Potential applications of Neem based products as

theHealth 2012; 3(4): 116-120

Review

Potential applications of Neem based products as biopesticides Bajwa AA 1 , Ahmad A 2 1 Institute

of Biochemistry & Biotechnology, University of the Punjab, Lahore, Pakistan, 2 School of Biological Sciences, University of the Punjab, Lahore, Pakistan Correspondence Aftab Ahmad School of Biological Sciences, University of the Punjab, Lahore, Pakistan. 54590 E-mail: [email protected] Keywords: Fungicidal, Insecticidal, Azadirachtin, Saponins, Antimicrobial Funding None Competing Interest None declared. Received: October 12, 2012 Accepted: December 23, 2012

Abstract Biopesticides are a good alternative to the synthetic pesticide. Both leaves and fruit of neem plant are known to have bitter taste having fungicidal, insecticidal and nematicidal properties. Azadirachtin, chemically a tetranorterpenoid component of neem acts on the mitotic cells and blocks the microtubule polymerization. Certain activities of genes and proteins are also altered by azadirachtin. As a botanical insecticide azadirachtin is effective against many biological processes. It may cause a reduction in feeding habit, suspend the molting process, larvae and pupae death and also cause sterility in the emerging adults, this all depend on the given dose. Apart from azadirachtin, many other components of neem also have insecticidal properties. Among them are saponins that are found to have antimicrobial activity that inhibits moulds and protecting plant from attack of insects. Likewise Nim80 and Neemas are also neem products that produce the insecticidal activity. In addition Parker oilTM is also effective insecticide which is a commercial product. There was maximum weight loss of treated insects when azadirachtin and NPV are used in combination. Commercial neem formulation has been assayed for the control of pine weevil in forestry, against rice leaf folder, root-knot nematodes and P. xylostella. Azadirachtin-A is extremely sensitive to the presence of sunlight so different mechanisms have been used to increase its photo stability. Introduction

ISSN (print): 2218-3299 ISSN (online): 2219-8083

Synthetic pesticides are of increasing concern as they accumulate in the environment, so much of attention has been diverted towards the natural compounds that could replace the synthetic ones.1 Biopesticides are good alternative to the synthetic ones as they cause a little environmental pollution, low toxicity level to humans and several other advantages.2,3 Neem is one of the most reliable botanical sources of biopesticides.1 Neem plant has been known for three decades for its potential against insect pests.4 Leaves and seed extract of neem plant have been observed for their deleterious effects on insects.5 The principle component that has insecticidal activity in neem extracts is a limonoid, azadirachtin. It has been evaluated as the most promising insecticide of botanical origin, used against more than 400 species of insects.6, 7 Neem seeds are potential source of biopesticedes that has been appraised against many important insect species8 as several active ingredients in neem makes it potential source of biopesticide against many important insect species. In most of the formulations of biopesticides is the tetranortriterpene, azadirachtin.4 Insecticide based on neem extracts have many effects on insects, it includes anti-

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oviposition, anti-feedant, regulation of growth and development etc.9, 10 The main source of azarirachtin is extracts from seeds of Indian neem plant.11 Due to azadirachtin marked insecticidal potential much research has been focused on it now a days.12 Azadirachtin mainly influences the feeding habits of insects, growth and reproduction and thus lowers their population density but still very little is known about the biochemical mode of action. Azadiractin not only has a unique mode of action against insects, it also has adverse effects on fungi, viruses, nematodes and protozoans.9 In addition larval growth is markedly reduced by azadirachtin. Usually anti-feedant effect is observed after treatment of azarirachtin.10 It is also responsible for providing mortality in Tinea dubiella (Stainton) and Tineola bisselliella (Humel). Azadirachtin acts on endocrine and neuroendocrine systems which are important to regulate the developmental processes of insects. By targeting these systems azadirachtin causes adverse development effects.12 As a biopesticide Azadirachtin is known to have very little effect on non-targeted organisms for example pollinators.9, 13, 14 It is completely non-toxic to vertebrates.15 Azadirachtin

Application of neem based products

Effects of Azadirachtin

Effects of Neem plant

Antiviral

Nematicidal

Insecticidal

Fungicidal

Antifeedancy

Growth & Reproduction

Antioviposition

Fungicidal

Figure 1: Different effects of Neem plant as biopesticide.

may not be effective against all pest insects and its effectiveness is dependent on used concentration.10 Persistence level of azadirachtin on food is unknown so there is need of further research in this area.4

Affects on acdyteroid and juvenile hormone

Direct affect on tissues

Azadiracta indica - Indian Neem Plant Azadiracta indica known as Indian neem plant is an evergreen, hard tree which is native to sub-continent. It can easily be grown in a nutrient poor soil and can survive even in dry harsh conditions. It is known to be friendly with other vegetation but it is disliked by insects. Both leaves and fruit of neem plant are known to have bitter taste having fungicidal, insecticidal and nematicidal properties.8 Recently neem plant has been gathered much attention because it is a potential source of drugs and eco loving pesticides.8 Leaves of neem plant have been used as substrate for producing vermicompost. The effect of vermicompost application on the growth and yield of brinjal plant has been studied. It has been revealed that application of neem vermicopmpost produce potential effects on plant growth.16 Chemical structure and function of Azadirachtin Azadirachtin has a molecular formula C35H44O16 and chemically it is a tetranorterpenoid. Azadirachtin have many reactive functional groups which are closely associated with each other which makes it a highly oxidized limonoid.17 The major component of neem seeds is azadirachtin A. Azadirachtin B is also present but it is present in very minute concentration. Azadirachtin has also been synthesized chemically. Simpler models of azadirachtin are being investigated which have simpler structure and same biological activity as compared with the complex one, synthetic derivatives of azadirachtin are also being investigated.18 Ever since the discovery of insecticidal activity of neem and azadirachtin as an active ingredient for its insecticidal activi-

Figure 3: Different effects of Azadirachtin.

ty, research on this compound is rapidly increasing.19 Up till now nine types of azadirachtin has been identified in neem seed extract. Their structure has also been elucidated. It has proved to be effective against many insect pests of various species in their developmental stages.8 The possible mode of action of azadirachtin is anti-feedancy which affects the acdysteroid and juvenile hormones and it directly affects tissues which are believed to be controlled by developmental hormones. Fungi, viruses and protozoa have also been reported sensitive to azadirachtin, apart from its action against insects.9 Besides its biopesticides properties, potential of azadirachtin have also been explored as inhibitor of sexual development of malaria parasite and larvicidal against mosquito larvae. It is also being investigated for having inhibitory effect on replication of dengue virus type-2.20 Mode of action Different molecular techniques have been used to determine the effect of azadirachtin at cellular and molecular level. It has been shown that azadirachtin acts on the mitotic cells and blocks the microtubule polymerization.22 It has been revealed that the anti-proliferating effect of azadirachtin is due to blocking of cell cycle and induction of apoptosis.23 In addition to this nuclear DNA is directly damaged by azadirachtin and also binds to a large protein complex including heat shock protein 60.24 It has been suggested that azadirachtin is highly reactive and have many cellular molecules as target in cytoplasm as well as in nucleus. Certain activities of genes and proteins are also altered by azadirachtin.25, 26 Different non-neem inhibitors of plant origin Several compounds are present in different plant parts including seeds, fruits, flowers, wood and leaves that acts as natural inhibitors. Magnifera indica is highly rich with polyphenol having antioxidant activity and also glycoside flavonoids.27 Cellulase activity of insects is inhibited by the flavonoids. Enzyme inhibition is easily understood by the structural evidences.28, 29 Green apples form tannins from polyphenol oxidase that is also responsible for inhibiting cellulase activity.30 Tannins interfere with the food intake of animal, their

Figure 2: Chemical structure of Azadirachtin. 21

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digestion and reproducibility. In addition to this Rosaceae family is also reported for its strong inhibitory effect against cellulase.31 Other neem derived compounds a. Saponins Saponins are the surface-active glycosides that naturally occur in certain plants, animals and microorganisms. Mainly they are produced by plants but lower amount in marine animals and some bacteria. Saponins are named because of their soap like characteristics. Usually saponins consist of a sugar moiety containing glucose, glactose, glucuronic acid, xylose, rhamnose or methyl pentose.32 A glycosydic linkage is present between sugar moiety and hydrophobic aglycone that may be a terpenoid or steroid in nature. The aglycone could contain one or more C-C unsaturated bonds. Many saponins are found to have antimicrobial activity that inhibits moulds and protecting plant from attack of insects. They can be taken as a defense system of plant. Saponins from oat have been reported to be effective against tissue damage and attack of pathogens.33, 34 b. Nim 80 and Azatin Shoot-borer are key forest pests in tropical areas, it belongs to genus Hypsipyla. H. robusta is present in old world tropic while in neotropic region H. grandelle is widely distributed.35 Both species suppress the apical dominance by boring into terminal shoots of young plants, causing stem forking and thus leading to high production of lateral branches. Extract of neem seeds obtained from Azadirachta indica have been shown to trigger certain harmful effects on a number of insect species.8, 9 Two products from neem; Nim 80 and azatin have been used to analyze that whether the neem products are causing antifeedant activity or acting as growth regulators. Studies have shown that both products do not show same activity against H. grandella larvae. Both neem products have been shown to produce the insecticidal activity or growth effects. At low concentration of azatin the growth rate was reduced while increasing the concentration lead to high mortality rate. Larvae become unable to feed when they were exposed to azatin. It has been shown that azatin acts as direct toxicant instead of inhibiting its growth. On the other hand Nim 80 has showed effectiveness against larval development.36 c. Parker oilTM and neemas Two other neem products Parker oilTM and neemas have been tested for their effectiveness against brown plant hopper. Their mortality rate, rate of food consumption and net survival rate has been investigated after application of these two neem products. It has been clearly observed that neem based products are more effective against brown plant hopper. In addition, these neem products have very low toxicity level against human beings.37 Azadirachtin function and effects As a botanical insecticide azadirachtin is effective against 118 | theHealth | Volume 3 | Issue 4

many biological processes. It may cause a reduction in feeding habit, suspend the molting process, larvae and pupae death and also cause sterility in the emerging adults, this all depend on the given dose. The mode of action of this toxin is obscure. Proteomic techniques have been used to analyze the changes in protein metabolism of Spodoptera litura (F) caused by azadirachtin. Until pupation 4th in-star larvae of Spodoptera litura was fed with an artificial diet containing 1ppm of azadirachtin. After that, pupae of 48 hours were collected and their protein samples were prepared. After measurement of total protein contents of samples results revealed that protein level was significantly influenced by azadirachtin. It has been revealed that these proteins are involved in many cellular functions. One of these proteins has been identified to function as ecdysone receptor that regulates the development of insect and its reproduction. It suggests that botanical insecticide azdirachtin effects the protein expression of insects.38 Effect of azadirachtin in combination with Nuleopolyhedrovirus Enzyme activity of mid-gut in Spodoptera litura Fabricius (Lepidoptera: Noctuidae) (Tobacco cutworm) have been evaluated by the effect of azadirachtin and nuleopolyhedrovirus (NPV). Enzyme activities of gut were decreased both when azdirachtin and NPV were used individually and in combination. Gut enzymes, acid phosphatases, alkaline phosphatises, adenosine triphosphatases and lactate dehydrogenasesactivities were reduced when S. litura larvae were fed with a diet of castor leaves which are pretreated with azadirachtin and NPV. Statistically there was a significant difference in the enzyme activities when treated individually or in combined doses. This effect was more prevalent in early in-stars. There was a maximum weight loss when azadirachtin and NPV were used in combination.39 Different application of neem based biopesticides a. Control of pine weevil in forestry The field and laboratory data has been collected which shows that, the use of neem extract as a significant antifeedant by the large pine weevil. Commercial neem formulation has been assayed for the control of pine weevil in forestry. In many Asian countries neem is still being used as pest controlling agent for protecting plant in rural environment. In the western world the formulation of neem extract have been approved to be used in the management of pests in USA and many other European countries. Currently no neem products have been licensed for use in UK forestry or agriculture, but the register of organic food standard of UK has mentioned the neem extract as an acceptable product that could be used within organic farming operations. At present, within UK formulations of neem extract have been approved for use as repellent mixed with various lotions shampoos and soaps for human use. It has been revealed that neem extract play a major role in protecting seedling confers from attack of H. abietis. These neem formulations are environment friendly and it can reduce the use of synthetic pesticides.40


b. Control of Rice leaf folder There are many compounds which have different chemical structures and diverse mode of action; all are classified insecticides of botanical origin. Azadirachtin is the most bioactive component of neem plant which is a complex limnoids.9 Rice leaf folder is an insect pest of rice (Oryza sativa). Insecticide based on neem, containing azadirachtin, has been proved to play important role in protection of crops. Azadirachtin have been tested along with five other limnoids and it has proved to be more effective against rice leaf folder than the other limnoids.38 Azadirachtin causes high anti-feedant activity. When neem was added to the food of these pests their growth was significantly reduced. Treatment of neem limnoids have been shown to produce pronounced results to the growth and development of C. medinalis larvae.39 c. Control of root-knot nematodes The most important nematode pest of both tropical and subtropical region of crop production is root-knot nematodes, Meloidogyne spp. There are more than 2000 plant spp including herbaceous, wood plants of mono and dicotyledons which are being affected by nematodes that are obligate parasite of these plants. M. incognita and M. javanica cause most of the damage to field crops, vegetables, fruit trees. Crude and refined neem formulations have been tested against these nematodes. The crude neem formulations are neem leaves and oil cake while the refined one is azadirachtin. It has been investigated that application of these two formulations reduces the number of eggs and egg mass when used as nematicides. Application of 0.1% w/w of azadirachtin reduces the invasion of juvenile nematodes. Azadirachtin have been proved for having nematicide activity.41 d. Control of P. xylostella Among vegetable crops cabbage is one of the most important one. P. xylostell L is one of the most important insect pests of cabbage as well as of some other important brassicae vegetables. The main issue related to the long term use of toxic insecticides and development of resistant in pests against pesticides.42 In addition to this negative health impacts and environmental concerns are also associated with the use of these synthetic pesticides. On the other hand neem based insecticides have played a potential role in the management of botanical pest. Neem based insecticides are non-toxic to humans and pests are usually do not become resistant to these pesticides.43 Neem extract have inhibitory effect on the oviposition of P. xylostella. It has been shown that when P. xylostella larvae are fed with leaves treated with azadirachtin it causes death of larvae within 7 days.44 Photosensitivity of azadirachtin Recently the use of synthetic pesticides such as carbamates, organophophates has been reduced and interest has been shifted toward environmental friendly and less toxic pesticides of plant origin. The most popular among them is

azdirachtin-A which is a mixture of isomers of tetraterpenoids obtained from seeds of neem tree. Azadirachtin-A is extremely sensitive to the presence of sunlight.45 When spray of azadirachtin is used for conifers and deciduous foliage, it is rapidly decomposed in the presence of light. Due to high photosensitivity the use of azadirachtin-A is limited. In the presence of sunlight half life of azadirachtin is also reduced. So for effective use of azadirachtin its stability should be increased. It has been suggested that addition of UVabsorbing compound to the formulation of azadirachtin sprays can make it more stable. These UV-absorbants can either absorb the UV-light or it can prevent the photo excitation of pesticides. Different UV-absorbers can be used for this purpose.45 Conclusion In the past few decades neem based pesticides are gaining more attention because of their non-toxic and environmental friendly nature. Azadirachtin and many other neem products have shown very impressive results as biopesticide against many pests of agricultural importance. There are several issues associated with the commercial use of neem based pesticides that are still to be investigated. Like photosensitivity of azadirachtin has to be managed for its long lasting effectiveness. Persistent level of azadirachtin in food and effective concentration of neem products is not defined yet. Also biochemical mode of action of azdirachtin is still not clear. So, much of research is still to be done in this field. References: 1. Raizada RB, Srivastava MK, Kaushal RA, Singh RP. Azadirachtin, a neem biopesticide: subchronic toxicity assessment in rats. Food Chem Toxicol. 2001; 39: 477-83. 2. Copping LG, Menn JJ. Biopesticides: a review of their action, applications and efficacy. Pest Manag Sci. 2000;56: 551-76. 3. Nathan SS, Kalaivani K, Murugan K, Chung PG. The toxicity and physiological effect of neem limonoids on Cnaphalocrocis medinalis (Guenée) the rice leaffolder. Pestic Biochem Physiol. 2005;81:113-22. 4. Abou-Tarboush FM, El-Ashmaoui HM, Hussein HI, Al-Rajhy D, Al-Assiry M. Effect of azadirachtin of neemix-4.5 on SWR/J mice. Saudi J Biol Sci. 2009;16: 69-76. 5. Senthil Nathan S, Young Choi M, Yul Seo H, Hoon Paik C, Kalaivani K, et al. Effect of azadirachtin on acetylcholinesterase (AChE) activity and histology of the brown planthopper Nilaparvata lugens (Stal). Ecotoxicol Environ Saf. 2008;70:244-250. 6. Isman MB. Neem and other Botanical insecticides: Barriers to commercialization. Phytoparasitica. 1997; 25:339-344. 7. Saber M, Hejazi MJ, Hasan SA. Effects of azadirachtin/neemazal on different stages and adult life table parameters of Trichogramma cacoeciae (Hymenoptera: Trichogrammatidae). J Econ Entomol. 2004;97: 905-10. 8. Schmutterer H. The Neem Tree, Azadirachta indica A. Juss and Other Meliaceous Plants: Source of Unique Natural Products for Integrated Pest Management, Medicine, Industry and Other Purposes. VCH, Weinheim, Germany. 1995. 9. Mordue (Luntz) AJ, Morgan ED, Nisbet AJ. Azadirachtin, a natural product in insect control. In: Gilbert, L.I., Iatrou, K., Gill, S.S. (Eds.), Comprehensive Molecular Science. Elsevier, Oxford. 2005;6:117-135.

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33. Morrissey JP, Osbourn AE. Fungal resistance to plant antibiotics as a mechanism of pathogenesis. Microbiol Mol Biol Rev. 1999;63:708-24. 34. Gus-Mayer S, Brunner H, Schneider-Poetsch HA, Rüdiger W. Avenacosidase from oat: puriﬁcation, sequence analysis and biochemical characterization of a new member of the BGA family of beta-glucosidases. Plant Mol Biol. 1994;26:909-21. 35. Schabel H, Hilje L, Nair KSS, Varma RV. Economic entomology in tropical forest plantations: An update. J Trop For Sci. 1999;11:303-15. 36. Mancebo F, Hilji L, Mora GA, Salazar R. Biological activity of two neem (Azadirachta indica A. Juss., Meliaceae) products on Hypsipyla grandella (Lepidoptera:pyralidae) larvae. Crop Protection . 2002;21:107-12. 37. Nathah SS, Choi MY, Paik CH, Seo HY, Kalaivani K. Toxicity and physiological effects of neem pesticide applied to rice on the Nila parvatalugens sta 1, the brown plant hopper. Ecotoxicol Environ Safety. 2009;72:1707-13. 38. Huang Z, Shi P, Dai J, Du J. Protein metabolism in Spodoptera litura (F.) is influenced by the botanical insecticide azadirachtin. Pest Biochem Physiol. 2004;80:85-93

20. Parida MM, Upadhyay C, Pandya G, Jana AM. Inhibitory potential of neem (Azadirachta indica A. Juss) leaves on Dengue virus type-2 replication. J Ethnophamacol. 2002;79:273-8.

39. Nathan SS, Kalaivani K, Chung PG. The effects of azadirachtin and nucleopolyhedrovirus on midgut enzymatic profile of Spodoptera litura Fab. (Lepidoptera:Noctuidae). Pest Biochem Physiol. 2005;83:46-57.

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40. Thackera JRM, Bryan WJ, McGinley C, Heritage S, Strange RHC. Field and laboratory studies on the effect of neem (Azadirachta indica) oil on the feeding activity of the lagre pine weevil (Hylobius abietis L.) and implications for pest control in commercial conifer plantations. Crop protection. 2003;22:75360.

22. Salehzadeh A, Akhkha A, Cushley W, Adams RL, Kusel JR, et al. The antimitotic effect of the neem terpenoid azadirachtin on cultured insect cells. Insect Biochem Mol Biol. 2003;33:681-9. 23. Huang JF, Shui KJ, Li HY, Hu Y, Zhong GH. Antiproliferative effect of azadirachtinA on Spodoptera litura Sl-1 cell line through cell cycle arrest and apoptosis induced by up-regulation of p53. Pestic Biochem Physiol. 2011;99:16-24. 24. Robertson SL, Ni W, Dhadialla TS, Nisbet AJ, McCusker C, et l. Identification of a putative azadirachtin-binding complex from Drosophila Kc167 cells. Arch Insect Biochem Physiol. 2007;64:200-8. 25. Mordue (Luntz) AJ, Morgan ED, Nisbet AJ,. Azadirachtin, a natural product in insect control. In: Gilbert, L.I., Iatrou, K., Gill, S.S. (Eds.). Comprehensive Molecular Science. Elsevier, Oxford, pp. 2005;6:117-35. 26. Lynn OM, Kim JE, Lee KY. Effect of azadirachtin on development and geneexpression of fifth instar larvae of Indianmeal moth, Plodia interpunctella. J Asia-Pacific Entomology. 2012;15:101-5. 27. Larrauri J A, Goñi I, Martín-Carrón N, Rupérez P, Saura-Calixto F. Measurement of health-promoting properties in fruits dietary fibres: Antioxidant capacity, fermentability and glucose retartdation index. J Sci Food Agric. 1996;71:515-9. 28. Varrot A, Leydier S, Pell G, Macdonald JM, Stick RV, et al. Mycobacterium tuberculosis strains possess functional cellulases. J Biol Chem. 2005;280:20181-4.

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41. Javed N, Gowen SR, Inam-ul-Haq M, Anwar SA. Protective and curative effect of neem (Azadirachta indica) formulations on the development of rootknot nematode Meliodogyne javanica in roots of tomato plants. Crop Protection. 2007;26:530-4. 42. Magaro JJ, Edelson JV. Diamondback moth (Lepidoptera:Plutellidae) in south Texas: a technique for resistance monitoring in the field. J Econ Entomol. 2007;83:1201-6. 43. Feng R, Isman MB, Selection for resistance to azadirachtin in the green peach aphid, Myzus persicae. Experientia. 1995;51:831-3. 44. Liang GM, Chen W, Liu TX. Effects of three neem-based insecticides on diamondback moth (Lepidoptera:puctellidae). Crop Protection. 2003;22:33340. 45. Sudaram KMS, Curry J. Effects of some UV light absorbers on the photo stabilization of azadirachtin, a neem based biopesticide. Chemosphere. 1996;32:649-59.