JOURNAL OF INTERNATIONAL ACADEMIC RESEARCH FOR MULTIDISCIPLINARY Impact Factor 1.393, ISSN: , Volume 2, Issue 2, March 2014

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1 STUDIES ON THE ISOLATION OF AZADIRACHTIN FROM NEEM OIL AND THEIR ROLE IN CONTROL PESTS OF RICE V. RAMAMURTHY* M.K. VADIVAZHAGI** *P.G & Research Dept. of Biochemistry, Marudupandiyar College, Vallam, Thanjavur, Tamil Nadu, India **P.G & Research Dept. of Biochemistry, Marudupandiyar College, Vallam, Thanjavur, Tamil Nadu, India ABSTRACT Evaluation of the activity of the cold expeller neem oil (Azadirachta indica A. Juss.) and the fractions derived through solvent partitioning, against Rhizoctonia solani and Bipolaris oryzae showed that the active antifungal fraction is a mixture of azadirachtin. Further, testing the azadirachtin mixture derived from the 90% hexane extract of neem oil against two phytopathogenic fungi revealed that various species were inhibited to different degrees. Direct preparative High Performance Liquid Chromatography (HPLC) of the active fractions and subsequent bioassay of the semi pure fractions indicated that the active fractions contained major compounds such as azadiradione. The pure Azadirachtin biopesticides based on fungus Rhizoctonia solani and Bipolaris oryzae were used on leaf folder of rice, which have reduced the population of these pests effectively both in laboratory and in the field. By using the combination of azadirachtin and solvent formulation the increase in mortality was studied and it was 100% after 96 h. In the field trials a significant effect on leaf folder was observed. On the basis of these results, biopesticides can safely be recommended for the control of rice pests with no harmful effect on its predators as in case with chemical pesticides. KEY WORDS: Biopesticide, HPLC, Azadirachtin, Rhizoctonia Solani And Bipolaris Oryzae. INTRODUCTION Rice (Oryza sativa) is the most important food crop of the world. In India, the yield of rice is very high as compared to other rice producing countries. Several factors contribute for such low yield and among them the most important are loses due to insect attack. The leaf folder (Cnaphalocrocis medinalis) is very serious insect pest of rice. Losses due to these insects usually occur 5-10% and sometime reaches to 60% (Pathak and Khan, 19941). For the control of these pests, chemical spray is most common practice. Concern about environment pollution, resistance to pesticides, residues in food and biodiversity make new and novel strategies for the control of pests like rice leaf folder. Critically important is to secure food 338

2 for a rapidly growing population. In view of these considerations, biopesticides offers a technically feasible and environmentally acceptable strategy for controlling agronomically important insects. The use of biopesticides in many countries is limited for a variety of reasons most notable among them is the poor efficacy of imported products under local conditions (Prior, 1989). Use of the crude extractives of seeds of neem for control of plant pathogenic fungi is known and has been amply documented (Locke, 1995). The antifungal activities of neem constituents relate to Azadirachtin (supposedly a mixture of a number of triterpenoids from seed oil) against Rhizoctonia solani, Bipolaris oryzae and Helminthosporium oryzae (Khan et al., 1974). Using some phytopathogenic fungi as test organisms, (Steinhauer, 1994) isolated a compound with antifungal activity from neem oil extracts, the identity of which is not known. Herein, we have attempted fractionation, isolation and identification of antifungal triterpenoids from neem oil, the results of which are presented. MATERIALS AND METHODS Extraction of neem compounds: Neem oil was collected in large sterilized container and brought to the laboratory. The collected neem oil was eluted 3:1 ratio with hexane and ether. Then the mixture was filled with a specially designed instrument (for heat extraction) container. After filled the mixture was heated 50 0 C to 60 0 C for 15 minutes. Then the neem oil extract was collected through Buchner funnel. The extract was purified by Column Chromatography Slurry method. Pure compounds were identified by HPLC analysis. Standard pure compounds were routinely purified in our laboratory through preparative HPLC, which forms the source. Rice field: The field trial experiment was conducted in premises of Pattukkottai taluk, Thanjavur district, Tamilnadu, during summer season of (March-July) Total plant area was divided into four treatments and four replicates according to randomized complete block design. The field was sown with supper ADT 43 cultivars of rice. Infestation of rice leaf folder started after days of rice plantation. The Azadirachtin was sprayed three times by looking the severity of infestation. The Azadirachtin was used in the various concentrations. For applying the Azadirachtin the following treatment were employed. 339

3 Test one (T1) = 50g/100lit. / Acre Test two (T2) = 100g/100lit. / Acre Test three (T3) = 200g/100lit. / Acre Test four (T4) = Control Laboratory bioassay: Laboratory experiments were conducted to check the effect of azadirachtin formulation on the larvae before applying in the field. The data was collected up to 96 hrs. The mortality percentage was calculated after 48, 72 and 96 h and it was recorded. Pest scouting: (Amer et al., 1999) Criteria of Azadirachtin evaluation were based on pre and post spray pest scouting. The mortality percentage data was calculated by looking the severity of infestation. Data was recorded after spray on 1, 2, 7 and 9 days. Antifungal activity: (Collins and Lyne, 1970) The Azadirachtin were tested separately for their fungal toxicity against Rhizoctonia solani, Helminthosporium oryzae and Bipolaris oryzae in the experiments. The 90% hexane extract of neem oil was also tested against two phytopathogenic fungal such as R. solani, H. oryzae and B. oryzae. The different concentrations of Azadirachtin viz., 5, 10, 15, and 20 per cent were added separately to the cooled potato dextrose agar medium. The amended PDA medium was dispersed in Petriplates and allowed to solidify. After solidification 5mm agar blocks cut from the actively growing margin of the pathogen R. solani, H. oryzae and B. oryzae were inoculated at the center of the plates. The plates were incubated at 30 2 C for five days. The radial growth was measured periodically and the mean growth rate was calculated. Control was maintained in each case without adding Azadirachtin. The percentage inhibition of growth was calculated as follows: Percentage of Growth Growth in control Growth in treatment inhibition = x 100 Growth in control RESULTS Isolation of Azadirachtin: The double refined oil extract from various solvents such as hexane, alcohol and ether. Showed that hexane solvent even effectively coagulated and separation of 4% azadirachtin compound. 340

4 Identification of Azadirachtin: Azadirachtin was purified by silica gel using column chromatography. Some fractionated substances were analyzed through HPLC. From the chromatogram, the peak value was calculated by Retention Time (RT) and identified the extract as Azadirachtin. Effect of Azadirachtin on pest of paddy: The paddy field was separated four plots and sprayed with various concentration of Azadirachtin, the number of larvae was counted in each replicate. The data was collected before spray and after (1, 2, 7 and 9 th day). The number of larvae per plant was calculated and presented in Figure 1. High number of larvae was observed in the first treatment (T1), on day 1 and with the passage of time the insect population was reduced due to effect of Azadirachtin and stem borer of the crop. It is usually observed after number of insecticide resistant species. The present study described the uses of Azadirachtin for the control of rice leaf folder and stem borer insect larvae. The experiment was continued up to 96 hours. Mortality due to Azadirachtin was 20% and 49% after 48 and 72 hours respectively in T2. Increased in morality was observed in T3 formulation and it was 100% after 96 hours (Fig. 2). The lower (T1) concentration Azadirachtin was exhibited 89% after 96 hours. In field trials on rice crop, spray data indicates that under natural infestation pest population in the field can never remain same. These plots sprayed Azadirachtin represented decrease in present infestation from 1 st to 9 th day. These results suggest that Azadirachtin were effective not in laboratory bioassay but also in the field against rice pests. Yield of rice crops in same conditions of attack was also studied. In treated very few insects were recorded on 9 th day. Similar results were observed in all other treatments. Mortality Percentage: The mortality percentage due to the effect of Azadirachtin was calculated. In all the treatments (T1, T2 and T3) higher percentage of mortality was observed than the control (T4). In T1 and T2 formulation, almost equal percentage of mortality was observed and showing the effect of Azadirachtin on insect population (Fig.3). Effect on Whiteheads: Whitehead was the damage caused by flowering. Stem borer larvae migrate to in between the leaf sheaths. It causes the entire panicle to dry. Average number of whiteheads was counted 341

5 in each replicates. A significant reduction of whitehead was observed in all treatments due to the effect of spray when it was compared to control. In control group the number of whitehead was very high. This demonstrates, that the effect of biopesticides on stem borer, which ultimately resulted in reduction of whiteheads (Fig. 4). Antifungal activity of Azadirachtin: The fungal pathogens R. solani, H. oryzae and B. oryzae were used to study the antifungal activity of azadirachtin. The results showed that the azadirachtin could inhibit the growth of the fungal strains. Among the fungal pathogen R. solani showed maximum growth inhibition when compared to H. oryzae and B. oryzae (Fig. 5). DISCUSSION Public concern about chemical residues on fruits, vegetables and other crops has led to a progressive increase of interest in alternative strategies for the control of diseases and pests. The application of biological control is increasing largely because of greater environment awareness and food safety concerns plus the failure of conventional chemicals due to an increasing, preliminary laboratory bioassay gave excellent results on found very effective against lepidopteron insects plots the crop looks healthier and gave better yield than nontreated, which recommends biopesticide as a best biological agent in crop protection. Analysis of the 90% hexane extract by analytical HPLC revealed the presence of major triterpenoids (Govindachari et al., 1995). The extracted compound is attributed to the triterpenoidal fraction. An attempt was made, therefore, to evaluate the different preparative HPLC fractions of the 90% hexane extract in order to identify the compound that impart Azadirachtin. Preparative HPLC (on a 5 cm - 25 cm, C18 column with MeOH: Hexane as an eluent in a stepwise gradient) resolved the 90% hexane extract into the peaks (Govindachari et al., 1995), after the elution of which the column was washed with 100% methanol in order to remove the non-polar components. Analysis of all the peaks employing analytical HPLC using a C18 column revealed that peaks contained mainly azadirachtins (Govindachari et al., 1996). Attempts are being made to collect these fractions in sizeable quantities to investigate further the individual fractions. The Peak was identified as 6-deacetylnimbin of 96% purity (by analytical HPLC). Peak (80% azadiradione as the major component), which resolved into at least three major components in analytical HPLC. 342

6 In field trials on rice crop, spray data indicates that under natural infestation pest population in the field can never remain same. Plots sprayed with Azadirachtin represented decrease in percent infestation from 1st to 9 th day. These results suggest that azadirachtin were effective not in laboratory bioassay but also in the field against rice pests. Yield of rice crop in same conditions of attack was also studied. In treated plots the crop looks healthier and gave better yield than non-treated, which recommends Azadirachtin as a best biological agent in crop protection. The effect of azadirachtin on rice leaf borer was also studied which resulted in reduction of whiteheads in all the replicates as compare to control. Azadirachtin based on fungal cry toxin, which binds to a specific receptor on the brush border membrane of insect mid gut epithelial cells, Bt toxin insert rapidly and irreversibly in to plasma membrane of gut cells (Malik et al., 2001) and either form an ion channel or act on some membrane components to open a pore, which resulted in cell lysis and eventually death of insect larvae (Knowles and Dow, 1993). Although neem oil has been used for control of phytopathogenic fungi, such as Rhizoctonia solani, Helminthosporium oryzae and Bipolaris oryzae rust pathogens, concentrations needed for complete field control were shown to be as high as 2% to 10%. High concentrations of neem oil are known to induce phytotoxicity (Locke, 1995). In our experiments the concentration for the antifungal activity assays was kept at 5 to 20 %. Cold expeller neem oil at control either brought about no inhibition (against R. solani, H. oryzae and B. oryzae) or minimal inhibition (5% against B. oryzae and 10 % against R. solani) against the test sample. While this confirms earlier reports (Locke, 1995), it may, in part, explain the reason for the use of higher concentrations of neem oil for field control. Assays of the antifungal activities of the mixture against the two test fungi revealed again that maximum inhibition was observed with H. oryzae and B. oryzae (5 %). It is also not surprising that differences existed in inhibition percentages among the test fungi. Hence, the concentration needed to inhibit each fungal species effectively has to be worked out independently. Both the natural Azadirachtin mixture from neem oil, as well as a mixture made up from pure Azadiradione was similar in their antifungal activity against the test fungi. Present studies have shown that the 90% hexane fraction contained mainly Azadirachtin. Hence, it was suspected that the Azadirachtin might be antifungal. Loss of activity after purification prompted us to recreate a mixture by combining pure compounds. Retention of activity of such mixtures of triterpenoids may indicate that there could be additive/synergistic effects of azadirachtin in 90% hexane extracts. 343

7 REFERENCES 1. Amer, M., Hussain, S.A.S., Khan, L., Khattak, M and Shah G.S The comparative efficacy of insecticides for the control of insect pest complex of cotton Gossypium hirsutum L. Pak. J. Biol. Sci., 2, Collins, C.H and Lyne, P.M Mirobiological Methods, 3 rd Edition. Butterworth and Co. Ltd. pp Govindachari, T.R., Gopalakrishnan, G and Suresh, G Isolation of various azadirachtins from neem oil by preparative high performance liquid chromatography. J. Liq. Chromtogr. Rel. Technol., 19, Govindachari, T.R., Suresh, G., and Gopalakrishnan, G A direct preparative high performance liquid chromatography procedure for the isolation of major triterpenoids and their quantitative determination in neem oil. J. Liq. Chromatogr., 18, Khan, M.W., Alam, M. M., Khan, A. M and Saxena, S.K Effect of water soluble fractions of oil cakes and bitter principles of neem on some fungi and nematodes. Acta Bot. Indica, 2, Knowles, B.H and Dow, J.A.T The crystal deltaendotoxins of Bacillus thuringiensis models for their mechanism of action on the insect gut. Bioassays, 15, Locke, J.C Fungi In: Schmutterer, H. [Ed.] The Neem Tree, Source of Unique Natural Products for Integrated Pest management, Medicine, Industry and Other Purposes. VCH, Weinheim, Germany. pp Malik, K.T., Mahmood, R and Riazuddin, S The receptor for Bacillus thuringiensis Cry 1Ac delta endotoxin in the brush border membrane of lepidopteran Helicoverpa armigera is aminopeptidase N. Pak. J. Biol. Sci.. 1, Pathak, M.D and Khan, Z.R Insect pests of rice. International rice research institute. Los Banos, Philippines. 10. Prior, C Biological pesticides for low external-input agriculture. Biocont. News and Inform., 10, Steinhauer, B Antifungal compounds from Azadirachta indica. In: Kleeberg, H. [Ed.] Practice Oriented Results on Use and Production of Neem Ingredients and Pheromones. Workshop (Wetzlar, Germany), pp Fig. 1: Population of rice leaf folder with effect of Azadirachitin T1(50mg/100lit T2(100mg/100lit T3(200mg/100lit T4(Control 0 Day 1 Day 2 Day 7 Days 9 Days 344

8 Fig. 2: Mortality persentage whiteheads with effect of different concentrations of Azadirachitin after exposure to different hours of duration hrs 72 hrs 96 hrs Fig. 3: Mortality percentage whiteheads with effect of different concentrations of Azadirachitin T1(50mg/100lit T2(100mg/100lit T3(200mg/100lit T4(Control 345

9 Fig. 4: The number of dead whiteheads with effect of different concentrations of Azadirachitin paddy leaf folder T1(50mg/100lit T2(100mg/100lit T3(200mg/100lit T4(Control Fig. 5: The antifungal activity of fungal strains with effect of different concentrations of Azadirachitin 30 minimal inhibition (mm) Concentration of Azadirachtin R. solani H. oryzae B. oryzae 346