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Crop Protection 29 (2010) 1428e1433

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Crop Protection

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Resistance of beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae)to endosulfan, organophosphorus and pyrethroid insecticides in Pakistan

Mushtaq Ahmad a,*, M. Iqbal Arif b

aNuclear Institute for Agriculture and Biology, Jhang Road, Faisalabad, Punjab, PakistanbCentral Cotton Research Institute, Multan, Pakistan

a r t i c l e i n f o

Article history:Received 15 February 2010Received in revised form29 July 2010Accepted 30 July 2010

Keywords:Spodoptera exiguaInsecticide resistancePakistanEndosulfanChlorpyrifosQuinalphosCypermethrinDeltamethrinBifenthrinFenpropathrinCotton

* Corresponding author.E-mail address: mushsoroya@gmail.com (M. Ahm

0261-2194/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.cropro.2010.07.025

a b s t r a c t

Field populations of beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), from Pakistan wereassessed for their resistance to the chlorinated hydrocarbon endosulfan, the organophosphates chlor-pyrifos and quinalphos, and the pyrethroids cypermethrin, deltamethrin, bifenthrin and fenpropathrin.Using a leaf-dip bioassay, resistance to endosulfan was high during 1998e2000 but declined to very low,to low levels during 2001e2007, following a reduced use of the insecticide. Organophosphates andpyrethroids were consistently used over the past three decades, and the resistance had been increasingto these insecticide classes. Generally, the resistance to chlorpyrifos and pyrethroids remained low from1998 to 2002e2003, but resistance increased to moderate to high levels from 2003e2004 to 2006e2007.For deltamethrin, resistance was very high during 2004e2007. Quinalphos resistance remained lowduring 1998e2006. Correlation analysis of LC50 and LC90 values showed a positive correlation betweenorganophosphates and pyrethroids, but no correlation between endosulfan and organophosphates orpyrethroids tested herein. These results suggest that the conventional chemistries should be replacedwith new chemistries for the successful management of S. exigua.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Beet armyworm, Spodoptera exigua (Hübner), is polyphagousand infests a wide range of crops in the tropical and sub-tropicalregions (Metcalf and Metcalf, 1992). It is an important insect pestof cotton, potato, tomato, soybean, okra, onion, chilli, and cloversin Pakistan. On Pakistani cotton it is usually found early in theseason. It is also called lesser armyworm, pigweed caterpillar andsmall mottled willow. It has the potential of causing outbreaksunder favourable conditions. Although a closely related speciesSpodoptera litura (F.) had frequent outbreaks in Pakistan, yet itremained localized and never occurred as an outbreak in largeareas.

S. exigua is an invasive pest species in Pakistan, which has beenobserved since late 1980s. However, it was in late 1990s whencomplaints of its control failures by insecticides were reported bygrowers and insecticide resistance was implicated as the potential

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cause of this poor control. Insecticide resistance has been docu-mented in S. exigua from China (Chen et al., 2002), Taiwan (Chouet al., 1984), Guatemala (Delorme et al., 1988), Mexico (Teran-Vargas, 1997), and the USA (Brewer and Trumble, 1989, 1994;Kerns et al., 1998; Mascarenhas et al., 1998). The present studywas initiated to determine the existence of resistance to endo-sulfan, organophosphates (OPs) (chlorpyrifos, quinalphos) andpyrethroids (cypermethrin, deltamethrin, bifenthrin, fenpropa-thrin) in S. exigua in Pakistan.

2. Materials and methods

2.1. Insects

Fifth or sixth instar larvae of S. exigua were collected primarilyfrom various locations within 50 km radius of Multan in thesouthern Punjab, Pakistan during 1998e2007. Each collection ofabout 300 larvae was made by walking through a 2-ha block ofa particular host crop in a zigzag manner to randomize collections.Larvae were fed in the laboratory on a semi-synthetic diet, whichconsisted of chickpea flour (300 g), ascorbic acid (4.7 g), methyl-

M. Ahmad, M.I. Arif / Crop Protection 29 (2010) 1428e1433 1429

4-hydroxybenzoate (3 g), sorbic acid (1.5 g), streptomycin (1.5 g),corn oil (12 ml), vitamin mixture (10 ml), yeast (48 g), and agar(17 g). Yeast and agar were dissolved in 800 ml of boiling waterand added to other constituents premixed in 500 ml of water.Adults were fed on a solution containing sugar (50 g), vitaminmixture (10 ml), methyl-4-hydroxybenzoate (1 g), and distilledwater (500 ml).

2.2. Insecticides

Commercial formulations of insecticides used in bioassays were:Thiodan (endosulfan, 350 g/l EC [emulsifiable concentrate]; BayerCropScience, Leverkusen, Germany), Lorsban (chlorpyrifos, 400 g/lEC; Dow AgroSciences, Indianapolis, IN, USA), Ekalux (quinalphos,250 g/l EC; Syngenta, Basle, Switzerland), Arrivo (cypermethrin,100 g/l EC; FMC, Philadelphia, PA, USA), Decis (deltamethrin, 25 g/lEC; Bayer), Talstar (bifenthrin, 100 g/l EC; FMC), and Danitol (fen-propathrin, 100 g/l EC; Sumitomo Chemical Company, Osaka,Japan).

2.3. Bioassays

Newly moulted second instar larvae from F1 laboratory cultureswere exposed to different insecticides using the leaf-dip methodrecommendedby the InsecticideResistanceActionCommittee (IRAC;http://www.irac-online.org/resources/methods.asp) (Anonymous,1990). Serial dilutions as ppm of the active ingredient of the testcompounds were prepared using distilled water. Five-centimetercotton (Gossypium hirsutum) leaf discs were cut and dipped into testsolutions for 10 s with gentle agitation, then allowed to dry on papertowel on both sides. Five larvae were released onto each leaf discplaced in a 5-cm-diametre Petri dish with adaxial side up. Eightreplicates of five larvae each were used for each concentration and5e10 serial concentrations were used for each test insecticide. Thesame number of leaf discs per treatment was dipped into distilledwater as an untreated check. Moistened filter papers were placedbeneath leaf discs to avoiddesiccation of leaves in Petri dishes. Beforeand after treatment, larvae were maintained at a temperature of 25(�2) �C with a photoperiod of 14 h.

2.4. Data analysis

Larval mortalities were recorded 48 h after treatment. Larvaewere considered dead if they failed to make a coordinated move-ment when prodded with a probe. Data were corrected for controlmortality using Abbott’s (1925) formula and analyzed by probitanalysis (Finney, 1971) using POLO-PLUS programme (LeOraSoftware, 2003). The lethal concentrations (LC) were calculatedand any two values compared were considered significantlydifferent if their respective 95% confidence limits (CL) did notoverlap. Resistance factors (RF) were determined at LC50s and LC90sby dividing the LC values of each insecticide by the correspondingLC values for theMultan-1 population. The 95% CLs for the RFs weredetermined according to Robertson and Preisler (1992). To inter-pret cross-resistance spectra among the insecticides tested, pair-wise correlation coefficients of log LC values of the commonpopulations for each insecticide were calculated by the Pearsoncorrelation formula according to Snedecor and Cockran (1989)using the MSTAT statistical computer programme (MSTAT-C,1989). As described previously (Ahmad and Arif, 2009), resistancewas generally classified as none (RF� 1), very low (RF ¼ 2e10), low(RF¼ 11e20), moderate (RF¼ 21e50), high (RF¼ 51e100) and veryhigh (RF > 100).

3. Results

3.1. Endosulfan

S. exigua is an early-season pest of cotton in Pakistan. Endosulfanwas used to be applied early in the season against sucking insectpests, and therefore S. exiguawas frequently exposed to endosulfan.A moderate to high resistance was found to endosulfan in five fieldpopulations of S. exigua tested during 1998e2000 (Table 1). TheShershah-1 population rather exhibited a very high resistance toendosulfan. Endosulfan resistance declined to low levels during2000e2002 and then to very low levels during 2003e2006. Thisdrop in endosulfan resistance may be associated with a decreaseduse of endosulfan against insect pests of cotton in Pakistan.

3.2. Organophosphates

Resistance to chlorpyrifos remained very low during 1998e2001and low during 2002e2003 in all nine populations of S. exiguatested (Table 1). During 2004e2007, chlorpyrifos resistanceincreased to moderate to high levels in the five populations tested.Like chlorpyrifos, quinalphos resistance was also very low during1998e2002 in the ten populations of S. exigua (Table 1). From 2002to 2006, quinalphos resistance was recorded as low in four pop-ulations and slightly moderate in three populations.

3.3. Pyrethroids

Pyrethroids have been consistently used against lepidopteranpests of cotton, especially in mixtures with OPs. Resistance tocypermethrin remained very low during 1998e2000 in eightpopulations and low during 2001e2002 in four populations ofS. exigua (Table 1). Cypermethrin resistance rose to moderate tohigh levels during 2003e2007 in all ten populations tested. Likecypermethrin, resistance to deltamethrin was very low during1998e2000 in seven populations and low to moderate during2001e2003 in six populations of S. exigua (Table 1). Deltamethrinresistance increased to high to very high levels during 2003e2007in the seven populations tested.

Resistance to bifenthrin remained very low during 1998e2002in the 11 populations of S. exigua tested (Table 1). Subsequently, itprogressively increased but at a slower pace than cypermethrin anddeltamethrin. Bifenthrin resistance was low in 2003 but moderateduring 2004e2005 and 2007 and high in 2006. Resistance to fen-propathrin remained very low during 1998e2001 in eight pop-ulations and then low during 2001e2002 in three populations ofS. exigua (Table 1). During 2003e2006, the fenpropathrin resistancewas moderate to high in all six populations tested.

3.4. Correlation between LC values of insecticides

Paired comparisons of the log LC50s and LC90s for the samepopulations of S. exigua showed positive and highly significantcorrelations between OPs chlorpyrifos and quinalphos, and amongpyrethroids cypermethrin, deltamethrin, bifenthrin and fenpropa-thrin (Table 2). Therewas also a highly positive correlation betweenOPs and pyrethroids tested, indicating a cross-resistance withinand between these two insecticide classes. On the contrary, nocorrelation and therefore no cross-resistance existed betweenendosulfan and OPs or pyrethroids in S. exigua.

4. Discussion

The Multan-1 population of S. exigua collected from okra inMay 1998 exhibited the lowest LC values and reasonably good

Table 1Toxicity of selected insecticides against field populations of Spodoptera exigua, Pakistan.

Insecticide Location Host Datetested

No.tested

Fit of probit linea LC50(mg l�1)(95% CL)

RF at LC50b

(95% CL)LC90 (mg l�1)(95% CL)

RF at LC90b

(95% CL)Slope � SE c2 df P

Endosulfan Multan-1 Okra May 98 280 2.32 � 0.23 3.56 5 0.61 6.61 (5.42e8.03) 1.0 23.6 (18.0e34.1) 1.0Lar-1 Cotton Jul. 98 240 2.71 � 0.29 3.58 4 0.47 211 (176e254) 32 (24e42) 628 (487e897) 27 (17e41)Shershah-1 Cotton Oct. 98 360 1.56 � 0.14 5.15 7 0.64 856 (671e1090) 130 (96e179) 5660 (3988e8981) 240 (147e406)Khokhran-1 Clover Feb. 99 360 1.68 � 0.15 3.80 7 0.80 280 (222e353) 42 (31e58) 1622 (1161e2518) 69 (42e113)Muzaffargarh Cotton Oct. 99 280 2.51 � 0.25 3.08 5 0.69 429 (355e517) 65 (50e85) 1388 (1075e1964) 59 (39e91)Shujabad-1 Clover Apr. 00 320 1.90 � 0.17 3.85 6 0.70 89.6 (72.0e111) 14 (10e18) 422 (310e636) 18 (11e29)Bosan-1 Okra Jun. 00 320 1.79 � 0.17 5.08 6 0.53 163 (130e204) 25 (18e33) 845 (611e1304) 36 (22e59)Khanewal-1 Cotton Aug. 00 240 3.08 � 0.33 1.29 4 0.86 91.5 (77.6e109) 14 (11e18) 239 (191e328) 10 (6.7e16)Kabirwala-1 Clover Jan. 01 240 3.00 � 0.32 1.78 4 0.78 102 (86.0e121) 15 (12e20) 273 (216e378) 12 (7.6e18)Lodhran-1 Okra May 01 320 1.88 � 0.17 3.71 6 0.72 93.8 (75.4e117) 14 (11e19) 451 (329e685) 19 (12e31)Vehari-1 Cotton Aug. 02 320 2.11 � 0.19 2.87 6 0.82 91.9 (74.9e113) 14 (10e19) 373 (280e546) 16 (10e25)Mailsi Potato Nov. 02 320 2.20 � 0.23 2.03 6 0.92 82.8 (65.7e103) 13 (9.3e17) 318 (241e462) 13 (8.6e21)Khanewal-2 Clover Apr. 03 280 2.12 � 0.21 5.01 5 0.41 65.5 (49.7e86.3) 9.9 (7.4e13) 264 (182e463) 11 (7.0e18)Lar-2 Okra Jul. 03 280 2.12 � 0.21 4.44 5 0.49 64.0 (52.0e78.8) 9.7 (7.2e13) 258 (192e386) 11 (6.8e17)Bosan-2 Okra Apr. 04 320 1.93 � 0.18 5.08 6 0.53 64.1 (51.8e79.9) 9.7 (7.2e13) 296 (216e455) 13 (7.7e20)Kabirwala-2 Cotton Oct. 04 280 2.14 � 0.24 0.98 5 0.96 53.6 (41.3e67.7) 8.1 (6.0e11) 212 (159e318) 9.0 (5.7e14)Multan-2 Potato Nov. 05 280 2.33 � 0.23 2.48 5 0.78 61.9 (50.9e75.3) 9.4 (7.1e12) 220 (168e318) 9.3 (6.0e14)Multan-3 Cotton Oct. 06 240 3.57 � 0.40 1.93 4 0.75 49.8 (42.5e58.3) 7.5 (5.9e9.8) 114 (92.3e153) 4.8 (3.3e7.2)Multan-4 Clover Apr. 07 280 2.07 � 0.20 2.58 5 0.76 122 (99.1e151) 18 (14e25) 508 (376e764) 22 (14e35)

Chlorpyrifos Multan-1 Okra May 98 280 2.27 � 0.22 3.98 5 0.55 6.07 (4.96e7.40) 1.0 22.3 (17.0e32.2) 1.0Lar-1 Cotton Jul. 98 280 2.27 � 0.23 3.82 5 0.58 23.1 (18.8e28.1) 3.8 (2.8e5.0) 84.6 (64.5e122) 3.8 (2.4e5.9)Shershah-1 Cotton Oct. 98 320 1.95 � 0.18 4.43 6 0.62 1.43 (1.15e1.77) 0.2 (0.2e0.3) 6.48 (4.79e9.67) 0.3 (0.2e0.5)Khokhran-1 Clover Feb. 99 360 1.52 � 0.13 5.70 7 0.58 8.31 (6.50e10.6) 1.4 (1.0e1.9) 57.7 (40.1e93.6) 2.6 (1.54e4.4)Muzaffargarh Cotton Oct. 99 280 1.99 � 0.23 2.63 5 0.76 15.8 (12.0e20.1) 2.6 (1.9e3.6) 69.6 (50.7e109) 3.1 (1.9e5.1)Shujabad-1 Clover Apr. 00 280 1.81 � 0.18 2.62 5 0.76 16.3 (13.0e20.6) 2.7 (2.0e3.6) 83.5 (59.4e134) 3.7 (2.2e6.2)Bosan-1 Okra Jun. 00 200 4.18 � 0.51 1.70 3 0.64 14.9 (12.8e17.2) 2.5 (1.9e3.1) 30.1 (24.9e39.7) 1.3 (0.9e2.0)Khanewal-1 Cotton Aug. 00 360 1.55 � 0.14 4.78 7 0.69 29.8 (23.3e37.9) 4.9 (3.6e6.7) 199 (140e318) 8.9 (5.4e15)Kabirwala-1 Clover Jan. 01 240 2.61 � 0.28 5.73 4 0.22 22.4 (16.1e31.1) 3.7 (2.8e4.9) 69.4 (46.6e141) 3.1 (2.0e4.9)Lodhran-1 Okra May 01 320 1.84 � 0.17 4.22 6 0.65 40.3 (32.3e50.3) 6.6 (4.9e9.0) 200 (146e304) 9.0 (5.6e15)Vehari-1 Cotton Aug. 02 240 3.11 � 0.34 3.40 4 0.49 70.8 (59.8e83.9) 12 (8.9e15) 183 (146e251) 8.2 (5.4e12)Mailsi Potato Nov. 02 360 1.72 � 0.15 4.55 7 0.71 72.6 (57.8e91.2) 12 (8.9e16) 401 (289e618) 18 (11e30)Khanewal-2 Clover Apr. 03 360 1.52 � 0.13 5.70 7 0.58 66.5 (52.0e85.1) 11 (8.0e15) 462 (321e749) 21 (12e36)Lar-2 Okra Jul. 03 280 2.35 � 0.23 4.13 5 0.53 112 (92.5e137) 18 (14e25) 395 (301e569) 18 (11e28)Bosan-2 Okra Apr. 04 280 2.05 � 0.22 3.73 5 0.59 240 (188e301) 40 (30e53) 1009 (744e1537) 45 (29e73)Kabirwala-2 Cotton Oct. 04 280 2.14 � 0.21 3.99 5 0.55 260 (212e320) 43 (34e54) 1034 (772e1541) 46 (30e71)Multan-2 Potato Nov. 05 320 1.90 � 0.18 3.99 6 0.68 453 (365e565) 75 (56e102) 2136 (1556e3268) 96 (60e159)Multan-3 Cotton Oct. 06 320 2.11 � 0.19 3.63 6 0.73 620 (505e761) 102 (78e133) 2515 (1895e3663) 113 (72e174)Multan-4 Clover Apr. 07 280 1.74 � 0.18 3.19 5 0.67 266 (210e337) 44 (32e60) 1445 (1012e2379) 65 (38e111)

Quinalphos Multan-1 Okra May 98 280 2.16 � 0.21 4.07 5 0.54 6.75 (5.49e8.28) 1.0 26.4 (19.9e38.9) 1.0Lar-1 Cotton Jul. 98 200 3.13 � 0.36 2.68 3 0.44 17.8 (15.0e21.2) 2.6 (2.0e3.5) 45.8 (36.2e64.2) 1.7 (1.1e2.7)Shershah-1 Cotton Oct. 98 240 3.16 � 0.35 4.15 4 0.39 13.7 (10.7e17.6) 2.0 (1.6e2.6) 34.9 (25.6e58.1) 1.3 (0.9e2.0)Khokhran-1 Clover Feb. 99 320 2.19 � 0.20 3.37 6 0.76 21.4 (17.5e26.2) 3.2 (2.4e4.2) 82.7 (62.7e120) 3.1 (2.0e4.9)Muzaffargarh Cotton Oct. 99 240 2.39 � 0.25 6.39 4 0.17 9.97 (6.88e14.5) 1.5 (1.1e2.0) 34.3 (21.8e80.5) 1.3 (0.8e2.1)Shujabad-1 Clover Apr. 00 280 1.91 � 0.23 4.43 5 0.49 12.0 (9.09e15.4) 1.8 (1.3e2.5) 56.2 (39.5e95.0) 2.1 (1.2e3.6)Bosan-1 Okra Jun. 00 280 2.35 � 0.23 2.19 5 0.82 16.5 (13.6e20.1) 2.4 (1.8e3.3) 57.9 (44.2e83.5) 2.2 (1.4e3.5)Khanewal-1 Cotton Aug. 00 240 1.99 � 0.22 1.36 4 0.85 26.1 (20.9e32.7) 3.9 (2.9e5.2) 115 (81.7e188) 4.4 (2.6e7.3)Kabirwala-1 Clover Jan. 01 360 1.53 � 0.13 5.30 7 0.62 33.8 (26.5e43.2) 5.0 (3.6e6.9) 232 (161e375) 8.8 (5.1e15)Lodhran-1 Okra May 01 320 1.75 � 0.16 5.66 6 0.46 35.5 (28.1e44.5) 5.3 (3.9e7.2) 191 (138e295) 7.2 (4.4e12)Vehari-1 Cotton Aug. 02 320 1.67 � 0.18 1.22 6 0.98 39.1 (28.8e51.5) 5.8 (4.1e8.2) 228 (161e369) 8.6 (5.1e15)Mailsi Potato Nov. 02 320 1.71 � 0.16 5.19 6 0.52 90.5 (71.7e114) 13 (10e18) 510 (362e808) 19 (11e32)Khanewal-2 Clover Apr. 03 320 1.92 � 0.18 3.54 6 0.74 77.1 (62.0e95.7) 11 (8.5e15) 358 (264e537) 14 (8.4e22)Lar-2 Okra Jul. 03 280 2.09 � 0.20 2.15 5 0.83 111 (89.8e137) 16 (12e22) 456 (340e680) 17 (11e27)Bosan-2 Okra Apr. 04 360 1.75 � 0.15 3.38 7 0.85 137 (109e171) 20 (15e28) 735 (532e1122) 28 (17e47)Kabirwala-2 Cotton Oct. 04 320 2.06 � 0.19 3.55 6 0.74 108 (87.9e133) 16 (12e21) 453 (338e671) 17 (11e27)Multan-2 Potato Nov. 05 280 2.22 � 0.22 3.38 5 0.64 133 (108e162) 20 (14e26) 502 (378e738) 19 (12e30)Multan-3 Cotton Oct. 06 320 1.92 � 0.18 3.54 6 0.74 154 (124e191) 23 (17e31) 716 (528e1074) 27 (17e44)

Cypermethrin Multan-1 Okra May 98 320 2.13 � 0.20 2.43 6 0.88 2.88 (2.35e3.53) 1.0 11.5 (8.68e16.8) 1.0Lar-1 Cotton Jul. 98 280 2.30 � 0.23 2.89 5 0.72 7.49 (6.15e9.12) 2.6 (2.0e3.5) 27.0 (20.6e39.0) 2.3 (1.5e3.7)Shershah-1 Cotton Oct. 98 320 2.20 � 0.20 3.08 6 0.80 10.6 (8.63e12.9) 3.7 (2.7e4.9) 40.4 (30.7e58.3) 3.5 (2.2e5.5)Khokhran-1 Clover Feb. 99 280 2.14 � 0.21 3.99 5 0.55 15.7 (12.8e19.3) 5.5 (4.1e7.3) 62.5 (46.8e93.0) 5.4 (3.4e8.7)Muzaffargarh Cotton Oct. 99 280 2.29 � 0.23 3.08 5 0.69 14.0 (11.4e17.0) 4.9 (3.6e6.5) 50.5 (38.5e73.1) 4.4 (2.8e6.9)Shujabad-1 Clover Apr. 00 320 1.84 � 0.17 3.81 6 0.70 10.8 (8.60e13.4) 3.7 (2.8e5.0) 53.6 (39.0e81.9) 4.7 (2.8e7.6)Bosan-1 Okra Jun. 00 280 1.91 � 0.26 3.97 5 0.55 18.3 (13.0e24.2) 6.4 (4.4e9.2) 85.9 (60.8e144) 7.5 (4.4e13)Khanewal-1 Cotton Aug. 00 320 1.99 � 0.18 4.21 6 0.65 16.8 (13.6e20.8) 5.8 (4.4e7.9) 74.2 (55.3e110) 6.5 (4.0e10)Kabirwala-1 Clover Jan. 01 320 1.49 � 0.16 3.49 6 0.75 26.6 (19.4e35.5) 9.2 (6.4e13) 193 (128e345) 17 (9.3e30)Lodhran-1 Okra May 01 360 1.55 � 0.14 4.70 7 0.70 35.0 (27.4e44.6) 12 (8.9e17) 234 (163e376) 20 (12e35)Vehari-1 Cotton Aug. 02 320 2.07 � 0.19 5.91 6 0.43 43.0 (34.9e52.9) 15 (11e20) 179 (135e262) 16 (10e25)Mailsi Potato Nov. 02 320 1.97 � 0.18 3.33 6 0.77 49.5 (40.0e61.3) 17 (13e23) 222 (164e333) 19 (12e31)Khanewal-2 Clover Apr. 03 320 1.62 � 0.17 4.03 6 0.67 69.7 (50.7e92.5) 24 (17e35) 433 (301e710) 38 (22e63)Lar-2 Okra Jul. 03 320 2.08 � 0.19 2.15 6 0.91 104 (84.4e128) 36 (27e49) 428 (320e630) 37 (23e60)Khokhran-2 Cotton Oct. 03 400 1.36 � 0.11 7.05 8 0.53 133 (102e172) 46 (33e65) 1158 (792e1901) 101 (59e177)Bosan-2 Okra Apr. 04 320 1.81 � 0.17 4.75 6 0.58 171 (137e214) 59 (44e80) 874 (633e1347) 76 (46e124)

M. Ahmad, M.I. Arif / Crop Protection 29 (2010) 1428e14331430

Table 1 (continued )

Insecticide Location Host Datetested

No.tested

Fit of probit linea LC50

(mg l�1)(95% CL)

RF at LC50b

(95% CL)LC90 (mg l�1)(95% CL)

RF at LC90b

(95% CL)Slope � SE c2 df P

Kabirwala-2 Cotton Oct. 04 360 1.65 � 0.14 5.77 7 0.57 148 (118e188) 51 (38e71) 883 (629e1384) 77 (47e130)Vehari-2 Okra May 05 320 1.93 � 0.18 3.85 6 0.70 197 (159e244) 68 (51e92) 906 (666e1370) 79 (49e128)Multan-2 Potato Nov. 05 320 2.06 � 0.19 2.43 6 0.88 191 (155e235) 66 (50e89) 799 (598e1176) 69 (44e112)Shujabad-2 Okra May 06 360 1.68 � 0.15 5.22 7 0.63 244 (193e307) 85 (63e117) 1406 (1010e2171) 122 (75e206)Multan-3 Cotton Oct. 06 280 2.32 � 0.23 3.80 5 0.58 287 (236e350) 100 (76e134) 1024 (777e1492) 89 (57e142)Multan-4 Clover Apr. 07 240 2.62 � 0.28 2.02 4 0.73 83.6 (69.4e101) 29 (22e39) 257 (199e368) 22 (14e35)

Deltamethrin Multan-1 Okra May 98 280 2.61 � 0.26 1.45 5 0.92 2.11 (1.76e2.54) 1.0 6.53 (5.09e9.17) 1.0Lar-1 Cotton July. 98 280 1.79 � 0.18 3.55 5 0.62 14.2 (11.3e17.9) 6.7 (5.0e9.0) 74.2 (52.8e119) 11 (6.9e18)Shershah-1 Cotton Oct. 98 320 2.05 � 0.19 3.15 6 0.79 11.0 (8.93e13.6) 5.2 (3.9e6.9) 46.5 (34.8e68.3) 7.1 (4.6e11)Khokhran-1 Clover Feb. 99 320 1.95 � 0.18 3.73 6 0.71 13.7 (11.0e17.0) 6.5 (4.9e8.6) 62.1 (45.6e93.9) 9.5 (6.0e15)Muzaffargarh Cotton Oct. 99 280 2.09 � 0.21 4.61 5 0.47 8.11 (6.58e10.0) 3.8 (2.9e5.1) 33.3 (24.7e50.2) 5.1 (3.2e8.0)Shujabad-1 Clover Apr. 00 320 2.00 � 0.18 3.79 6 0.71 10.9 (8.82e13.5) 5.2 (3.9e6.8) 47.8 (35.5e71.0) 7.3 (4.6e11)Bosan-1 Okra Jun. 00 320 2.25 � 0.21 2.23 6 0.90 12.5 (10.3e15.3) 5.9 (4.5e7.8) 46.6 (35.5e67.1) 7.1 (4.6e11)Khanewal-1 Cotton Aug. 00 360 1.79 � 0.15 5.32 7 0.62 14.4 (11.5e17.9) 6.8 (5.1e9.1) 74.6 (54.6e112) 11 (7.2e18)Kabirwala-1 Clover Jan. 01 280 2.26 � 0.22 3.22 5 0.67 28.3 (23.1e34.5) 13 (10e17) 104 (79.2e152) 16 (10e24)Lodhran-1 Okra May 01 360 1.53 � 0.13 5.27 7 0.63 34.4 (26.9e43.9) 16 (12e22) 236 (164e382) 36 (22e61)Vehari-1 Cotton Aug. 02 360 1.64 � 0.14 4.66 7 0.70 29.8 (23.6e37.7) 14 (11e19) 179 (128e279) 27 (17e45)Mailsi-1 Potato Nov. 02 360 1.46 � 0.13 3.72 7 0.81 31.6 (24.6e40.7) 15 (11e21) 237 (162e391) 36 (22e62)Khanewal-2 Clover Apr. 03 280 1.83 � 0.18 2.91 5 0.71 60.0 (47.7e75.3) 28 (21e38) 301 (216e475) 46 (28e75)Lar-2 Okra Jul. 03 360 1.68 � 0.14 3.81 7 0.80 62.7 (49.7e79.1) 30 (22e40) 365 (261e565) 56 (34e89)Khokhran-2 Cotton Oct. 03 280 1.85 � 0.22 2.41 5 0.79 137 (101e180) 65 (46e93) 677 (478e1116) 104 (61e174)Shershah-2 Clover Feb. 04 320 1.86 � 0.17 4.22 6 0.65 172 (138e214) 82 (61e108) 840 (613e1280) 129 (80e204)Bosan-2 Okra Apr. 04 320 1.90 � 0.17 4.08 6 0.67 220 (177e274) 104 (77e138) 1041 (759e1590) 159 (98e251)Kabirwala-2 Cotton Oct. 04 400 1.48 � 0.12 4.66 8 0.79 209 (164e268) 99 (74e136) 1532 (1066e2451) 235 (143e392)Multan-2 Potato Nov. 05 360 1.53 � 0.13 5.30 7 0.62 242 (190e310) 115 (85e158) 1662 (1160e2677) 255 (154e429)Multan-3 Cotton Oct. 06 280 2.12 � 0.21 4.52 5 0.48 260 (211e320) 123 (93e160) 1049 (781e1570) 161 (101e247)Multan-4 Clover Apr. 07 360 1.76 � 0.15 3.42 7 0.84 141 (113e177) 67 (50e89) 757 (548e1158) 116 (72e183)

Bifenthrin Multan-1 Okra May 98 320 1.99 � 0.18 3.19 6 0.78 2.23 (1.80e2.75) 1.0 9.82 (7.33e14.5) 1.0Lar-1 Cotton Jul. 98 280 2.23 � 0.22 2.44 5 0.79 4.84 (3.92e5.91) 2.2 (1.6e2.9) 18.2 (13.9e26.4) 1.9 (1.2e3.0)Shershah-1 Cotton Oct. 98 320 1.93 � 0.18 4.05 6 0.67 2.58 (2.08e3.20) 1.2 (0.86e1.6) 11.9 (8.77e17.8) 1.2 (0.74e2.0)Khokhran-1 Clover Feb. 99 280 2.40 � 0.24 3.78 5 0.58 7.18 (5.91e8.69) 3.2 (2.4e4.3) 24.5 (18.9e34.9) 2.5 (1.6e3.9)Muzaffargarh Cotton Oct. 99 320 2.15 � 0.20 2.62 6 0.85 4.84 (3.95e5.92) 2.2 (1.6e2.9) 19.1 (14.4e27.6) 1.9 (1.2e3.1)Shujabad-1 Clover Apr. 00 240 2.58 � 0.27 4.34 4 0.36 5.55 (4.18e7.38) 2.5 (1.9e3.3) 17.4 (12.1e31.8) 1.8 (1.1e2.8)Bosan-1 Okra Jun. 00 280 2.27 � 0.22 3.14 5 0.68 7.88 (6.45e9.60) 3.5 (2.7e4.8) 28.8 (21.9e42.0) 2.9 (1.9e4.7)Khanewal-1 Cotton Aug. 00 360 1.62 � 0.14 4.45 7 0.73 3.41 (2.68e4.32) 1.5 (1.1e2.1) 21.2 (15.1e33.1) 2.2 (1.3e3.6)Kabirwala-1 Clover Jan. 01 320 2.11 � 0.19 3.28 6 0.77 6.23 (5.08e7.65) 2.8 (2.1e3.8) 25.3 (19.0e36.9) 2.6 (1.6e4.1)Lodhran-1 Okra May 01 320 2.20 � 0.20 3.27 6 0.77 9.72 (7.95e11.9) 4.4 (3.3e5.9) 37.1 (28.2e53.4) 3.8 (2.4e6.1)Vehari-1 Cotton Aug. 02 320 1.80 � 0.17 4.96 6 0.55 9.51 (7.58e11.9) 4.3 (3.1e5.8) 49.0 (35.6e75.1) 5.0 (3.0e8.2)Mailsi-1 Potato Nov. 02 240 2.01 � 0.23 5.15 4 0.27 15.4 (10.3e22.0) 6.9 (5.0e9.4) 66.8 (42.1e155) 6.8 (4.1e11)Khanewal-2 Clover Apr. 03 360 1.58 � 0.14 4.67 7 0.70 19.2 (15.1e24.4) 8.6 (6.2e12) 124 (86.5e198) 13 (7.4e22)Lar-2 Okra Jul. 03 360 1.84 � 0.16 3.32 7 0.85 27.9 (22.4e34.7) 13 (9.2e17) 139 (102e208) 14 (8.6e23)Khokhran-2 Cotton Oct. 03 200 2.55 � 0.33 1.99 3 0.57 33.9 (27.1e41.8) 15 (11e21) 108 (81.0e166) 11 (6.7e18)Shershah-2 Clover Feb. 04 320 2.34 � 0.22 2.64 6 0.85 47.5 (39.2e57.7) 21 (16e28) 167 (128e239) 17 (11e27)Bosan-2 Okra Apr. 04 320 1.91 � 0.18 3.74 6 0.71 54.9 (44.2e68.3) 25 (18e33) 257 (188e391) 26 (16e43)Kabirwala-2 Cotton Oct. 04 320 1.83 � 0.17 4.00 6 0.68 43.8 (35.0e54.8) 20 (14e27) 220 (160e337) 22 (13e37)Lodhran-2 Clover Mar. 05 360 1.80 � 0.16 4.04 7 0.78 67.6 (54.2e84.5) 30 (22e41) 348 (254e526) 35 (22e58)Vehari-2 Okra May 05 320 1.97 � 0.18 3.23 6 0.78 76.2 (61.5e94.2) 34 (25e46) 340 (253e505) 35 (22e56)Multan-2 Potato Nov. 05 280 1.94 � 0.23 4.66 5 0.46 73.4 (55.2e94.3) 33 (23e46) 336 (241e544) 34 (20e57)Shujabad-2 Okra May 06 320 1.88 � 0.17 4.48 6 0.61 115 (92.8e144) 52 (38e70) 552 (401e850) 56 (34e93)Multan-3 Cotton Oct. 06 320 2.08 � 0.19 3.62 6 0.73 175 (143e216) 78 (58e106) 725 (544e1063) 74 (46e119)Multan-4 Clover Apr. 07 320 2.10 � 0.19 3.08 6 0.80 87.7 (71.4e108) 39 (30e54) 358 (269e523) 36 (23e60)

Fenpropathrin Multan-1 Okra May 98 320 2.01 � 0.18 2.79 6 0.83 4.54 (3.67e5.59) 1.0 19.7 (14.7e28.9) 1.0Lar-1 Cotton Jul. 98 240 3.06 � 0.33 1.51 4 0.82 11.0 (9.24e13.0) 2.4 (1.8e3.1) 28.8 (22.9e39.4) 1.5 (1.0e2.2)Shershah-1 Cotton Oct. 98 240 2.83 � 0.30 3.01 4 0.56 18.7 (15.7e22.4) 4.1 (3.1e5.4) 53.2 (41.9e74.1) 2.7 (1.7e4.2)Khokhran-1 Clover Feb. 99 320 1.87 � 0.17 4.32 6 0.63 19.4 (15.6e24.2) 4.3 (3.1e5.8) 94.3 (69.2e143) 4.8 (2.9e7.8)Muzaffargarh Cotton Oct. 99 360 1.33 � 0.12 4.23 7 0.75 24.2 (18.5e32.0) 5.3 (3.7e7.5) 223 (144e407) 11 (6.1e21)Shujabad-1 Clover Apr. 00 280 1.90 � 0.19 2.51 5 0.77 31.7 (25.3e39.5) 7.0 (5.2e9.5) 149 (108e232) 7.6 (4.6e13)Bosan-1 Okra Jun. 00 320 1.91 � 0.18 3.78 6 0.71 36.7 (29.5e45.6) 8.1 (5.9e11) 172 (127e259) 8.7 (5.3e14)Khanewal-1 Cotton Aug. 00 320 1.88 � 0.17 4.48 6 0.61 35.5 (28.4e44.1) 7.8 (5.8e11) 170 (125e256) 8.6 (5.3e14)Kabirwala-1 Clover Jan. 01 320 2.35 � 0.22 2.73 6 0.84 40.3 (33.2e48.9) 8.9 (6.6e12) 141 (109e200) 7.2 (4.6e11)Lodhran-1 Okra May 01 320 1.81 � 0.17 4.68 6 0.59 78.7 (62.8e98.4) 17 (13e24) 401 (292e616) 20 (12e34)Vehari-1 Cotton Aug. 02 360 1.67 � 0.14 5.19 7 0.64 75.4 (59.8e95.3) 17 (12e22) 443 (316e692) 22 (13e37)Mailsi Potato Nov. 02 280 2.32 � 0.23 3.56 5 0.61 77.5 (63.7e94.5) 17 (13e23) 277 (210e405) 14 (8.8e22)Khanewal-2 Clover Apr. 03 240 2.63 � 0.32 1.17 4 0.88 111 (88.8e136) 24 (18e32) 340 (261e501) 17 (11e27)Lar-2 Okra Jul. 03 320 1.78 � 0.16 4.93 6 0.55 181 (144e227) 40 (30e55) 946 (681e1469) 48 (29e82)Bosan-2 Okra Apr. 04 320 1.94 � 0.18 4.01 6 0.68 215 (174e267) 47 (35e64) 986 (723e1493) 50 (30e81)Kabirwala-2 Cotton Oct. 04 280 1.95 � 0.19 7.21 5 0.21 216 (150e306) 48 (35e65) 980 (622e2096) 50 (30e82)Multan-2 Potato Nov. 05 320 1.84 � 0.17 4.36 6 0.63 310 (248e387) 68 (51e95) 1549 (1131e2359) 79 (48e132)Multan-3 Cotton Oct. 06 320 1.84 � 0.17 3.71 6 0.72 368 (295e460) 81 (59e108) 1836 (1333e2818) 93 (56e150)

a Probit model fitted using POLO-PLUS programme (LeOra Software, 2003).b RF ¼ LC50 or LC90 resistant population/LC50 or LC90 reference population.

M. Ahmad, M.I. Arif / Crop Protection 29 (2010) 1428e1433 1431

Table 2Pairwise correlation coefficient comparisons between LC values of the insecticides tested on the field populations of Spodoptera exigua, Pakistan.

Insecticide Endosulfan Chlorpyrifos Quinalphos Cypermethrin Deltamethrin Bifenthrin

LC50 LC90 LC50 LC90 LC50 LC90 LC50 LC90 LC50 LC90 LC50 LC90

Chlorpyrifos �0.424ns �0.458ns

Quinalphos �0.261ns �0.350ns 0.8920.01 0.8880.01

Cypermethrin �0.180ns �0.219ns 0.9020.01 0.8750.01 0.9600.01 0.9500.01

Deltamethrin �0.158ns �0.161ns 0.8950.01 0.8960.01 0.9460.01 0.9140.01 0.9720.01 0.9580.01

Bifenthrin �0.287ns �0.339ns 0.9210.01 0.9210.01 0.9100.01 0.8980.01 0.9520.01 0.9380.01 0.9410.01 0.9290.01

Fenpropathrin �0.174ns �0.191ns 0.8920.01 0.8650.01 0.9330.01 0.8600.01 0.9860.01 0.9410.01 0.9520.01 0.8910.01 0.9230.01 0.9030.01

Superscripts denote significance of the regression.

M. Ahmad, M.I. Arif / Crop Protection 29 (2010) 1428e14331432

slopes for all the seven insecticides tested herein (Table 1);hence, the Multan-1 population was used here as a referencestrain and its LC values were used as baselines to calculateresistance factors for the respective insecticides. The baseline LCvalues for endosulfan, chlorpyrifos and quinalphos were similarbut twice more than the pyrethroids cypermethrin, deltamethrinand bifenthrin. The LC values of fenpropathrin were higher thanthe rest of pyrethroids.

During the study period from 1998 to 2007, S. exigua resistanceto endosulfan had been declining over the years whereas it wasincreasing to OPs and pyrethroids. This is due to the fact that OPsand pyrethroids were consistently being used in the Pakistaniagriculture while endosulfan use was decreasing during the period.Compared with the Pakistani populations of S. exigua from thesame area during the same period (Table 1), the S. litura resistanceto chlorpyrifos, quinalphos and bifenthrin did not increase overtime and it usually remained lower than S. exigua (Ahmad et al.,2007).

A moderate to high resistance was found to OPs and pyrethroidsin S. exigua during 2000s in our study (Table 1). A very high resis-tance was also reported to profenofos and pyrethroids in this pestfrom China (Chen et al., 2002; Wang et al., 2002; Mu et al., 2005),and to pyrethroids from Guatemala (Delorme et al., 1988) andMexico (Teran-Vargas, 1997). Simultaneously, a moderate to highresistance to pyrethroids was found in the Pakistani populations ofS. litura (Ahmad et al., 2007; Saleem et al., 2008).

Correlation analysis demonstrated that there was cross-resis-tance within the same insecticide classes i.e., OPs or pyrethroids inS. exigua (Table 2). Similarly, insecticides belonging to OPs orpyrethroids exhibited positive cross-resistance in the same class inS. litura from Pakistan (Ahmad et al., 2007). A highly positivecorrelation also existed between OPs and pyrethroids in S. exigua inthe current study. Owing to their dissimilar modes of action, thiscross-resistance between OPs and pyrethroids may be mediated byone or more common mechanisms of metabolic detoxification.Hence, OPs and pyrethroids should not be mixed or rotated for thecontrol of resistant populations of S. exigua. No cross-resistancewasfound between endosulfan and OPs or pyrethroids in S. exigua(Table 2), which was in concurrence with results for S. litura(Ahmad et al., 2007). This implies that endosulfan can be rotatedwith OPs and pyrethroids for the management of insecticideresistance in S. exigua.

Synergism studies indicate that metabolic detoxification bycytochrome P450 monooxygenases and esterases may be partiallyinvolved in imparting resistance to pyrethroids in the Pakistanipopulations of S. exigua (Ahmad, M., unpublished data). Pyrethroidresistance of a Chinese strain of S. exigua was associated with thedecreased cuticular penetration, nerve insensitivity and increasedactivities of mixed-function oxidases, esterases and glutathioneS-transferases (Wang et al., 2002). Mechanisms of delayed cuticularpenetration (Liu and Shen, 2003a) and enhanced metabolicdetoxification by cytochrome P450 monooxygenases (Liu and Shen,

2003b) were responsible for imparting resistance to lambdacyha-lothrin in another Chinese strain of S. exigua. Esterase metabolismand reduced penetration were the causes of resistance to delta-methrin in S. exigua from Guatemala (Delorme et al., 1988). Bothcytochrome P450 monooxygenases and esterases were partiallyresponsible for a very high level of resistance to OPs and pyre-throids in S. exigua from Mexico (Garza-Urbina and Teran-Vargas,1998). Methomyl resistance in field populations of S. exigua wasfound to be due to an insensitive cholinesterase (Byrne andToscano, 2001).

The present studies demonstrate that, for several Pakistani pop-ulations of S. exigua, resistance is now rampant tomajor conventionalchemistries such as endosulfan, OPs and pyrethroids. Similarly,resistance is widespread in another armyworm S. litura in Pakistanagainst all the conventional chemistries (Ahmad et al., 2007; Saleemet al., 2008). Growers are already experiencing control failures ofarmyworms via the old chemistries. It is therefore advisable that theconventional insecticides be replaced with the new andmore potentchemistries having novel modes of action. Our advice, disseminatedthrough the department of agricultural extension, growers havealready started using emamectin benzoate, flubendiamide and insectgrowth regulators (IGRs), particularly ecdysone agonists methox-yfenozide and benzoylphenyl ureas (BPUs) lufenuron, flufenoxuronand chlorfluazuron, for the control of these armyworms with greatsuccess. It is recommendedthat thesenewchemistries shouldbeusedin rotation in conjunction with other Integrated Pest Management(IPM) tactics.

Acknowledgements

The technical assistance of Rashid Mehmood, Munir Ahmad,Abid Hameed Khan, Tanveer Hussain, Tabassam Nurin, Hina Ranaand Qudsia Naz is much appreciated.

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