, also suggest that resistance to neonicotinoid insecticides is MFO or esterase based (Table 2). The use of synergists in combination with mixtures also indicates a similar mechanism of resistance to neonicotinoids and IGRs in the selected population (Table 2). The physiological mechanism by which IGRs synergized the neonicotinoids at specific ratios is unclear and it could be due to the disruption of general physiological mechanisms. Further electrophysiological and biochemical investigations should be carried out to identify the synergistic mechanism in insects. Such an approach would be very helpful for better understanding the modes of action of insecticides and may contribute to improving pest control in the field. Mixtures should be used very carefully to counter the development of resistance in insects. They may give rise to multiple resistance mechanisms that extend to other chemical classes which may be difficult to manage as has occurred in the Plutella xyllostella in South East Asia (Sayyed et al., 2004). The use of synergistic mixtures as a resistance management strategy is a good option in the short run but they are not the best tool as a long-term management strategy. Potentiating mixtures can be used to reduce the cost of crop protection by use of lesser amounts of active ingredients, and labor costs by reducing spray applications. Antagonistic mixtures should not be used as they can exacerbate the problem by increasing insecticide application rates, accelerating the development of multiple resistance and adverse effects to the environment. Before recommending mixtures, study should be carried out that a particular mixture at the specific ratio displays the maximum synergism and is safe to natural enemies and the environment. Alternate strategies such as mosaics, rotations and non-chemical control measures should be adopted to avoid the development of multiple resistances in insect pests.
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