Azadirachta indica: One Tree's Arsenal Against Pests
By Kirk Howatt (Colorado State University)


ABSTRACT

The tree Azadirachta indica is native to parts of South Asia where it has been used for many things. Of primary interest to research scientists is its activity as an insecticide. Many of the tree's secondary metabolites have biological activity, but azadirachtin is considered to be of the most ecological importance. Studies have shown a wide spectrum of activity and species affected. Research has increased in the past few years as the desire for safe pest control methods increases and it becomes apparent that this tree will be able to play a role in integrated pest management systems.


INTRODUCTION

Azadirachta indica has been used for centuries as the country store of developing nations. Earliest reference to it is in Sanskrit writings that are over 4,000 years old (Larson, 1990). Parts of this tree have been used for medicine, shade, building materials, fuel, lubrication, and most of all as pesticides. It is the use of this tree as an insecticide that now draws interest from industrialized countries. It is seen as an environmentally safe alternative to synthetic pesticides. To date over 195 species of insects are affected by this trees extracts at concentrations ranging from 0.1 to 1,000 ppm, and insects that have become resistant to synthetic pesticides are controllable with these extracts (Lindquist et al., 1990; Menn, 1990).


THE NEEM TREE

Azadirachta indica, commonly referred to in many countries as the neem tree, is a member of the Meliaceae family. This broad-leaved evergreen can reach heights of 30 meters with a trunk girth of 2.5 meters and live for over two centuries. Its deep root system is well adapted to retrieving water and nutrients from the soil profile, but this deep root system is very sensitive to waterlogging. The neem tree thrives in hot, dry climates where shade temperatures often reach 50 degrees celsius and annual rainfall ranges from 400 to 1,200 millimeters. The tree can withstand many environmental adversities including drought and infertile, stony, shallow, or acidic soils. The neem produces ellipsoidal drupes, that are about two centimeters in length, borne on axillary clusters. These fruits contain kernels that have high concentrations of secondary metabolites (National Research Council, 1992). There is evidence, but no scientific correlation, that trees grown in climates with lower rainfall produce kernels with higher content of metabolites (Schmutterer, 1990a).

The neem tree is believed to have originated in Assam and Burma of South Asia, but other reports suggest various areas of Pakistan, Sri Lanka, Thailand, Malaysia, and Indonesia (National Research Council, 1992). The tree also grows well in other tropical and subtropical areas around the world (Verkerk et al., 1993). This is very important to commercial neem extract production so that a broad raw material base for industrial refinement can be established. Neem trees have successfully been established in Australia, Haiti, West Africa, the Dominican Republic, Ecuador, Puerto Rico, the Virgin Islands, and in the continental United States in Florida, California, Oklahoma, and Arizona (Jacobson, 1990; Schmutterer, 1990a; Verkerk et al., 1993). The trees growing in Arizona are part of a breeding and selection program aimed at developing a variety that will be frost tolerant to temperatures as low as 18 degrees below zero celsius. Such a development would allow this tree to be established in many more regions. The seed for this project was obtained from natural tree populations growing in northern India where the climate is cooler than most areas where neem grows (Jacobson, 1990).

Cultivation of the neem tree is also an important consideration as the tree is established in new regions. Very little problems arise in vegetative propagation. Transplanting seedlings, saplings, or root suckers achieves a high success rate (National Research Council, 1992). Seeds are more desirable to use when transporting a long distance for ease of packing, however, minor problems have been observed when growing these trees from seeds. It was found that dry or unripe seeds would rot in soil. Large scale establishment of neem trees required germination in sand, transplanting to clay pots after a month, and then planting in the field when the seedlings reached 30 to 45 centimeters in height (Jacobson, 1990).


NEEM CHEMISTRY

The chemicals that have pesticidal activity can most efficiently be extracted from neem seed kernels. Neem trees begin their reproductive stage at about three to five years of age but don't become fully reproductive until they are ten years old. From this time on, the tree will yield an average of about 20.5 kilograms of fruit per year, with maximum production reaching 50 kilograms per year (National Research Council, 1992). Of the fruit yield, only about ten per cent is attributed to seed kernels, and desired biologically active compounds comprise only ten grams per kilogram of kernel weight. This means that an adult neem tree will only produce about 20 grams of pesticidal compounds in a season (Schmutterer, 1990b).

Many biologically active compounds can be extracted from neem, including triterpenoids, phenolic compounds, carotenoids, steroids, and ketones. The tetranortriterpenoid azadirachtin has received the most attention as a pesticide because it is relatively abundant in neem kernels and has shown biological activity on a wide range of insects. Azadirachtin is actually a mixture of seven isomeric compounds labeled as azadirachtin-A to azadirachtin-G with azadirachtin-A being present in the highest quantity and azadirachtin-E regarded as the most effective insect growth regulator (Verkerk et al., 1993). Many other compounds have been isolated that show antifeedant activity as well as growth regulating activity on insects. Polar and non-polar extractions yield about 24 compounds other than azadirachtin that have at least some biological activity (Schmutterer, 1990b; Jacobson, 1990). This cocktail of compounds significantly reduces the chances of tolerance or resistance developing in any of the affected organisms. However, only four of the compounds in neem have been shown to be highly effective in their activity as pesticides: azadirachtin, salannin, meliantriol, and nimbin (Jacobson, 1990; National Research Council, 1992).

These compounds can be extracted by many methods. Leaching with water is the oldest method and is still used by some firms to selectively extract azadirachtin. On the other hand, most companies are using more non-polar solvents to obtain a more varied mixture of chemicals. Hexane, pentane, ethanol, methanol, esters, and dichloromethane are used in extractions as well as mixtures of these solvents with water (Lee et al., 1988; National Research Council, 1992; Schmutterer, 1990b). Once extracted, several separation techniques are often incorporated to isolate compounds. For instance, in the isolation and identification of 7-deacetyl-17á-hydroxyazadiradione, researchers used insect bioassays to guide reverse phase HPLC fractionation, IR spectrum analysis, 13C NMR and 1H NMR spectrum analysis, and mass spectrum to determine the structure of the active compound (Lee et al., 1988).

By using laboratory techniques, it is possible to closely mimic azadirachtin as it has been identified from the neem tree. Anderson et al. (1990) and Kolb et al. (1991) each describe processes in which they synthesized roughly half of separate ends of the azadirachtin molecule. These subunits form a compound that has similar but less activity than the natural molecule. The activity of synthetic azadirachtin compares close enough to natural products to verify that azadirachtin is the primary toxic compound in neem (Verkerk et al., 1993). Because of the great number of reactions involved in each process, synthetic azadirachtin will be very costly to produce. For this reason, companies developing azadirachtin as a commercial pesticide are working with natural products. W. R. Grace & Co., NPI, and Safer Ltd. are all trying to produce low cost, yet effective, neem-based pesticides (Isman et al., 1990; Walter et al., 1990; Wood, 1990). Research is discovering that initial by-products of azadirachtin extraction have significant efficacy on pests also. Neem seed oils have detrimental effects on viruses, mites, and early larval stages of some insects, while the solid seed residue has enough residual chemical content to have activity on soilborne fungal pathogens and plant parasitic nematodes (Larew, 1990; Locke, 1990; Schmutterer, 1990b).


NEEM EFFECTS

The mode of action of neem extracts is not understood very well. It is quite possible that the different chemicals or different ratios of chemicals found in neem trees have varied effects on insects. There is also evidence given in many research studies, a few of which will be cited later, that insect species react quite differently to compounds from the neem tree.

More research has been conducted to find the primary mode of action of azadirachtin than of any other chemical in the neem tree. This is because of interest in it as a product for commercial use. Azadirachtin alone probably has several modes and sites of action (Koul, 1991). Primary of which is an interference with the neuroendocrine system in insects which controls the synthesis of ecdysone and juvenile hormone. It has been indicated by Schmutterer (1988) that interference involves the inhibition of the release of these hormones. Indication of this was an accumulation of large quantities of stainable neurosecretory material in the corpora cardiaca of Locusta migratoria. In this insect, azadirachtin regulated juvenile hormone titer to prevent vitellogenin production in females, causing sterility. This and other research has convinced many people that azadirachtin definitely has antihormonal activity. However, other evidence indicating that control of hormone concentrations is controlled indirectly leads to the conclusion that azadirachtin is not a true antihormone.

The effect of azadirachtin as an antihormone on juvenile hormone titer was also investigated in the variegated cutworm by Koul et al. (1991). Their goal was to either eliminate or reproduce the effect of azadirachtin on metamorphic abnormalities by artificially raising the concentrations of juvenile hormones I and II or BEPAT, a juvenile hormone esterase inhibitor. They were unable to achieve any desired outcome, but ligation experiments did indicate that the region of activity was in the head capsule. The possibility was proposed that an inhibition of the synthesis of a neurosecretory protein could alter titer levels (Koul et al., 1991). So while azadirachtin has activity on hormone levels, it may be an indirect relationship indicating that azadirachtin is not a true antihormone. Evidence at this time is not conclusive on the matter of primary mode and site of action, and researchers involved admit that much more investigation is necessary to unwind the mystery (Schmutterer, 1988; Schmutterer, 1990a).

Other research has indicated a more direct role in the inhibition of molting. Direct cytotoxic effects on imaginal discs and epidermal cells result in primary lesions that prevent molting (Koul et al., 1991). Azadirachtin has also been proved to be a chitin synthesis inhibitor, but the role of this inhibition as the primary mode of action has not been investigated (Schmutterer, 1988).

Neem extracts have many effects on insects. The antifeedant and growth regulating effects are the most valued in pest management as these are the most intense effects on the widest range of insects. Other secondary effects that have been studied include repellency, antioviposition, sterility, fecundity reduction, loss of flying ability, disrupting sexual communication, and reducing guttural motility (National Research Council, 1992; Schmutterer, 1990a).


EFFICACY STUDIES

Research has shown that many organisms are sensitive to neem extracts. These include insects from several orders, mites, nematodes, snails, fungi, and viruses (Bhatnagar et al., 1990; Locke, 1990; National Research Council, 1992). Insect control is now the primary use of neem and has been found to be effective against insects by several methods stated earlier. The growth regulation and feeding deterrence of azadirachtin are receiving the most attention, and other effects are studied secondarily as the experiment enables. This is not so much because these responses are less important, but not as many insects show sensitivity.

Insects have shown the most sensitivity to azadirachtin as a growth regulator. Metamorphic stages are affected in such a way that death often occurs during the molting process. These results are not only dose dependent, but also, response increases with earlier larval stages. Not all species react the same though. A few insects show no mortality or metamorphic abnormalities until the final molt to an adult insect, at which time very high rates of death are observed. Molting inhibition can be seen at very low topical and ingestion rates, one ppm. Even though this was in a laboratory, field rates are effective at rates much lower than those required to elicit other responses (Isman et al., 1990; Schmutterer, 1990b; Stark et al., 1990; Wood, 1990).

Antifeeding effects have received much attention especially in crops that suffer from excessive insect damage. Response by insects to neem extract applications varies greatly across the spectrum of sensitive insects. Even within an order this can be seen. The desert locust is believed to be the most sensitive insect to antifeedant effects of azadirachtin, but the migratory grasshopper feeds undeterred on cabbage treated with 500 ppm, a rate that would deter many other insect species. Growth reduction as a preliminary indication of food refusal can be seen at 0.1 ppm azadirachtin, but antifeedant activity often requires higher concentrations, usually over 200 ppm. In Rhodnius prolixus, antifeedant activity is observed at 600 times the amount needed to disrupt development. Gustatory and non-gustatory sensilla as well as reduced guttural motility may contribute to deterrent responses (Koul et al., 1991; Schmutterer, 1990b; Wood, 1990; Zehnder et al., 1990).

With a large number of organisms being affected by neem extracts, concern was expressed for the welfare of beneficial organisms under management programs using neem tree extracts. It has been found, however that predator and parasitoid insects are relatively unaffected when their life cycle involves exposure to neem extracts. Evidently, azadirachtin does not affect these insects in the same way or not enough chemical is taken up in their diet to cause behavioral or metamorphic abnormalities. Some parasitoids showed slight toxic affects when emerging from treated mummified hosts, but these parasitoids were likely exposed to a much higher dose than normal. Further longevity studies are warranted to determine if extracts have any effects on reproduction or alter fitness of natural enemies (Hoelmer et al., 1990).

The effects of neem on other desirable organisms have led to similar conclusions. In a study conducted by Shapiro et al. (1994), mortality of the gypsy moth was evaluated in the presence of a virus pathogen and also when the moth and virus were subject to neem treatments. Not only did the extracts have no adverse effect on viral activity but, when applied concurrently, moths died sooner. A neem product has shown no toxicity to honeybee workers at rates of 500 ppm. Earthworms actually benefit from soil application of neem by-products with increased weight gain and more progeny (Schmutterer, 1990b). And spiders, butterflies, ants, and ladybugs also show no detrimental effects from exposure to neem tree extracts (National Research Council, 1992).


APPLICATION PROBLEMS

One of the main problems of using neem treatments is the durability of azadirachtin in field conditions. The activity of neem-based products subsides rapidly, lasting four to eight days, meaning that many applications will likely be needed in a season. The primary means of this is photodegradation by ultra-violet light. But leaf pH can also affect detoxification rates, and rain can wash residue off leaf surfaces. Derivation of natural product stabilizes azadirachtin and may provide an avenue for greatly increasing its residual activity (Wood, 1990). Also, activity can be extended in plants, such as potato and tomato, that demonstrate systemic activity. This protects azadirachtin from light and through translocation enables protection of new growth which is often preferred by insects (Klocke et al., 1991, Verkerk et al., 1993).

Systemic activity in plants also relates to a greater chance of phytotoxicity. Potato, onion, cabbage, and chrysanthemum have demonstrated various types and extent of phytotoxicity. In most instances this is undesirable, but the stunting that occurs on chrysanthemums can actually take the place of plant growth regulators that are sprayed for the same effect on plants grown in greenhouses (Oetting et al., 1990; Schmutterer, 1990a).

Azadirachtin content in neem kernels and quickness of activity are further considerations in the commercialization of neem extracts. To provide a consistent product, refining kernels with similar levels of compounds is essential. On the contrary, a Canadian company discovered that samples of neem oil from Indian sources ranged from undetectable amounts, less than 50 ppm, of azadirachtin to 6,800 ppm (Isman et al., 1990). Farmers using synthetic pesticides also are used to quick acting chemicals. They may not be patient enough to wait for the activity of neem-based products to produce results (Schmutterer, 1990b).


CONCLUSION

While neem tree products have some shortcomings as a conventional alternative, they fit in well as a tool to be used in integrated pest management systems. As more and more synthetic chemicals are being pulled from the market, neem is an environmentally benign alternative. It has significant effect on pests without harming beneficial organisms. Toxicology studies have indicated it to be quite safe to mammals also (Schmutterer, 1990b). Researchers, however, still have much work ahead of them to characterize the responses of sensitive insects in the field.


REFERENCES

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