Aragalur

Thalaivasal Pest control

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Monitoring Techniques for Detecting Insecticide Resistance in Helicoverpa armigera (Hubner)

S. Gouthaman and A. Regupathy
Department of Entomology
Tamil Nadu Agricultural University
Coimbatore-641 003. INDIA

ABSTRACT In the absence of susceptible strain of Helicoverpa armigera in India, indirect method was used to fix discriminating doses (DD) for other methods of bioassay viz., vial, bouquet, spray tower and larval dip for commonly used insecticides viz., endosulfan, quinalphos, chlorpyriphos, fenvalerate and cypermethrin. The DD for other methods were extrapolated by multiplying the DD available for susceptible cultures of NRI, UK and Australia by topical application with the factor of ratio of LD99 of topical and other methods. Validation of extrapolated DDs was done by testing on different H. armigera populations. The extrapolated DDs inflicted mortality with standard error (SE) varying from 3.9 to 7.1 when batches of 50 insects were used. The variation could be reduced with more number of insects per test.

KEY WORDS: Discriminating dose, insecticide resistance, Helicoverpa armigera.

In India, resistance was first recorded in major cotton growing region of Andhra Pradesh in 1987 (McCaffery et al., 1989). Helicoverpa resistance to different chemicals is widely recognized and documented (Phokela et al., 1989; Pasupathy and Regupathy 1993; Arms et al., 1992, Regupathy et al., 2003). The effective resistance management depends on the successful monitoring programme. In most practical situations the best monitoring method is the use of discriminating doses i.e. the dose that kills 99% of susceptible individual (Roush and Miller, 1986).

In the monitoring programme the discriminating doses for fenvalerate, cypermethrin, quinalphos and endosulfan have been fixed using the susceptible cultures available in Australia for topical application method (Forrester and Cahill, 1987; Forrester et. al., 1993; Gunning et al., 1984). The high-tech nature of topical application prevents many field level workers and marginal farmers adopting this due to low literacy rate in India.

In the absence of susceptible lines for Helicoverpa in India, extrapolated discriminating doses for other bioassay methods are to be arrived. With this in view an attempt was made to extrapolate the discriminating doses for other methods from that of topical application.

MATERIALS and METHODS

Mass culturing of H. armigera

Various populations of H. armigera larvae were reared in a semi-synthetic diet as described by Sathiah, (1987).

Preparation of Insecticide Solutions

The insecticide solutions required for the discriminating dose assays were prepared from technical grade insecticides (of known purity) diluted with analytical grade acetone. The technicals used for different bioassay methods were, endosulfan (92%), chlorpyriphos (95%), quinalphos (71.5%), cypermethrin (94.8%) and fenvalerate (96.2) obtained from Thudialur Co-operative Agro. Service Pvt. Ltd., Coimbatore, Coramandal Indag Pvt. Ltd., Chennai, Syngenta India Ltd., Mumbai, National Organic Chemical Industries Ltd., Mumbai and Rallis India Ltd., Bangalore respectively.

For larval dip method the respective formulations viz., endosulfan (Thiodan 35 EC), quinalphos (Ekalux 25 EC), fenvalerate (Sumicidin 20 EC), cypermethrin (BILCYP 25 EC) and chlorpyriphos (Durmet 20 EC) were used.

Bioassay methods

In all the methods third instar larvae (30-40 mg) were used.

Topical assay

Serial dilutions of technical grade insecticides in analytical grade acetone were prepared and 1ul repeating dispenser (PB 600-01, Hamilton Co Ltd) fitted with 50 µl syringe and "Rheodyne needle" was used to deliver 1 µl drop to the thoracic dorsum of each larva.

Vial residue bioassay/Contact toxicity method

Glass scintillation vials of 20 ml capacity were evenly coated with 1 ml acetone solutions of technical grade insecticides (Plapp 1971). Larvae were released into the vials individually and covered with a muslin cloth.

Foliar residue bioassay Bouquet method/ Terminal bud bioassay

Chickpea shoots were taken and terminal branches were removed retaining only five compound leaves on each shoot. These shoots were surface sterilized in 0.5% sodium hypochlorite, rinsed in sterile water and shade dried (Rose Victoria, 1991). These shoots were dipped in respective concentration of the chemical which was prepared with analytical grade acetone containing Triton-X 100 surfactant (0.25 g/lit) for about 30 sec. and the excess fluid was drained off. The petioles of the shoots with cotton swab were kept immersed in water in a penicillin vial and allowed for shade drying. Each penicillin vial having the treated shoots was kept separately in a large plastic (19x10 cm) container to which third instar larvae were released individually. The mouth of the container was covered with muslin cloth and the larvae were allowed to feed on the shoots. After allowing 24 h of feeding, the larvae were transferred to semi-synthetic diet. A control was maintained with chickpea shoots treated with acetone plus Triton-X 100 alone.

Spray (potter's) tower bioassay

One ml of various concentrations of the technicals prepared in acetone was sprayed at a pressure of 2 kg/cm2 with the help of a spray tower on to the H. armigera larvae in a Petri dish. Each time a batch of 10 larvae were anesthetized with the help of CO2 and sprayed. The Petri dishes containing the larvae were dried for 5 min and the treated larvae were transferred individually into vials containing semi-synthetic diet.

Larval dip bioassay

Aqueous dilutions of formulated (emulsion concentrates) insecticides were prepared and batches of third instar larvae were submerged for 5 sec. as described by Watkinson et al (1984). A group of 50 larvae were dropped into 100 ml of the appropriate dilution in 500 ml beaker and gently swirled for 5 sec. to ensure complete wetting. The solution plus larvae were then poured through a fine nylon mesh suspended over an empty beaker. The solution was decanted and larvae separated by this process. After shade drying for about 5 min. the treated larvae were then transferred individually into semi synthetic diet. Control insects were treated with water alone.

Bioassays were carried out at 26 ± 1° under approximately 12h: 12h LD Photo period. Mortality was recorded at 24h interval up to 6 days. The log dose/concentration probit mortality curve was fitted to extrapolate LC/LD99 after making Abbott's correction.

The tentative discriminating doses of endosulfan, quinalphos, cypermethrin, fenvalerate and chlorpyriphos for different methods were extrapolated considering the following discriminating doses calibrated for topical application.

Endosulfan: 10.0 µg/µl (Approximate LD99 for susceptible strains calibrated in Australia).
Cypermethrin: 1 µg/ml (LD99 for Delhi strain ) (Phokela et al., 1989).
Quinalphos: 0.75 µg/ml (LD99 for NRI Lab susceptible strain).
Fenvalerate: 0.2 µg/ml (LD99 for susceptible strain in Australia) (Forester and Cahill, 1987).

The above discriminating doses for topical application were multiplied by the factor of ratio of LD99 of topical and other methods.

Validation of different methods

Various field populations or a particular location at periodic intervals were dosed with the extrapolated discriminating doses and mortality was recorded. The extrapolated discriminating doses for different methods viz., topical contact, foliar, spray tower and larval dip tested with batches 50-100 larvae and the mortality was recorded at an interval of 24 h for 6 days.

Corrected control mortality was calculated using Abott's formula (Abott, 1925) and binomial standard error was calculated by using the formula

P (100 - P) /(n-1)
P = % of larva surviving in the discriminating dose
n = Number of larva tested

Linear correlation was worked out for the combination of different methods.

RESULTS and DISCUSSION

Lethal dose

The LD99 of the tested insecticides by topical application varied from 2.24 µg - 36.70 µg; (Tables 1,2, 3, 4, and 5) the lowest for chlorpyriphos and the highest value for endosulfan.

The LD99 values were higher than the discriminating doses of these compounds used for assessing the resistance nature indicating the high degree of resistance of H. armigera population evaluated for these compounds.

Lethal concentration

The LC99 for the above chemicals by vial method was 94.40 µg for endosulfan, 36.49 µg for fenvalerate, 32.13 µg cypermethrin, 17.90 µg for quinalphos and 4.50 µg for chlorpyriphos. In the case of bouquet method the LC99 varied between 59.35 and 2571.20 ppm. The LC99 obtained by larval dip method varied from 123.02-3706.50 ppm. The LC99 obtained by spray tower method varied from 363.08-14003.67 ppm (Tables 1,2, 3, 4, and 5). Irrespective of the methods the order of toxicity of the insecticides was same i.e. chlorpyriphos > quinalphos > cypermethrin > fenvalerate > endosulfan.

The lowest values were observed for chlorpyriphos and highest for endosulfan in all the bioassay methods. The steepness of log - dose - probit - mortality (ldpm) lines for the various insecticides by different methods was more or less same as indicated from the slope function. The slope function was 1.78-1.92 for fenvalerate (Table 2), 1.81-1.92 for cypermethrin (Table 3) and 1.57-1.77 for quinalphos (Table 4). In the case of chlorpyriphos, the slope function was very low (2.12) for the spray tower method whereas for other methods the `b' value was 2.71 for vial, 2.89 for larval dip, 3.04 for topical and 3.03 bouquet method. In the case of endosulfan the slope function ranged from 2.16-3.48, the highest for larval dip and lowest for the topical (2.16) whereas that of the contact and spray tower were 2.41 and 2.45 respectively. The LC99 value was always the least in the vial method followed by bouquet, larval dip and spray tower method for all insecticides indicating the less variation in the response of H. armigera for different methods of bioassay.

Tentative Discriminating Doses

Provisional discriminating doses that kill 99 per cent were derived by extrapolation. The LD99: LC99 ratios between topical and other methods were worked out by taking topical method as unity (Table 6).

The LD99: LC99 ratio between topical and vial methods was 2.06 for endosulfan, 1.27 for fenvalerate, 2.58 for cypermethrin, 1.47 for quinalphos and 2.01 for chlorpyriphos. The ratios of LD99: LC99 of topical: bouquet methods were 70.05, 39.33, 75.08, 21.38 and 26.59 for endosulfan, fenvalerate, cypermethrin, quinalphos and chlorpyriphos respectively. The ratios of LD99: LC99 of topical: larval dip was 100.99, 54.41, 120.78, 57.64 and 54.92 for endosulfan, fenvalerate, cypermethrin, quinalphos and chlorpyriphos respectively. The ratios between topical and spray tower method were 781.60, 116.22, 267.74, 113.15 and 162.09 for endosulfan, fenvalerate, cypermethrin, quinalphos and chlorpyriphos respectively.

The discriminating doses of 1.0 µg, 0.75 µg, 0.2 µg, and 10 µg for cypermethrin, quinalphos, fenvalerate and endosulfan used for topical application were multiplied by the respective above ratios.The tentative discriminating doses derived for endosulfan were 25 µg, 700 ppm, 1015 ppm and 3800 ppm, for fenvalerate 0.25, 8 ppm, 10 ppm, 23 ppm, for cypermethrin 2.6 µg, 75 ppm, 125 ppm, 270 ppm and for quinalphos 1.1 µg, 16 ppm, 45 ppm and 85 ppm for the vial, bouquet, larval dip and spray tower method respectively.

The tentative discriminating doses were used to assess the resistance nature of different Helicoverpa populations viz., laboratory cultured TNAU (Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India) population and field collected populations from TNAU, Madurai, Chidambaram and Thalaivasal. In the case of chlorpyriphos LD/LC 99 values were used for comparing the different methods (Table 7).

Lab cultured TNAU population exhibited survival rate of 8-16% for endosulfan, 44-50% for cypermethrin, 76-84% for fenvalerate, 28-34% for quinalphos and 8-10% for chlorpyriphos. The survival rate of Madurai population for endosulfan was 10-16%, cypermethrin 30-36%, fenvalerate 68-72%, quinalphos 20-24% and chlorpyriphos 10-14%. The Chidambaram population showed the survival rate of 30-34% for endosulfan, 50-54% for cypermethrin, 76-80% for fenvalerate, 32-40% for quinalphos, and 16-20% for chlorpyriphos. The Thalaivasal population exhibited the survival rate of 18-24% for endosulfan, 28-34% for cypermethrin, 80-84% for fenvalerate and 18-22% for quinalphos. TNAU field collected population showed the survival rate of 8-16% for endosulfan, 44-48% for cypermethrin, 78-82% for fenvalerate, 30-36% for quinalphos, and 8-12% for chlorpyriphos.

Though the degree of resistance was found to be varying among the different populations and with the insecticides, the assessment made by different methods did not indicate much variation except in one or two cases.

The linear correlation co-efficients (r) were worked out to establish the relationship among different assay technique (Table 8). The variation in the survival rate as assessed by different assays was not much; the `r' values being 0.943-1.0 for endosulfan, 0.953-1.0 for cypermethrin, 0.883-0.989 for fenvalerate, 0.987-1.0 for quinalphos and 0.970-1.0 for chlorpyriphos.

Perfect relationship (r = 1) was obtained in cases topical vs. larval dip for endosulfan, topical vs. spray tower for cypermethrin, bouquet vs. spray tower for quinalphos, bouquet vs. larval dip, bouquet vs. spray tower and larval dip vs. spray tower for chlorpyriphos. The `r' values obtained for vial vs. bouquet for endosulfan and larval dip vs spray tower for fenvalerate were significant at p=0.05. The `r' values for all other combination were significant at p=0.01.

It indicated that any one of the above methods could be used for monitoring the resistance by field level functionaries. Among these the larval dip method was found to be easy-to-adopt. A ready to use field kit has been designed and is being tested by different institution.

REFERENCES

Abbott, W. S. 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol., 18: 265-267.

Armes, N. J., D. R. Jadhav and A. B. S. King. 1992. Pyrethroid resistance in the podborer, Helicoverpa armigera in southern India. pp 239-244. In: Proceedings of British Crop Protection Conference, Pests and Diseases. British Society of Chemical Industry. Brighton, DC.

Forrester, N.W. and M. Cahill. 1987. Management of insecticide resistance in Helicoverpa armigera (Hubner) in Australia. pp 127-137 In: Combating resistance to Xenobiotics, biological and chemical approaches (Eds M. G. Forel, D. W. Holloman, B.P.S. Khambay and K. M. Sawicki). Ecllis Hordwood Publishers.

Forrester, N.W., M.Cahill, L.J.bird and K.J.Layland. 1993. Management of pyrethroid and endosulfan resistance in Helicoverpa armigera (Lepidoptera : Noctuidae) in Australia. Bull. Ent. Res. Suppl., No. 1.

Gunning, R.V., C.S.Easton, L.R.Greenup and V.E.Edge. 1984. Pyrethroid resistance in Heliothis armigera (Hubner) (Lepidoptera : Noctuidae) in Australia. J. Econ. Entomol., 77 : 1283-1287.

McCaffery, A. R., A. B. S. King, A. J. Walker and H. El - Nayir. 1989. Resistance to synthetic pyrethroids in the boll worm, Heliothis armigera from Andhra Pradesh, India. Pesticide Res. J., 27: 65-76

Pasupathy, S. and A. Regupathy. 1993. Status of insecticide resistance in the American bollworm, Helicoverpa armigera in Tamil Nadu. Pesticide Res. J., 6: 117-120

Phokela, A., S. Dhingra and K. N. Mehrotra. 1989. Pyrethroid resistance in Heliothis armigera Hubner.I. Resistance to cypermethrin. Proc. Ant Acad. Sci. India, 59 (B) IV: 373-380.

Plapp, F. W. Jr. 1971. Insecticide resistance in Heliothis: Tolerance in larvae of H. virescens as Compared with H. zea to organophosphate insecticides. J. Econ. Entomol., 64(5): 999-1002

Regupathy, A., Keshav Kranthi, Joginder Singh, Arif Iqbal, Yidong wu, Derrek Russell. 2003. Patterns of Resistance levels in India, Pakistan and China. World Cotton Research Conference 3, 9-13, March, 2003, Cape Town, South Africa. Abst.. PS.30.9.

Rose Victoria. 1991. Biological control of podborers in chickpea Cicer arietinum L. and pigeonpea Cajanus cajan (L). Mill sp. M.Sc.(Ag.) Thesis. Tamil Nadu Agricultural University, Coimbatore 641 003

Rousch, R.T. and G.L. Miller. 1986. Considerations for design of insecticide resistance monitoring programs. J. Econ. Entomol., 79: 293-298.

Sathiah, N. 1987. Studies on increasing the effectiveness of Nuclear Polyhedrosis Virus against podborer, Heliothis armigera (Hubner) on Chickpea. M.Sc(Ag.) Thesis., Tamil Nadu Agricultural University, Coimbatore 641 003

Watkinson, I.A., J. Wireman and J. Robinson. 1984. A sample test for field evaluation of the susceptibility on insect pests to pesticides. pp 559-564 In: Proceedings, British Crop Protection Conferences Pests and Diseases British Society of Chemical Industry. Brighton, UK.