019, Tamil Nadu, India.

Transcription

1 International Journal of Latest Research in Science and Technology Volume 5, Issue 5: Page No 22-27, September-October ISSN (Online): PHYTOPESTICIDAL ACTION OF HYPTIS SUAVEOLENS, MELOCHIA CORCHORIFOLIA AND THEIR BIOACTIVE MOLECULES ON COTTON BOLLWORM, HELICOVERPA ARMIGERA (HUBNER) (LEPIDOPTERA: NOCTUIDAE) M. Pavunraj 1*, V. Ramesh 2, P. Murali 3 S. Janarthanan 4 and S. Igancimuthu 5 1 Post Graduate & Research Department of Zoology, Vivekananda College, Affiliated to Madurai Kamaraj University (MKU), Tiruvedakam West, Madurai, District , Tamil Nadu, India. 2 Department of Botany, Vivekananda College, Affiliated to Madurai Kamaraj University (MKU), Tiruvedakam West, Madurai, District , Tamil Nadu, India. 3 N.M.S.S. Vellaichamy Nadar College, Affiliated to Madurai Kamaraj University (MKU), Nagamalai, Madurai, District , Tamil Nadu, India. 4 Department of Zoology, University of Madras, Guindy Campus, Chennai , Tamil Nadu, India. 5 Entomology Research Institute, Loyola College, Chennai , Tamil Nadu, India. Abstract The phytopesticidal effects of crude extracts, fractions and an isolated compound from the leaves of Hyptis suaveolens and Melochia corchorifoliawas tested for their antifeedant activity against the third instar larvae ofhelicoverpa armigera. Among various solvent extracts tested, crude ethyl acetate extract of both the plants were found to be very effective on the larvae of H. armigera. Subsequently, crude ethyl acetate extracts of both the plants were column fractionated.column fraction-2 of H. suaveolens and column fraction-9 of M. corchorifolia at 1,000 ppm exhibited maximum antifeedant activity against the larvae of H. armigera. Molecular analysis of both the active larval antifeedant fractions revealed a single major compound, caryophyllene in H. suaveolensand â-sitosterol in M. corchorifolia. These two compounds were used further to prepare different biopesticidal formulations using neem and pongam oils. The formulations tested against the larvae of H. armigera conferredhigh antifeedantactivities at 500 ppm concentrations. Key words - Hyptis suaveolens, Melochia corchorifolia,phytopesticidal formulation,helicoverpa armigera, Lepidopteran pest I. INTRODUCTION The cotton bollworm, Helicoverpa armigera (Noctuidae: Lepidoptera) is a polyphagous pest damaging a wide range of economically important crops such as cotton, chickpea, pigeon pea, tomato, maize, sorghum, pearl millet etc [1&2]. It is a major pest and responsible for significant reduction in the yield [3]. Depending upon the crop, H. armigera inflict damage in the range of percent of the crop yield [3&4]. The present agriculture system relies largely on chemical pesticides and fertilizers. Farmers are spending lot of money for chemical pesticides in our country. They are applying heavy amounts of chemical pesticides and this unrestrained application of chemicals has created many environmental and health problems. Due to the unrestrained application of synthetic chemicals this pest has evolved a high degree of resistance to organophosphates, carbamates and pyrethroid insecticides and has become less feasible for its management [5]. The increasing number of pest species, pesticide resistance in pests and secondary pest outbreak are some significant side effects of chemical pesticides. Some of them induce malignancy in non-target organisms, while most give rise to development of resistant in insect population, or cause resurgence of minor/alternate pests. In the search for safe and more congenial alternatives, attention has been focused on botanicals. The potential of several secondary chemical compounds in plants to disrupt specific physiological mechanisms involved in nutrition, reproduction, metamorphosis and behavior of insects could be exploited in the control of insect pest as an eco-friendly alternative to the conventional pesticide use. The increasing cost of crop protection and urbanization has placed the agriculture in a critical position. In this context there is a need for searching eco-friendly compounds especially from plant sources. Several secondary metabolites of plants have been reported to be useful in controlling insect pests that they are known to affect various physiological and behavioural activities of insects [6]. There exist some reports about some other plants having such antifeedant and insecticidal properties [7-13]. In this context, the present investigation two well-known traditional medicinal plants of India such as H. suaveolens and M. ISSN:

2 corchorifolia were used to investigate their insect antifeedant activities. II. MATERIALS AND METHODS Plant collection and preparation of leaf extracts Leaves of mint weed, H. suaveolens L. Poit (Lamiaceae) and chocolate weed, M. corchorifolia L. (Sterculiaceae) were collected during 2006 in and around Chennai, Tamil Nadu, India. Plants were identified and voucher specimens of these plants (LC/ERI/Her.No.557 and 558) have been deposited in the herbarium collection at Entomology Research Institute, Loyola College, Chennai, India. The leaves were shade-dried at room temperature and coarsely powdered in a powdering machine. One kilogram powder of each plant was extracted sequentially with hexane, chloroform and ethyl acetate at room temperature after 48 h of soaking in an aspirator bottle with the respective solvents. The extracts were filtered through Whatman No.1 filter paper and condensed by vacuum evaporator. The crude extracts were stored at 4 C until used. Fig. 2 Percent antifeedant activity of different crude extracts of M. corchorifolia against 3 rd instar larvae of H. armigera Antifeedant activity The antifeedant activity of hexane, chloroform and ethyl acetate crude extracts of leaves of H. suaveolens and M. corchorifolia was studied at 1% concentration using leaf disc [15] under no-choice [16] method. Treatment solutions were prepared by dissolving the crude extract in 5 % acetone solution with tween-20 at a concentration of 0.05 % [17]. The fresh cotton leaf discs of 4 cm diameter were punched using cork borer and dipped in the crude extracts separately and air dried for 5 min. Third instars larvae, pre-starved for 2h, were released on the treated leaf discs. Leaf discs treated with 5 % acetone solution with 0.05 % of tween-20 were considered as control. Five replicates were maintained for each treatment with 10 larvae per replicate). Progressive consumption of leaf area by the larvae was recorded after 24 h in treatment and control using leaf area meter (Delta-T Devices, Serial No F 96 UK). The percentage of antifeedant activity was calculated using the formula of [18]: Isolation of fractions Fig. 1 Percent antifeedant activity of different crude extracts of H. suaveolens against 3 rd instar larvae of H. armigera Insects Third instar larvae of H. armigera were obtained from the laboratory culture at Entomology Research Institute, Loyola College, Chennai, India. The general rearing of larvae was carried out on semisynthetic diet at laboratory conditions (28 1 C; h photoperiod; 65 70% R.H) [14]. The effective crude ethyl acetate extracts of leaves of H. suaveolens and M. corchorifolia were separately subjected to column chromatography. A glass column was packed with silica gel (Acme s silica gel mesh) in hexane and the crude extract was eluted with hexane (100%), hexane and ethyl acetate (in the ratios between 95:5 and 5:95) and ethyl acetate (100%). All the collected fractions were run on thin layer chromatography (TLC) and similar fractions were pooled together. A total of 15 column fractions of H. suaveolens and 13 column fractions of M. corchorifolia were collected. Screening of fractions for antifeedant activity All the 15 column fractions of extracts of H. suaveolens and 13 column fractions of M. corchorifolia were tested for their antifeedant activity against 3 rd larvae of H. armigera at 1,000 ppm concentration. The procedure described for crude extracts was followed for larval antifeedant activity. Molecular analysis of active antifeedant fraction Column fraction-2 of H. suaveolens and fraction-9 of M. corchorifolia with a single spot identified in the TLC profile which were also exhibiting higher larval antifeedant activity was further characterized using GC MS (Shimadzu- QP2010 chromatography system with software version 2.53), IR (Shimadzu FT IR 8000 series in the region between cm 1 ), 1 H NMR (at 23 C in CDCl 3 using a Burker 300 MHz spectrometer), 13 C NMR (Burker 300, 75MHz spectrometer in CDCl 3 as solvent). The molecular structure ISSN:

3 was predicted for the fractions of antifeedant substance based on the spectrum of infrared spectroscopy and 1 H and 13 C NMR. Preparation and evaluation of pesticidal formulations using the bioactive molecules The following formulations were prepared by mixing these two active compounds namely caryophyllene and â-sitosterol isolated from H. suaveolens and M. corchorifolia respectively. The active compounds were mixed with neem and pongam oils. Suitable emulsifier (DMA-NE) and stabilizer (UNITOP chemicals) were used. extracts.in linear regression analysis, significant and high correlation was noted between concentration and antifeedant activity with [y=17.94x (R²=0.958)] against H. armigera. Formulation 1 Formulation 2 Control Caryophyllene â-sitosterol Emulsifier mg mg 1.00 Neem oil 4.45 Neem oil 4.45 Stabilizer 0.10 Pongam 4.45 Pongam 4.45 Distilled water Emulsifier Stabilizer 0.10 Total = 10 Emulsifier 1.00 Stabilizer 0.10 Total = 10 Total = 10 These formulations at 62.5, 125, 250 and 500 ppm doses were then assayed for their larval antifeedant activities with the third instar larvae of H. armigera. Statistical analysis The data related to antifeedant activity were presented in graph. Concentration dependent larval mortality was calculated by linear regression analysis. Values are percentage mean of five replicates. III. RESULTS Antifeedant activity of crude extracts and column fractions H. suaveolens leaves derived hexane, chloroform and ethyl acetate extracts showed antifeedant activities against the 3 rd larvae of H. armigera at different concentrations are illustrated in Figure 1. The results showed that the ethyl acetate extract of H. suaveolens was the most effective treatment that recorded the antifeedant activity of 88.16% against H. armigera followed by chloroform (65.28%) and hexane (31.65%) extracts of H. suaveolens at 5% concentration. The ethyl acetate extract exhibited the higher linear relationship between concentration and antifeedant activity [y=17.12x (R²=0.971)] followed by chloroform and hexane extracts. All the treatments showed good R 2 value of more than 0.94 for concentration dependent activity. The results of the antifeedant activities of different crude solvent extracts of M. corchorifolia against H. armigera are presented in (Fig. 2). The ethyl acetate extracts of M. corchorifolia recorded a significantly maximum antifeedant activity of 76.47% over chloroform and hexane Fig. 3 Percent antifeedant activity of different fractions of ethyl acetate extracts of H. suaveolens at 1,000 ppm concentration against 3 rd instar larvae of H. armigera Antifeedant activities were screened at 1,000 ppm cencetrations of all the column fractions. Among the fifteen column fractions of H. suaveolens screened, the highest antifeedant activity was recorded in fraction-2 (55.16%), which was eluted with hexane: ethyl acetate at the ratio of 90:10 (Figure 3) followed by fractions 15, 11 and 6 compared with control. There were thirteen fractions isolated from ethyl acetate extract of M. corchorifolia and among them fraction-9 recorded the highest antifeedant activity (48.21%) followed by fractions 6, 13 and 10. The effective fraction-9 was eluted with 60:40 ratio of hexane:ethyl acetate (Fig 4). Molecular analyses of fraction-2 of H. suaveolens and fraction-9 of M. corchorifolia The GC MS analysis of fraction-2 of H. suaveolens showed a single compound at RT.19.7 sec. KBr IR cm 1 : 3030, 2923, 2852, 1462, 1378, 908, 760. Max ISSN:

4 Fig. 4 Percent antifeedant activity of different fractions of ethyl acetate extracts of M. corchorifolia at 1,000ppm concentration against 3 rd instar larvae of H. armigera GC-MS: 204 (M + ), 189 [M CH 3 ] +, 175 [M CH 3 CH 2 ] +, 161 [M CH 3 (CH 2 )] +, 147 [M CH 3 (CH 2 ) 2 ] +, 133 [M CH 3 (CH 2 ) 4 ] +, 120, 105, 93, 79, 69, 55, [CH 3 ) 2 C CH] +, 40 [CH 3 C CH] + The GC MS spectrum showed it as a single compound at M (C 15 H 24 ). The compound was identified as caryophyllene in comparison of its mass spectrum with that of caryophyllene from the GC-MS library NIST. The superimposability was 96%. The IR spectrum revealed the existence of gemdimethyl group (1378 cm 1 ) and the presence of unsaturation (1462, 908, 760 cm 1 ). The characteristic fragments found in the mass spectrum also confirmed the structure. The structure of the compound was in comparison with the spectral data from earlier report of Peerzada (1997). correspond to those reported in the literature (Friedland, et al. 1959). MS spectrum of the sterol also showed the presence of â-sitosterol. The mass spectral fragmentation pattern of the â- sitosterol, the fragmentation pathways coincide with those reported in the literature (Friedland et al. 1959; Eneroth et al. 1964; Bindseil et al. 2001; Patric, 2006). 1 H NMR (ä, CDCLC 3, 400 MHz) (sterol methyls), 3.55 (1H, m, H-3) and ( olefinic proteins) EI-MS m/s 414 (M + ), 396, 381, 351, 329, 316, 303, 289, 273, 255, 231, 213, 199, 186, 173, 159, 145, 132, 119, 105, 94, 81, 68, 57, 43. C 29 H 5 O = 414, M+ 414, [M H 2 O] + 396, [M H 2 O CH 3 ] + 381, [M sidechain] + 273, [M sidechain H 2 O] 255, [M sidechain ring D fragment 42] + 231, 231-H 2 O 213, M/Z 303 more intense than M/Z 300 is characteristic of â-sitosterol. H HO â sitosterol Antifeedant activitiy of formulations prepared using bioactive compounds Caryophyllene The fraction-9 of M. corchorifolia on drying and crystallization from methanol gave a colorless crystal solid with melting point 137 C (yield 200 mg). It gave positive for Lieberman Burchard test (sterol substance in chloroform + AC 2 + H 2 O + H 2 SO 4 green colour). A single spot (RF = 0.43) was observed on TLC over silica gel with 1:1 sulphuric acid spray heated at 110 C for 5 min. The compound was identified as â-sitosterol. The structure of compound was confirmed in comparison with its spectral data and through the literature values. A direct comparison was also performed with authentic sample (mp, mmp, Co-TLC and superimposable IR). KBr IR cm 1 : 3442 & 1062 (Hydroxy), 2936, 2867, 1646, 1463, 1381, 1240, 1023, 969, 956, (trans di-substituted double bond), 838, (Trisubstituted double bond), 800, 739. Max The IR spectrum showed the presence of hydroxyl 3421 cm 1, transtituted double bond in sterols 1646, 838 cm 1 and trans olefinic linkage in side chain = 1646, 838, 800, H NMR of â-sitosterol showed the sterol methyl in the region q and H-3 at ä 3.30 as multiplet. The olefinic proteins appeared in region ä , the upfield peaks due to side chain double bond. The 1 H NMR values The results of antifeedant activity of different oil formulations prepared with active compounds isolated from the selected plants against the larvae of H. armigera are shown in Fig 5. Effective antifeedant activity at a concentration of 500 ppm of formulation-1 was 94.35% and formulation-2 was 87.70%. Concentration-dependent antifeedant activity was recorded against the target larval pests. R 2 were greater than 0.99 for all the two formulation against tested larval pests. These two oil formulations also produced various kinds of abnormalities in larva, pupa and adults of H. armigera (Fig 6). IV. DISCUSSION Plants are the potential source of new pesticidal molecules and search for new insecticides compounds from plants have been intensified during the past few decades [19]. Plant toxic compounds are the limiting factors for herbivore attack on plants and their products [20]. Plant secondary chemical compounds are responsible for disruption of certain physiological mechanisms involved in feeding, reproduction, metamorphosis and behavior of insects and hence they are exploited in eco-friendly insect pest management [21]. There are plenty of literatures available on insecticidal, antifeedant, oviposition deterrent, ovicidal, repellent and growth inhibiting properties of botanical pesticides [22, 9, 23, 14, 12 & 24]. ISSN:

5 Fig. 5 Percent antifeedant activity of two different oil formulations prepared using bioactive molecules against 3 rd instar larvae of H. armigera Our results have shown that the crude ethyl acetate extracts of H. suaveolens andm. corchorifolia at 1% concentration possess very active feeding deterrents against the larvae of H. armigera. These results are in agreement with the reports of Pavunraj et al. [10, 14 & 25] who observed that the ethyl acetate extracts of Lippia javanica, Pergularia daemia and Alangium salvifolium had higher antifeedant activities on S. litura and H. armigera. The combination of neem and pongam oils along with active molecules at a concentration of 1,000 ppm exhibited very good antifeedant activity against the larvae of H. armigera. These findings coincide with results of Maria Packiam and Ignacimuthu [26], who reported that PONNEEM, a neembased botanical oil formulation was responsible for more than 80% antifeedant activity against S. litura larvae. Vendan et al. (2010) have reported that neem oil based formulation of active fraction isolated from ethyl acetate extract of Hydnocarpus alpina presented higher antifeedant activity against S. litura. Pavunraj et al. [13] reported that effective fraction from Melochia corchorifolia with 1:1 ratio of neem and pongam oils showed antifeedant activity against four lepidopteran pests. Due to growth inhibitory effect of the formulations, treated larvae showed abnormalities in larval, pupal and adult structures. The morphological deformities at various life stages of lepidopteran pests due to botanicals treatment were already reported) [27& 12]. Fig.6 Different malformations in H. armigera due to the treatments of oil formulations prepared using bioactive compounds. The biological compounds present in the plants have a vital role in regulating the growth of insect pests by way of working as juvenile hormone of insect pests. As a result they arrest the function of ecdysone. The larvae are therefore unable to moult and continue as larval instar [28]. In the present study, larvae treated with formulation-1 exhibited the maximum percentage of deformities in larval, pupal and adult forms. Telang et al. [29] stated that, malformed adult insects were produced as a result of plant toxin treatments and the insects were also short-lived and infertile. These effects could be considered important in the pest population reduction. The results of the present investigations are in agreement with aforementioned findings. Thus it is concluded that the effectiveness of neem and pongam oil based formulations with the bioactive molecules such as caryophyllene from H. suaveolens and â-sitosterol from M. corchorifolia are effective as feeding deterrents to H. armigera. Based on these results, an application of both the formulations could potentially prevent the damage of larvaeof H. armigera. It should be noted that the test subjects were confined to petridishes in the laboratory condition without any information on other relevant factors such as starvation tolerance, natural disperse ability, morphological structure and vegetation coverage of the crops, etc to the larvae exposed to these formulations. These formulations could be considered as eco-friendly botanical pesticides for crop protection after filling those gap studies as mentioned above. V. CONCLUSION The commercially available neem based biopesticides are prepared with azadirachtin. The cost of neem oil and pongam oil is cheaper than that of azadirachtin and these oils are easily available and can be used at low cost. The synergistic activity of these oils with the bioactive molecules caryophyllene from H. suaveolens and â-sitosterol from M. corchorifolia tested in the present investigation has given encouraging results and these newly developed formulations can be used for cost-efficient and environmentally friendly and sustainable agriculture from the pest attack in future. ISSN:

6 ACKNOWLEDGMENTS The authors thank the Council of Scientific and Industrial Research (CSIR), (Ref: No. 37(1204)/04/EMR-II), New Delhi for financial support. milkweed, Pergularia daemia on the cotton bollworm, Helicoverpa armigera and tobacco armyworm, Spodoptera litura. Phytoparasitica, (2): p Isman M.B, O. Koul, A. Lucyzynski, J. Kaminski. Insecticidal and antifeedant bioactivities of neem oils and their relationship to Azadirachtin content. J Agric Food Chem, : p REFERENCES 1. King, A.B.S. Heliothis/Helicoverpa (Lepidoptera: Noctuidae). In: Insect pests of Cotton Mathews. G.A., and Tunstall, J.P., (Eds.). CAB Publishing Inc., Wallingford, Oxon, UK. [1994] p Shanower, T.G., J. Romeis, Insect pests of pigeon pea and their management. Ann Rev Entomol : p Reed W, C.S. Pawar, Heliothis: a global problem. In Proceedings of the International Workshop on Heliothis Management, November 15 20, ICRISAT, Patancheru, India, [1982] p Sehgal V.K, R. Ujagir. Effect of synthetic pyrethroids, neem extracts and other insecticides for the control of pod damage by Helicoverpa armigera (Hubner) on chickpea and pod damageyield relationship at Pantnagar in northern India. Crop Protection.9: 1990: P Armes NJ, D.R. Jadhav, K.R. Desouza. A survey of insecticide resistance in Helicoverpa armigera in the Indian subcontinent. Bull Entomol Res, 1996, 86(5): p Arnason JT, B.J.R. Philogene, P. Morand, Insecticides of plant origin. In: American Chemical Society Symposium Series Vol. 387: [1989] Washington, DC. 7. Sharma, R.N, S. Deshpnde, G. Nanda. Biochemical analysis of acetone extract of Cassine glauca having antifeedant effect on castor semilooper (Achaea janata). Indian J. Agric. Sci, : p Raja N, A. Jeyasankar, V. Jeyakumar, S. Ignacimuthu. Efficacy of Hyptis suaveolens against lepidopteran pests. Curr. Sci, : p Pavunraj M, K. Subramanian, C. Muthu, S.P. Seenivasan, V. Duraipandiyan, S.M. Packiam, S. Ignacimuthu, Bioefficacy of Excoecaria agallocha (L.) leaf extract against the armyworm,spodoptera litura (Fab.) (Lepidoptera: Noctuidae). Entomon : p Pavunraj M, S. Ignacimuthu, K. Karthikeyan, S.M. Purushothaman. Antifeedant activity of Lippia javanica (Burm. f.) Spreng. Leaf extracts on tobacco cutworm, Spodoptera litura (Fab.) Lepidoptera Noctuidae). Indian J. Plant Prot, (1): p Ezhil V.S, S.Lingathurai, M. Gabriel Paulraj, S. Ignacimuthu, Bioefficacy of neem oil formulation with Hydnocarpus alpina leaf extract against Spodoptera litura. Int. J. Curr. Res, : p Baskar K, R. Maheshwaran, S. Kingsley, S. Ignacimuthu. Bioefficacy of plant extracts against Asian army worm Spodoptera litura Fab. (Lepidoptera: Noctuidae). J. Agric. Technol, : p Pavunraj M, M. Gabriel Pulraj, S. Selva Kumar, M.R.K..Rao, S. Ignacimuthu. Feeding deterrence, larvicidal and haemolymph protein profiles of an Indian traditional medicinal plant Alangium salviifolium (L.F.) Wangerin on cluster caterpillar, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Archives of Phytopathology and Plant Protection. 2012: p Pavunraj M, S. Ignacimuthu, S. Janarthanan, V. Duraipandiyan, S. Vimalraj, C. Muthu, N. Raja. Antifeedant activity of a novel 2- (4, 7-Dihydroxy - heptyl) quinone from the leaves of the 16. Maggi M.E, A. Mangeaud, M.C. Carpinella, C.G. Ferrayoli, G.R.Valladares, S.M. Palacios. Laboratory evaluation of Artemisia annua L. extract and Artemisinin activity against Epilachnapaenulata and Spodoptera eridania. J. Chem. Ecol, : p Saxena S.C, R.S. Yadav. A new plant extract to suppress the population of yellow fever and dengue vector Aedes aegypti L. (Diptera: Culicidae). Curr. Sci, : p Bentley M. D, D.E. Leonard, W.F. Stoddard, L.H. Zalkow. Pyrrolizidine alkaloids as larval feeding deterrents for spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae). Ann Entomol Soc Am, : p Villani M, F. Gould. Screening crude plant extracts as feeding deterrents of the swireworm, Melanotus communis. Entomol. Exp. Appl, : p Rosenthal G.A, E.A. Bell. Naturally occurring, toxic nonprotein amino acids, In: Rosenthal GA, Janzen DH, (eds), Herbivores their interaction with secondary plant metabolites. Academic Press, N.Y. [1979] p Joseph TM, Antifeedant and growth inhibitory effects of neem seed kernel extract on Ailanthus defoliator, Eligma narcissus indica.entomon, : p Koul O, M.P. Jain, V.K.Sharma. Growth inhibitory and antifeedant activity of extracts from Melia dubia to Spodoptera litura and Helicoverpa armigera larvae. Indian J. Exp. Biol, : p Goùawska S. Deterrence and toxicity of plant saponins for the pea aphid Acyrthosiphon pisum Harris. J. Chem. Ecol, : p Pavunraj M, K. Baskar, S. Ignacimuthu. Efficacy of Melochia corchorifolia L. (Sterculiaceae) on feeding behavior of four lepidopteran pests. Int. J. Agric. Res, 2012a. 7(2): p Pavunraj M, M. Gabriel Pulraj, S. Selva kumar, M.R.K. Rao, S. Ignacimuthu. Feeding deterrence, larvicidal and haemolymph protein profiles of an Indian traditional medicinal plant Alangium salviifolium (L.F.) Wangerin on cluster caterpillar, Spodoptera litura(fabricius) (Lepidoptera: Noctuidae). Arch. Phytopathol. plant protect, 2012b. 45 (17): p Maria Packiam S, S. Ignacimuthu. Phytopesticidal oil formulations for insect pest management - A review. Int. J. Interdisci. Res. Rev, (2): p Alvarez C.O, A. Neske, S. Popich, A. Bardon. Toxicity effect of annonaceous acetogenins from Annona cherimolia (Magnoliales: Annonaceae) on Spodoptera frugiperda (Lepidoptera: Noctuidae). J. Pest Sci, : p Koul O, M.B. Isman. Effect of azadirachtin on the dietary utilization and development of the varieted cutworm, Peridroma saucia. J. Insect Physiol, : p Telang M, A. Srinivasan, A. Patankar, A. Harsulkar, V. Joshi, A. Damle, V. Deshpande, M. Sainani, P. Ranjekar, G. Gupta, A. Birah, S. Rani, M. Kachole, A. Giri, V. Gupta. Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura. Phytochemistry, : p ISSN: