Title of Invention

A PHYTOPESTICIDAL FORMULATION 'PONNEEM' FOR CONTROLLING INSECT PESTS

Abstract

The primary object of the invention is to prepare a phytopesticdal formulation possessing antifeedant and insecticidal properties. PONNEEM consists of 44.5% Pongam oil, 44.5% Neem oil, 8% emulsifier, 1% stabilizer, 0.123% azadirachtin and 2% isopropyl alcohol. PONNEEM along with other formulations was tested against Spodoptera litura and Helicoverpa armigera for antifeedant and larvicidal activities. Quantitative and qualitative variation in the amount of protein of S. litura and H. armigera were also analyzed. PONNEEM exhibited the best antifeedant and larvicidal activities against insect pests due to synergistic effect. PONNEEM reduced the protein content of insect pests. Field trials using PONNEEM in groundnut ecosystem showed higher control of insect pests and protected more natural enemies. PONNEEM showed no toxic effects in rats. The synergism of PONNEEM was better than neem and pongam oils alone. PONNEEM can be a better alternative to chemical pesticides. It is safe and environment- friendly.

Full Text

FIELD OF INVENTION: This invention relates to the development of a new biopesticide formulation with antifeedant and insecticidal properties to control insect pests thereof. PRIOR ART In order to feed the fast growing population chemical pesticides are used continuously to prevent the damage caused by insect pests. But indiscriminate use of chemical pesticides over a long period has not only proved to be harmful to soil micro flora, animals and human life, but also has contributed to a number of side effects, viz. development of resistance by the insects/ weeds/pests resurgence and out break of new pests, toxicity to non-target organisms like beneficial insects and humans. An insecticide has traditionally targeted nervous system functions that are common to many species including humans. Dreadful facts of chemical pesticides demand for eco-friendly alternate method for crop protection. Plants are known to contain innumerable biologically active compounds. They yield valuable herbal products, which are often used in the treatment of various ailments. Plant oils and plant parts have also been traditionally used to control insects. Hence an attempt has been made to develop a formulation which is biodegradable and environment friendly using Pongam and Neem oils. Accordingly the present invention provides a new formulation, which contains oils from Azadirchta indica (Neem), Pongamia glabra (Pongam) and pure azadirachtin, emulsifier, stabilizer and isopropyl alcohol. OBJECTS OF THE INVENTION: The primary object of the invention is to propose a process for preparation of PONNEEM, a new biopesticide with antifeedant and insecticidal property and a new formulation thereof. Another object of this invention is to propose a process for preparation of PONNEEM, a new biopesticide with antifeedant and insecticidal property and a formulation thereof to overcome the disadvantages of the prior art. Further object of this invention is to propose a process for preparation of PONNEEM, a new biopesticide with antifeedant and insecticidal properties and new formulation thereof, which exhibited antifeedant and insecticidal activities. Still further object of this invention is to propose a process for preparation of PONNEEM, a new biopesticide with antifeedant and insecticidal properties and a new formulation thereof, which is not harmful to beneficial organisms and humans. STATEMENT OF THE INVENTION: According to this invention there is provided a process for preparation of PONNEEM, a new biopesticide comprising steps of: -Taking 44.5% Pongam oil + 44.5% Neem oil in a stainless steel vessel with a stirrer and stirring at 120 rpm for 10 minutes. Then adding 8% emulsifier + 1% stabilizer to the oils and again stirring at 120 rpm for 10 minutes. - Lastly adding 0.123% Azadirachtin + 2% isopropyl alcohol and again mixing thoroughly by using a stirrer at 120 rpm for 10 minutes. - Then the final formulation 1 was obtained which is named PONNEEM. Further according to this invention there is provided a new biopesticide with antifeedant and insecticidal properties which comprises PONNEEM consisting 44.5% Pongam oil + 44.5% Neem oil + 0.123% azadirachtin (w/v) + 8 % emulsifier + 1% stabilizer and 2% isopropyl alcohol. BRIEF DESCRIPTION OF THE PROCESS: Further objects and advantages of this invention will be more apparent from the ensuing description when read in conjunction with the following details and plates. NATURE OF COMPONENTS: EMULSIFIER: The emulsifying agent used for the present formulation was DMA-NE from Unitop, which is a powerful emulsifying agent for neem and pongam oils. The appearance of the emulsifier is amber colour liquid and a blend of anionic and nonionic surfactant. It is easily dispersible in water. The inert chemical nature does not have deteriorating effect on the basic azadirachtin present in the neem oil and karajin and pongamol in pongam oil. It is biodegradable and environment friendly emulsifier and does not contain any hazardous chemicals and solvents like toluene, benzene, etc. STABILIZER: The composition of the new formulation contains other ingredients such as stabilizer, which is used to increase the stability of the oil formulation during storage. Unistab-P, a powerful acid scavenger and stabilizer from Epoxy oil was used in the new formulation. The appearance of the oil is pale yellow, viscous clear liquid. It is a safe compound, non-corrosive, non-toxic, which is mainly based on soybean oil. ISOPROPYL ALCOHOL: It is used in the present formulation as UV protectant and as an intermediator between oil and emulsifier. It is useful in solubility of azadirachtin. PONGAM OIL Pongam oil is thick yellow orange to brown and is extracted from seeds of Pongamia glabra. The oil has a bitter taste and a disagreeable aroma. The oil is used as a fuel for cooking and lighting lamps and also used as lubricant, water paint binder, pesticide and in soap making and tanning industries. The oil is known to have value in folk medicine for the treatment of rheumatism, as well as human and animal disease. It is effective in enhancing the pigmentation of skin affected by leucoderma or scabies. The oil contains flavonoid including 1.25% karanjin and 0.85% pongamol. The acute oral toxicity to rats exceeds 4000mg/kg and dermal toxicity exceeds 2000mg/kg body weight. NEEM OIL: Neem oil is greenish brown in colour extracted from the seed kernel of Azadirachta indica. The oil has repulsive odour. The oil contains several terpenoids, steroids, alkaloids, flavonoid, glycosides, etc. The isolated constituents are Margosic acid, Nimbin, Nimbidin, Nimbinin, kaemperol, Azadirone, Quercursertin, B-Sitosterol, Praisine, Vanilic acid, Nimbecetin, Meliacins, Azadirachtin etc. The main active ingredient of neem is azadirachtin, a tetranortriterpenoid. It exhibits antifeedant, insect repellent and insect sterilization properties. It interferes with ecdysone, the key insect moulting hormone and prevents larvae and pupae from completing the moulting process. Acute oral toxicity to rats exceeds 5000mg/kg and dermal toxicity exceeds 2000mg/kg of body weight. AZADIRACHTIN: Azadirachtin, one of the major phago-repellents of neem seed, was originally isolated by Butterworth and Morgan (1968) and its structure was proposed by Zanno et al (1975). Aza has feeding deterrent properties, insect growth regulator. The larval adult development is disrupted due to which no moulting taking place. Aza does not have bad effects on mammals, birds, useful insects and the environment. DETAILED DESCRIPTION OF THE INVENTION WITH REFERENCE TO VARIOUS FORMULATIONS: Five different formulations were prepared as follows. Formulation 1: 44.5% Pongam oil + 44.5% Neem oil + 0.123% azadirachtin (w/v) + 8 % emulsifier + 1% stabilizer and 2% isopropyl alcohol. Preparation: 44.5% Pongam oil + 44.5% Neem oil were taken in a stainless steel vessel with a stirrer and were stirred at 120 rpm for 10 minutes. Then 8% emulsifier +1% stabilizer were added to the oils and again it was stirred at 120 rpm for 10 minutes. At last 0.123% Azadirachtin + 2% isopropyl alcohol were added and again it was mixed thoroughly by using a stirrer at 120 rpm for 10 minutes. Then the final formulation 1 was obtained which is named as PONNEEM. Formulation 2: 66.23% pongam oil + 22.67% neem oil + 0.123% azadirachtin (w/v) + 8% emulsifier + 1% stabilizer + 2% isopropyl alcohol. Preparation: 66.23% Pongam oil + 22.67% Neem oil were taken in a stainless steel vessel with a stirrer and were stirred at 120 rpm for 10 minutes. Then 8% emulsifier + 1% stabilizer were added to the oils and again it was stirred at 120 rpm for 10 minutes. At last 0.123% Azadirachtin + 2% isopropyl alcohol were added and again it was mixed thoroughly by using a stirrer at 120 rpm for 10 minutes. Then the final formulation 2 was obtained. Formulation 3: 22.67% Pongam oil + 66.23% neem oil + 0.123% azadirachtin (w/v) + 8% emulsifier + 1% stabilizer + 2% isopropyl alcohol. Preparation: 22.67% Pongam oil + 66.23% Neem oil were taken in a stainless steel vessel with a stirrer and were stirred at 120 rpm for 10 minutes. Then 8% emulsifier +1% stabilizer were added to the oils and again it was stirred at 120 rpm for 10 minutes. At last 0.123% Azadirachtin + 2% isopropyl alcohol were added and again it was mixed thoroughly by using a stirrer at 120 rpm for 10 minutes. Then the final formulation 3 was obtained. Formulation 4: 89% neem oil + 0.123% azadirachtin (w/v) + 8% emulsifier + 1% stabilizer + 2% isopropyl alcohol. Preparation: 89% Neem oil was taken in a stainless steel vessel with a stirrer and were stirred at 120 rpm for 10 minutes. Then 8% emulsifier + 1% stabilizer were added to the oils and again it was stirred at 120 rpm for 10 minutes. At last 0.123% Azadirachtin + 2% isopropyl alcohol were added and again it was mixed thoroughly by using a stirrer at 120 rpm for 10 minutes. Then the final formulation 4 was obtained. Formulation 5: 89%pongam oil + 0.123% azadirachtin (w/v) + 8% emulsifier +1% stabilizer +2% isopropyl alcohol. Preparation: 89% Pongam oil was taken in a stainless steel vessel with a stirrer and was stirred at 120 rpm for 10 minutes. Then 8% emulsifier + 1% stabilizer were added to the oils and again it was stirred at 120 rpm for 10 minutes. At last 0.123% Azadirachtin + 2% isopropyl alcohol were added and again it was mixed thoroughly by using a stirrer at 120 rpm for 10 minutes. Then the final formulation 5 was obtained. BIOLOGICAL ACTIVITY OF DIFFERENT FORMULATIONS Antifeedant activity of different formulations:- Antifeedant activity of different formulations was studied using leaf disc no choice method. Fresh castor leaf discs of 4-cm diameter were dipped in 5, 10, 15, 20, and 25 ppm concentration of oil formulations against III and IV instar larvae of S. litura. Leaf discs treated with water and Nimbecidine were used as control and reference control respectively. In each petri dish, single third and fourth instar larvae of S. litura were introduced individually for antifeedant activity (Plate-1). Progressive consumption of leaf area by insect after 24 hrs was recorded in control and treated discs using a leaf area meter (Delta-T Devices, Serial No. 15736 F96, UK). Ten replicates were maintained for each concentration and percentages of antifeedant activity were calculated using the following formula of Ben Jennet et al (2000). Area protected in control leaf - Area protected in treated leaf = Area protected in control leaf + Area protected in treated leaf x 100 The data collected were subjected to angular transformation and two-way analysis of variance to derive statistical significance. BIOLOGICAL ACTIVITY OF PONNEEM 1. Antifeedant activity of PONNEEM against the polyphagous pests: PONNEEM, Neem oil, Pongam oil and other commercially available neem based formulations were evaluated for their antifeedant activity using choice and no-choice method in the following manner: 3 cm castor leaf discs (S.litura) and cotton leaf discs (H. armigera) were thoroughly washed with tap water and treated individually with 10 ppm concentration of above mentioned formulations. Treated leaf disc was placed inside the petridish having wet filter paper to avoid early drying of the treated leaf disc. In each petridish single IV instar larva of S. litura and H. armigera was released individually for no-choice method and four larvae were released for choice method (Plate-2). For no-choice method the experiment was repeated 300 times and for choice method, the experiment was repeated 50 times. Progressive consumption of leaf area by the larva after 24hrs was recorded using leaf area meter (Delta-T Devices, Serial No. 15736 F96, UK). After treatment the larva was reared continuously to assess the impact of oil formulations during the development. The percentage of antifeedant index was calculated using the formula of Ben Jennet et al (2000). Values are Mean ± Standard deviation; 300 replications for no-choice method; 50 replications for choice method. Among all tested neem based formulations, PONNEEM showed the best antifeedant activity in no-choice method and choice method (Table-3) and the tested larvae were maintained to observe any malformation during development. Larval, pupal intermediate and wing deformities were observed during the development (Plate-3). 2. LarvicidalActivity ofPONNEEM against Spodoptera litura (Tobacco caterpillar): The test was conducted in plastic troughs in which castor leaves treated with test concentration at 10 ppm against IV instar larvae of 5. litura was placed after shade drying. Batches of 25 prestarved larvae were released into each trough. Each treatment was replicated thrice. Larvae released on untreated leaf discs served as untreated check. Treated fresh leaves were provided every 24 hours and the numbers of larvae dead were observed at 24 hours interval upto seven days. Number of larvae pupated and number of adults emerged were recorded. The percent mortality in laboratory studies was corrected using Abbot's (1925) formula. Values are Mean of 3 replications with 25 larvae for each replication. 3. Larvicidal Activity ofPONNEEM against Helicoverpa armigera (Cotton bollworm): The test was conducted using petri dish in which cotton leaves treated with test concentration (l0ppm) were placed after shade drying. In each petri dish one prestarved larva was released. Ten IV instar larvae constituted one replication and the treatments were replicated thrice. Larvae released on untreated cotton leaves serves as untreated check. Treated fresh leaves were provided daily and numbers of larvae dead were observed at 24 hours interval upto seven days. Number of larvae pupated and adults emerged were recorded. The percent mortality in laboratory studies was corrected using Abbot's (Abbot, 1925) formula. Test mortality (%) - Control mortality (%) XI00 Percent corrected mortality = 100 - Control mortality (%) Table 5. Larvicidal activity of botanical formulations against IV instar larvae of Helicoverpa armigera at l0ppm Values are Mean of 3 replications with 10 larvae for each replication PONNEEM exhibited the maximum larvicidal activity against S. litura and H. armigera at 6th day after the treatment and the emergence of adult was much lower when compared to other neem based formulations (Tables-4&5). The azadirachtin and karanjin present in the oil formulation inhibited the growth of larvae at larval, pupal and adult stages during the development. Azadirachtin, a main active ingredient present in the formulation inhibited the molting hormone activity during the development and showed various deformities in selected pest. The presence of toxic flavonoids, karanjin and pongamol in pongam oil affected the larvae during the development thereby increasing the protection. 4. BIOCHEMICAL STUDY PONNEEM and commercially available neem based formulations were used to study the quantitative and qualitative variations in the protein of IV instar larvae of S. litura and H. armigera after the treatment Collection of haemolymph from the larvae The haemolymph was collected from 10 larvae of S. litura and H. armigera from each treatment separately at 96hrs after treatment. Haemolymph samples were drawn from the larvae by wounding the 2nd proleg and draining the haemolymph into eppendorf tube having crystals of phenylthiourea solution on ice. Later it was centrifuged at 12,000rpm for 10 minutes and the clear haemolymph samples without any tissue debris and haemocytes were collected from both treated and control larvae. After this the sample was stored at -20°C for experiment. Quantification of Protein The quantity of total protein content/ml of haemolymph was estimated according to BRADFORD (1976) method using Bovine serum albumin as the standard. 2\A of haemolymph was mixed with equal volume of protein extraction buffer (Tris HCL pH 7.2). From this l was mixed with 5ml of Bradford reagent and incubated for 10 min and the OD was taken at 595nm. The amount of protein present in the sample was calculated using the following formula. Amount of concentration of standard X = X OD of the Sample OD of the standard PONNEEM treated larvae of S. litura and H. armigera showed significant reduction in the amount of haemolymph protein compared to the control and other treatments (Table 6). Table 6. Quantitative changes in haemolymph protein (mg/ml) of Spodoptera litura and Helicoverpa armigera Products tested Spodoptera litura Helicoverpa armigera Formulation - 1 (PONNEEM) 132.7 ± 3.26 152.5 ± 5.36 Formulation -2 233.7 ± 3.14 138.2 ±3.11 Formulation-3 412.3 ± 2.79 101.5 ± 2.36 Formulation-4 151.2±2.67 110.2 ± 1.76 Formulation -5 350.5 ± 3.45 115.4 ± 2.41 Neem Gold 984.4 ± 5.10 96.5 ± 2.67 NeemAzal 283.7 ± 3.94 105.2 ± 4.16 Nimbecidine 206.3 ± 3.52 297.3 ± 5.11 Control 451.8 ± 3.6 356.4 ± 2.01 Values are mean ± SD often replicates Qualitative Estimation of Protein By SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) Haemolymph samples of both control and the treatments were analyzed by SDS-PAGE (Laemmli 1970) using 10% separating gel and 5% stacking gel. L00µg of protein was loaded in each well. 2jil of haemolymph protein samples were mixed with 30jil of sample buffer. The sample was digested in a boiling water bath for 2-3 min, cooled and centrifuged at 10,000 rpm for 5 min in a refrigerated centrifuge at 4°C and the supernatant was loaded into the well. The electric power supply was adjusted to 60 volts until the marker dye entered into the separating gel; after that the voltage was increased to 120 volts. The electrophoretic run was stopped when the dye front reached the anodic end of the gel. Marker protein of known molecular weight was also loaded along with the sample proteins separately for comparison. After running, the gel was stained with Coomassie brilliant blue R-250.The gel was kept in the stain overnight and destained with the destaining solution until the clear background was obtained. The destained gels were scanned using laser densitometer scanner and the scanned gels were stored in 7% acetic acid. PONNEEM treated S.litura and H. armigera did not show much variation in protein profile (Plate-4). TOXICOLOGICAL STUDY ON RATS Male albino rats weighing 100 to 120gm were used for the present study. The rats were provided with pellet feed (manufactured by Hindustan Liver Limited, Bombay, India). Food and water were given fresh ad libitum. Acute Toxicity Studies; Rats were divided into eight groups of six animals each. PONNEEM was administered by oral incubation to different groups at doses of 25, 50, 75, 100, 125,150 and 200ppm (0.5ml/100gm body wt.), while the control group received vehicle (water) only. The animals were observed for toxic symptoms and mortality for 72 hrs. PONNEEM formulation did not induce any toxic effect or mortality up to the dose level of 200 ppm. During acute toxicity studies in albino rats, the PONNEEM did not produce any external symptoms or mortality up to the dose of 200 ppm when given orally Sub Acute Toxicity: The animals were divided into 6 groups of six animals each. The oil was administered for 15 days at a dose of 5, 10, 20, and 30 ppm. The control group received vehicle (water) throughout the experimental period. The change in body weight was recorded weekly, and simultaneously observed for any mortality. All the animals were sacrificed by cervical decapitation and the vital organs like liver, kidney and spleen were carefully dissected and weighed for estimation of glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH). In sub acute toxicity, the activity of liver dysfunction marker enzymes Glutamate oxaloacetate transaminase (GOT), Glutamate Pyruvate transaminase (GPT) and Lactate dehydrogenase (LDH) activity in serum, liver, kidney and spleen did not show much variation. Animal behaviour, food and water intake were normal during the sub acute toxicity studies. Transaminases (GPT and GOT) and Lactate dehyrogenase are good indices of liver and kidney damage (Tables-7-10). The oil did not induce any damage to liver, kidney and spleen, which could be inferred from normal activity of these enzymes. concentrations. Concentration of PONNEEM I Weight in Gms Body Liver Kidney Spleen 5ppm 133.75±6.29ab 5.59±.82a 1.12±.02a 0.40±.04a lOppm 146.00±13.5b 5.95±.79a 1.15±.02a 0.48±.02b 20ppm 130.0O±7.07a 5.43±.55a 1.18±.23a 0.45±.04ab 30ppm 141.25±7.50ab 5.99±.29a 1.13±.05a 0.50±.05b Control I 141.25±4.78ab | 5.73±.59a | 1.22±.04a | 0.48±.01b Values represent Mean ± SD. Values followed by the similar letter(s) are not significantly different (P= 0.05) by DMRT. Table 8. Effect of PONNEEM formulation on Glutamate oxalate Transaminase (GOT) in rats at different concentrations Concentration of PONNEEM SAMPLES (IU/L) Serum Liver Kidney Spleen 5ppm 41.42±3.44a 3.74±0.4a 9.68±0.83b 179.52±13.05a lOppm 40.66±2.62a 3.81±0.44a 10.45±0.54b 198.63±13.15b 20ppm 41.17±2.97a 3.86±0.39a 8.61±0.64a 167.62±5.52a 30ppm 41.05±3.38a 3.87±0.50a 9.73±0.09b 167.1 l±8.21a Control 1 41.39±3.06a | 4.16±0.27a | 9.48±0.75ab 1 209.86±7.54b Values represent Mean ± SD. Values followed by the similar letter(s) are not significantly different (P= 0.05) by DMRT. Table 7. Effect of PONNEEM formulation on body weight and vital organs in rats at different concentrations. Concentration of PONNEEM Weight in Gms Body Liver Kidney Spleen 5ppm 133.75±6.29ab 5.59±.82a 1.12±.02a 0.40±.04a lOppm 146.00±13.5b 5.95±.79a 1.15±.02a 0.48±.02b 20ppm 130.00±7.07a 5.43±.55a 1.18±.23a 0.45±.04ab 30ppm 141.25±7.50ab 5.99±.29a 1.13±.05a 0.50±.05b Control I 141.25±4.78ab | 5.73±.59a | 1.22±.04a | 0.48±.01b Values represent Mean ± SD. Values followed by the similar letter(s) are not significantly different (P= 0.05) by DMRT. Table 8. Effect of PONNEEM formulation on Glutamate oxalate Transaminase (GOT) in rats at different concentrations Concentration of PONNEEM SAMPLES (IU/L) Serum Liver Kidney Spleen 5ppm 41.42±3.44a 3.74±0.4a 9.68±0.83b 179.52±13.05a L0ppm 40.66±2.62a 3.81±0.44a 10.45±0.54b 198.63±13.15b 20ppm 41.17±2.97a 3.86±0.39a 8.61±0.64a 167.62±5.52a 30ppm 41.05±3.38a 3.87±0.50a 9.73±0.09b 167.1 l±8.21a Control 1 41.39±3.06a | 4.16±0.27a | 9.48±0.75ab 1 209.86±7.54b Values represent Mean ± SD. Values followed by the similar letter(s) are not significantly different (P= 0.05) by DMRT. Randomized block design experiments for field evaluation: Randomized block design experiments were conducted in a selected farmer's field under irrigated conditions. In the selected field, groundnut JL-24 variety was used. Plot size was fixed in 40 sq.m. (1 cent). Six treatments were given with the three replications to assess the pest and natural enemy complex under the influence of chemical pesticides, commercially available botanical pesticides and a new formulation (PONNEEM). Pest surveillance and population of natural enemy were conducted 5 days after treatment. The treatments were carried out by using the power sprayer. PONNEEM showed higher activity in protection of groundnut and higher natural enemy population when compared to commericial available biopesticides and chemical pesticide. Chemical pesticide Monocrotophos treated plots showed maximum reduction of aphids, thrips, leaf miner and tobacco caterpillar. Natural enemies such as spiders, ladybird beetles, ants and damselflies were reduced maximum in Monocrotophos treated plots. In neem based formulations maximum population of natural enemies were observed. PONNEEM treated plots showed maximum population of natural enemies compared to other treatments. Also PONNEEM treated plots recorded good reduction in pest population (Tables-11&12). Values represent mean ± SD of three replications. Values followed by same letter (s) in a column are not significantly different (p=0.05) by DMRT. ADVANTAGES OF THIS INVENTION Among the different biopesticides formulations prepared, formulation 1, named PONNEEM exhibits significant antifeedant, larvicidal and insecticidal activities at l0ppm concentration against Helicoverpa armigera and Spodoptera litura, polyphagous insects of crop plants. The larvae after feeding fail to moult due to the insecticidal activity of the formulation and ultimately die. Since this formulation is having antifeedant and insecticidal activity against the potential pests Helicoverpa armigera and Spodoptera litura, it is therefore useful for controlling other pests also; hence this biopesticide in useful in the pest management. PONNEEM formulated using Pongam and Neem oils does not show any phytotoxicity and is not harmful to other beneficial organisms. EXAMPLE: · 44.5% Pongam oil and 44.5% Neem oil were taken in a vessel and mixed well using a stirrer at 125 rpm for 10 minutes. · Then 8% emulsifier and 1% stabilizer were added to the mixture and mixed well again using a stirrer at 125 rpm for l0minutes. · Then 0.125% azadirachtin and 2% isopropyl alcohol were added to the mixture and mixed well using a stirrer at 125 rpm for 10 minutes. · The final yield is PONNEEM, a new biopesticide. It is to be noted that the present invention is susceptible to modification, changes and adaptations by those skilled in the art. Such modifications, changes adaptations are intended to be within the scope of the present invention, which is further set forth under the following claims. Plate-1 Antifeedant activity - leaf disc no choice method a) Larva of Spodoptera litura feeding on castor leaf disc treated with PONNEEM (l0ppm) b) Larva of Helicoverpa armigera feeding on cotton leaf disc treated with PONNEEM (lOppm) Plate - 2 Antifeedant activity - leaf disc choice method a) Larvae of Spodoptera litura feeding maximum on control castor leaf discs compared and PONNEEM treated leaf discs b) Larvae of Helicoverpa armigera feeding on control cotton leaf discs and PONNEEM treated leaf discs Plate - 3 a) Dead larvae of Spodoptera litura due to treatment with PONNEEM b) Dead larvae of Helicoverpa armigera due to treatment with PONNEEM c) Larval-pupal intermediate of Spodoptera litura due to treatment with PONNEEM d) Malformed pupae of Helicoverpa armigera due to treatment with PONNEEM e) Malformed adults of S. litura due to treatment with PONNEEM f) Malformed adults of Helicoverpa armigera due to treatment with PONNEEM Plate 4 Electrophorogram of haemolymph protein profile A) Spodoptera litura; B) Helicoverpa armigera M - Marker protein C - Control 1 - Formulation 1 (PONNEEM) 2 - Formulation 2 3 - Formulation 3 4 - Formulation 4 5 - Formulation 5 6 - Neem Gold treatment 7 - NeemAzal treatment 8 - Nimbecidine treatment

Documents:

657-CHE-2006 FORM-18.pdf

657-che-2006-abstract.pdf

657-che-2006-claims.pdf

657-che-2006-correspondence-po.pdf

657-che-2006-description(complete).pdf

657-che-2006-drawings.pdf

657-che-2006-form 1.pdf

657-che-2006-form 9.pdf