Neem: Mode of Action of Compounds Present in Extracts and Formulations of Azadirachta indica Seeds and Their Efficacy to Pests of Ornamental Plants an

Neem: Mode of Action of Compounds Present in Extracts and Formulations of Azadirachta indica Seeds and Their Efficacy to Pests of Ornamental Plants and to Non-Target Species

D. Casey Sclar
Colorado State University
Fort Collins, Colorado 80523

Summary: Overview of growth habits and culture of Azadirachta indica.
Commercial and experimental sources of Neem formulations. The mode of
action of azadirachtin and other principal chemical compounds present in
Neem extracts. The efficacy of various Neem formulations to arthropod
pests of ornamental plants and to non-target organisms. A discussion of
future directions in Neem research is presented.

Key Words:Neem, Azadirachtin, Antifeedant, Insect Growth Regulator,
Ornamental Plants, Botanical Insecticides.

I. Introduction:
In recent years, several reviews have been published which outline
the use of the Neem tree, Azadirachta indica (Meliaceae) as a botanical
insecticide (Jacobson, 1989; Koul et al. 1990; Schmutterer, 1990;
Ascher, 1993). The mode of action of azadirachtin, the principal
insecticidal constituent of Neem oil has only recently been elucidated
(Mordue & Blackwell, 1993). Little attention has been paid to recent
experiments involving Neem's use as an insecticide for arthropod pests
of ornamental plants (Cranshaw et al., 1993). This paper was written to
accomplish three functions: First, to acquaint the reader with the Neem
tree and sources of Neem oil available commercially and experimentally.
Second, to outline the mode of action of azadirachtin and the other
compounds present in Neem oil. Finally, a report on experiments
involving the use of Neem to control ornamental plant pests and efficacy
to non-target organisms is presented.

II. The Neem Tree:
The Neem tree, Azadirachta indica (Meliaceae) is native to
Southeast Asia and grows in many countries throughout the world
(Schmutterer, 1990; Ascher, 1993). It is closely related to the
Chinaberry tree, Melia azedarach (Meliaceae) (also called Persian
Lilac). M. azedarach contains several biologically active compounds.
Although more widely distributed than A. indica because of its hardier
nature and more pleasing appearance, the use of M. azedarach as a
natural insecticide is limited because it contains tetranitroterpenoid
compounds known as meliatoxins that are highly toxic to mammals (Ascher,
1993).

A. indica propagates readily from cuttings, stumps, tissue culture
or seed. Seed propagation in nurseries followed by direct planting
into the field is the accepted method to produce plantation stands
quickly and efficiently (Jacobson, 1989). It is widely used as a shade
tree in many areas (as is M. azedarach) because it tolerates a wide
variety of field conditions (Koul, 1990; Schmutterer, 1990). The tree
tolerates heat up to 50oC, and poor, shallow, even saline soils (Koul,
1990; Schmutterer, 1990; Ascher, 1993). A. Indica grows rapidly; 4-7
meters in its first five years of growth and 5-11m for the following
five years. It will bear fruit within three years and reach a maximum
fruiting yield of 50kg seed/year ten years after planting
(Jacobson,1989; Koul, 1990; Ascher, 1993). A. indica is sensitive to
injury at temperatures around 0oC, which limits its distribution in
temperate regions of the world (Jacobson, 1989; Koul, 1990; Ascher,
1993).
The Neem tree has many medicinal uses. Notable among these are its
use as an antiseptic and diuretic. It has been used to cure many
illnesses from diabetes to syphilis, and is widely relied upon by
herbalists in its native habitat (Jacobson, 1989; Koul, 1990). The use
of A. indica as a source of natural insecticide was discovered
approximately 30 years ago (Ascher, 1993).
III. Sources of Neem:
III.1 Within Plant Distribution
Seeds of the Neem tree contain the highest concentration of
azadirachtin and all other biologically active chemical compounds
present in A. indica. (Jacobson, 1989; Koul, 1990; Schmutterer, 1990).
Other tissues of A. indica known to contain these compounds at lower
levels are the bark, leaves and heartwood (Ascher, 1993). Because they
contain the highest concentrations of biologically active compounds,
most experimental and commercial preparations of Neem are seed extracts
(Jacobson, 1989). Aqueous, methanolic and ethanolic extracts of Neem
seeds show biological activity in the laboratory and the field,
although at a varying extent to different target organisms (Ascher,
1993).
III.2 Commercial and Experimental Sources of Neem
The first commercial Neem insecticide, Margosan-O_, was registered
by the EPA for non-crop use in the United States in July, 1985
(Jacobson, 1989). Since that time, the EPA has exempted Margosan-O_
from food crop tolerances and several other commercial Neem insecticides
have been developed worldwide (Ascher, 1993). Table 1 lists current
commercial and experimental suppliers of Neem extracts and formulations.
Table 1: Commercial and Experimental Sources of Neem Within the United
States (Adapted from Larson, 1993; Mordue & Blackwell, 1993)
Company Name and Address Trade Name Formulation (a)
AgriDyne Technologies, Inc. Turplex (formerly Azatin)C
417 Wakara Way
Salt Lake City, UT 84108
Gharda Chemicals Neemguard C
Bombay, India
Grace/Sierra, Inc. Margosan-O C
Iron Run Industrial Park
570 Grant Way
P.O. Box 789
Fogelsville, PA 18051
ITC Ltd. Wellgro, RD- Repelin C
Andhra Pradesh, India
Ringer Corp. Bioneem, Neemesis C
9959 Valley View Road (former Safer Ltd. products
Eden Prairie, MN 55344 consolidated here)
Rohm & Haas Co. RH - 9999 E
Independence Mall West
Philadelphia, PA 19105
Trifolio M GmBH Neemazal C
D-6335
Lahnau 2, Germany
USDA-ARS N/A E
National Center for Agricultural
Utilization Research
Peoria, IL 61601
Valent USA Corp. AD 1000 E
1333 N. California Boulevard #600
P.O. Box 8025
Walnut Creek, CA 94526-8025
West Coast Herbochem Neemark C
Bombay, India
a Key: C = Commercial, E = Experimental

Experimental sources of Neem remain cryptic. Within the United
States of America, only the USDA-ARS is a consistent supplier of raw,
unprocessed Neem seeds. Individual research laboratories may have
sources not known by the author.
IV. Modes of Action of Neem Oil Compounds:
IV.1 Overview
All biologically active Neem compounds are suspected to be derived
from one parent compound, the tetracyclic triterpenoid tirucallol (Fig.
1). All other products formed are considered successive rearrangement
and oxidation products of tirucallol (Ascher, 1993). It is generally
accepted that the tetranotriterpenoid (also called limonoid) compound
azadirachtin (Fig. 1) is responsible for the majority of biological
effects observed in organisms exposed to Neem compounds (Isman, 1990;
Mordue & Blackwell, 1993; Verkerk & Wright, 1993). However, 25
different biologically active compounds have been isolated from Neem
seeds (Lee et al., 1991). Other compounds present in Neem oil are
responsible for some of the biological activity observed (See - IV.4:
Other Effects of Neem).
Figure 1. The structures of tirucallol and azadirachtin
(Ascher,1993)
Within the azadirachtin molecule, the decalin fragment is
responsible for the insect growth regulation and development effects
observed, while the hydroxy furan fragment causes the antifeedant
effects more widely observed among target species (Fig. 2)(Aldhous,
1992). The IGR and antifeedant effects of azadirachtin are independent
of each other, but both remain relative to concentration (Koul & Isman,
1991).
Figure 2. The decalin and hydroxy furan fragments of azadirachtin
(Aldhous,
1992)
IV.2 Antifeedant Effects of Azadirachtin
The antifeedant effects of azadirachtin are well known (for reviews
see Jacobson, 1989; Schmutterer, 1990; Ascher, 1993; Mordue and
Blackwell, 1993). Both primary and secondary antifeedant effects have
been observed in the case of azadirachtin (Ascher,1993). Primary
effects include the process of chemoreception by the organism (e.g.
sensory organs on mouthparts which stimulate the organism to begin
feeding) whereas secondary processes are effects such as gut motility
disorders due to topical application only (Schmutterer, 1990;
Ascher,1993). Inhibition of feeding behavior by azadirachtin results
from blockage of input receptors for phagostimulants or by the
stimulation of deterrent receptor cells or both (Mordue & Blackwell,
1993). In a recent study by Yoshida and Toscano (1994), the relative
consumption rate of Heliothis virescens larvae treated with azadirachtin
was 25% of the control, attributing to the lowest assimilation
efficiency of all natural insecticides tested. In another study, larvae
of Heliothis virescens consumed less food, gained less weight, and were
less efficient at converting ingested and digested food into biomass
(Barnby & Klocke, 1987). Sensitivity between species to the
antifeedant effects of azadirachtin are profound. Order Lepidoptera
appear most sensitive to azadirachtin's antifeedant effects, with
Coleoptera, Hemiptera and Homoptera being less sensitive (Mordue &
Blackwell, 1993).
IV.3 Insect Growth Regulatory Effects of Azadirachtin
The insect growth regulatory effects of azadirachtin (in contrast
to its antifeedant effects) are remarkably similar among species (Mordue
& Blackwell, 1993). Various developmental, post-embryonic, reproductive
and growth inhibitory affects have been observed, causing malformation
and mortality in a dose-dependent manner (Ascher, 1993).
Schmutterer (1990) suggested that azadirachtin modifies the
programs of insects by influencing hormonal systems, especially that of
ecdysone. The effects of azadirachtin are both dose and time
dependent, prevent both ecdysis and apolysis, and can cause death before
or during molting, possibly inducing "permanent" larvae (Mordue &
Blackwell, 1993). Exogenous application of growth hormones did not
deter the effects of azadirachtin, leading researchers to suggest that
the most probable site of action of azadirachtin is at the site of
synthesis and release of Prothoracicotropic hormone (PTTH) (Koul and
Isman, 1991). The main action of azadirachtin appears to be at the
release sites of PTTH from the corpora cardiaca. Azadirachtin appears
to block the release of neurosecretory material from the corpora
cardiaca resulting in a reduced turnover rate. This affects the rate of
synthesis of PTTH by brain neurosecretory cells (Barnby & Klocke, 1990;
Mordue & Blackwell, 1993). Marco et al. (1990) stated that azadirachtin
caused a significant depletion of immunoreactive ecdysteroids in
Tenebrio molitor pupae. T. molitor has no PTTH glands and yet is still
sensitive to the ecdysteroid antagonistic effect exhibited by exposure
to azadirachtin. A possible explanation for this phenomenon could
involve epidermal cells or oenocytes being affected as both are
suggested as alternative sites of ecdysteriod production. It should be
noted that all these effects are working in conjunction with blockages
in JH (juvenile hormone) and allotropin titers, collectively resulting
in both molting and reproductive aberrations. It is assumed that
azadirachtin has direct effects on a variety of tissues and organs. This
suggests either a number of different modes of action or a specific
toxic lesion to all cells which manifests itself more obviously in some
cells than others (Mordue & Blackwell, 1993). All insect growth
regulatory effects of azadirachtin are suggested by Schmutterer (1990)
to be indirectly influenced by temperature, with greater activity seen
at higher temperatures.
IV.4 Other Effects of Neem (Unconventional Effects)
It has been noted that the presence of azadirachtin alone is not as
toxic as all Neem oil components present together (Verkerk & Wright,
1993). Other compounds present in Neem seed extracts besides
azadirachtin exhibit biological activity in myriad ways (Ascher, 1993).
Blaney et al. (1990) found that salinnin and nimbin, two other compounds
present in Neem seed extracts, exhibit an entirely different mode of
action than azadirachtin. Effects which may be exhibited by one or more
compounds present in Neem seed extracts include: oviposition repellency,
egg sterility, longevity, fitness and inhibition of chitin biosynthesis
(Ascher, 1993).
Use of a commercial formulation of Neem (RD-Repelin) successfully
deterred aphids attempting to land, probe or oviposit (Hunter and
Ullman, 1992). Lowery & Isman (1993) suggest that this deterrence
results from a variety of compounds working in concert with one another,
producing different behavioral responses which vary in magnitude between
species. Schmutterer (1990) reported reduced fecundity and longevity in
aphids treated with Neem seed extract. Treatment with Neem oil resulted
in a reduction of eggs produced and increased incubation time for eggs
of the spider mite Tetranychus urticae (Dimetry et al., 1993). Neem
inhibited adult eclosion and reproductive potential in Liriomyza
trifolii, as well as longevity of adults surviving treatment as eggs or
larvae (Parkman and Pienkowski, 1990). A reduction in transmission of
aphid-borne viruses in some species has also been observed (Hunter &
Ullman, 1992; Mordue & Blackwell, 1993).
It is suggested that Neem compounds which are present in solid form
in or upon leaf surfaces are responsible for these effects. Evidence
supporting this hypothesis is the study of Pathak & Krishna (1991) in
which Eucalyptus oil volatiles adversely affected growth and
reproduction of Corcyra cephalonica whereas exposure to Neem oil
volatiles had no effect.
Other effects of Neem are more behavioral in nature. Saxena et al.
(1993) reported that Neem disrupted mating signals in Nilaparvata lugens
(Homoptera: Delphacidae). Some insects failed to produce calls, while
others emitted unrecognizable calls.
V. Use of Neem Oil for Ornamental Plant Pest Control:
V.1 Use of Neem to Control Greenhouse Ornamental Plant Pests
Larew (1990) reviewed the use of Neem against pests of greenhouse
crops. The resistance of greenhouse pests to many insecticides and the
introduction of new pests (e.g. the Sweetpotato Whitefly - Bemesia
tabaci) to the greenhouse environment continue to complicate the
production of ornamental greenhouse crops. Although Schmutterer (1990)
suggested that the use of Neem may not be suitable for crops with high
quality demands, many successes have been reported using Neem
formulations to control pests on greenhouse ornamental plants.
Chrysanthemum Leafminer (Liriomyza trifolii) is an extremely
devastating pest of indoor greenhouses. Using Neem extracts, greenhouse
population levels of this pest were significantly reduced (Parkman and
Pienkowski, 1990). Leafminer infestations occur primarily by the
introduction of infested cuttings into the greenhouse. Sanderson et al.
(1989) observed decreased larval population and adult emergence level
after drenching boxed cuttings with Neem oil before shipping. Ascher et
al. (1992) used the commercial Neem formulation Azatin (now called
Turplex) to successfully reduce nymphal populations of Frankliniella
occidentalis, a thrips species difficult to control with conventional
insecticides.
Whiteflies represent perhaps the biggest challenge to growers of
greenhouse ornamental crops. Lindquist et al. (1990) reported the
efficacy of Neem against both susceptible and binfenthrin-resistant
populations of the Greenhouse Whitefly (Trialeurodes vaporarium). Price
and Schuster (1991) conducted field trials using Neem and various other
synthetic insecticides to control populations of Bemesia tabaci on
Poinsettia plants. Their data show that although Neem was slower to
display an initial effect, it ultimately yielded a level of control
comparable to that of many synthetic insecticides. This effect was
present without the phytotoxicity to leaves and bracts commonly
associated with the use of synthetic insecticides on Poinsettias.

V.2 Use of Neem to Control Pests of Landscape Ornamental Plants
Not since the review in Jacobson (1989) has the use of Neem for
control of landscape plant pests been addressed. During that time, new
developments have surfaced in the use of Neem for this purpose.
Schmutterer (1990) noted antifeedant effects of azadirachtin to Japanese
Beetle (Popillia japonica), a major pest of landscape plants in North
America. A reduction in field populations of aphids of various species
was reported by Lowery et al. (1993) at a level of control similar to
that of pyrethrum, another botanical insecticide. In a subsequent
paper, Lowery & Isman (1993) state that the settling and probing
behavior of the resistant aphid species Myzus persicae was not deterred
by Neem compounds, although the behavior of two other aphid species
(Sitobion avenae and Rhopalosiphum padi) was successfully its use.
Stark (1992) suggested that Neem would be useful as part of a turfgrass
IPM program. Recently, Cranshaw et al. (1993) reported the efficacy of
several commercial formulations of Neem against eggs and larvae of the
Elm Leaf Beetle (Xanthogaleruca luteola) in laboratory and field trials.
VI. The Efficacy of Neem to Non-Target Organisms:
Neem's efficacy to non-target and beneficial organisms has been
documented in previous and recent literature (Jacobson, 1989;
Schmutterer, 1990; Ascher, 1993; Mordue & Blackwell, 1993). Table 2
summarizes the effects of various formulations of Neem to several
different organisms. Because the amount of azadirachtin and other
compounds present in Neem oil is often not quantified by researchers,
the long-assumed benign effects of Neem to non-target organisms listed
below may be questionable (Stark, 1992). For this reason, data
presented in Table 2 is of qualitative nature only. As stated
previously, Neem is widely utilized in the tropics by humans for
medicinal purposes, and is assumed to have no detrimental effects to
humans with the exception of one trial in which an aflatoxin-
contaminated carrying agent is suspected to have been present (Jacobson,
1989; Schmutterer, 1990).
VII. Conclusion: Future Directions in Neem Research:
In the future, researchers will continue to elucidate the modes of
action of azadirachtin at a cellular level, and investigate the
mechanisms of biological activity exhibited by the other chemical
components found in Neem oil (Mordue & Blackwell, 1993). Other areas
which may be focused upon are insect resistance to azadirachtin and Neem
oil, the possible use of Neem as a systemic chemical and the stability
of Neem compounds in the field . Neem offers promise in the fight
against pesticide resistance, because of the diverse mode(s) of action
of azadirachtin and other Neem-associated compounds (Jacobson, 1989;
Ascher, 1993). It was recently hypothesized that the type of host plant
involved in a given situation may effect Neem's efficacy against a
particular pest (Lowery et al., 1993). What impact this will have in
future experiments remains to be seen.
Table 2: A selective summary of the effects of compounds present
in Neem extracts against non-target organisms and beneficial insects.
Organism Effect Level Reference Comments

Predaceous Spiders:
Lycosa pseudoannulata NE (Schmutterer, 1990)
Chiracanthium mildei NE (Schmutterer, 1990)
Predaceous Mites:
Phytoseiulus persimilis NE (Schmutterer, 1990) Some mortality
observed
but significantly
less than
that of target
organism

Oribatid Mites: OA (Stark, 1992)
Predaceous Coccinellids:
Delphastus pusillus NE (Schmutterer, 1990) No effect when
either
(Hoelmer et al., 1990) plant or prey
eggs treated
Predaceous Hemiptera:
Perillys bioculatus OA (Mordue & Blackwell,
1993)
Honeybees:
Apis mellifera NE (Schmutterer, 1990) No effect in
colonies of
> 200 individuals
Hymenopterous Parasitoids:
Aleurodiphilus sp. OA (Price & Schuster,
1991)
Apanteles glomeratus NE (Schmutterer, 1992) Below 40ppm/ AZ
Aphidius cerasicola NE (Schmutterer, 1990)
Cotesia congregata OA (Mordue & Blackwell,1993)
Dieratiella rapae NE (Schmutterer, 1990)
Encarsia sp. OA (Price & Schuster, 1991)
Telenomous remous OA (Schmutterer, 1990) Reduced longevity
Collembola: NE (Stark, 1992)
Rainbow Trout OA (Jacobson, 1989)
(Schmutterer, 1990)
a Key: NE = No biological effect observed, OA = Organism affected by
treatment
Recent reviews validated the theory that Neem has systemic action
(Ascher, 1993; Mordue & Blackwell, 1993). Xie et al. (1991) used soil
drenches of Neem to control laboratory populations of the Western Corn
Rootworm, Diabrotica virgifera virgifera and noted persistent effects
to adults feeding on plants as well as to the subterranean grubs and
pupae treated.
A foliar spray application of most commercial Neem formulations
persists 5-7 days under field conditions (possibly longer due to some
systemic effects) (Schmutterer, 1990). Even though breakdown of
azadirachtin occurs in UV light, its metabolites may still have
bioactivity (Ascher, 1993). Dihydroazadirachtin, a compound obtained by
hydrogenation of the C-22, 23 double bond of the hydroxy furan fragment
of azadirachtin, currently shows promise as a more stable compound for
better field persistence (Mordue & Blackwell, 1993). In previous
studies, hydrogenation of the hydroxy furan fragment resulted in no
decrease in bioactivity of the molecule (Blaney et al., 1990).
The types of substitutions made to the azadirachtin molecule are
important in the efficacy of the compound to target organisms. Several
different bioactive forms of the azadirachtin molecule exist.
Substitutions at some positions on the molecule will increase efficacy
to a particular target organism whereas other substitutions will result
in decreased efficacy to the same species (Simmonds et al., 1990).
Pure chemical synthesis of the azadirachtin molecule was once thought to
be impractical because of the difficult steps involved and the size of
the molecule. However, the group of Ley and Simmonds at Imperial
College in London have recently reported the success at the synthesis of
both the decalin fragment and the hydroxy furan fragment, with only the
linkage of the two fragments remaining to form the entire molecule of
azadirachtin in vitro (Aldhous, 1992). Future chemical synthesis of the
azadirachtin molecule and chemical mimics hold promise for the discovery
of safe pesticides with faster knockdown activity. This synthesis may
yield the ability to create and test each chemical component of Neem oil
in an isolated environment. Experiments of this nature would allow
researchers to continue to unravel the mystery surrounding the activity
of Neem compounds. Research of this type enhances the existing
knowledge of how Neem controls insects and mites, allowing better use of
this product by consumers, growers, farmers, and researchers.
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