Abe 2

DEVELOPMENT OF A BOTANICAL INSECTICIDE FROM AMBON AND
SURROUNDING AREAS (INDONESIA) FOR LOCAL USE
By
Johanna Audrey Leatemia
B.Sc. (Honors), Bogor Agricultural University, 1987
M . S o , University of Guelph, 1994
A T H E S I S S U B M I T T E D IN PARTIAL F U L F I L L M E N T O F
THE REQIUREMENTS FOR THE D E G R E E OF
DOCTOR OF PHILOSOPHY
in
THE FACULTY OF GRADUATE STUDIES
Faculty of Agricultural Sciences
W e accept this thesis as conforming

to the required standards
T H E UNIVERSITY O F BRITISH C O L U M B I A
April, 2003
©Johanna Audrey Leatemia, 2003

In presenting this thesis in partial fulfilment of the requirements for an advanced
degree at the University of British Columbia, I agree that the Library shall make it
freely available for reference and study. I further agree that permission for extensive
copying of this thesis for scholarly purposes may be granted by the head of my
department or by his or her representatives. It is understood that copying or
publication of this thesis for financial gain shall not be allowed without my written
permission.
Department of pfa^ bteiu faculty c\ N

The University of British Columbia
Vancouver, Canada
Date
DE-6 (2/88)
ABSTRACT
Intensive use of synthetic insecticides to control insect pests had lead to
many problems such as pest resistance and resurgence, effects on non-target
organisms, human exposure and environmental impacts. These negative effects
have provided the impetus for the development of alternatives including botanical
insecticides. Some tropical plant families (e.g. Meliaceae and Annonaceae) have
been shown to possess promising insecticidal properties. The general objectives of
this study were to identify sources of botanical insecticides from plants growing in
Ambon and surrounding areas (Indonesia) that might be of value for commercial
development, and to develop simple methods of production for local use in these
areas.
Crude ethanolic seed extracts of Annona muricata L , A. squamosa L.
(Annonaceae), Lansium domesticum Corr., and Sandoricum koetjape (Burm. F.)
Merrill (Meliaceae) collected from different locations and years in Maluku, Indonesia,
were screened for inhibition of larval growth against the polyphagous lepidopteran,
Spodoptera litura (Fabr.). Extracts of A. squamosa were the most active ones.
These extracts were significantly more active (20-fold) than A. muricata. There were
geographic as well as annual differences among the extracts of both species.
Extracts of L. domesticum and S. koetjape did not show sufficient bioactivity to be
considered further.
Laboratory evaluation was carried out to assess the efficacy of the promising
extracts. Aqueous emulsions of ethanolic seed extracts and crude aqueous seed
extracts of A. squamosa were tested via bioassays against the diamondback moth
ii
Plutella xylostella L. and the cabbage looper, Trichoplusia ni (Hubner). The extracts
showed toxic as well as antifeedant effects against both species.
Greenhouse trials were conducted to assess the efficacy of the extracts
against diamondback moth larvae. Toxicity of crude aqueous extracts was evaluated
against some commercially available biocontrol agents. An aqueous emulsion of an
ethanolic seed extract at a concentration of 0.5% (w/v) was more (2.5 fold) effective
than 1 % Rotenone, a commercial insecticide. Crude aqueous seed extracts showed
efficacy comparable to pyrethrum (0.1% a.i), a widely used botanical insecticide.
Chrysoperla carnea (Stephens) larvae were the least susceptible to the extracts
followed by Orius insidiosus (Say.) adults, while Trichogramma brassicae (Bezd)
adults were the most susceptible.
A preliminary economic analysis was conducted to evaluate the feasibility of
producing the crude aqueous seed extracts as a botanical insecticide for local use.
Production and utilization of crude aqueous seed extracts of A. squamosa was just
slightly more economical than using synthetic insecticide.
Good efficacy of crude seed extracts of A. squamosa both in the laboratory
and the greenhouse against lepidopteran pests and a slight economic benefit will
make this species a promising candidate for development as a simple botanical
insecticide for local use in Ambon (Indonesia).
iii
TABLE OF CONTENTS
TITLE i
ABSTRACT ii
TABLE OF CONTENTS iv
LIST O F T A B L E S vii
LIST O F F I G U R E S ix
ACKNOWLEDGEMENTS xi
CHAPTER 1. GENERAL INTRODUCTION AND LITERATURE

REVIEW 1
1.1. General Introduction 2
1.2. Literature Review 5
1.2.1. Insect-plant interactions 5
1.2.2. Botanical insecticides in present use 8
1.2.3. 1.2.3. Promising botanical insecticides 10
1.2.4. Annonaceous acetogenins 17
1.2.4.1. Modes of action 18
1.2.4.3. Structure-activity relationships 18
1.2.4.4. Pesticidal properties 19
1.2.4. Ecology and Botany of Annona spp, Lansium

domesticum and Sandoricum koetjape 25
1.3. Thesis objectives 33
CHAPTER 2. SCREENING FOR INSECTICIDAL ACTIVITY OF

CRUDE SEED EXTRACTS OF ANNONA SPP. (ANNONACEAE),
LANSIUM DOMESTICUM AND SANDORICUM KOETJAPE
(MELIACEAE) AGAINST LEPIDOPTERAN LARVAE 34
2.1. Introduction 35
2.2. Materials and Methods 38
2.2.1. Plant Extracts 38
iv
2.2.2. Insects 38
2.2.3. Screening of plant extracts 41
2.2.4. Dose response experiment 43
2.2.5. Data Analysis 44
2.3. Results 45
2.3.1. Screening of plant extracts 45
2.3.2. Dose response experiment 50
2.4. Discussion 51
CHAPTER 3. LABORATORY EVALUATION OF CRUDE SEED

EXTRACTS OF A SQUAMOSA VIA BIOASSAYS 54
3.2.1. Aqueous extracts 58
3.2.2. Aqueous emulsions 58
3.2.3. Insects 59
3.2.4. Drench bioassay • 59
3.2.5. Leaf dip bioassay 60
3.2.6. Leaf disc choice bioassay 62
3.2.7. Leaf residual bioassay 64
3.3. Results 66
3.3.1. Drench biossay 66
3.3.2. Leaf dip bioassay 67
3.3.3. Antifeedant activity of crude aqueous
extracts to P. xylostella 70
3.3.4. Residual effect of crude seed extracts to P. xylostella ... 71
3.4. Discussion 73
CHAPTER 4. EFFICACY OF CRUDE SEED EXTRACTS OF
A. SQUAMOSA AGAINST DIAMONDBACK MOTH, P. XYLOSTELLA
IN THE GREENHOUSE AND TOXICITY OF THE EXTRACTS TO
NATURAL ENEMIES IN THE LABORATORY 79
4.2.1. Efficacy of crude seed extracts in the greenhouse 82
4.2.2. Toxicity of crude aqueous extracts to natural enemies ... 86
4.2.2.1. Aqueous extracts 86
4.2.2.2. Insects 86
4.2.2.3. Toxicity to larval C. carnea 87
4.2.2.4. Toxicity to adult O. insidiosus 88
4.2.2.5. Toxicity to adult T. brassicae 89
4.3. Results 90
4.3.1. Efficacy of crude seed extracts in the greenhouse 90
4.3.2. Toxicity of crude aqueous extracts to natural enemies ... 92
4.3.2.1. Toxicity to C. carnea larvae 92
4.3.2.2. Toxicity to O. insidiosus adults 94
4.3.2.3. Toxicity to T. brassicae adults 95
4.4. Discusssion 96
CHAPTER 5. FEASIBILITY OF PRODUCING CRUDE AQUEOUS

SEED EXTRACTS OF SQUAMOSA AS A SIMPLE BOTANICAL
INSECTICIDE FOR LOCAL USE IN AMBON (MALUKU),
INDONESIA-A PRELIMINARY ECONOMIC ANALYSIS 101
5.2. Cost analysis 104
5.3. Results 106
5.4. Discussion 111
CHAPTER 6. SUMMARY OF CONCLUSIONS 115
References 119
LIST OF TABLES
CHAPTER 2.
Table 2.1. Growth inhibitory effect of crude ethanolic seed extracts

of A. squamosa (sweetsop) from different locations and years of
collection on neonate S. litura (250 ppm = 0.025% fwt, n = 20) 46

of A. muricata (soursop) from different locations and years of
collection on neonate S. litura (5000 ppm = 0.5% fwt, n = 20) 47

of L. domesticum from different locations on neonate S. litura
(5000 ppm = 0.5% fwt, n = 20) 49

of S. koetjape from different locations on neonate S. litura
(5000 ppm = 0.5% fwt, n = 20) 49
Table 2.5. Effect of crude ethanolic seed extracts of A squamosa

incorporated into artificial diet on different larval instars of S. litura 50
CHAPTER 3.
Table 3.1. Range of concentrations of aqueous extracts (AE) and

aqueous emulsions (AS) of A. squamosa tested in 2 insect bioassays .. 61
Table 3.2. Efficacy of crude aqueous seed extracts of A. squamosa

from Namlea ('96, '98 and '99) in drench bioassays 66

from Namlea ('96, '98 and '99) to larvae of P. xylostella in
leaf dip bioassays 67

pooled from all locations in two insect bioassays 68
Table 3.5. Efficacy of aqueous emulsions of ethanolic seed extracts

of A. squamosa pooled from all locations in two insect bioassays 68
CHAPTER 4 .
Table 4.1. Mortality of diamondback moth (P. xylostella) larvae sprayed

with an aqueous emulsion of ethanolic seed extracts of A. squamosa
or dusted with rotenone in a greenhouse trial 90
Table 4.2. Mortality of diamondback moth (P. xylostella) larvae

2 days after spraying with crude aqueous seed extracts of
A. squamosa or pyrethrum in two greenhouse trials 91
Table 4.3. Mortality of 1 -instar C. carnea after 24 h when exposed

st
to crude aqueous seed extracts of A. squamosa in a direct spray test ... 92
Table 4.4. Cumulative mortality of 1 -instar C. carnea exposed to crude

st
aqueous seed extracts of A. squamosa in a residual contact test 93
Table 4 . 5 . . Toxicity of crude aqueous seed extracts of A. squamosa

to adult O. insidiosus exposed for 24 h in a residual contact test 94
Table 4.6. Mortality of adult T. brassicae after 24 ht when exposed to

crude aqueous seed extracts of A. squamosa in residual contact tests 95
CHAPTER 5
Table 5.1. Comparison of costs of using synthetic insecticide with

producing and using botanical insecticide to control insect pests
on cabbage plants when farmers own the trees and usually
sell the fruits 107
Table 5.2. Comparison of costs of using synthetic insecticide with

producing and using botanical insecticide to control insect pests on
cabbage plants when farmers collected seeds 109
viii
LIST OF FIGURES
CHAPTER 1.
Figure 1.1. Chemical structures of promising botanical insecticides 12
Figure 1.2. Chemical structures of Annonaceous acetogenins with
promising insecticidal properties 21
Figure 1.3. Tree, fruits and seeds of A., muricata 28
Figure 1.4. Tree, fruits and seeds of A. squamosa 30
Figure 1.5. Fruits and seeds of L. domesticum and seeds of S. koetjape .... 32
CHAPTER 2.
Figure 2.1. Maps of Indonesia and Ambon 39
Figure 2.2. Maps of Seram, Haruku, Saparua and Buru islands 40
Figure 2.3. Artificial diet used in chronic growth bioassays 42
Figure 2.4. Tree-to-tree variation in bioactivity of crude ethanolic

seed extracts of Annona squamosa (Namlea, 1999) on larval growth
of Spodoptera litura (250 ppm) and Trichoplusi ni (100 ppm) 48
CHAPTER 3 .
Figure 3.1. Leaf disc choice test 63
Figure 3.2. Mortality of 3 and 4 -instar P. xylostella on leaf dip

rd th
bioassay with aqueous extracts of A. squamosa 69
Figure 3.3. Feeding deterrency of crude aqueous seed extracts of

A.squamosa on 4 - instar P. xylostella overtime in the leaf disc choice test .... 70
th
Figure 3.4. Residual effect of aqueous seed extracts of

A. squamosa on 3 -instar P. xylostella
rd
71
ix
Figure 3.5. Residual effect of aqueous emulsions of ethanolic
seed extracts of A. squamosa on 3 -instar P. xylostella
rd
72
CHAPTER 4.
Figure 4.1. Layout of greenhouse experiments 83
Figure 4.2. Cabbage plants in greenhouse experiments 85
x
ACKNOWLEDGEMENTS
Thanks and praises be to God Almighty in Jesus Christ my Lord for His grace,
guidance and wisdom.
I would like to express my sincerest thanks to my supervisor, Dr. Murray B .
Isman, for his guidance, supervision and financial support, during my study. I would
also like to extend my gratitude to Dr. Judith Myers, Dr. Robert (Bob) Vernon and
Mr. Chris P. Bennett for their advice and valuable discussions. I would like to thank
Dr. Rick Barichello for his helpful suggestions.
Thank you to Nancy Brard for rearing the insects and for her technical help.
Thanks to Dr. Joanne Wilson and Dr. Debbie Wheeler for their technical assistances
and helpful suggestions. Thank you to Yette de Kock and Felicia Adam for their help
in collecting the seeds. Let me thank Martin Kim and Christi Wakefield for their
technical help. Thank you to all the people in the laboratory especially to Yasmin
Akhtar for the friendship and for making my work at this place enjoyable.
Acknowledgement is due to Canadian International Development Agency
(CIDA) for my scholarship provided through the Eastern Indonesia University
development Project (EIUDP).
I am grateful for my husband, Jesaja A. Pattikawa for his patience and
support. Thanks to my dear son, Jerry Pattikawa and my nephew, Asye Samson for
making it possible for me to finish this thesis. Sincere thanks goes to my mother,
sisters and brothers in Indonesia for their support and prayers.
This thesis is dedicated to my late father in memory of his love.
xi
CHAPTER 1
GENERAL INTRODUCTION AND LITERATURE REVIEW

1.1. GENERAL INTRODUCTION
Due to the photosynthetic capability of green plants, they are the basis of
nearly all food chains, i.e. they produce food for many organisms, which in turn
provide food for other organisms. Plants not only produce carbohydrates and
proteins, but also an enormous array of compounds, which are not directly required
for their growth and development. These compounds are called plant secondary
metabolites, and include thousands of alkaloids, phenolics, terpenoids, and
flavonoids. These compounds are generally thought to be involved with interactions
between the plant and its surrounding environment, including deterrence of
herbivores and other detrimental organisms, and attraction of beneficial organisms
such as pollinators and natural enemies of the herbivores. These plant secondary
metabolites can be deleterious to organisms or at least affect herbivore behaviour by
attracting or deterring them.
Humans have used plant secondary metabolites since ancient times. They
have been used for medicinal purposes to treat a wide range of diseases. Many
plant compounds also have been used as botanical pesticides to kill insects,
parasites and weeds. Their use as botanical pesticides was largely abandoned with
the advent of synthetic pesticides. The majority of synthetic pesticides, however, are
acutely toxic with broad-spectrum activity. The intensive use of these chemicals has
led to many problems including pest resistance and resurgence, effects on non-
target organisms, environmental contamination and human exposure. These
undesirable effects have provided the impetus for the development of alternatives.
2
In the search for alternatives, many scientists have turned back to the plant kingdom
with the hope of utilizing pesticidal properties of plant natural compounds. Many
plant natural compounds have been shown to possess biological activity and some
even act as acute toxins. However, most have been shown to have other, non-acute
modes of action including disrupting insect growth and development (growth
regulators) and deterring feeding (antifeedants). Such novel modes of action would
be desirable for pest control, due to their greater selectivity and possible reduction in
pest resistance.
Development and commercialization of new plant-derived (botanically)
pesticides is the main goal of this field of research. Ryania, a botanical insecticide
still in use, is a unique example of a commercially successful botanical insecticide
which was discovered by screening plant extracts for bioactivity (Crosby, 1971).
Neem is a botanical insecticide that has been registered recently in the USA. This
insecticide is based on the active principle azadirachtin, a modified triterpene
derived from the neem tree, Azadirachta indica (Meliaceae). The development of
these insecticides from crude extracts to a formulated and registered commercial
product demonstrated that it is possible to bring botanical extracts to the commercial
market. Neem also has unique modes of action, acting as both an antifeedant and
insect growth regulator. Due to their greater selectivity and safety to non-target
organisms, such novel modes of action are highly desirable. Therefore, this
suggests that botanical insecticides can play a role as alternatives for controlling
insect pests.
3
Insects are notoriously adaptable and are likely to eventually overcome most
control methods. Therefore, the best strategy is to implement an integrated pest
management (IPM) system. In an IPM system, more than one type of control method
is used e.g. biological control, resistant varieties, cultural control (e.g. crop rotation,
trap crops and good sanitation), with chemical control as the last resort. IPM is an
ecological approach to pest management in which all available, necessary
techniques are consolidated into a unified program, such that pests can be managed
in a way that economic damaged is avoided and collateral damage to the
environment is minimized. Botanical insecticides with slow acting activities and/or
non-toxic modes of action may not be highly effective on their own, but may be ideal
candidates for use in an IPM system.
Indonesia, as a tropical country with an extremely diverse flora, that may have
many promising candidates for the development of botanical insecticides for local or
potentially broader commercial use. The sources should therefore be identified from
plants growing locally, with further evaluation of promising candidates to assess their
efficacy and potential use in the future.
4
1.2. LITERATURE REVIEW
1.2.1. Insect- plant interactions
Plants are well protected from herbivores in their natural environment.
Protection can be mediated by plant structures such as thorns and spines or by plant
chemicals. The chemicals in plants considered "essential for plant growth and
development" are referred to as primary metabolites. The compounds that are not
essential for the basic growth of the plants, and whose functions are not always
known, are commonly referred to as secondary metabolites or allelochemicals
(Bernays and Chapman, 1994).
Plant secondary metabolites can be toxic to animals, fungi or
microorganisms. They may also have other ecological functions such as providing
protection from abiotic factors or helping in competition with other plants. Their
profiles are also characteristic of plant species and can be used in species
identification (Bernays and Chapman, 1994).
Many plant secondary compounds are toxic, not only to potential herbivores,
but also to the plant itself. Therefore, they are usually either compartmentalized and
separated from cytoplasm, or they are stored in an inactive form. Some alkaloids are
sequestered in epidermal tissue or vacuoles or in latex, others are found only in the
vacuoles of young tissue. Some, such as nicotine are produced in the roots and
transported to the aerial parts; they occur at high levels in xylem. Others are
deposited in cell walls, and in trees may end up in the bark. Non-protein amino acids
are usually in highest concentration in seeds. Coumarins tend to be localized in oil
5
glands or in cells of the epidermis. Acetylenes and other-lipid soluble compounds
may be secreted into the surface wax. Terpenoids are always secreted in specific
sites (Bernays and Chapman, 1994).
The use of plant natural products for insect control probably originated in
prehistoric times. Thousands of plant secondary metabolites (allelochemicals) have
been isolated and identified and hundreds of these have demonstrated biological
activity against insects (Crosby, 1971). These plant natural products could be
deleterious or at least behaviorally deterrent to one or more species of insects.
However, there are very few botanical insecticides used commercially or extensively.
According to Isman (1994) this disparity exists because we tend to search for
botanicals with acute toxicity. However, in nature, chemically mediated insect-plant
interactions are far more subtle. Most allelochemicals do not kill insects completely
but instead discourage insect herbivory, either by deterring feeding (antifeedant) and
oviposition or by inhibiting larval growth. Isman et al. (1996) defined antifeedant as
a peripherally mediated behavior-modifying substance (i.e. acting directly on
chemosensilla) resulting in feeding deterrence. Antifeedants have some advantages
over toxic compounds with respect to natural enemies, pollinators and other non-
target organisms as well as the development of resistance. However, some
drawbacks of antifeedants are the great interspecific differences in bioactivity
(Champagne et al., 1989; Nawrot, et al., 1991; Isman, 1993; Gonzales-Coloma et
al., 2002); and the fact that an insect can become desensitized to a feeding
deterrent (Schoonhoven, 1982; Blaney et al., 1990; Bomford and Isman, 1996).
6
Plant extracts usually consist of mixtures of constituents. Advantages of using
complex mixtures as crop protectants are that: natural mixtures may act
synergistically (Berenbaum, 1985); they may show greater overall bioactivity
compared to the individual constituents (Berenbaum et al. 1991; Chen et al. 1995);
and insect resistance and desensitization is much less likely to develop with
mixtures (Feng and Isman, 1995; Bomford and Isman, 1996).
Pest resistance is one of the problems associated with intensive use of
synthetic insecticides. Diamondback moth, Plutella xylostella (Lepidoptera:
Plutellidae), a cosmopolitan and major insect pest of cruciferous plants, is the first
crop pest in the world to develop resistance to DDT (Ankersmit, 1953; Johnson,
1953). This first resistant population was reported in Indonesia, and has now
become resistant to most synthetic insecticides in many countries (Talekar and
Shelton, 1993). Diamondback moths also have the distinction of being the first insect
to develop resistance in the field to the microbial insecticide Bacillus thuringiensis
(Kirsch and Schmutterer, 1988: Tabashnik et al., 1990; Hama, 1992: Shelton and
Wyman, 1992). Insecticide resistance and control failures are common in tropical
climates such as parts of Southeast Asia, Central America, the Carribean, and the
southeastern United States (Talekar and Shelton, 1993). Alternative control
methods for pests such as the diamondback moth are therefore needed. Utilization
of plant natural products to control insect pests (botanical insecticides) is one of the
alternatives.
7
1.2.2. Botanical insecticides in present use
Botanical insecticides are often safer than synthetic insecticides because of
their minimal persistence, although mammalian toxicities vary. They had been used
since the late 1800's but only five have been registered for use in the USA. These
are ryania, sabadilla, neem, rotenone, pyrethrum (Isman, 1995). Ryania is the
ground stemwood of Ryania speciosa (Flacourtiaceae). The active principle,
ryanodine, is a muscle poison that interferes with calcium release. Ryania is mostly
used by organic growers of apples and pears to control codling moth. Sabadilla is
the powdered seeds of the lily, Schoenocaulon officinale (= Veratrum sabadilla), a
plant native to South America. The active principles, cevadine alkaloids, are very
toxic to mammals (rat LD o of 12.5 mg/kg) but commercial sabadilla contains only
5
0.8% alkaloids, making it relatively safe for use. It is registered for a wide range of
pests of vegetable, fruit and berry crops, but used infrequently by organic growers.
Pyrethrum is an extract ofthe flowers of Tanacetum cinerariaefolium (Asteraceae). It
is the major botanical insecticide used in the world. It has a fast knockdown effect on
flying insects, which explains why it remains a common ingredient in many
household insecticide sprays. Pyrethrin esters, the active principles, are neurotoxins
that act by blocking voltage-dependent sodium channels in neural membranes. Its
out-of-doors efficacy is limited due to rapid degradation by ultraviolet light. Rotenone
is derived from the roots and tubers of legumes in the genera Derris and
Lonchocarpus. It has been used traditionally by indigenous peoples of the tropics as
a fish poison and is still used extensively in North America as a commercial
piscicide. Rotenone is an inhibitor of cellular respiration, specifically blocking NADH
8
oxidation. It is mainly used as a broad-spectrum home and garden insecticide (as a
dust) but is also acceptable for organic food producers. Neem is a botanical
insecticide recently registered in the U.S.A. It is a derivative of the seeds of the
Indian neem tree, Azadirachta indica (Meliaceae). The active principle, azadirachtin,
is different from other botanicals with more acute toxicities. It has both insect growth
regulator and potent antifeedant properties. Neem was first registered for non-food
(Margosan-O®) uses in 1989, and received approval from the U.S. Environmental
Protection Agency for use on food crops in 1993.
To be commercially available, a botanical insecticide must meet a number of
criteria, both biological and practical. Biological criteria include efficacy; selectivity
favoring natural enemies and other non-target organisms; favorable mammalian
toxicology; biodegradability; and a lack of phytotoxicity. Practical criteria include:
sourcing (i.e sustainable availability); potential to standardize active ingredients
from sources with natural variation, and the potential to protect the technology
(Isman, 1995). Some of these criteria could be major problems for
commercialization of new botanical insecticides (Isman, 1997) including:
(1) . sustainable availability of the natural resources; Promising plant extracts should
be available from natural sources on a sustainable basis to make them economical
to use. Otherwise, cultivation of the plants is needed.
(2) . standardization and quality control; Plant extracts, as natural products, contain
mixtures of compounds and are highly variable, making standardization difficult. It is
difficult to standardize a product when it contains many active constituents of
differing proportions and bioactivity. Quantification of active ingredient(s) is needed
9
for commercial and regulatory purposes. Analysis will be more difficult and
expensive if several active compounds require quantification.
(3). registration; In order to register a product in the USA, the EPA requires detailed
characterization of the active ingredient including its effects on the environment and
on non-target organisms. Therefore, it may take a long time and be expensive to
provide such information for a botanical insecticide which contains mixtures of active
ingredients.
1.2.3. Promising botanical insecticides
Screening of plant extracts for insecticidal activity is an important step in
discovering promising botanical insecticides. Tropical plants are considered an
excellent source of plant natural compounds due to the greater selection pressure by
herbivores in warm climates (Jacobson, 1989; Schmutterer, 1992a; Isman, 1995).
Screening of plant families with reputedly insecticidal properties is a more effective
way of searching for promising botanical insecticides. The most promising botanicals
for use at the present time and in the future are species of the families Meliaceae,
Rutaceae, Asteraceae, Piperaceae and Annonaceae (Jacobson, 1989; Isman,
1995).
10
Meliaceae
The Meliaceae (mahagony) is a tropical family of woody-plants (Cronquist,
1981). It has been the focus of much research due to its limonoid content.
Limonoids (triterpene derivatives), natural products of the Meliaceae, Rutaceae and
other Rutales, have a wide range of biological activities including insect antifeedant
and growth regulation, antifungal, bactericidal, antiviral, and medicinal effects on
animals and humans (Champagne et al., 1992). Azadirachtin, a limonoid from the
neem tree (Azadirachta indica) is well known for its potent antiffedants and insect
growth regulator properties (Schmutterer, 1990). Botanical insecticides (i.e
Margosan-O® and Azatin®) derived from the seeds of the neem tree have been
commercially available.
The chinaberry, Melia azedarach, seeds contain several limonoids
(Srivastava, 1986; Lee et al., 1989). Chiu (1987) reported that seed oil of chinaberry
was effective against orange spiny whitefly, Aleurocanthus spiniferus and the citrus
red mite, Panonychus citri, but harmless to predatory mites, Amblyseius spp.
Methanolic extracts of the seed kernels had feeding deterrent and toxic properties
against young oriental armyworm, Mythimna separata (Chiu, 1989). However, the
drawback of M. azaderach is the high toxicity of its fruit to warm-blooded animals
which may limit its use as an insecticide (Schmutterer, 1992a). Another limonoid,
toosendanin (Figure 1.1.b), was isolated from the bark of M. azedarach and M.
toosendan (Chiu, 1989).
11
(CH )4-
2
e. a-Terthienyl f. Pipercide
Figure 1.1. Chemical structures of promising botanical insecticides.
12
Toosendanin is a strong antifeedant, stomach poison and growth inhibitor against
Pieris rapae. Crude toosendanin at 500-800 ppm provided effective control of this
pest in field trials (Chiu, 1989). It is the active ingredient of a botanical insecticide
that has been registered for use against a broad spectrum of fruit and vegetable
pests in China (Zhang, et al., 1992).
Another promising genus of Meliaceae is Aglaia. Nineteen species of Aglaia
have been screened against the variegated cutworm, Peridroma saucia, seven of
which significantly inhibited growth. A. odorata (Chinese rice flower bush) was the
most active of those tested (Satasook et al., 1994). Twig extracts of A. odorata
showed antifeedant properties and disrupted the development ofthe cabbage worm,
Pieris rapae. Rocaglamide (Figure 1.1.c) a highly substituted benzofuran, was
isolated using bioassay-guided fractionation (Janpraset et al., 1993). This compound
inhibits larval growth and is insecticidal to the variegated cutworm, Peridroma saucia
and the Asian armyworm, Spodoptera litura (Satasook, et al., 1992; Janpraset, et al.,
1993). Rogaclamide is 4 times less active than azadirachtin as a growth inihibitor,
but 400 times less active as an antifeedant against the cutworm, P. saucia (Arnason
et al., 1993). A patent application has been filed in Thailand for the use of this
extract for insect control, and commercialization is being considered.
Some other promising genera in the family Meliaceae that have been
screened and showed good insecticidal activity include Chisocheton, Cedrella,
Toona, Turraea and Trichilia (Isman et al., 1995).
13
In general, there are no isolated compounds from other Meliaceae that
approach the outstanding potency of azadirachtin as an insect growth disruptor.
Therefore, only a handful of species of Meliaceae are promising as botanical
insecticides.
Rutaceae
Limonin and nomilin are limonoids isolated from several Citrus species of the
family Rutaceae, especially orange and grapefruit seeds. Klocke and Kubo (1982)
recognized the potential use of citrus limonoid for use as insect antifeedants. Nomilin
is about 10 times more active than limonin as a larval growth inhibitor against
Heliothis zea and Spodoptera frugiperda. Limonin (Figure 1.1.d) and nomilin are
significantly less active as antifeedants and growth inhibitors against Ostrinia
nubilalis (Arnason et al., 1987) and Peridroma saucia (Champagne et al., 1989).
However, limonin is a potent antifeedant to the Colorado potato beetle, Leptinotarsa
decemlineata (Alford et al., 1987), and field trials have shown its promising
efficacy(Murray et al., 1995).
Asteracea
The sunflower familiy (Asteraceae) is best known as the source of pyrethrins
from Tanacetum spp. Sesquiterpene lactones isolated from a number of species of
this family have shown excellent feeding deterrence to pest insects. For example
isoalantolactone deters feeding of granary weevil, Sitophilus granarius, khapra
14
beetle, Trogoderma granarium and confused flour beetle, Tribolium confusum
(Steibl etal., 1983).
Floral and foliar extracts of Mexican marigold, Tagetes minuta have been
shown to be effective against adult Mexican bean weevils, Zabrotes subfasciatus
(Weaver et al. 1994). a-Terthienyl (Figure1.1.e), a thiophene isolated from genus
Tagetes and other genera, is a photoactivated compound effective against the
malaria mosquito, Anopheles gambiae (Arnason et al., 1989). It has also served as
a lead chemical for synthetic studies aimed at producing compounds with enhanced
activity (Philogene et al., 1986).
Piperaceae
The Piperaceae (pepper family) is well known for their use as spices and
medicinal plants and have long been used for insecticides in traditional agriculture.
The active compounds isolated from this family include acutely acting amides and
slower acting, growth reducing lignans (Addae-Mensah et al., 1977). Three
isobutylamides, pipercide (Figure1.1.f), dihydropipercide and guineesine were
isolated from black pepper, Piper nigrum (Miyakado et al.,1989). These compounds
were toxic to insects such as the European corn borer, Ostrinia nubilalis; adzuki
bean weevil, Callosobruchus chinensis; mosquito (Series atropalpus and Culex
pipiens), and the European earwig, Forficula auricularia (Miyakado et al., 1992;
MacKinnon et al., 1997; Assabgui et al., 1997). Another compound, dillapiol, a
phenylpropanoid, has been isolated from P. aduncum. Dillapiol is potentially useful
15
as a synergist with other botanical insecticides due to its potent activity as a
polysubstrate monooxygenase inhibitor (Assabgui et al., 1997).
A standardized extract of 18% amides is effective against the diamondback
moth, Plutella xylostella in field trials, and looks promising for the control of mosquito
larvae and stored product pests. A patent application has been filed in Thailand and
full commercialization is in progress (Wiriyachitra,1991).
Annonacea
The Annonaceae (custard-apple family) is a family of almost exclusively
tropical trees and shrubs. Plant parts of some species of this family have been used
traditionally as insecticides. For example, the powdered seeds and leaf juices of
Annona spp are used to kill head and body lice, and the bark of Goniothalamus
macrophyllus is used to repel mosquitoes (Secoy and Smith, 1983; Morton, 1987).
The insecticidal properties of these plants were originally attributed to a series
of benzylisoquinoline alkaloids (reviewed in Crosby, 1971). Many investigations with
respect to phytochemicals of this family focused upon the alkaloids. About 320 plant
secondary compounds including various carbohydrates, amino acids, proteins,
lipids, polyphenols, essential oils, terpenes, alkaloids and aromatic compounds from
150 species belonging to 41 genera were summarized from 288 publications in 1982
by Leboeuf et al. Uvaricin, the first Annonaceous acetogenin was discovered in
1982. It was isolated from, roots of Uvaria accuminata using bioactivity-directed
fractionation and is an in vivo antileukemic agent (Jolad et al., 1982). Since then, the
phytochemical investigations of Annonaceae have centered on acetogenins.
16
1.2.4. Annonaceous acetogenins
Annonaceous acetogenins are a class of natural compounds found
exclusively in the plant family Annonaceae. The annonaceous acetogenins have
attracted extensive interest due to their biological activities including anthelmintic,
antitumor, antimalarial, antimicrobial, antiprotozoal, and pesticidal activities. Up to
1999, nearly 400 of these compounds had been isolated from seeds, bark, leaves
and roots of 37 spesies in the genera Annona, Asimina, Goniathalamus, Rollinia and
Uvaria (Alali et al., 1999; Johnson et al., 2000).
Annoanaceous acetogenins are a series of C-35/C-37 natural products
derived from C-32/C-34 fatty acids that are combined with a 2-propanol unit. They
are usually characterized by a long aliphatic chain bearing a terminal methyl-
substituted a, 3-unsaturated v-lactone ring (sometimes rearranged to a ketolactone).
In the biogenesis, double bonds along the fatty acid chain seem to epoxidize and
cyclize to form tetrahydrofuran (THF) rings. They are classified into mono-THF (e.g.
annonacin, goniothalamicin, isoannonacin); adjacent bis-THF (e.g. uvaricin, annonin
l= squamocin, asimicin); non-adjacent bis-THF (e.g. gigantecin, bullatalicin,
sylvaticin); tri-THF (e.g. goniocin); and nonTHF-ring (e.g. goniotriocin, trilobalicin);
and nonclassical acetogenins (e.g. muconin, pyranicin), followed by subclassification
of y-lactone, substituted v-lactone, or ketolactone variations (Alali et al., 1999). The
sources, biogenesis, isolation, chemistry, synthesis and biological activities of
Annonaceous acetogenins have been extensively reviewed by a research group led
by Professor Jerry McLaughlin at Purdue University, USA (Alali et al.,1999; Zeng, et
al., 1996; Gu et al., 1995; Fang, et al., 1993; Rupprecht et al, 1990).
17
1.2.4.1 Modes of action
The Annonaceous acetogenins are the most powerful of the known inhibitors
of complex I (nicotinamide adenine dinucleotide (NADH): ubiquinone
oxidoreductase) in mammalian and insect mitochondrial electron transport systems
(Londerhausen et al., 1991; Lewis et al., 1993; Ahammadsahib et al., 1993;
Hollingworth et al., 1994). This action is analoguous to that of rotenone. Annonin I
(=squamocin), an acetogenin isolated from A. squamosa seed, is 2 -4 fold more
inhibitory than rotenone (Londerhausen et al., 1991) to mitochondria of Locusta.
Morre et al. (1995) demonstrated that acetogenins are potent inhibitors of NADH
oxidase of the plasma membranes of cancer cells. These actions decrease
oxidative, as well as, cytosolic ATP production. The consequence of such ATP
deprivation is apoptosis (programmed cell death) (Wolvetang et al., 1994). Oberlies
et al (1997) have found that acetogenins also inhibit cancer cells that are multidrug
resistant. Annonaceous acetogenins have been shown to be effective against
pesticide-resistant German cockroaches (Alali et al., 1998).
1.2.4.2. Structure-activity relationships
Structure-Activity Relationships (SAR) of Annonaceous acetogenins with
respect to their pesticidal properties have been studied by He et al. (1997). Forty-
four Annonaceous acetogenins, isolated by bioactivity-guided fractionations, were
evaluated in the yellow fever mosquito, Aedes aegypti larvae assay. The SAR was
summarized as following: (i). the adjacent or non-adjacent bis-THF ring acetogenins
are more potent than the mono-THF ring compounds which possess the same
18
number of hydroxyl groups; and (ii). the potency of activity is related to the number
and the positions of the hydroxyl groups and the positions of the THF rings. They
observed that the adjacent bis-THF ring compounds with three hydroxyl groups (e.g.
bullatacin and trilobin) are the most potent. Bullatacin and trilobin have been shown
to be 12 and 1.8 fold more potent, respectively, than rotenone (He et al., 1997).
1.2.4.3. Pesticidal properties
Annonin I (Figure1.2.a), an active compound isolated' from A. squamosa
(sweetsop) seeds, has been shown to have insecticidal properties against many
insect pests. In 1987, Moeschler et al. patented annonin as an insecticidal
compound. Annonin I (=squamocin) is a slow acting toxin particulary effective
against lepidopteran larvae. The L D 50 of this compound in larvae of the
diamondback moth, Plutella xylostella was found to be 20 ug/cm , but one-tenth of

2
that concentration reduced larval growth by 90% (Mitsui et al., 1991). An aqueous
solution of annonin I (40 ppm) caused complete mortality of diamondback moth
larvae (Londerhausen, et al., 1991). Kawazu et al. (1989) reported that annonin I
(squamocin) and neoannonin show strong ovicidal and larvacidal activity at 125-140
ug/2 g of diet for Drosophila melanogaster. To date more than twenty-five
acetogenins have been isolated from the seeds of A. squamosa; among which
annonin I (=squamocin) and squamostatin-A were two major constituents (Araya et
al., 2002; Fujimoto et al., 1988, 1994).
Asimicin (Figure 1.2.b), an active compound isolated from bark of the paw
paw tree, Asiminina triloba has been shown to have insecticidal effect against the
19
Mexican bean beetles, Epilachna varivestis, melon aphid, Aphys gossypii, yellow
fever mosquito larvae, Aedes aegypty and nematode, Caenorhabditis elegans and
antifeedant effect against the striped cucumber beetle, Acalymma vittatum
(Mickolajzcak et al.,1988, 1989b; Alkofahi et al.,1989). In 1988, Mikolajczak et al.
patented the entire group of Annonaceous acetogenins as pesticides and asimicin
was claimed as an example for the first structurally defined pesticidal acetogenin. A
divisional patent then protected the composition of matter of asimicin (Mikolajczak et
al., 1989b).
Extraction of seeds of A. glabra yielded both asimicin and annonin I
(=squamocin). These acetogenins are reported to be equitoxic to several
lepidopterans, a leafhopper, and a ladybird beetle, but asimicin is about three times
more toxic than squamocin against the adzuki bean weevil, Callosobruchus
chinensis (Mitsui et al., 1991). Annonacin, a mono-THF ring acetogenin, isolated
from seeds of A. glabra has been reported to have an antifeedant effect on
Leptinotarsa decemlineata and squamocin is toxic to L. decemlineata and Myzus
persicae. Both acetogenins are not mutagenic (Guandano et al., 2000).
Bullatacin, an acetogenin isolated from the bark of A. bullata has been shown
to be toxic to cotton aphids, southern corn rootworm, Diabrotica undecimpunctata at
24 ppm, and two-spotted spider mites,Tetranychus urticae at 10 ppm (Hui et al.,
1989). He et al. (1997) demonstrated that bullatacin and trilobacin are more potent
than rotenone, a classic complex I mitochondrial inhibitor, in a structure-activity
relationship (SAR) study using yellow fever mosquito larvae, A. aegypty.
20
6H OH
a. Annonin I
c. Annonacin
Figure 1.2. Chemical structures of Annonaceous acetogenins with promising
insecticidal properties.
21
Goniothalamicin, a mono-THF ring acetogenin, isolated from stem bark of
Goniothalamus giganteus has been shown to cause 100% mortality to blowfly
larvae,Colliphora vicina at 1% concentration (Alkofahi et al., 1988).
Alali et al. (1998) reported that annonaceous acetogenins are equipotent or
superior to commercial baits against both insecticide-susceptible and insecticide-
resistant strains of the German cockroach, Blatella germanica. The resistance ratios
are near 1, suggesting equipotency against the resistant strain.
Besides the many studies that have been done with pure acetogenins as
mentioned above, many other studies have shown that crude Annonaceous extracts
are effective against agricultural and urban pests. Mikolajczak et al. (1988) and
Alkofahi et al. (1989) reported that a partitioned ethanolic extract (F005: methanol
fraction) of paw paw bark is effective against the Mexican bean beetles, melon
aphids, and blowfly larvae. The extract was more (1.6 fold) effective than rotenone
against mosquito larvae at 10 ppm. This extract caused 100% mortality against
nematodes (C. elegans) at 10 ppm after 72 h, while pyrethrins showed no
nematocidal activity at the same dose and time period. Partitioned ethanolic bark
extract (F005) of paw-paw has been shown to be effective against the Colorado
potato beetle, L. decemlineata at 250 ppm (McLaughlin et al., 1997). A mixture of
this extract with pyrethrum showed effective synergism and enhanced efficacy
against white flies on cotton leaves. The mixture of this extract with neem extracts
also showed a synergistic effect on the Colorado potato beetle. Standardized crude
extracts ofthe bark of A. triloba shows promise as a garden pesticide.
22
Partitioned ethanolic seed extract (F005) of soursop (A. muricata) has been
shown to be effective against the Colorado potato beetle, larvae and eggs of white
flies, and green peach aphids. Mixture of the extract and its seed oil is more effective
than the extract alone (McLaughlin et al., 1997). Bioactivity-directed fractionations of
seed and foliar extracts resulted in the isolation of more than 11 mono-THF ring
acetogenins typified by annonacin (Figure1.2.c).
Seed oil of A. squamosa has been reported to reduce survival of the
leafhopper, Nephotetix virescens and transmission of rice tungro virus (Mariapan
and Saxena, 1983, 1984; Mariapan et al., 1988). The sweetsop seed oil is more
effective than neem oil in reducing the survival of the leafhopper and its transmission
of the virus. Mixture of sweetsop and neem seed oils is more effective than
individual oils (Mariapan and Saxena, 1984). Reddy and Urs (1988) demonstrated
that a petroleum ether extract of sweetsop oil reduced oviposition of rice brown
planthopper, Nilaparvata lugens. Methanolic extracts of sweetsop and pond apple
(A. glabra) seeds have been shown to be effective against rice brown planthopper
(Prijono et al.,1994).
Aqueous seed extracts of four Annona species have been tested for their
insecticidal activities against the cabbage head caterpillar, Crocidolomia binotalis. A.
glabra had the highest insecticidal activity, followed by A. squamosa, A. reticulata,
with A. muricata being the least effective (Basana and Prijono, 1994). Methanolic
seed extracts of A. glabra are effective against Phaedonia inclusa (Coleoptera:
Chrysomelidae) (Prijono and Hindayana, 1993) and aqueous extracts are effective
against C. binotalis (Prijono et al.,1997). Seed extracts of eleven species of
23
Annonaceae species have been tested against C. binotalis. Acetone seed extracts
of A. glabra and A. squamosa show strong insecticidal activity and both extracts are
more active than Derris elliptica root extracts. Aqueous seed extracts of both species
also show activity against test insects. Acetone seed extracts of three other Annona
species {A. montana, A. reticulata and A. muricata) show low to moderate lethal
effect against test insects (Prijono et al., 1997).
Ethereal twig extracts of A. senegalensis have been shown to be highly toxic
against milkweed bugs (Jacobson, 1989). Ethanolic fruit extracts of A. reticulata, A.
glabra, and A. purpurea have a juvenilizing effect on adults of the striped cucumber
beetle, Diabrotica sp. (Jacobson, 1989). Ewete et al. (1996) reported that an
ethanolic extract of Dennetia tripelata reduces growth of European corn borer larvae.
Distilled stem oil of D. tripelata is toxic to the American cockroach, Periplaneta
americana (Iwuala, et al., 1981). Leaves of Polyalthia longifolia are toxic to larvae of
mosquitoes, Culex quinquefasciatus (Murty et al., 1997). Pods and seeds oiXylopia
aethiopia deter feeding by termites, Reticulitermes speratus (Escoubas et al., 1994).
Annonaceous acetogenins are more inhibitory than rotenone, not only to
mitochondria of insects (Londerhausen et al., 1991; He et al., 1997) but also to
mammalian mitochondria (bovine heart muscle), which would account for the
significant toxicity of pure annonins to vertebrates (Londerhausen, et al.,1991).
Regardless of the vertebrate toxicity of the pure acetogenins, partitioned ethanolic
extracts of paw paw show little effect when fed to mice at 1 % concentration in their
diet and they do not cause skin irritations. Also the compounds are emetic, so the
effect of any ingestion would be prevented by emesis (McLaughlin et al., 1997).
24
Crude extracts of some species of Annonaceae have also shown potential pesticidal
properties as pointed out in this review. Seeds of Annona spp. (e.g. A. muricata =
soursop and A. squamosa = sweetsop) are waste products of these edible fruits and
could become sources of botanical insecticides particularly in tropical areas where
the trees are available locally.
1.2.5. Ecology and botany of Annona spp, Lansium domesticum and
Sandoricum koetjape
Annonaceae, the custard-apple familiy, is the largest family in the order
Magnoliales with about 130 genera and 2300 species. About a third of the species
belong to only 5 genera, Guatteria (250), Uvaria (175), Xylopia (160), Polyalthia
(150), and Annona (120). The family is well developed in both the Old and the New
World, as trees, shrubs or lianas and almost exclusively confined to tropical regions.
Asimina (ca 10 spp) is a notable exception, centering in the southeastern United
States and extending as far north as New York and Michigan (Cronquist, 1981).
Some are grown as ornamentals, while others are known for their edible fruits and
perfume. The genus Annona is the most important, since among its 120 species,
seven species and one hybrid are grown commercially. All are native to the
American tropics. The other closely related genus with some commercial fruits is
Rollinia. The important commercial species are Annona cherimola Mill, A.
diversifolia Saff., A. glabra L, A. montana Magfady, A. muricata L, A. reticulata, L,
A. squamosa L, A. squamosa x A. cherimola (atemoya), and Rollinia orthopetala
R.DC. (Nakasone and Paull, 1998).
25
A. muricata (soursop) is the most tropical and produces the largest fruit
among the Annona species. It is native to the American tropics, with the Carribean
being the area of origin. It was introduced very early to the warm lowlands of eastern
and western Africa and to southeast China. It is commonly found on subsistence
farms in South-east Asia and was established very early in the Pacific islands. It is
grown extensively in Mexico in orchards as large as 20 ha (Nakasone and Paull,
1998). In Maluku (Indonesia), the trees are scattered all over the islands, not on
plantations but in houseyards. Soursop is generally a small to medium, slender and
upright or low branching and bushy tree, growing to heights of 4.5-9 m
(Figure1.3.a). The trees require heat and humidity, lots of water, and do not tolerate
low temperatures. In the tropics, soursop grows from sea level to 1000 m. Soursop
trees are commonly grown from seed, but they can also be propagated by grafting
and budding methods. The leaves are glossy, dark green, obovate to elliptic and
12.7-20 cm long. When crushed, the leaves emit a strong odour. Flowers are
solitary, yellow, 2.5-4 cm long with three thick, fleshy petals and three minute inner
petals alternating with outer petals. Fruits are large, elongated, somewhat ovaloid
varying in size from less than 0.45 kg to more than 4.5 kg. The fruit is covered in
small knobby soft spines that easily break off when the fruit is ripe (Figure1.3.b).
The thin, inedible, leathery green skin cuts easily to yield the large mass of cream
colored, fragrant, juicy and somewhat fibrous, edible flesh (Naksone and Paull,
1998). The fruit's common name in Indonesia is "sirsak" which comes from the
Dutch "zuur zak" meaning "sour sack". A typical soursop contains anywhere from
30-100 dark-brown seeds, each about 2 cm long and 1 cm wide (Figure 1.3.c)
26
enclosed in a separate "pocket" of flesh. The soursop is usually processed into ice
creams, sherbets and juices, but more often eaten as fresh fruit.
A. squamosa (sweetsop) is native to the West Indies and is the most widely
distributed of the Annona species. This species is the most commonly grown
Annona sp. in the tropical regions of the Americas, Africa, Asia and the Pacific. It is
more tolerant to cold temperatures than soursop, and is found thriving in some
subtropical areas, such-as the coastal areas of south Florida. Sweetsop is
commonly grown from seed, but they can also be propagated by grafting and
budding methods. The tree is small, 3.0-4.6 m high, with slender branches (Fig
1.4.a). The leaves are oblong-lanceolate, 10-15 cm long. Fruit is heart-shaped
(Figure 1.4.b), 5-10 cm in diameter, yellowish green in color and packed with seeds
(Naksone and Paull, 1998). Sweetsop is a compound fruit, produced by the fusion of
many florets. The exterior parts of adjacent carpels are not completely fused and
these rounded protuberances frequently separate, exposing the white flesh upon
ripening. The pulp is creamy white in color and slightly granular in texture. The
edible pulp has a thin envelope surrounding each of the many seeds. A typical
sweetsop contains 100-150 shiny, black-brown seeds, each about 1.5 cm long and
0.5 cm wide (Figure 1.4.c). Eating a sweetsop can be quite hard work but
rewarding. The taste is sweetly bland but distinctive. The fruit's common name in
Indonesia is "srikaya" which comes from the Malay "seri kaya" meaning "rich in
grace" (Piper, 1989, Eismen, 1988).
27
The family Meliaceae has six or seven fruit species native to India, Malaysia,
Indonesia, Borneo and the Philippines. The best-known edible fruit species is
Lansium domesticum Corr., which has two major types, notably langsat and duku.
Santol or kecapi (Sandoricum koetjape (Burm. F.) Merrill is also well known. L.
domesticum originated in the area from peninsular Thailand to Borneo where wild
species are still foundalong side areas of major cultivation. Along with the
Philippines, it is also cultivated in Vietnam, Myanmar, India, Sri Lanka, Australia,
Surinam, and Puerto Rico. Kecapi is native to Indonesia, Malaysia, Borneo and the
Philippines and is cultivated in a narrower area than L. domesticum, including
Indonesia, the Phillipines, Thailand and Vietnam. Langsat, duku and kecapi can be
grown on a number of soil types. Langsat and especially duku require high soil
moisture, with adequate rainfall being essential to prevent flower and fruit drop. The
more drought-tolerant kecapi can survive and bear fruit down to 800 mm of rainfall
per annum. Damage occurs at temperatures less than 6°C in both langsat and duku.
The trees can withstand 40°C and grow better at a mean temperature of 22°C.
Kecapi is more cold-tolerant and grows up to 100 m in Java, but does best in a wet
monsoon climate (Nakasone and Paull, 1998).
The langsat and duku can grow to 30 m, although in cultivation they
are only 5-10 m tall. The bark is irregularly fluted, mottled grey and orange with a
sticky milky sap. The glossy leaves are alternate, 30-50 cm long, on petioles up to 7
cm long. Langsat leaves are faintly hairy underneath, while duku are hairless.
Kecapi is semideciduous, can grow to the same height as langsat, and also has a
29
30
milky sap. The leaves are glossy green above and light green below, alternate
trifoliate on long petioles. Langsat and duku bear many-flowered racemes
sometimes in grouping of two to five on the trunk and large branches. Kecapi flowers
are similar in size to langsat and occur on loose-hanging bunches arising on the
branches. Duku fruit are round (40-50 mm), while langsat are slightly ovoid (30-50
mm). There are 15 to 25 fruits per langsat raceme (Figure 1.5.a) and 4 to 12 in
duku. The pale green immature fruit with white latex ripens to a pale yellow,
frequently with brown blemishes. Langsat pericarp is thin with a sticky sap, while
duku has a thicker pericarp and no sap. The pericarp peels easily to reveal a clear,
white translucent and juicy adhering aril. Langsa tends to vary from sweet to sour,
with duku being sweet. Both fruits have five separate segments, with one to five
seeds in langsat (Figure 1.5.b) and one or two in duku. Langsat is more commonly
grown than duku in Ambon. The kecapi is a golden-yellow-skinned berry (50-100
mm dia), which is firm and downy. The skin is thick, soft and hairy. The edible flesh
is thin (15 mm), white, juice and translucent, surrounding three to five seeds (15-20
mm long) (Figure 1.5.c). These fruits are usually consumed fresh (Nakasone and
Paull, 1998).
31
Figure 1.5. (A) Fruits and (B) seeds of L domesticum
(C) seeds of S . keotjape
Photos by author.
32
1.3. THESIS OBJECTIVES
The overall objectives of my study were:
i. to identify sources of botanical insecticides from plants growing in Ambon and
surrounding areas (Indonesia) that might be of value for commercial
development
ii. to develop simple methods of production for local use in these areas
The specific objectives were:
1. to collect and extract some reputedly insecticidal plants and their related
species from different locations in Ambon and surrounding areas
2. to determine variability/variations among the extracts and to evaluate efficacy
of the extracts via bioassays
3 to conduct greenhouse/field trials using these extracts to control local insect
pests
4. to evaluate the feasibility of producing a simple botanical insecticide for local
use
33
CHAPTER 2
SCREENING OF CRUDE SEED EXTRACTS OF ANNONA SPP. (ANNONACEAE),
LANSIUM DOMESTICUM AND SANDORICUM KOETJAPE (MELIACEAE) FOR
INSECTICIDAL ACTIVITY AGAINST
LEPIDOPTERAN LARVAE
34
2.1. INTRODUCTION
A s an alternative to synthetic insecticides, botanical insecticides offer a more
natural, "environmentally friendly" approach to pest control. Screening of plant
extracts for deleterious effects on insects is one of the approaches used in the
search for novel botanical insecticides (Schmutterer, 1992a; Arnason, et al., 1993;
Isman, 1995). The most promising botanicals for use at the present time and in the
future are species of the families Meliaceae, Rutaceae, Asteraceae, Annonaceae,
and Piperaceae (Jacobson, 1989; Isman, 1995).
The Meliaceae (mahogany) is a tropical family of woody-plants comprising
approximately 51 genera and 550 species (Cronquist, 1981). Seed (Mikolajczak and
Reed, 1987; Mikolajczak et al., 1989a) as well as foliar (Champagne, et al., 1993)
extracts of several Meliaceous species have been reported to have toxic and potent
growth-reducing activity to insects. Many species of this family have been screened
due to the outstanding bioactivity of azadirachtin, a limonoid from the neem tree
(Azadirachta indica), which is both a potent antifeedant and insect growth regulator
(Schmutterer, 1990). Limonoids (triterpene derivatives), natural products of the
Meliaceae, Rutaceae and other Rutales, have a wide range of biological activities
including insect antifeedant and growth regulator, antifungal, bactericidal, antiviral,
and medicinal effects on animals and humans (Champagne, et al., 1992).
The Annonaceae (custard-apple family) is a large family of almost exclusively
tropical trees and shrubs comprising about 130 genera and 2300 species (Cronquist,
1981). Plant parts of some species of this family have been used traditionally as
insecticides. For example, the powdered seeds and leaf juices of Annona spp are
35
used to kill head and body lice, and bark of Goniothalamus macrophyllus is used to
repel mosquitoes (Secoy and Smith, 1983; Morton, 1987). Annonaceous
acetogenins extracted from tree leaves, bark and seeds have pesticidal and/or
insect antifeedant properties (Alkofahi et al., 1989; Ratnayake et al., 1992;
McLaughlin et al., 1997). A group of C-32/C-34 fatty-acid derived natural products,
Annonaceous acetogenins, are among the most potent inhibitors of complex I
(NADH: ubiquinone oxidoreductase) in the mitochondrial electron transport system
(Londerhausen et al., 1991; Ahammadsahib et al., 1993; Lewis et al., 1993). To
date, nearly 400 of these compounds have been isolated from the genera Annona,
Asimina, Goniothalamus, Rollinia, and Uvaria (Alali et al., 1999; Johnson et al.,
2000). Their biological activities include cytotoxicity, in vivo antitumor, antimalarial,
parasiticidal, and pesticidal effects (Rupprecht et al., 1990; Fang et al., 1993; Alali et
al., 1999).
Soursop (Annona muricata L.), sweetsop (A. squamosa L.) (Annonaceae),
langsat {Lansium domesticum Corr.) and Sandoricum koetjape (Burm. F.) Merr.
(Meliaceae) are abundant as fruit trees in Ambon (Maluku), Indonesia. These trees
are sources of fresh fruit and/or fruit juices and could generate tons of waste seeds.
These waste products might potentially be developed into simple, locally available
botanical insecticides.
Experiments in this chapter form the basis of this thesis. Initially, crude seed
extracts of these four species were screened for their insecticidal bioactivities
against the Asian armyworm, Spodoptera litura (Fabricius) and the cabbage looper,
Trichoplusia ni (Hiibner). Differences in bioactivity are compared from different
36
locations in Ambon (Maluku), Indonesia and surrounding areas. The most active of
these species is then evaluated for its efficacy against Plutella xylostella L. and T. ni.
37
2.2. MATERIALS AND METHODS
2.2.1. Plant extracts
Thirty-eight plant samples (seeds) of Annona muricata, A. squamosa,
Lansium domesticum and Sandoricum koetjape were collected from different
locations between 1996 and 1999 in Ambon (Maluku), Indonesia (Figures 2.1 and
2.2). Seeds were pooled from different trees at each location. Seeds were air-dried,
ground, and 100 g of each sample extracted with 95% ethanol (5 x 200 ml) over 5
days. The extracts were vacuum-filtered (Whatman No. 1) and evaporated in vacuo
using rotovapor. The dried extracts were resuspended in a small volume of 95%
ethanol and transferred to pre-weighed vials. After evaporation of the ethanol the
vials were re-weighed to determine extract weight.
2.2.2. Insects
Asian armyworms, Spodoptera litura and cabbage loopers, Trichoplusia ni
used in this study were obtained from laboratory cultures reared on artificial diet
(F9796, Bioserv, Inc., Frenchtown, NJ) and maintained at 22 ± 1°C and a
photoperiod of 16L:8D. A laboratory-reared colony of S. litura has been maintained
at U.B.C for 7 years. The original colony was started from insects provided by
Hokkaido University and has been supplemented with new insects from Seoul
University and more from Hokaido University. A laboratory colony of T. ni has been
maintained for over 12 years. The original colony was started with pupae provided
by Safers Soap Ltd., Victoria, BC .
38
Figure 2.1. Maps of Indonesia (A) and Ambon island (B).
Seeds of A. muricata, A. squamosa, L. domesticum and
S. koetjape were collected from these (•) locations.
Figure 2. 2. Maps of Seram, Haruku, and Saparua
Islands (A) and Buru island (B). Locations of seed
collections (•)
2.2.3. Screening of plant extracts
Extracts were screened for growth inhibitory effects on neonate larvae of S.
litura via a chronic growth bioassay. Ethanolic seed extracts were incorporated into
the artificial diet at concentrations of 0.025% fresh weight (250 ppm) and 0.5% fresh
weight (5000 ppm) for A. squamosa and the other species, respectively, by the
method of Isman and Rodriguez (1983). Concentrations were determined from
preliminary experiments. Control diets were treated with carrier solvent (ethanol)
alone. Two newly hatched neonate larvae were placed in an individual cell in a
plastic assay tray (No. 9067, BioServe Inc., Frenchtown. NJ) with approximately 1g
of treated or control diet. Larvae were maintained in a growth chamber at 26°C and
a photoperiod of 16L: 8D. After 3 days, one of the two larvae was removed, leaving
one larva per cell (n = 20 for each treatment). This was to ensure that there was one,
healthy larva per compartment. Larval weights were determined individually after 10
days, the mean larval weights for each treatment expressed as a percentage of
control (Figure 2.3).
Seed extracts from five different trees of A. squamosa collected from Namlea
in 1999 were tested for growth inhibitory effect against S. litura (0.025% fresh weight
or 250 ppm) as well as against T. ni (0.01% fresh weight or 100 ppm). This
experiment was done twice and data combined for analysis.
41
Figure 2. 3. Artificial diet used in chronic growth bioassays.
The extract is incorporated into the artificial diet at prescribed
concentration and neonate larvae placed on the diet and allowed
to feed for 10 days. Larval weights are determined after 10 days
and compared to larvae fed on control diet.
42
2.2.4. Dose response experiment
Seed ethanolic extracts of A. squamosa collected from Namlea (1996), which
showed the most inhibitory effect (Table 2.1), were used for dose response
experiments. The chronic growth bioassay was carried out using a series of five
different concentrations of extracts on each larval instar of S. litura to investigate
whether different instars differ in their susceptibility to the extracts. Ethanolic seed
extracts were incorporated into artificial diet at the following concentrations 10, 25,
50, 100, 150 ppm for 1 - and 2 -instars; 50, 100, 250, 500, 750 ppm for 3 -instar
st nd rd
and 250, 500, 750, 1000, 2000 ppm for 4 - and 5 -instar larvae. Control diets were
th th
treted with carrier solvent (ethanol) alone. Bioassay was performed with neonate
larvae as described above. Bioassays with other larval instars (2 , 3 , 4 and 5 ) nd rd th th
were carried out as following. Freshly moulted insects were collected and the
bioassays conducted as before, with one insect per cell (n=20). Each experiment
proceeded until the control larvae reached late 5 /early 6 instars (this is the stage
th th
reached after 10 days, starting as neonate larvae). For 2 n d

instars, this equated to 7
days, 3 instar 6 days, 4 instars 4 days and 5 instars 3 days. Insects were
r d th th
weighed after this time and larval weights compare to larvae fed on control diets.
The EC50 (effective concentration to inhibit growth by 50% relative to control) was
calculated by extrapolating from the linear regression equation.
43
2.2.5. Data analysis
Growth inhibitory effect data was subjected to Analysis of Variance (ANOVA)
on the basis of the actual numbers observed since the variances of the sample
means were determined to be homogenous. Differences between treatment means
were analyzed using the Least Significant Difference (LSD) test (Snedecor and
Cochran, 1989) in SAS 1999 and dose response data was analyzed using linear
regression in Microsoft Excel 1997.
44
2.3. RESULTS
2.3.1. Screening of plant extracts
Extracts of both A. squamosa (sweetsop) and A. muricata (soursop)
(Annonaceae) showed bioactivity against S. litura. Extracts of A. squamosa inhibited
growth by 33-92% (Table 2.1) while A. muricata inhibited growth by 4-80% (Table
2.2). There were significant differences in growth inhibition among the extracts of
both species collected from different locations and years. (Tables 2.1 and 2.2).
Extracts of A. squamosa were screened at a dietary concentration of 250 ppm
against S. litura, 20 times less than the concentration of A. muricata used. A.
squamosa collected from Namlea yielded the most inhibitory extracts, but there was
variation among 3 different years of collection (Table 2.1).
There were also significant differences in larval growth for both S. litura and
T. ni among extracts of A. squamosa collected from different trees at one location at
the same time (Figure 2.4). A pooled extract showed significantly more inhibitory
effect (80%) than most single-tree extracts 45-55%) (Figure 2.4). Extracts were
tested against S. litura at 250 ppm, which was 2.5 times higher than the
concentration tested against T. ni.
Extracts of L. domesticum and S. koetjape (Meliaceae) were relatively
ineffective, inhibiting larval growth by 0-22% (Table 2.3) and 3-51% (Table 2.4)
respectively. There were no significant differences between locations of collection for
each species (Tables 2.3 and 2.4).
45
Table 2.1. Growth inhibitory effect of crude ethanolic seed extracts oi Annona
squamosa (sweetsop) from different locations and years of collection on
neonate Spodoptera litura (250 ppm = 0.025% fwt, n = 20)
Location Larval growth (% relative to control)

(village, island, year) Mean ± S E
Negeri Lama, Ambon, 1996 (1) a
66.9 ± 7.2 a*
Batugantung, Ambon, 1996 (2) 59.8 ±6.3 ab
Tantui, Ambon, 1997 (3) 55.2 ± 6.1 abc
Batugantung, Ambon, 1999 47.2 ± 9.0 bed
Batugantung, Ambon, 1998 45.6 + 4.3 bed
Batugantung, Ambon, 1997 42.8 ± 6.1 ed
Namlea, Buru, 1998 (4) 42.4 ± 7.5 ed
Kudamati, Ambon, 1997 (5) 33.7 ± 6.9de
Latuhalat, Ambon, 1996 (6) 32.4 ± 4.2def
Kate-Kate, Ambon, 1997 (7) 23.7 ± 4.9efg
Namlea, Buru, 1999 15.9±2.4fg
Namlea, Buru, 1996 8.3±2.8g
*Means followed by the same letter do not differ significantly at p<0.05 by the Least
Significant Difference (LSD) test.
a
Number in the brackets indicated location in the maps (Figures 2.1 and 2.2)
46
Table 2.2. Growth inhibitory effect of crude ethanolic seed extracts of Annona
muricata (soursop) from different locations and years of collection on neonate
Spodoptera litura (5000 ppm = 0.5% fwt, n = 20).

(village, island, year) Mean ± SE
Wainitu, Ambon, 1996 (8) a
96.0±9.1.a
Kayu Putih, Ambon, 1996 (9) 95.9 ± 8.5 a
Batugantung, Ambon,1997 83.5 ± 9.9 ab
Kilang, Ambon, 1997 (10) 79.7 ± 6.7 ab
Wakal, Ambon, 1996(11) 75.8 +7.5 b
Tuhaha, Ambon, 1996 (12) 69.4 ± 8.2 be
Kamarian, Ceram, 1997 (13) 69.2 ±7.0 be
Latuhalat, Ambon, 1996 54.7 ±. 6.5 cd
Waii, Ambon, 1996 (14) 50.0 ± 6.7 de
Wasu, Haruku, 1997 (15) 46.7 ± 5.6 de
Namlea, Buru, 1997 41.9 ±9.1 def
Piru, Ceram, 1996 (16) 36.7 ± 5.9 defg
Hative Besar, Ambon, 1996 (17) 34.2 ± 6.4 efg
Amahusu, Ambon, 1996 (18) 31.1 ±9.4 efg
Hative Besar, Ambon, 1999 25.0 ± 4.2 fg
Amahusu, Ambon, 1999 20.72 ± 3.4 g
Diponegoro, Ambon, 1999 (19) 20.4 ± 4.0 g
Mamala, Ambon, 1996 (20) 18.3 ± 4.2 g
Kusu-Kusu, Ambon, 1999 (21) 17.8 ± 2.8 g
* Means followed by the same letter do not differ significantly at p<0.05 by the Least
a
Numbers in the brackets indicated locations in the maps (Figures 2.1 and 2.2)
47
70
Figure 2.4. Tree-to-tree variation in bioactivity of crude ethanolic seed
extracts oi Annona squamosa (Namlea, 1999) on larval growth of
Spodoptera litura (250 ppm) and Trichoplusi ni (100 ppm) (n=40). p = pooled
extracts from trees 1-5. Means followed by the same letter do not differ
significantly at p<0.05 by the Least Significant Difference (LSD) test.
48
Table 2.3. Growth inhibitory effect of crude ethanolic seed extracts of Lansium
domesticum from different locations on neonate Spodoptera litura (5000 ppm
= 0.5% fwt, n = 20)

(village, Ambon, 1996) Mean ± SE
Soya (22) 117.7 ±34.9.a*
Amahusu 116.1 ±32.4.a
Kilang 112.2±37.7.a
Kusu-kusu 77.6 ± 16.8.a
a
Table 2.4. Growth inhibitory effect of crude ethanolic seed extracts of

Sandoricum koetjape from different locations on neonate Spodoptera litura
(5000 ppm = 0.5% fwt, n = 20)

(village, Ambon, 1997) Mean ± SE
Soya 97.4 ± 33.2 a*
Galala (23) 73.4 ± 14.2 a
Tantui (24) 48.8 ± 7.0 a
a
49
2.3.2. Dose response experiment
Larval growth was significantly reduced in a dose dependent manner, when
different larval instars of S. litura were fed on artificial diet containing seed extracts
of A. squamosa. I found that the first two instars were equally sensitive to the extract
(EC s of 192 and 202 ppm, respectively). The 3 and 4 instars were much less
50
rd th
sensitive (EC50S of 533 and 705 ppm, respectively) and the 5 instar was relatively
th
insensitive (EC 50 of 1708 ppm) (Table 2.5).
Table 2.5. Effect of crude ethanolic seed extracts of A. squamosa

incorporated into artificial diet on different larval instars of S. litura. 3
Larval instar ECso (ppm) b rvalue of regression

1 st
191.7 0.87
202.0 0.94
3rd 0.98
533.1
th
4
704.9 0.70
5 th
1707.5 0.94
degression lines were calculated from five points, n=20 for each point.
b
EC = effective concentration to reduce larval growth by 50% relative to the control
50
after 10 days of feeding
50
2.4. DISCUSSION
Screening of ethanolic seed extracts of two species of Annona from
Indonesia showed that both possess bioactivity against S. litura. However, A.
squamosa (sweetsop) was twenty-fold more active than A. muricata (soursop)
(Tables 2.1 and 2.2). My results are comparable to those of Prijono et al. (1997)
who showed that acetonic seed extracts of A. squamosa were about thirty-fold more
active than those of A. muricata against the cabbage head caterpillar, Crocidolomia
binotalis. The insecticidal activity of the seed extracts of A. squamosa is attributable
to annonins (i.e. annonin I = squamocin), adjacent bis-tetrahydrofuran (THF) ring
acetogenins (Londerhousen et al., 1991; Sahai et al., 1994; Zafra-Polo et al., 1996)
while that of A. muricata is attributable to mono THF ring acetogenins typified by
annonacin (Rieser, et al., 1993; Rieser, 1996). Structure-activity relationship (SAR)
studies have shown that acetogenins having two THF rings are more potent than
those having only one and the adjacent bis-THF acetogenins are the most potent
ones (Ahammadsahib, et al., 1993; Landolt, et al., 1995; He et al., 1997; Oberlies, et
al., 1997). This SAR may explain the much lower activity of soursop compared to
sweetsop seed extracts observed in this study.
The sweetsop extracts collected from Namlea showed the most inhibitory
effect but there is variation (5 fold) among three different years of collection (Table
2.1). Extracts collected from different trees at one location and at the same time also
showed tree-to-tree variation with respect to inhibitory effect on S. litura and T. ni. T.
ni was 2.5 times more susceptible than S. litura (Figure 2.4). Isman (1993) reported
that different insects showed wide differences in their susceptibilities to the natural
51
insecticide, azadirachtin. Extracts from pooled trees were significantly more active
than that from most single trees (Figure 2.4). There were geographic as well as
annual differences among the extracts of both species (Tables 2.1 and 2.2). Similar
variability was reported by Johnson et al. (1996) in which there were monthly
variations of twig extracts collected from a single paw-paw tree {Asimina triloba,
Annonaceae) as well as between-tree variation. Geographical variations were also
reported of neem seed extracts with respect to azadirachtin content (Ermel et al.
1987) and bioactivity (Singh, 1987). As natural products, these extracts are
subjected to environmental (i.e. type of soil, soil nutrients, temperature, humidity) as
well as genetic factors, which could be responsible for this variability.
Ethanolic seed extracts of L. domesticum and S. koetjape yielded minimal
bioactivity at 5000 ppm against S. litura (Table 2.3 and 2.4). These results contrast
with previous screening results (Mikolajczak et al., 1989a), which showed that
ethanolic seed extracts of L. domesticum and S. koetjape at 2000 ppm resulted in
99% larval growth inhibition in S. frugiperda. Variability among individuals of the
same tree species and differences in sensitivity of test species used could account
for these differences. Studies have shown that even closely related insects can
show widely different susceptibilities to the same extract or compound (Isman,
1993). Unlike the sweetsop and soursop, there is limited local variation in bioactivity
of seed extracts of both L. domesticum and S. koetjape (Tables 2.3 and 2.4), but
this may be due to the small number of locations from which collections were made.
Extracts of A. squamosa collected from Namlea in 1996 and tested on
different larval instars of S. litura showed negative growth correlation with dietary
52
concentration. I found that the first two larval instars of S. litura were equally
sensitive to the extracts. The 3 and 4 instars were much less sensitive and the 5
rd th th
instar was relatively insensitive to the extracts (Table 2.5). A similar trend was
reported by Wheeler (1999) when crude extracts of Trichilia americana (Meliaceae)
were tested against different larval instars of the same species. The age of the
insects should therefore be considered when testing insecticidal activity of any
compound or extract.
My study shows that crude seed extract of A. squamosa is a promising
candidate as a botanical insecticide. Simple methods for preparation of extracts and
their toxicity to other pest species and natural enemies are discussed in the following
chapters.
53
CHAPTER 3
LABORATORY EVALUATION OF CRUDE SEED EXTRCATS OF A. SQUAMOSA
VIA BIOASSAYS
54
3.1. INTRODUCTION
Botanical insecticides represent one alternative to synthetic insecticides due
to the negative effects of the latter, i.e. pest resistance, secondary pest outbreaks
and effects on the environment and non-target organisms. Botanical insecticides
developed from plant extracts are less persistent in the environment and are often
safer than synthetic chemicals. The steps involved in the development of botanical
insecticides from plant extracts begin with screening of candidates for deleterious
effects on insects followed by standardization of promising extracts via bioassays
(Arnason et al.,1993; Isman, 1995). Extracts that contain promising active
compounds can be used directly, but these can also form the basis for synthetic
derivatives with similar or even better insecticidal properties.
Simple crude extracts from plants have been used as insecticides in many
countries for centuries (Crosby, 1971). Crude plant extracts consist of mixtures of
active compounds. Advantages of using complex mixtures as pest control agents
are that natural mixtures may act synergistically (Berenbaum, 1985), they may show
greater overall bioactivity compared to the individual constituents (Berenbaum et
al.,1991; Chen et al.,1995), and insect resistance is much less likely to develop with
mixtures (Feng and Isman, 1995). These reasons support the use of crude,
chemically unrefined plant extracts, containing mixtures of bioactive plant
compounds rather than the use of the pure individual compounds. Also, the former
will be simpler and cheaper to prepare if the plant materials are locally available.
Previous investigations of annonaceous acetogenins, the bioactive principles
of the plant family Annonaceae, have shown that many have pesticidal and/or
55
antifeedant properties (Alkofahi et al., 1989; Ratnayake et al., 1992; Mc Laughlin et
al., 1997). Crude seed extracts of A. squamosa, promising plant extracts from
Maluku, Indonesia (see chapter 2), were subjected to bioassays, in order to assess
their toxicity and deterrent action as well as residual effect on the diamondback
moth, Plutella xylostella and the cabbage looper, Trichoplusia ni.
A drench bioassay can be used to assess contact toxicity to very small larvae.
In this bioassay, insects are drenched directly with the test solutions and mortality is
assessed after a prescribed period (Sparks et al., 1998). A common assay used to
assess contact as well as stomach toxicity of a compound in older larvae is a leaf dip
bioassay. In this assay, a leaf or leaf disc is dipped in a solution of the extract being
tested or dipped in the solvent alone (= control). Test insects are fed these discs and
mortality recorded. A leaf disc choice bioassay is commonly used to assess
antifeedant activity. In this assay, leaf discs are treated with a solvent containing the
extract being tested or with the solvent alone. Discs are then presented to test
insects and the amount of each disc eaten is measured (leaf area or weight) or the
number of insects on or adjacent to each disc is recorded. If the insect shows a
preference for the control discs, the compound is acting as an antifeedant (Lewis
and Van Emden, 1986 and Cole 1994).
The diamondback moth, Plutella xylostella, (Lepidoptera: Plutellidae) is a
cosmopolitan insect pest of cruciferous plants and can be especially destructive
(Talekar and Shelton, 1993). The first-instar larvae mine in the spongy mesophyl
tissue, whereas older larvae feed from the lower leaf surface and usually consume
all tissue except the wax layer on the upper surface, thus creating a window in the
56
leaf. Diamondback moth was reported as the first crop pest in the world to develop
resistance to DDT in Java, Indonesia (Ankersmit, 1953; Johnson, 1953) and now
has become resistant to most synthetic insecticides used against it in the field in
many countries (Talekar and Shelton, 1993). Diamondback moths had also
developed resistance in the field to the bacterial insecticide Bacillus thuringiensis
(Kirsch and Schmutterer, 1988; Tabbashnik et al.,1990; Hama, 1992; Shelton and
Wyman, 1992;). The cabbage looper, Trichoplusia ni is one of the most important
pests of crucifers including broccoli, cabbage, cauliflower, Chinese broccoli, Chinese
cabbage, etc. This species also attacks several other vegetable crops including
lettuce, beet, peas, celery, tomato, and many weedy plants. Additional hosts are
flower crops such as chrysanthemum, hollyhock, snapdragon, and sweat pea and
field crops such as cotton and tobacco. It is the larval stages that damage the crop.
The first two larval stages feed on the lower side of the leaf, eating through the
upper epidermis, leaving "windows" in the leaf. Older larvae chew larger holes in the
leaves. This species has established resistance to many insecticides including DDT,
carbaryl, parathion, methomyl, and others (Capinera, 2001).
The following experiments used techniques such as those outlined above to
evaluate the efficacy of crude seed extracts of A. squamosa. Toxicity of the extracts
was assessed using drench and leaf dip bioassays against P. xylostella and T. ni.
Antifeedant activity was investigated using leaf discs choice bioassays. Residual
action of the extracts was assessed using leaf residual bioassays against P.
xylostella.
57
3.2. MATERIALS AND METHODS
3.2.1. Aqueous extracts
Ground seeds of A. squamosa pooled from Namlea ('96,'98,'99) as well as
those pooled from several locations were extracted in distilled water according to the
concentration needed (% w/v = gr ground seeds/100ml water). Seeds were pooled
from several locations (between 1996-1999, see Table 2.1) and between 2000-
2001. The suspension was stirred for ca. 2 hours then filtered (Whatman No.1).
(seeds that collected between 1996-1999, see Table 2.1) Powdered laundry
detergent (Sunlight® 0.05% w/v) was added as an emulsifier. Detergent was used as
an emulsifier in order to develop a simple preparation using locally available
materials that ultimately could be used by farmers.
3.2.2. Aqueous emulsions
Aqueous emulsions of ethanolic seed extracts of A. squamosa was made by
adding distilled water to ethanolic extracts according to the concentration needed (%
w/v). Ethanolic seed extracts were obtained from previous extractions (see Chapter
2). These ethanolic seed extracts were pooled from all locations (Table 2.1).
Powdered laundry detergent (0.05% w/v) was added as an emulsifier.
58
3.2.3. Insects
Diamondback moth, P. xylostella larvae used in this study were obtained from
a laboratory colony reared on cabbage plants {Brassica oleracea var. Stonehead)
and maintained at ambient room temperature and a photoperiod of 16L: 8D. The
colony has been maintained for 3 years. The original colony was started with insects
collected from cabbage plants in Totem field at the University of British Columbia
campus. Cabbage loopers, T. ni, were also obtained from a laboratory colony (see
chapter 2).
3.2.4. Drench bioassay
Neonate and 2 n d
instar larvae of diamondback moth, P. xylostella as well as
neonate T. ni from the laboratory colony were used in drench bioassays. Ten larvae
were placed in a 59.2 ml plastic cup (B200 Solo cup company, Urbana, III.) lined with
42.5 mm dia. Whatman (No.1) filter paper. Larvae were drenched with 223 ul of
aqueous extracts, aqueous emulsions or control solutions (Sparks et al. 1998). The
range of concentrations of aqueous extracts and aqueous emulsions tested for each
larval instar is presented in Table 3.1. Larvae were provided with two cabbage leaf
discs (Brassica oleracea var. Stonehead; 1cm dia) 1 h after treatment. Water plus
powdered laundry detergent (0.05 % w/v) was used as a control. Six to eight
replicates were used for each treatment. The cups were placed in a plastic box lined
with moistened towel paper in a growth chamber at 26°C and a photoperiod of
16L:8D. Mortality of the insects was recorded after 24 hours. These experiments
were done four times and the data combined for analysis.
59
3.2.5. Leaf dip bioassay
Third and 4 -instar larvae of the diamondback moth obtained from the
th
laboratory colony were used in leaf-dip bioassays. The following methods were used
for aqueous seed extracts of A. squamosa pooled from Namlea ('96, '98, '99). Each
of five concentrations (Table 3.1) was tested on twenty larvae. Water plus powdered
laundry detergent (0.05% w/v) was used as a control. Cabbage leaves (Brassica
oleracea var. Stonehead) were washed with water and allowed to air-dry. Leaf discs
(2 cm dia), cut from these cabbage leaves with a cork borer, were dipped in one of
five concentrations of aqueous extracts (Table 3.1) for ca. 5 seconds and allowed to
air-dry. One disc was placed in a 5 cm dia petri dish lined with moistened Whatman
(No. 1) filter paper and a larva was placed individually in the petri dish. Petri dishes
were placed in a plastic box lined with moistened towel paper in a growth chamber
at 26°C and a photoperiod of 16L:8D. Mortality ofthe insects was recorded after 24,
48 and 72 hours. The experiments were repeated four times and the data combined
for analysis.
The following methods were used for aqueous extracts as well as aqueous
emulsions of ethanolic extracts of A. squamosa pooled from all locations.
Concentrations tested are given in Table 3.1. Water plus powdered laundry
detergent (0.05% w/v) was used as a control. Cabbage leaf discs (1.5 cm dia) were
dipped in one of five concentrations of aqueous extracts or aqueous emulsions of
ethanolic extracts (Table 3.1) and allowed to air-dry. Control discs were dipped in
control solutions. Three discs were placed in 59.2 ml plastic cups lined with
moistened Whatman (No.1) filter paper. Ten 3 -instar or five 4 -instar larvae were
rd th
60
placed in each cup. Five and 10 replicates were used for each treatment for 3 and rd
4 th
larval instar, respectively. The cups were placed in a plastic box lined with
moistened paper towels in a growth chamber at 26°C and a photoperiod of 16L8D.
Mortality of the insects was recorded after 24 hours. The latter technique of leaf dip
bioassay was used in order to increase sample size. The experiments were
conducted two to four times and the data combined for analysis.
Table 3.1. Range of concentrations of aqueous extracts (AE) and aqueous

emulsions (AS) of A. squamosa tested in 2 insect bioassays
Extracts Location Bioassay Insect (instar) Concentrations
(% w/v)
AE Namlea Drench T. n/(1 )
st
1 -16
(pooled) P. xylostella (1 )
st
0.125 -2
(2 )
nd
0.25-4
Leaf-dip P. xylostella (3 )
rd 0.25-4
(4 )
th 0.625- 10
All Drench T.ni (1 st ) 1.25-20

s
0.0625 - 1
(2 )
nd
0.125-2
rd 1.25-20
(4 ) th 2.5,-40
AS All Drench T.n/(1 )

st
0.05-0.8
st 0.00625-0.1
2 ) nd
0.0125-2
rd
0.0625 - 1
(4 ) th 0.125-2
61
3.2.6. Leaf disc choice bioassay
Leaf disc choice tests were carried out to determine antifeedant activity of
aqueous seed extracts of A. squamosa pooled from Namlea ('96, '98, '99). Fourth
instar larvae of P. xylostella were starved for 4 hours. Cabbage leaf discs (1.5 cm
dia) were dipped in 2, 4, 6, 8 or 10 % (w/v) of aqueous extracts for ca. 5 seconds
and allowed to air-dry. Control discs were dipped in solutions of water plus detergent
(0.05% w/v). A choice test was performed in a 9 cm dia. petri dish lined with
moistened Whatman (No. 1) filter paper. The arena was divided into equal quadrants,
each quadrant containing a treated or control disc placed alternately. Ten 4 -instar
th
larvae of P. xylostella were placed in the center of each dish (Figure 3.1). There
were 4 replicates (40 larvae) for each treatment. The petri dishes were placed in a
plastic box lined with moistened paper towels in a growth chamber at 26°C and a
photoperiod of 16L:8D. Numbers of larvae on or adjacent to each disc were
recorded at 2, 4, 6, 8, 10, and 12 hours. A Feeding Deterrency Index (DI) for each
treatment was calculated using the following formula (Isman et al. 1990): DI = (C-
T)/(C+T) x 100, where: DI = deterrency index; C = number of larvae on control discs;
T = number of larvae on treated discs.
62
CO
3 O
o- C© **
0, ~
O ©
— Q) •
•Q * - XJ
A © CD
> CD O
£ c
TJ CO o
5
5 9
o c>
o
—, J5 !=
to _
•— o «
•o oo ©
= -5 2
© « 2
0.2*5
CO £ 3
=5 8z
§ 5fU
CO CO t l "O
C O gg C
© .fc 3 co
to co *r o
• o> Z. *"!
£ .E © oo
r * 7"! **
to <2 © -
© c
*- o £CD oi
© u ** *-
o c c •
J= co T3 CD
© "D
° -3
U CO © O
CO 3
'•5 o" ©
•s J= ©
© u © >-
© CO © »
-J © © 5
• CO
© > o
© «- « .©
!= T3
.SP =• "Z "°
« JC
u- O
63
3.2.7. Leaf residual bioassay
This experiment was conducted to evaluate the residual effect of aqueous
emulsions of ethanolic extracts as well as aqueous extracts on 3 -instar P.rd
xylostella. Cabbage plants were sprayed with 0.5, 1 or 2 % (w/v) of aqueous
emulsions or 7.5, 15 or 30% (w/v) of aqueous extracts (1 plant per treatment) to the
point of run off (approx. 40 ml per plant) using 500 ml plastic bottle sprayers.
Control plants were sprayed with solutions of water plus powdered laundry detergent
(0.05 % w/v). Plants were held in the laboratory at ambient room temperature and a
photoperiod of 16L8D. Three leaf discs (1.5 cm dia) were cut from each treated
plant and placed in 59.2 ml plastic cups at 0, 1, 2, and 3 days after spraying. Each
cup had a moistened Whatman (No.1) filter paper on the bottom to prevent the leaf
from desiccation. Ten 3 -instar larvae were placed in each cup and five replicates
rd
were used for each treatment. The cups were placed in a plastic box lined with
moistened towel paper in a growth chamber at 26°C and a photoperiod of 16L8D.
Mortality of insects was recorded after 24 hours. The experiment was done twice
and the data combined for analysis.
3.2.8. Data analysis
In drench, leaf dip, and leaf residual bioassays, percent control mortalities
were corrected using Abbot's transformation (1925). L C 50 (lethal concentration
causing 50% mortality) in drench and leaf dip bioassays, was calculated using probit
analysis (Finney, 1971). Estimated L C 50 values were considered significantly
different when the 95% confidence intervals did not overlap. Statistical software
64
(Anonymous, 2000) was used for data analysis. Analysis of variance (ANOVA) was
performed on the basis of the actual numbers observed, for deterrency index in the
leaf disc choice bioassay, since the variances of the sample means were determined
to be homogenous. For the residual effect bioassay, an ANOVA was performed on
the arcsine-transformed percentage mortality. Differences between treatment
means were analyzed using the Least Significant Difference (LSD) test (Snedecor
and Cochran, 1989). Actual numbers observed are reported in the results.
65
3. 3. RESULTS
3.3.1. Drench bioassay
Drench bioassays were carried out to determine LC50 (lethal concentration
that cause 50% mortality of population) for aqueous seed extracts as well as
aqueous emulsions of ethanolic seed extracts of A. squamosa. There were no
significant differences in susceptibility between 1st and 2 -instar larvae when tested
nd
with crude aqueous extracts pooled from Namlea (LC50S of 0.53 and 0.97%) (Table
3.2) nor for aqueous emulsions of ethanolic extracts pooled from all locations (LC s 50
of 0.02 and 0.03%) (Table 3.5) However, 2 -instar larvae were significantly (p<0.05)
nd
less susceptible when tested with crude aqueous seed extracts pooled from several
locations (Table 3.4) The drench experiments showed that neonate cabbage
loopers, T. ni were 12 to 28 times less susceptible to the extracts than neonate
diamondback moth larvae (Tables 3.2, 3.4 and 3.5).

from Namlea ('96, '98 and '99) in drench bioassays
Insect (larval instar) LC50 (95 % CI)* Control mortality ± SE
(% w/v) (%)
T.n/(1 ) st
6.64 (4.09- 10.78 5.63 1.42
P. xylostella (1 ) st
0.53 (0.33-0.84) 12.38 ±2.57
(2 )
nd
0.97 (0.75- 1.26) 13.60 + 2.23
* LC 50 values differ significantly where 95% Cis do not overlap
66
3.3.2. Leaf dip bioassay
Leaf dip bioassays showed that mortality of 3 and 4 -instar P. xylostella fed
rd th
on discs treated with aqueous extracts pooled from Namlea, increased with
increasing concentration of extracts over time (Figure 3.2 and Table 3.3). Mortality
at 24 h was lower than 60% for both instars at the highest concentrations
(respectively, 4 and 10 % w/v), and increased to higher than 80% after 72 h (Figure
3.2). There were no significant differences in LC o between the 3 and 4 instars

5
r d th
(5.15 and 8.65%) at 24 h after treatment (Table 3.3). The same trend was also
observed for the same instar tested with aqueous extracts pooled from all locations
(LC s of 12.96 and 35.20) (Table 3.4). However, L C

50 50 values of 4 -instar larvae at
th
48 and 72 h were significantly (p<0.05) higher than those of 3 -instar (Table 3.3).
rd
Table 3.3. Efficacy of crude aqueous seed extracts of A. squamosa from

Namlea ('96, '98 and '99) to larvae of P. xylostella in leaf dip bioassays
Larval Instar Assessment LC o (95 % CI)*
5 Control Mortality ± S E
time (hours) (% w/v) (%)
3 rd
24 h 5.15(3.14-8.46) 2.50 ± 1.44
48 h 1.69 (1.31 -2.19) 10.00 ±6.77
72 h 0.88 (0.68- 1.15) 12.50 ±5.95
m 8.65 (6.64- 11.27) 0
4
24 h
48 h 4.20 (3.49 - 5.07) 1.25 ± 1.25
72 h 2.03 (1.72-2.40) 5.00 ± 2.04
67
Fourth instar larvae P. xylsotella was significantly (p<0.05) less susceptible than 3 - rd
instar larvae when tested on aqueous emulsions of ethanolic seed extracts (Table
3.5). Aqueous emulsions of ethanolic seed extracts are more (9-50 fold) potent than
crude aqueous seed extracts (Tables 3.4 and 3.5).
Table 3.4. Efficacy of crude aqueous seed extracts of A. squamosa pooled

from all locations in two insect bioassays
Insect Bioassay LC o (95 % CI)*
5 Control Mortality ± SE
(larval instar) (% w/v) ' (%)
r.A?/(1 ) st
Drench 5.19 (4.26-6.31) 2.00 ±0.88
Drench 0.18(0.14-0.25) 9.17 ±1.99
(2 ) nd
Drench 0.57 (0.45-0.73) 5.00 ±1.58
(3 ) rd
Leaf-dip 12.96(7.71-21.80) 5.00±2.24
(4 ) th
Leaf-dip 35.20(16.7-73.79) 2.50 ±1.06
Table 3.5. Efficacy of aqueous emulsions of ethanolic seed extracts of A.

squamosa pooled from all locations in two insect bioassays
Insect Bioassays LC 50 (95% CI)* Control Mortality ± SE
(larval instar) (% w/v) (%)
T.n/'(1 ) st
Drench 0.27 (0.19-0.38) 3.85 ± 1.25
Drench 0.02 (0.01 - 0.03) 11.25 ± 2.51
(2 )
nd
Drench 0.03(0.02-0.03) 5.33 ±1.42
(3 )
rd
Leaf-dip 0.39(0.33-0.46) 3.00 ±1.53
(4 )
th
Leaf-dip 0.67(0.57-0.78) ' 1.00 ±1.00
68
Figure 3.2. Mortality of 3 rd
and 4 -instar P. xylostella
th
on leaf
bioassay with aqueous extracts of A. squamosa
3.3.3. Antifeedant activity of crude aqueous extracts to P. xylostella
There were no significant differences in deterrency index between 2, 4, and
6% extracts after 2 to 12 h of exposure (Figure 3.3). However, the 10% extracts
exhibited significantly (p <0.05) higher antifeedant activity than the 2, 4 and 6 %
extracts, while 8 and 10 % extracts were not significantly different in their antifeedant
activity (Figure 3.3).
x
0)
T3
C
>»
o
c
0)
l-
l_
0>
CD
Q
Time (hour)
Figure 3.3. Feeding deterrency of crude aqueous seed extracts of A.

squamosa on 4 -
th
instar P. xylostella over time in the leaf disc choice test.
Means followed by the same letters within the same time do not differ significantly at
p<0.05 by the Least Significant Difference (LSD) test.
70
3.3.4. Residual effect of crude seed extracts to P. xylostella
Bioassays were carried out to evaluate the residual effect of both aqueous
extracts and aqueous emulsions of ethanolic extracts of A. squamosa at 0-3 days
after spraying on 3 -instar P. xylostella. There were no significant differences in the

rd
efficacy of aqueous extracts 1,2 and 3days after spraying for each concentration
tested (Figure 3.4). At 3 days after spraying, mortality with the 30% aqueous extract
was significantly lower than at day 0, but was not significantly different from that at 1
and 2 days after spraying (Figure 3.4).
100
7.5 15 30
Concentration (% w/v)
Figure 3.4. Residual effect of aqueous seed extracts of A. squamosa on 3 -

rd
instar P. xylostella. Means followed by the same letters within a treatment

concentration do not differ significantly at p<0.05 based on the Least Significance
Difference (LSD) test.
71
A similar trend was observed when aqueous emulsions of ethanolic extracts
were tested on 3 -instar larvae (Figure 3.5). However, when larvae fed on leaf discs
rd
3 days after spraying with 2% solutions, mortality was significantly lower than that at
0, 1 and 2 days after spraying (p<0.05) (Figure 3.5).
Figure 3.5. Residual effect of aqueous emulsions of ethanolic seed extracts of
A. squamosa on 3 -instar P. xylostella.

rd
Means followed by the same letters
within a treatment concentration do not differ significantly at p<0.05 based on the
Least Significance Difference (LSD) test.
72
3.4. DISCUSSION
My bioassays results showed that crude seed extracts of A. squamosa have
both toxic (Tables 3.2 - 3.5) as well as antifeedant properties (Figure 3.3). In leaf
dip bioassays, I observed that high concentrations of extracts caused high mortality
of larvae even though only a very small portion of leaf discs were consumed. This
suggests that insect death was due to a combination of starvation and toxicity of the
extracts. Alkofahi et al. (1989) and Mikolajczak et al. (1989) reported that asimicin,
an acetogenin from the bark of paw-paw tree, A. triloba has both toxic and
antifeedant properties. Natural insecticides such as azadirachtin are also known to
possess both antifeedant and toxic (insect growth regulatory) properties
(Schoonhoven,1982; Schluter et al.,1985; Isman et al.,1990; Schmutterer, 1990).
The combination of these properties is advantageous since insects could develop
desensitization toward antifeedants. Therefore, the efficacy of extracts that also
have toxic properties is likely to be less affected by desensitization, since ingestion
of the compound will lead to the death of the insect. Crude aqueous seed extracts
of A. squamosa showed significant antifeedant activity at a concentration of 10%
(Figure 3.3) This concentration was much higher compared to those of Perera et al.
(2000) who reported significant antifeedant activities of 0.01% aqueous emulsion of
azadirachtin-based insecticide (Azatin® 3 % azadirachtin) and 0.01% aqueous
emulsion of neem seed oil (1% azadirachtin) against 4 -instar P. xylostella. This
th
suggests that the extracts have weak antifeedant activity.
73
My results suggest that there is a trend of decreasing susceptibility with
increasing larval stages (Table 3.2-3.5). First and second instars of the
diamondback moth, P. xylostella were the most susceptible stages to the crude
extracts of A. squamosa; therefore the extract should be targeted to the earlier larval
stages of this pest. A similar trend was reported by Kohyama (1986) when an insect
growth regulator, MK-139 (teflubenzuron) was tested against diamondback moth
larvae. Abdul-Kadir et al. (1999) observed that susceptibility of P. xylostella larvae
decreased with increasing larval instar when treated with three baculoviruses.
The LC50S for the aqueous extracts decreased significantly over time for both
3 and 4 -instar larvae (Table 3.3), suggesting slow-acting toxicity of the extracts. A
rd th
similar trend was reported by Basana and Prijono (1994) and Prijono et al
(1994,1997) when crude seed extracts of A. squamosa were tested against the
cabbage head caterpillar, Crocidolomia binotalis and the rice brown planthopper,
Nilaparvata \ugens. Moeschler et al.(1987) also reported 100% mortality of P.
xylostella 14 days after treatment with 40 ppm of pure annonin, an active compound
extracted from A. squamosa. McLaughlin et al. (1997) demonstrated slow toxicity of
crude extracts of A. muricata and A. triloba on 4 -instar larvae of the Colorado

th
potato beetle, Leptinotarsa decemlineata. The highest mortality of the beetles was
observed after 7 days.
I obtained LC s for aqueous seed extracts of A. squamosa on 3 -instar P.

50
rd
xylostella at 48 and 72 h of 1.69% and 0.88% respectively (Table 3.3). These values
are 3 to 6 times higher than those of Prijono et al. (1997) who reported LC s for 50
aqueous seed extracts of A. squamosa on 3 -instar C. binotalis at 2 and 3 d of 0.29

rd
74
and 0.28% (2.90 and 2.76 g seeds/I water). Variability among individuals of the
same tree species and differences in sensitivity of test species used could account
for these differences.
Due to differences in the extracts tested, it is not possible to directly compare
the results of my study with those reported by other workers. Some results, however
have also been reported in regard to toxicity of other alternative controls (i.e.
microbial) against P. xylostella. Mohan and Gujar (2001) reported LC50 values for
various Bt strains against 2 -instar DBM of 0.17 - 2.56%. They concluded that
nd
commercial formulations with strains producing a mixture of Cry 1 and Cry 2 toxins
will be more effective than conventional insecticides. Amiri et al. (1999) observed
that destruxins (cyclic depsipeptides produced by entomogenous fungus
Metarhizium anisopliae) had insecticidal and antifeedant properties against P.
xylostella larvae. They reported that LC50S of destruxins against 2 -instar DBM were
nd
1 7 - 3 0 and 53 - 376 ppm in leaf dip and contact assays, respectively. Efrapeptins
(metabolites of pathogenic fungus Tolypocladium) were reported to have
insecticidal, antifeedant and growth inhibitory activities against DBM larvae (Bandani
and But, 1999). They obtained LC s of these metabolites against 3 -instar DBM of
50
rd
3 ppm using a leaf dip assay and 150 - 980 ppm in a topical assay.
I obtained L C 50 values for aqueous emulsions of ethanolic extracts of 0.02-
0.67% on neonate to 4 -instar DBM (Table 3.5). These values are much lower
th
compared to those of Mariapan and Saxena (1983) who reported that crude seed oil
(methanolic extracts) of A. squamosa at concentrations of 5 - 50% caused 75 - 98%
mortality of leafhopper, Nephotettix virescens. Differences in sensitivity of test
75
species used and variability among the extracts could account for these differences.
However, this also suggests that crude extracts consist of more constituents than oil
alone. A similar trend was observed when a mixture of partitioned ethanolic seed
extracts of A. muricata and its oil was tested against the Colorado potato beetle,
larvae and eggs of white flies, and green peach aphids. The mixture of extract and
its seed oil is more effective than the extract or oil alone (McLaughlin et al., 1997).
Aquoeus emulsions of ethanolic seed extracts were more potent (9-50 fold)
than crude aqueous seed extracts (Tables 3.4 and 3.5) strongly suggesting that
more of the active principles were extracted by ethanol than by water. Prijono et al.
(1997) demonstrated that acetonic extracts of A. squamosa were 11-17 times more
potent than crude aqueous seed extracts. Sookvanichsilp et al. (1994) reported that
four organic solvent extracts of A. squamosa seeds and leaves at 10% in propylene
glycol caused varying degrees of irritation to rabbit eyes and skin, depending on the
polarity of the solvent. The most polar extracts (ethanolic) exerted the mildest toxicity
to rabbit eyes and no toxicity to rabbit ear skin. In another study, Saxena et al.
(1992) found that a methanolic extract of A. squamosa was toxic to fish, but not to
other non-target aquatic organisms.
More than twenty acetogenins have been isolated from the seeds of A.
squamosa, among which squamocin (annonin I) and squamostatin-A were the major
constituents (Fujimoto et al., 1994; Sahai et al.,1994; Zafra Polo et al., 1996; Araya
et al. 2002). The biochemical mode of action of this toxin is via inhibition of complex I
(NADH I: ubiquinone oxidoreductase) in mitochondria, analogous to the action of the
insecticide rotenone (Londershausen et al., 1991; Ahammadsahib et al., 1993; Lewis
76
et al.,1993). Londershausen et al. (1991) also reported that Annonin I is more
inhibitory than rotenone, not only to mitochondria of insects (e.g. Locusta) but also to
mammalian mitochondria (bovine heart muscle), which would account for the
significant toxicity of pure annonins to vertebrates. Regardless of the vertebrate
toxicity of the pure acetogenins, partitioned ethanolic extracts of paw paw (A triloba)
show little effect when fed to mice at 1 % concentration in their diet and they do not
cause skin irritations. Also the compounds are emetic, so the effect of any ingestion
would be prevented by emesis (McLaughlin et al., 1997). Irrespective of the
vertebrate toxicity of the pure compounds and irritation by the organic solvent
mentioned above, sufficiently potent crude aqueous extracts as shown in this study
and a previous study (Prijono et al., 1997) support the use of crude aqueous
extracts for insect pest control.
Crude seed extracts of A. squamosa showed significant residual action for 2
d and started to decrease at 3 d after treatment (Figs. 3.4 and 3.5). Soares and
Quick (1992) reported that residual activity of B. thuringiensis var. kurstaki (Javelin®
and Dipel® 4L) against DBM larvae dropped below 80% of original activity within 2 d
of application while residual activity of MVP® (Cellcap encapsulation of Bt) was still
high at 7 d after application in the field. Shelton et al. (1998) showed that residual
activity of fungi, Beauvaria bassiana (Mycotrol® WP) against DBM larvae was > 50%
7 d after application in a screenhouse. In general, botanical and microbial
insecticides are non-persistent in the environment. The short residual activities of
these alternative products are both advantageous and disadvantageous to their
application. With short residual activity, these alternatives might be less harmful to
77
non-target organisms and the environment, but on the other hand their efficacy to
the target pests might also be lower. I observed that crude seed extracts of A.
squamosa had short (2 - 3 d) residual activity against diamondback moth. Because
of the slow-acting toxicity of these extracts (Figure 3.2 and Table 3.3) somewhat
longer residual activity would be required for effective control.
Nevertheless, good efficacy of the crude seed extracts mentioned above and
local availability of seed of A. squamosa as waste products of these edible fruits in
Maluku (Indonesia) as well as simple preparation methods to obtain crude aqueous
seed extracts will make them promising candidates for local use by farmers.
Utilization of crude extracts that contain natural mixtures as pest control agents has
some advantages such as: the mixtures may act synergistically (Berenbaum, 1985);
they may show greater overall bioactivity than individual constituents (Berenbaum et
al., 1991; Chen at al., 1995); and insect resistance is much less likely to develop
with mixtures (Feng and Isman, 1995).
Efficacy of the extracts in the greenhouse and their toxicity to natural enemies
is presented in the following chapter.
78
CHAPTER 4
EFFICACY OF CRUDE SEED EXTRACTS OF A. SQUAMOSA AGAINST
DIAMONDBACK MOTH, P. XYLOSTELLA IN THE GREENHOUSE AND
TOXICITY OF THE EXTRACTS TO NATURAL ENEMIES
IN THE LABORATORY
79
4.1. INTRODUCTION
Demonstrations of greenhouse or field efficacy are an important step in the
development of pesticides, including botanical insecticides from plant extracts. A
promising plant extract that shows high efficacy in the laboratory must also be tested
in the greenhouse and/or in the field to evaluate its efficacy in a real pest situation.
Following bioassays in the laboratory, I conducted experiments to assess the
effectiveness of crude seed extracts of A. squamosa against the diamondback moth
in the greenhouse.
For a botanical insecticide to be compatible in an Integrated Pest
Management (IPM) program, it is desirable to have selectivity favoring natural
enemies. Therefore, it is necessary to evaluate the toxicity of a promising plant
extract to natural enemies. I investigated the toxicity of crude aqueous seed extracts
of A. squamosa against the commercially available biological control agents,
Chrysoperla carnea Stephens, Orious insidiosus Say., and Trichogramma brassicae
Bezd.
The green lacewing, C. carnea is an important beneficial predator species.
Adults feed only on nectar, pollen and aphid honeydew, but their larvae are active
predators (Henn and Weinzierl, 1990). They are reported to attack several species
of aphids, spider mites, thrips, whiteflies, eggs of leafhoppers, moths and leafminers,
small caterpillars, and beetle larvae. Lacewing larvae are considered generalist
beneficials but are best known as aphid predators (Hoffmann and Frodsham, 1993).
C. carnea appears to have some natural tolerance to several chemical insecticides,
although there may be considerable variation. Populations tolerant to pyrethroids,
80
organoposphates, and carbaryl have been selected in the laboratory (Pree et al.,
1989).
The insidiosus flower bug, O. insidiosus is a common predaceous insect
found on many agricultural crops, on pasture land, in orchards and is successfully
used as a biological control agent in greenhouses. Both nymphs and adults feed on
a variety of small prey including thrips, spider mites, insect eggs, aphids, and small
caterpillars (Jacobson and Kring 1995).
Trichogramma spp. are minute endoparasitoids that attack the eggs of
various species of lepidopteran insects, many of which are economically important
pests (Nagakartti and Nagaraja, 1977). They are the most widely used parasitoids
for the biological control of insect pests (Ridgway et al., 1981J. T. brassicae ,
originally from Europe, is used to control European corn borer and is now available
commercially in North America.
81
4. 2. MATERIALS AND METHODS
4. 2.1. Efficacy of crude seed extracts in the greenhouse
Experiments were conducted at the Faculty of Agricultural Sciences
greenhouse, at the University of British Columbia campus, to determine efficacy of
aqueous emulsions of ethanolic seed extracts as well as crude aqueous seed
extracts of A. squamosa on diamondback moth larvae. Cabbage, Brassica oleracea
L. var Stonehead was planted in plastic pots (10 cm diameter) in a mixture of 70%
peat moss, 20% perlite and 10% pasteurized soil. Osmocote® (13-13-13) slow
release fertilizer was added to the soil mixture (1,200 mg/m mixture). Plants were
3
watered and fertilized with water-soluble nutrients (Peter's Excel® 15-5-15 CalMag)
as required. Six week-old plants were used in the experiments.
Aqueous emulsions of crude ethanolic seed extracts of A. squamosa pooled from
several locations (see chapter 3) were tested at concentrations of 0.5, 1, and 2 %
w/v. Powdered laundry detergent (Sunlight®, 0.05% w/v) was added as an
emulsifier. Water plus detergent was used as a control and 1% Rotenone (Later's
Rotenone Garden Dust®, Later Chemicals Ltd. Richmond, BC) used as a positive
control. Treatments were laid out in a randomized block design, with 4 blocks and 10
plants/treatment/block. Plants were arranged along 2 benches, where each bench
(1.5 m wide and 14 m long) consisted of 2 blocks. Plants were spaced 30 cm within
rows and 50 cm between rows in each treatment. Spaces between treatments and
between blocks were 75 cm and 1 m respectively (Figure 4.1 and 4.2). Each plant
82
c
t/5
CN
o _
^: C
O o
LO o .CO
Q
d)
N ®
•E
o 2>°
o
O <>
l
o
o
CO
00
=1
_ CD
GO re ^
c u
— c
CM
co
re g>
CO O )
t c
d> re
E |5
E
E
re a)
1
CM
IS
(A (0
c c
a) re
| CL
CO 8 w
Q.
X C
E o> E
o
O LO S *
o
CO
o £
o
o c </>
CO
22 re
oo-g
^9 °
LO
c -a
> ro
CN
_l O
E • U
LO o
o CN ^ OH
LO <* S 3
'E £ .2
oo
= •=2
B ~ o
u. 5 o
E
LO
ro
oo
was infested with seven 3 -instar diamondback moth larvae, obtained from the
laboratory colony (see chapter 3). A pre-spraying count was made one day after
infestation and the plants were then sprayed with either one of the extract
concentrations or the control solution on the same day. Plants were sprayed using
910 ml plastic spray bottles to the point of run off (approx. 40 ml per plant) and one
bottle was used for each treatment. Rotenone (dust) was applied directly to cabbage
foliage from the canister. Numbers of surviving larvae were recorded at 2 and 7
days after spraying. Differences between numbers of larvae counted before and
after spraying were assumed to result from larval mortality. The experiment was
conducted in July 2001.
Crude aqueous seed extract of A. squamosa pooled from all locations were
tested at concentrations of 7.5, 15 and 30%. The extracts were prepared the same
way as mentioned in chapter 3 except that fine muslin cloth was used as a filter.
Powdered laundry detergent (0.05%) was added as an emulsifier. Water plus
detergent was used as a control and Pyrethrum (0.1% pyrethrins) used as a positive
control. Emulsion of Pyrethrum (0.1% a.i) in water was prepared from Premium
Pyrocide 175 (20% a.i) and powdered laundry detergent (0.05% w/v) was added as
an emulsifier. The layout of the experiment was the same as that mentioned for
aqueous emulsions of ethanolic extracts (Figure 4.1). Each plant was infested with
ten 3 -instar larvae. A pre-spraying count was made one day after infestation and
rd
the plants were then sprayed with the various extract concentrations, control solution
or positive control on the same day. Plants were sprayed using 910 ml plastic bottle
sprayers to the point of run off (approx. 40 ml per plant) and one bottle was
84
CO
!- TJ
3 CD
— CD
3 CO
« CD
i? 3
3 O «
co .2
JZ CJ)
CO
CO CO o
CD
£
"TT CO
E
° 3 O
SI 2
CD * C
i
CD CO c
o 'c•o=
CD O OO
C 03 co
^ CD" O-
E
CO
CO 3
CT
CO
Sl«c
. O •*-
^ w °
* © co
CD O O
C (o
3 CD i_
.EP O "S
LL CO LU
85
used for each treatment. Numbers of surviving larvae were recorded 2 days after
spraying. Differences between the numbers of larvae counted before and after
spraying were assumed to result from larval mortality. This experiment was
conducted twice (March and April, 2002) and data combined for analysis.
4. 2.2. Toxicity of crude aqueous extracts to natural enemies
4.2.2.1. Aqueous extracts
Aqueous extracts were prepared as mentioned in chapter 3. Water plus
powdered laundry detergent (0.05% w/v) was used as a control.
4.2.2.2. Insects
Eggs of the Mediteranean flour moth, Ephestia kuehniella (Lepidoptera:
Pyralidae) were used as prey for Chrysoperla carnea and Orius insidiosus. Sterilized
eggs of E. kuehniella were purchased from a commercial biological supply company
(Westgro, Delta, BC) and stored in the refrigerator until used.
Eggs of green lacewings, C. carnea (Neuroptera: Chrysopidae) were
purchased from Westgro. Eggs were placed in a growth chamber at 26°C and
16L8D until larvae emerged. First-instar larvae were used in the experiments.
Adults and nymphs of O. insidiosus (Hemiptera: Anthocoridae) were
purchased from Westgro and maintained on E. kuehniella eggs. Adults were used in
the experiments.
86
Pupae of the parasitoid, Trichogramma brassicae (Hymenoptera:
Trichogrammatidae) in the form of parasitisized eggs of E. kuehniella were
purchased from Westgro. Pupae were placed in a growth chamber (26°C and
16L8D) until adults emerged. Adults were used in the experiments.
4.2.2.3. Toxicity to larval C. carnea
A direct spray test was carried out to determine toxicity of crude aqueous
seed extracts of A. squamosa to green lacewing, C. carnea larvae. Five 1 -instar

st
larvae (< 24 h-old) were placed in 59.2 ml plastic cups (B200 Solocup Company,
Urbana, III.) lined with 42.5 mm dia (Whatman No.1) filter paper. Larvae were
sprayed with 0, 5, 7.5, 15 or 30% crude aqueous extracts using 50 ml plastic spray
bottles and producing a fine spray with an approximate diameter of 50 mm. One
spray bottle was used for each concentration and spray was applied in ascending
order. There were 7 replicates for each treatment. After treatment larvae were
removed to clean sealed 59.2ml plastic cups lined with a moistened (Whatman No.1)
filter paper. Larvae were provided with E. kuehniella eggs on strips made of 3M
Post-it® message pads (each strip holds about 175 eggs; Corrigan and Laing, 1991)
for the duration of the experiment. Two strips were placed in each cup. The cups
were then placed in a plastic box lined with moistened paper towels in a growth
chamber at 26°C and a photoperiod of 16L:8D. Mortality of the insects was recorded
after 24 and 48 hours.
For the residual contact test, 20 ml scintillation vials (Kimble Glass Inc., NJ)
were coated with 225 ul of 0, 5, 7.5, 15, or 30% aqueous extracts. This volume was
87
enough to coat the inside of the vials thoroughly. After 3-4 h of drying at room
temperature, five 1 -instar larvae (< 24 h-old) were placed inside each vial and
st
provided with E. kuehniella egg strips. The vials were plugged with cotton balls.
There were 5 replicates for each treatment. The vials were then placed in a plastic
box lined with moistened towel paper in a growth chamber at 26°C and a
photoperiod of 16L8D. Mortality of the insects was recorded after 24 and 48 hours.
The experiments were done twice and data combined for analysis.
4.2.2.4. Toxicity to adult O. insidiosus
Residual contact tests were carried out to determine toxicity of crude aqueous
seed extracts of A. squamosa against adult O. insidiosus. Glass vials (7 ml) were
coated with 50 pi of either 0, 5, 7.5, 15 or 30% (w/v) aqueous extracts using a
micropipette. Vials were left to dry at ambient room temperature and used at 0, 1, 2,
3 or 7 days after treatment (DAT). Five unsexed adult O. insidiosus (<48h-old) were
placed in each vial and provided with E. keuhniella eggs on a strip for the duration of
experiment. The vials were plugged with cotton balls. There were 10-18 replicates in
each of treatments. The vials were then placed in a plastic box lined with moistened
towel paper in a growth chamber at 26°C and a photoperiod of 16L8D. Mortality of
the insects was recorded after 24 hours.
88
4.2.2.5. Toxicity to adult T. brassicae
Residual contact toxicity was assessed for crude aqueous seed extracts of A.
squamosa to adult 7. brassicae. Glass vials (7 ml) were coated with 50 ul of 0, 5,
7.5, or 30% (w/v) of aqueous extracts. Vials were left to dry at room temperature and
used at 1, 3, or 7 days after treatment. Five unsexed adult T. brassicae (<24 h-old)
were placed in each vial. A streak of 50% diluted honey was placed inside the vials
as food for the insects. The vials were plugged with cotton balls. There were 10
replicates for each of treatments. The vials were then placed in a plastic box lined
with moistened towel paper in a growth chamber at 26°C and a photoperiod of
16L8D. Mortality of the insects was recorded after 24 hours.
4.2.3. Data Analysis
For greenhouse trials, percent mortalities were corrected using Abbott's
transformation (1925). Statistical software (Anonymous, 2000) was used for data
analysis. Analysis o f variance (ANOVA) was performed on the arcsine-transformed
percentage mortality for both greenhouse trials and toxicity to natural enemies.
Means were compared by the Least Significant Difference (LSD) test (Snedecor and
Cochran, 1989) for both greenhouse trials and toxicity to natural enemies. Actual
numbers observed are reported in the results.
89
4.3. RESULTS
4.3.1. Efficacy of crude seed extracts in the greenhouse
There were no significant differences in mortality among 3 concentrations of
aqueous emulsions of ethanolic extracts tested both 2 and 7 days after treatment
(DAT). Mortality of larvae at 3 concentrations tested were significantly (p<0.05)
higher than that for 1% Rotenone (p<0.05) (Table 4.1). Mortality did not increase
between 2 and 7 DAT for all treatments but Rotenone(Table 4.1).
Table 4.1. Mortality of diamondback moth (P. xylostella) larvae sprayed with
an aqueous emulsion of ethanolic seed extracts of A. squamosa or dusted
with rotenone in a greenhouse trial
Corrected Mortality (%) ± SE
Treatment 2 DAT 3
7 DAT
Aq. emulsions (% w/v) 0.5 82.24 ±4.02 a 83.44 ± 4.50 a*
1 85.84 ±4.36 a 86.03 ±4.46 a
2 90.33 ± 3.99 a 90.41 ±3.77 a
Rotenone (%) 1 35.81 ±5.26 b 58.78 ± 4.99 b
Control 8.09 ±5.0 9.54 ±4.63
* Means followed by the same letters within a column do not differ significantly at
p<0.05 based on the Least Significance Difference (LSD) test.
a
DAT= day after treatment.
90
There were no significant differences in larval mortality between 7.5 and 15 %
of crude aqueous seed extracts and Pyrethrum (0.1% a.i) (Table 4.2). The lowest
extract concentration of 5% had significantly (p<0.05) lower mortality than higher
concentrations and Pyrethrum, but efficacy was still high (84% larval mortality)
(Table 4.2).
Table 4.2. Mortality of diamondback moth (P. xylostella) larvae 2 days after
spraying with crude aqueous seed extracts of A. squamosa or pyrethrum in
two greenhouse trials
Treatment Mortality (%) ± SE
Aqueous extracts (% w/v) 5 84.17 ± 2.36 b*
7.5 91.20 ± 1.78 a
15 93.90 ± 1.20 a
Pyrethrum (% a.i) 0.1 96.19 ± 1.20 a
Control 0.28 ±2.92
*Means followed by the same letters do not differ significantly at p<0.05 based on
the Least Significance Difference (LSD) test.
91
4.3.2. Toxicity of crude aqueous extracts to natural enemies
4.3.2.1. Toxicity to C. carnea larvae
For C. carnea larvae sprayed directly with crude aqueous seed extracts of A.
squamosa, there were no significant differences in mortality for concentrations
ranging between 5 and 15% after 24 h (Table 4.3). However, the 30% extract was
significantly more toxic (p<0.05) than 5 and 7.5% extracts after 24 h (Table 4.3).
Toxicity of the extracts was rather low (9 - 29%) after 24 h (Table 4.3). However,
mortality appeared to increase after 48 h at all concentrations (43-57%), but control
mortality was also high (46%). Therefore no statistical analysis was performed on
that data.
Table 4.3. Mortality of 1 -instar C. carnea after 24 h when exposed

st
to crude aqueous seed extracts of A. squamosa in a direct spray test
Concentration Corrected mortality (%) ± S E

(%w/v)
5 9.29 ± 6.21 a
7.5 9.29 ± 4.42 a
15 22.14 ± 4.48 ab
30 28.57 ± 4.46 b
Control 8.57 ± 4.04
* Means followed by the same letters do not differ significantly at p<0.05

based on the Least Significance Difference (LSD) test.
92
I found no significant differences in mortality of C. carnea larvae that
contacted extract residue on treated vials at 5, 7.5 and 15 % after 24 h. However,
mortality on the 30% concentration was significantly (p<0.05) higher than that for 5%
(Table 4.4). Mortality increased after 48 h for all treatments, but again there were no
significant differences among treatments (Table 4.4). The extracts had very low
toxicity at all concentrations tested (2 - 12%) after 24 h and low toxicity (15- 22%)
after 48h (Table 4.4).
Table 4.4. Cumulative mortality of 1 -instar C. carnea exposed to crude

st
aqueous seed extracts of A. squamosa in a residual contact test

Cumulative corrected mortality (%) ± SE
Concentration (%w/v) 24 h 48 h
5 2.08 ±2.08 a 15.28 ±5.48 a
7.5 8.75 ± 3.15 ab 19.44 ±4.87 a
15 6.68 ± 2.84 ab 21.94 ±4.42 a
30 12.08 ±3.11 b 22.08 ± 5.05 a
Control 1.67 ± 1.67 11.67 ±4.58
*Means followed by the same letter within a column do not differ significantly at
p<0.05 by the Least Significant Difference (LSD) test.
93
4.3.2.2. Toxicity to O. insidiosus adults
Toxicity of aqueous seed extracts of A. squamosa to adult O. insidiosus that
contacted extract residues on treated vials decreased with increasing age of residue
(Table 4.5). However, there were no significant differences in LC50 values at 0, 1, 2
and 3 days of residue (Table 4.5). Toxicity at 7 DAT was significantly (p<0.05) lower
than that for 0 to 2 DAT (Table 4.5).
Table 4.5. Toxicity of crude aqueous seed extracts of A. squamosa to adult O.
insidiosus exposed for 24 h in a residual contact test
Age of residue LC 50 (95% CI) Control mortality ± SE

(day) (%w/v) (%)
0 1.89 (0.81 -4.40)* 11.25 ±2.56
1 2.73(1.35-5.52) 8.89 ±3.32
2 3.85 (2.37-6.24) 17.14±4.12
3 6.07 (4.31-8.54) 9.23 ±2.88
7 8.79 (6.69- 11.54) 10.00 ±3.33
*LC values differ significantly where 95% Cis do not overlap

50
94
4.3.2.3. Toxicity to T. brassicae adults
Mortality of T. brassicae adults that contacted extract residues at treated vials
at either 1, 3 or 7 DAT were significantly (p<0.05) higher than controls for all
treatments Table 4.6). However, both of the controls (water and water + detergent)
caused moderate toxicity to the insects. All the treatments except controls caused
complete mortality (100%) of the insects (Table 4.6).
Table 4.6. Mortality of adult T. brassicae after 24 ht when exposed to

crude aqueous seed extracts of A. squamosa in residual contact tests
Mortality (%) ± SE
Age of residue (day)
Concentration 1 3 7
(% w/v)
5 100 a* 100 a 100 a
7.5 100 a 100 a 100 a
15 100 a 100 a 100 a
30 100 a 100 a 100 a
Control 1 a
40 ±5.16 b 46 ± 9.90 b 50 + 8.56 b
Control 2 b
42 + 4.67 b 48 ± 6.80 b 48 ± 5.33 b
*Means followed by the same letter within a column do not differ significantly at
p<0.05 by the Least Significant Difference (LSD) test. Control 1 = water +
a
0.05% powdered laundry detergent. Control 2 = water

b
95
4.4. DISCUSSION
My greenhouse trials showed that aqueous emulsions of crude ethanolic seed
extracts as well as crude aqueous seed extracts of A. squamosa had very high
efficacy (Tables 4.1 and 4.2). Mortality of P. xylostella larvae was significantly high
(> 80%) when sprayed with concentrations as low as 0.5 and 5% (w/v) of aqueous
emulsions of ethanolic seed extracts and crude aqueous seed extracts, respectively
(Table 4.1 and 4.2). Aqueous emulsions of ethanolic seed extracts were
significantly 2-3 fold more effective than 1% Rotenone (Table 4.1) in reducing larval
populations. This is consistent with the finding of Londerhausen et al. (1991), who
reported that annonin I, a bioactive constituent of A. squamosa seed, is 2-4 fold
more inhibitory than rotenone to mitochondria of insects (e.g Locusta). Mikolajczak
et al. (1988) and Alkofahi et al. (1989) reported that a partitioned ethanolic extract of
pawpaw bark was more (1.6 fold) effective than rotenone against mosquito larvae at
10 ppm.
Larval mortality did not increase from 2 - 7 DAT when sprayed with aqueous
emulsions of ethanolic extracts. I observed very high mortality of larvae at 2 DAT,
and most of the few surviving larvae had pupated by 7 DAT. This suggests that the
extract is effective against larvae but not pupae and should be targeted at the earlier
larval stages.
Crude aqueous seed extracts at concentrations of 7.5 and 15% provided very
high efficacy (> 90%) against P. xylostella larvae and were as effective as pyrethrum
(0.1% pyrethrins). At the lowest concentration of 5%, the extracts showed high
efficacy (84%) (Table 4.2). Partitioned ethanolic extracts of paw paw bark caused
96
100% mortality against nematodes (Caenorhabditis elegans) at 10 ppm after 72 h,
while pyrethrum showed no nematicidal activity at the same dose and time period
(Mikolajczak et al., 1988; Alkofahi et al. (1989). McLaughlin et al. (1997) reported
that a mixture of pyrethrum and paw paw extract shows effective synergism and
enhanced additive efficacy against white flies on cotton leaves. The mixture of this
extract with neem extracts also showed a synergistic effect on the Colorado potato
beetles. Mariapan and Saxena (1984) demonstrated that 20% seed oil of A.
squamosa caused 98% mortality of rice leafhopper, Nephotettix virescens, which is
significantly higher than that of 20% neem oil. A mixture of A. squamosa and neem
oils is more effective in reducing the survival of the leafhopper and its transmission
of rice tungro virus than the individual oils (Mariapan and Saxena, 1984). These
findings suggest that combination of natural insecticides would be beneficial.
Venkataramireddy et al. (1990) reported that 0.1% petroleum ether extracts of
leaves of A. squamosa are prominent in reducing populations of brinjal spotted leaf
beetle {Henosepilachna vigintiopunctata) grubs by > 90% which is similar to that of
neem (A. indica) leaf extracts in a greenhouse trial. Before its commercialization,
neem had been studied thoroughly both in the laboratory and in the field. Field
efficacy of neem extracts against P. xylostella has been reported by several
investigators. Prijono and Hasan (1993) showed that neem oil (60 g aza/ha) gave
protection comparable to that from Sumicidin® 200 EC (fenvalerate 50 g/ha). Klemm
and Schmutterer (1993) reported that 25 - 30 g seed kernel per I water (0.25 -0.3%)
reduced populations of P. xylostella larvae significantly. Some workers also
reported that neem seed extracts were more or as effective as some synthetic
97
insecticides used in field trials against DBM larvae (Adhikary, 1981; Fagoonee,
1987; Sombatsiri and Temboonkeat, 1987; Dreyer, 1987, Isman et al, 1991). DBM
has already developed resistance to most synthetic insecticides used (Talekar and.
Shelton, 1993) and the microbial, Bacillus thuringiensis (Tabashnik et al., 1990,
1992). Therefore, alternative controls for this pest are needed. Good efficacy of
neem against DBM larvae mentioned above, along with my results, suggest that
botanical insecticides have promising efficacy against DBM larvae. However, further
experiments on the efficacy of A. squamosa extracts against DBM larvae in the field
are necessary.
In spite of the good efficacy of crude aqueous extracts against DBM larvae,
these extracts do not favor some natural enemies. Experiments on toxicity of crude
aqueous extracts to natural enemies showed that C. carnea larvae were the least
susceptible (Tables 4.3 and 4.4) followed by O. insidiosus adults (Table 4.5), with T.
brassicae adults being the most susceptible (Table 4.6).
Direct spray and residual contact tests on C. carnea showed that the extracts
were less toxic when contacted several hours after application (Tables 4.3 and 4.4).
Mortalities were 2 - 1 2 % after 24 h and 15 - 22% after 48h when larvae were
exposed to residues in the vials (Table 4.4). The mortality I observed was much
lower compared to those of Hamilton and Lashomb (1997), who reported LC50S for
Rotenone to C. carnea larvae exposed to foliar residues of 12.68 g/l and 3.29 g/l
after 24 and 48 h, respectively. They also showed that Esfenvalerate and Oxamyl
were much more toxic (33 and 11 times, respectively) than Rotenone to the larvae.
Rumpf et al. (1997) reported that Diflubenzuron at a concentration of 0.0025%
98
caused 100% mortality in C. carnea during the molt into the 3 instar. My results
showed that crude aqueous seed extracts of A. squamosa can be slightly toxic to C.
carnea larvae, but suggest that there will be a window of opportunity for using green
lacewings and crude aqueous seed extracts of A. squamosa in an IPM program.
However, further tests on residual contact with longer residual times are required.
O. insidiosus adults were quite susceptible to the extracts. However, toxicity
of the extracts decreased with increasing age of residue(Table 4.5). Elzen (2001)
compared toxicity of 10 insecticides to O. insidiosus using a residual insecticide
bioassay. He reported that tebufenozide (0.28% a.i) caused 21.7% and 19.8 %
mortality in male and female adults, respectively, while cyfluthrin (0.056% a.i)
caused 28.4% mortality in male adults, after 72 hours. These insecticides were
significantly less toxic to O. insidiosus than malathion (Elzen. 2001). We observed
that crude aqueous seed extracts at 5% concentration caused 24% mortality at 7
DAT (LC 50 of 8.79) (Table 4.5). This is comparable to that of cyfluthrin at 3 DAT
(Elzen. 2001). We found that crude aqueous seed extracts of A. squamosa had
moderate to high toxicity to O. insidiosus. However, when exposed to extract
residues after 7 days, the toxicity decreased significantly (p<0.05). Again, further
tests on residual contact with longer residual times are desirable before considering
the use of A. squamosa seed extracts in conjunction with biocontrol agents for pest
management.
The extracts were very toxic (100% mortality) to T. brassicae adults at
concentrations as low as 5% with moderate toxicity of the controls (water plus 0.05%
w/v powdered laundry detergent and water only) (Table 4.6). Brunner et al. (2001)
99
reported that biorational pesticides such as soap, oil, and B. thuringiensis products,
caused no toxicity to Colpoclypeus florus (Hymenoptera: Eulophidae) but had a
direct impact on Trichogramma platnerivla topical application. However, insecticidal
soap residues were non-toxic at 1 DAT. My results suggest that the method used to
assess toxicity of the extracts to T. brassicae was probably inappropriate and
therefore needs to be redesigned if this work is to be repeated. More experiments
are needed to assess toxicity of crude aqueous seed extracts of A. squamosa to
more species of natural enemies.
100
CHAPTER 5
FEASIBILITY OF PRODUCING CRUDE AQUEOUS SEED EXTRACTS OF
A.SQUAMOSA AS A SIMPLE BOTANICAL INSECTICIDE FOR LOCAL
USE IN AMBON (MALUKU), INDONESIA-A PRELIMINARY
ECONOMIC ANALYSIS
101
5.1. INTRODUCTION
Botanical insecticides are often safer than synthetic insecticides because of
their minimal persistence, although mammalian toxicities vary. They have been used
since the late 1800's but only 5 have been registered in the USA. These are
pyrethrum, rotenone, ryania, sabadilla and neem (Isman, 1995). To be commercially
available, a botanical insecticide must meet a number of criteria, both biological and
practical. Biological criteria include efficacy; selectivity favoring natural enemies and
other non-target organisms; favorable mammalian toxicology; biodegradability; and a
lack of phytotoxicity. Practical criteria include sourcing (i.e sustainable availability);
potential to standardize for active ingredients from sources with natural variation;
and the potential to protect the technology (Isman, 1995, 1997).
Tropical plants are considered an excellent source of plant natural
compounds due to the greater selection pressure by herbivores in warm climates
(Jacobson, 1989; Schmutterer, 1992a; Isman, 1995). Indonesia, the world's largest
archipelago, straddles the equator for more than 8,500 kilometers and consists of
more than 17,000 islands. Ambon is one of the main islands of the Maluku region
and along with many other islands in its vicinity possess rich botanical resources.
Insect pest problems in tropical regions are often more obvious, since temperature is
favorable and host plants are available all year round. In 1987, the Indonesian
government banned 57 broad-spectrum pesticides, eliminated state subsidies on
other pesticides, and instituted Integrated Pest Management (IPM) as a national
strategy to combat agricultural pests. This policy initiative was undertaken because
outbreaks of major insect pests that devastated crops were shown to be caused or
102
exacerbated by excessive use of synthetic pesticides (Gallagher, 1992). The
national IPM strategy and the increasing price of synthetic insecticides due to the
elimination of state subsidies are driving forces behind the use of alternative controls
including botanical insecticides.
A. squamosa (sweetsop) trees as sources of fresh fruits are abundant as
backyard trees in many villages in Ambon (Maluku) Indonesia, and the seeds are
waste products. Crude aqueous seed extracts of A. squamosa showed promising
efficacy against lepidopteran pests in laboratory and greenhouse tests (see chapters
3 and 4). Sustainable availability and good efficacy support the concept of
development of these crude extracts as simple botanical insecticides for local use by
farmers.
This chapter presents a preliminary economic analysis to compare the cost of
producing the extracts with the cost of using synthetic insecticides. This comparison
was attempted in order to evaluate the economic feasibility of producing and using
the extracts locally by the smallholder vegetable farmers in Ambon (Maluku),
Indonesia.
103
5.2. Cost-Benefit Analysis
In this analysis of a simple scenario, I compared the cost of producing and
using crude aqueous seed extracts of A. squamosa with the cost of using synthetic
insecticides to control insect pests on vegetable crops in Ambon, Indonesia.
The following assumptions were made in the analysis.
1. Each smallholder farmer has a 0.1 ha vegetable field
2. Farmers have A. squamosa trees in their vicinity (i.e. village)
3. Farmers prepare the extracts by themselves
4. Farmers have sprayers and spray the plants themselves
5. Labor cost (opportunity cost) is based on the daily wage rate for unskilled
labor
6. Interest rate was estimated at 35%
7. Recommended concentration of crude aqueous seed extracts of A.
squamosa is based on the results of laboratory and greenhouse
experiments..
Components of costs in producing crude aqueous seed extracts of A.
squamosa (sweetsop) were: materials used for mixing the extracts; labor; fruits; and
additive (Table. 5.1). Price of the fruits was calculated as if they were sold in the
fresh market. In this case the farmers did not get that amount of money since they
consumed the fruits in order to use the seeds. If farmers never sold the fruits, and
just consumed them, then the cost will be lower. A tree bears about 40 to 60 fruits
104
and these are available all year round. To produce crude aqueous seed extracts for
a 0.1 ha vegetable field (Table 5.1) a farmer needs only 2 to 3 trees in his backyard.
Another scenario used in comparison was that the farmers do not have the
trees but just collect the seeds from within their vicinity (i.e. village area). In this
case, I calculated labor cost of collecting the seeds but not the price of the fruits
(Table 5.2). This cost will be a little bit lower if female and children are doing the
collection. I assumed that seeds were collected once and could be used for 2
sprays. The dried seeds could be stored for more than 1 year. Under laboratory
conditions seeds stored for more than two years maintain their insecticidal
properties.
Components of costs in using synthetic insecticide are materials used for
mixing the insecticide; labor; and the actual insecticide (Table 5.1). The insecticide
used in this comparison was a synthetic pyrethroid, Decis® 2.5 EC (deltamethrin 25
g/l). It is available in pesticide kiosks in Ambon and has been used by farmers. My
greenhouse trials showed that crude aqueous seed extracts of A. squamosa were as
effective as pyrethrum in controlling diamondback moth, P. xylostella on cabbage
plants (chapter 4).
Prices of materials, fruits, insecticide and additive and labor costs used in this
analysis were gathered as secondary data from Ambon. The data were collected by
interviewing sixteen farmers in three villages in Ambon.
105
5.3. RESULTS
Comparison of cost analysis between using synthetic insecticide and crude
aqueous seed extracts of A. squamosa showed that the latter were 2.5 % (Table
5.1) and 9% (Table 5.2) more economical than the former.
106
CO
4-1 o
'5 o
o
CD o •a
o c
CO
CO
CD t_
<*- CD CL
o
ical Ins cti de
CD o CO
c
CO
uatm
O
co ract: CL
CD
m CD
n E
a:
ded for 50 1 volu

o 00
CO
CD CD *
o
><
CD 4-: °
da
wa
X CD O CO
CO CD a) o C CO
! o
3 o CD 3 O
ha
X
E
eds =
C O
T3 E ^
see
C o E 5 o CO
'ork
w CL CO
o
3 CO d oo co"
or
4—» CD
"D CO c CO CO c
ed =
50 1/
CD
Rp.
seed/
C CD CO TO
j= o CO
ueoi
Bot
LO
CO CD Q.
00
-•—<
3
O LO - r
—J
cr
CO O o II CD CM CD
CO
II
CO CM
•e CD oCO L - 0
o
ha
TO -5 CO
o CD C L— L_
CD E
CD
C o CD
C
rayi o" CO
II
CD o
o O
E
—
T3 co c T3
O
cL
4—
o CD o O
= f CD 4—
Cr Q_ o. o CD LO LO co
C
o 00 < CO 00 or LO LO CO CM CM
or 00
T—
O
2 *
Q. °
^ 12 T3
CD
o
LO
."S CD
* I o o
CD CO
•S £
O CD
UJ 3 C LO
CD JD CD
CO "5^ CM
£ § c LO
O CO
T3
s g
CD
>,
L_
O •= CM
CO 4-» o CL CO o CD
CD 5 O .co
o
CO o CO o C
£
1
_co "D LO
o
c co CD d
8
and
JZ CL CD
CL
ing
._ *•* CD o 0
0
*->
{§
c
4-* Q
o
o o
o
co •g CO
0 g
CD Q. >» CM
o" CD
C E o
CD
E LO —
-C <0 O
_g
1*
O CD
C DJ CD
cL CM £
0
X LO
•>, (0 c
CL O CO
o
is O LO C
>
or
CH Q- LO
o QT CM
g> co
c o
w c CO
4-<
= O CO
"~ (0 O O
4-> O
CO CD
CD CO
O
a c
CD
TD
CO
L_
CL
4-1 "D
c CO
o
c o CD
T3
c
o CD
o
CD Q.
E CO C E
o _2
4-»
CO CD CD
o CO
-g
c Oo o E o
L—
E
4-»
o
o
CO
CD c >
o
4—»
4-> CD E o o
CD
O o
CD »
c CD
73 CO CO CD
o CD
or o CO
c
u
CD
CO o
z CM CO
c
CD
O
l^ O o
. CD
jco C CO
CD t CO
CD O
E if)
O £ C
CD d
CO II o E ^
ro o
—
d CO CO
c IT) .£=
O o o o -4—*
~t o
CD LO
o CZ s£ O 'CL
CD
-•-» CD oo" CM o in o O CO
CD
E oo, E •~ o
cL Q .
LO
0T CO
oo oo 3 •-
C
o
CM
II or O
o
o
CD
LO
« L
ro LO
o
o
11
II
O o CD CM
LO c ®
ro ~t © O d d o oo CO -Q
l-o L— CD o CO o CD CO o CO m CO CM -° ro
<o LO
o
1 IO
^—'
c LO c CD
CD CM m o *
5T 2 oo" oo J= CL
CD d Q CT>" CL
CL CN CO
CM
CL CD
O CD CL CD
II CD CO
CD CO CM
ri.
O
c c CL CL
or or
CO
o LO CL CM CL
E 2 co
or or
O or 1L
o
or
CM
l-o
CO
' or o ^
CO * i
CD
(5 P o
CD > o o
CD £ ,_;
o CO
CL CO
c
2 8
CO CD O
c o CD £ co"
o E
E cl trf CL
o ro ro 00
o o o CD CO CD
in m
N- £ o or
o o d d o d
cci co £ ° II
d d o o o o OJ i_ CD T —
o o CD CO o d LO LO LO CO CD ~0
LO CD >> o CM r-- CO
O) CD" °
§ 'T5
O CO
LO_ CO oo ~ • • ro
f 1ro
CM" 1_ CO IT) CD CO o>
05
CD CL CM CM" CN CD
O CL CL CO
CO
CL
or
c
CL CL
O
CM CL
or CL
& a.
lor or or
CD
or
CO
CD
or
"
"
CD
S CL 3
CD
ro oc
JO O
E CO <-> CD
c
CO
x:
-*—*
CD
c
LO
CM, I*"
CO c c •- CD •
l_
ro q
Q. CO o CL ro CO J9 T3
CO >> E ro CD O g o
ro 'o
CL oo, oo, - ° — "ft
CO CD
CD
CD
CL m + eo 2, CD Q CD
+ CO
E CD CO,
O + £ CO £
CO -Q CO TD t; CO —
LO,
o JD CD o X
° ^ ro
CD -4—'
> o
CD
-Q
CO _C o X ro CO
O +-»
+
•4—'
ro E c "ro CM +± LO 2>
CL
Ss
T3 o CJ _ro o
<
CM CD m O P 8
<
CN?
CL H CO
oo
o ^ (0^
*
o
CD 00 CO
o
CD
"D o
o
o LO
o T3
O C OI
CD d CO
CO o CO CO >»
c o >s CO
CO c CO
I
—
15 •a o
CL
o O
co 0 CO
'E CO CL
c CO
co LO
CD
CO o
CO or o I— 5^
CO
CO CO
T3
E
o l _
4-; ° o l _ c
CD
4-»
o CO
X
CD
0
o
d
«;
CL
CO
CO CO > o CO
O) X
•o T3 O LO CM
"E c CO
o
<? 3
c
E
E
CD CO JZ
CO LO
T3 o CO
3
CD
CO
CL CD o
o
CO o o
o o "O
CO
00
"O
u
CD CO
CO or CD
•o c LO CD
c CO ZJ
CO
"co
CO
CO
d
o CD JZ
d
o "3 o
T3
CD
CO
T3
CO c o •c o CO CO LO CD CD
CO
CD
ZJ o o c ZJ ZJ CO CD
LO TD
CD
LO
CM
CD
0
CO
c
L—
CO CO
"co
c o
CT CO
OJ E CD ^ T3 o i— CD CO
o
CO CO i_
o 'E CD E CD
CD
O CL
CD
CD CO
CO
L_ CL
CO
JZ c CO
3 CO T3 CO
c c O or
CO
c CL
CO
or o
CO
T3 2
o
o
T3 CO LO,
O "C o o o CD LO
sq
CD CO o < 00 Q LO CD
CO
CO T- CM
CD LO
CO CO
T3
CD o
CD LO
o
CD CD CO o
O C
=D ITS p
O o
o CO
JZ
T3 d
U
CD
U *<3 JD c
>. 0
LO
OI o
CO CO o TJ in
1_ CD O CO
l _ ,>% s 0 II
c
CD CO "co o CL CO o 0 >< o CM"
E
c o o CO
•o
o
o LO c
LO
d o
and
LU CL CL
ing
CL
o d 0 o
un II
-g CO
c CD oi o
o o
o CO
o 0
o
or
c CD co OJ
CO L—
E o E
LO"
II
>
'o c o o 0 CM
CD X LO
o co ZJ
CO
ll CD c
CL CL
d LO c o> CL in
DI
c
co
4-1
Q
O or or or
CM
c
"co (0
3
Mo- a.
CD
CO D) CO
•*-» (0 4-<
CO CO
ja o
o
o J2 o
o CO CD
CO
o u
c c C o CO
1_
o o CD
C
T3 CL
CO
CO CO -o c
)= CO o g 0
CO CD CO
CD
E
o 0
*4-»
CL T3
E o C
CO
1—
_ZJ
•g
E £• o
c >o o
4-»
CO CD
0 ° IO CD
E 0 o
*4-»
a) E o E o JZ
U
CO
"co o c CO 0
JD
o o CO
01 •- CO
CD
o c
* o
A j=
or
CO o
I- o
OJ
co
CD
it;; O
O
jo
ZJ
E CO
If)
CD
CO
o
CD
d
+-> II
rz
CD
E> o un CO CO
JZ
-«—»
c ^5 c
CO
sz
0 ) d o in o
"53 d o o
o in d E oo, E
o
un
CD
I-a c CO
"L oo o
c g CO o o
o o o
o
CD
o
ro
ZJ "
4
-— » m CO o d I-
s
LO
_C0 CD CN
CM d o oo 0 CM in
i_
CL C L d o CO in oo
l-o c II or
-*—»
o 0 CO LO
o CN If)
CM
o.
-°
CH I -
s
CD I -
ro in
S
CD CD CM" CO oq
o cn ©
CL CM £
'1
so
CD
c in CO
CD
O CO CM ^ CL
CD"
CL
O
d C CL c CL CL cL OH
O
CL
O
CD II m
or CM OH O
CM
CH II \0H lot JL
CO
CO
.c .c
-f—'
-
4— '
c £Z
o o o
o E E m
o r-
o
d ^:
d
o
«ro
-
CD
o
CO
o
ro
0
CO
in co
o>
o o I -
in oo
S
in CO
o in
CD CO d
m CD I - to"
in CC DN oo C D
S
CO •«t
a>" l _
CO CO CO
CM
CD CL CM CN"
CL CL CO
O
CL c
CO
CL cL OH OH
OH O
CM
CH OH |£ JL
ro
CD CD
E
-•—< 0 s
CO
CO m
-
4—' £Z CN,
c c c
CD o o
L_
CL CO
>»
E ro
CL
< "
4
-—' ro
CO
CO CM, CO 0
CD
l _ X o oo,
+ C O o>
CL CD
+ CO CO CD CD
2>
0 s
ro E +
CL
If) O
CO CO 0
00, O +
CD o
-Q
CO o X -*—<
_i
> o + CD -Q
ro _£Z o x -o CO
o c ro CM 0 - s
2> <
ro CQ E "ro in 0- +
o
s
T3
T3 -4—»
ZJ ro •4—1 '
CM 0
m
<
O CM
CL O 'CL c oo
!— CD
o
in CD
oo CO
5.4. DISCUSSION
Producing and using crude seed extracts of A. squamosa as a simple
botanical for local use by farmers may not give dramatic economic benefits in
comparison with using synthetic insecticides. My preliminary economic analysis
showed that this technology was just slightly more economical than using synthetic
insecticides (Table 5.1 and 5.2). It is labor intensive but local labor costs are low.
Crude plant extracts are less persistent in the environment and are often safer than
synthetics. Thus in addition to the small economic benefit, environment and human
health concerns will enhance the benefit of using botanical insecticides. To
encourage adoption of this technology, it would be important to persuade farmers of
the wide benefits, e.g. through extension campaigns.
Although more expensive to obtain, availability, ease of preparation, faster
knockdown activity and efficacy of most synthetic insecticides make them more
attractive to farmers. In developing countries of the tropics and subtropics,
production of vegetables (e.g. crucifers) is mostly on small farms employing
intensive use of land, labor, and pesticides. Because of the need to produce fresh
vegetables for the residents of larger cities on a daily basis, farms are usually
located on the outskirts of such population centers or in cleared areas in highlands
with easy access to cities. Cultivation of fresh vegetables is an important source of
income, and production of healthy looking, damage-free vegetables for the city
dwellers is an important consideration in all cultivation practices, especially plant
protection (Talekar and Shelton, 1993). Vegetables have short production periods
and are high-investment crops therefore, farmers will not risk losing their harvest
111
because of insect pest damage. Consequently, they use synthetic chemicals more
intensively, frequently applied using high volume application rates with backpack
sprayers and few safety features for applicators.
In most developing countries, introduction of synthetic insecticides, many of
which are imported from developed countries, face few, if any, of the registration
hurdles common in the West and Japan. As a result, most insecticides (some of
which are not registered in the country of origin), are readily available at an
affordable cost. In Indonesia, pesticides used to be subsidized by the state,
especially for staple food crops such as rice. Because of the absence or poor
enforcement of restrictions on pesticide use, insecticides registered for rice are often
also applied to vegetables. Resistance of insect pests is one problem associated
with excessive use of synthetic chemicals. For example the diamondback moth, P.
xylostella, the most important insect pest of cruciferous plants, was reported as the
first crop pest in the world to develop resistance to DDT. This first resistant
population was observed in Java, Indonesia (Ankersmith, 1953; Johnson, 1953). It
now has become resistant to most synthetic insecticides used and even to the
microbial B. thuringiensis (Tabashnik et al., 1990, 1992: Shelton and Wyman, 1992;
Talekar and Shelton, 1993). Alternative control methods as used in the IPM program
represent one way of reducing the use of synthetic chemicals.
In 1987, the Indonesian government banned 57 broad-spectrum pesticides
and eliminated state subsidies on other pesticides, and instituted Integrated Pest
Management (IPM) as a national strategy to combat agricultural pests. This policy
initiative was undertaken because outbreaks of major insect pests were shown to be
112
caused or exacerbated by excessive use of synthetic pesticides (Gallagher, 1992).
The Indonesian National IPM Program is the most intensive ecological agricultural
training program in the world. By early 1998, nearly 1 million Indonesian smallholder
farmers had graduated from season-long IPM Farmer Field Schools (FAO, 1998).
This community-based IPM program is empowering farmers by equipping them with
the knowledge necessary to make their own crop management decisions (FAO,
1998). The program was conducted mostly for rice farmers in Java and Bali but
recently has been expanded to include other crops such as soybean and vegetables
(Hammig et al. 1997). This kind of program is ideal for other parts of Indonesia
including Ambon (Maluku) as well as for other developing countries. The ongoing
economic crisis in Indonesia adds urgency to the task of providing farmers with a
sustainable and affordable solution to the problems of pest control.
Simple crude plant extracts have been used as insecticides in many countries
for centuries (Crosby, 1971). A few farmers in villages in Ambon island, have been
using crude plant extracts such as derris root, chili peppers, or firewood dust for crop
protection. Some also compost a mixture of spicy plants with manure and alcohol
and then mix this with soil for protection against soil pests (personal interviews). This
kind of indigenous knowledge of local farmers is an important asset. It indicates the
potential willingness of farmers to be educated, motivated and encouraged to
develop and implement alternative controls in their farming systems. Strengthened
by "social marketing" the agriculture extension service has a key role to play in this
process.
113
My results showed that crude aqueous seed extracts of A. squamosa had
good efficacy against diamondback moth larvae in the laboratory (see chapter 3)
and the greenhouse (see chapter 4). Local availability of the seeds and good
efficacy of the extracts as well as indication of economic benefit (Tables 5.1 and
5.2) make A. squamosa a feasible candidate for further development as a botanical
insecticide for local use in villages in Ambon (Maluku), Indonesia. However, field
trials are needed to assess efficacy (e.g. effectiveness, residual action) in real pest
situations as well as for comprehensive cost-benefit economic analysis. The net
benefit over the synthetic insecticides could be assessed by measuring impacts on
actual market and consumable yields. Demonstration plots, in farmer fields in the
villages in Maluku where the fruits are available, are essential if this technology is to
be adopted by local farmers. Cooperation between universities (education
institutions) and agricultural extension services (Ministry of Agriculture) will facilitate
adoption.
Another possibility is producing crude ethanolic seed extracts as a small-scale
industry, which would then be sold to farmers for use as an aqueous emulsion. My
results showed that aqueous emulsions were more potent than crude aqueous
extracts, therefore smaller amounts of extracts would be needed. However, more
studies are required to evaluate the feasibility of this choice.
114
CHAPTER 6
SUMMARY OF CONCLUSION
SUMMARY OF CONCLUSIONS
The overall objectives of this thesis were to identify sources of botanical
insecticides from plants growing in Ambon and surrounding areas (Indonesia) and to
develop simple methods of production for local use as botanical crop protectants.
Seeds of A. muricata and A. squamosa (Annonacea); S. koetjape and Lansium
domesticum (Meliaceae) from different locations and years of collection were
extracted and screened for growth-inhibiting activity against S. litura. Screening for
bioactivity of these extracts was presented in chapter 2. Both Annona species
showed good activity while both species of Meliaceae yielded minimal bioactivity. A.
squamosa was twenty-fold more active than A. muricata, but there were geographic
as well as annual differences among extracts of both species. A. squamosa
collected from Namlea yielded the most inhibitory extracts, reducing growth of larvae
over 10 days to 42% or less, compared to control larvae. However, there were
annual as well as tree-to-tree variations of the extracts from that location. Crude
extracts of A. squamosa showed promising bioactivity and their efficacy was
investigated.
Efficacy of crude seed extracts was evaluated via bioassays and discussed in
chapter 3. Aqueous emulsions of ethanolic extracts and crude aqueous extracts
were tested for their toxicity and deterrent action against P. xylostella and T.ni in the
laboratory. Drench, leaf dip and leaf disc choice assays showed that the crude
extracts have both toxic and antifeedant properties. The combination of these
properties is advantageous since insects could develop desensitization toward
116
antifeedants. Therefore, the efficacy of these extracts that also have toxic properties
is likely to be less affected by desensitization, since ingestion of the compound will
lead to the death of the insect. Advantages of using crude extracts that consist of
mixtures of compounds are that natural mixtures may act synergistically
(Berenbaum, 1985); they may show greater overall bioactivity compared to the
individual constituents (Berenbaum et al. 1991: Chan et al. 1995; McLaughlin 1997),
and insect resistance is much less likely to develop with mixtures (Feng and Isman,
1995). Aqueous emulsions of ethanolic extracts were more potent than crude
aqueous extracts. However, the latter were sufficiently potent and easier to prepare.
Therefore, the use of crude aqueous extracts is encouraged.
Efficacy of crude extracts against P. xylostella in the greenhouse and toxicity
to natural enemies in the laboratory were presented in Chapter 4. Aqueous
emulsions of ethanolic extracts were more effective than 1% Rotenone and a
concentration of 0.5% (w/v) caused 83% mortality of larvae. Efficacy of crude
aqueous seed extracts was comparable to pyrethrum (0.1% pyrethrins), and the
lowest concentration (5% w/v) tested yielded 84% larval mortality. In spite of the
good efficacy against P. xylostella, crude aqueous extracts do not favor some
natural enemies. T. brassicae adults were the most susceptible to the extracts,
followed by O. insidiosus adults, with C. carnea larvae the least susceptible.
Residual contact tests showed that toxicities of the extracts decreased over time
suggesting that there will be a window of opportunity for using the extracts in an IPM
program.
117
Seeds of A. squamosa as waste products of these edible fruits are available
locally in Ambon (Maluku) Indonesia. The economic feasibility of producing the
crude aqueous seed extracts as crop protectants for local use was discussed in
chapter 5. The costs of production and utilization of the extracts are slightly more
economical compared to the costs of using synthetic insecticides. The economic
benefit along with the environmental and human health concerns will make the
production and utilization of the aqueous seed extracts of A. squamosa more
desirable.
This study showed that crude aqueous seed extracts of A. squamosa have
good efficacy both in the laboratory and the greenhouse against some lepidopteran
pests and are feasible to develop as crop protectants for local use in Ambon
(Maluku), Indonesia. However, some studies are required including efficacy of the
extracts in the laboratory and in the field against more insect pests, toxicity to more
species of natural enemies in the laboratory and in the field, and a comprehensive
cost-benefit economic analysis of production and utilization of the extracts locally.
118
REFERENCES
Abbot, W. S. 1925. A method of computing the effectiveness of an insecticide. J.

Econ. Entomol. 18: 265-276.
Abdul-Kadir, H. B., Payne, C. C , Crook, N. E., Fenlon, J . S., and Winstanley, D.

1999. The comparative susceptibility of the diamondback moth Plutella xylostella
and some other major lepidopteran pests of brassica crops to a range of
Baculoviruses. Biocon. Sci. Tech. 9: 421-433.
Addae-Mensah, I., Tort, F.G., Oppong, I., Baxter, I. And Sanders, J . 1977.N-
isobutyl-trans-2-trans-4-eicosadienamide and other alkaloids of fruits of Piper
guineese. Phytochemistry16: 483-485.
Adhikary, S. 1981. The Togo experience in moving from neem research to its
practical application for plant protection. In: Natural Pesticides From the Neem Tree
(Azadirachta indica A. Juss). Proceedings of the first International Neem
Conference, Rottach-Egern,Germany. H. Schmutterer, K.R.S. Ascher, and H.
Rembold (Eds). German Agency for Technical Cooperation, Eschborn, Germany,
pp. 215-222.
Ahammadsahib, K. I., Hollingworth, R. M., McGovren, P. J . , Hui, Y. H., and

McLaughlin, J . L., 1993. Inhibition of NADH: ubiquinone reductase (mitochondrial
complex I) by bullatacin, a potent antitumor and pesticidal Annonaceous acetogenin.
Life Sci. 53: 1113-1120.
Alali, F. Q., Kaakeh, W., Bennett, G. W. and McLaughlin, J . L. 1998.

Annonaceous acetogenins as natural pesticides: potent toxicity against insectide-
suceptible and resistant German cockroaches (Dictyoptera:Blattelidae). J. Econ.
Entomol. 91: 641-649.
Alali, F. Q., Liu, X. X., and McLaughlin, J . L., 1999. Annonaceous Acetogenins:
Recent Progress. J. Nat. Prod. 62: 504-540.
Alford, A. R., Cullen, J . A., Storch, R. H. and Bentley, M. D. 1987. Antifeedant

activity of limonin against the Colorado potato beetle (Coleoptera: Chrysomelidae).
J. Econ. Entomol. 80: 575-578.
119
Alkofahi, A., Rupprecht, J . K., Anderson, J . E., McLaughlin, J . L., Mikolajczak,
K. L., and Scott, B. A., 1989. Search for new pesticides from higher plants. In:
Insecticides of Plant Origin. J.T. Arnason, B.J.R. Philogene, and P. Morand (Eds.).
American Chemical Society Symposium Series 387, Washington, D.C. pp. 25-43.
Alkofahi, A., Rupprecht, J . K., Smith, D. L., Chang, Ch,-J. and McLaughlin, J . L.
1988. Goniothalamicin and annonacin: bioactive acetogenins from Goniothalamus
giganteus (Annonacea). Experientia 44: 83-85.
Amiri, B., Ibrahim, L., and Butt, T.M., 1999. Antifeedant properties of destruxins
and their potential use with the Entomogeneous fungus Metharizium anisopliae for
improved control of crucifer pests. Bio. Scie. Tech. 9: 487-498.
Ankersmith, G.W. 1953. DDT resistance in Plutella maculipennis (Curt.)

(Lepidoptera) in Java. Bull. Entomol. Res. 44: 421-425.
Anonymous, 2000. Analytical Software Statistix (7) User's Manual. Tallahassee,

F.L. 359 pp.
Araya, H., Sahai, M., Singh, S., Singh, A.K., Yoshida, M., Hara, N. and Fujimoto,
Y. 2002. Squamocin-Oi and squamocin- O2, new adjacent bis-tetrahydrofduran
acetogenins from the seeds of Annona squamosa. Phytochemistry §V. 999-1004.
Arnason, J . T., Philogene, B. J . R., Donskov, N. and Kubo, I. 1987. Limonoids

from the Meliaceae and Rutaceae reduce feeding, growth and development of
Ostrinia nubilalis. Entomol. Exp. Appl. 43: 221-226.
Arnason, J . T., Philogene, B. J . R., Morand, P., Imrie, K., Iyengar, S., Duval, F.,
Soucy-Breau, C , Scaiano, J . C , Werstiuk, N. H., Hasspieler, B. and Downe, A.
E. R. 1989. Naturally occurring and synthetic thiophenes as photoactivated
insectides. In: Insecticides of Plant Origin. J.T. Arnason, B.J.R. Philogene and P.
Morand (Eds.). American Chemical Society Symposium Series 387. Washington,
D.C. pp.164-172.
120
Arnason, J . T., MacKinnon, S., Durst, A., Philogene, B. J . R., Hasbun, C ,
Sanchez, P., Poveda, L., San Roman, L., Isman, M. B., Satasook, C , Towers, G.
H. N., Wiriyachitra, P., and McLaughlin, J . L., 1993. Insecticides in tropical plants
with non-neurotoxic modes of action. In: Phytochemical Potential of Tropical Plants.
K.R. Downum, J. Romeo and H. Stafford (Eds.). Plenum Press, New York, pp. 107-
131.
Assabgui, R., Lorenzetti, F., Terradot, L., Regnault-Roger, C , Malo, N.,

Wiriyachitra, P., Sanchez-Vindas, P.E., San Roman, L., Isman, M.B., Durst, T
and Arnason, J.T. 1997. Efficacy of botanicals from the Meliaceae and Piperaceae.
In: Phytochemicals for Pest Control. P.A. Hedin, R.M. Hollingworth, E.P. Masler, J.
Miyamoto and D.G. Thompson (Eds.). American Chemical Society Symposium
Series 658. Washington, D.C. pp. 38-48.
Bandani, A. R., and Butt, T. M. 1999. Insecticidal, antifeedant and growth inhibitory
activities of efrapeptins, metabolites of the fungus Tolypocladium. Bio. Sci. Tech. 9:
499-506.
Basana, I. R., and Prijono, D. 1994. Insecticidal Activity of Aqueous seed extracts
of four species of Annona (Annonaceae) against cabbage head caterpillar,
Crocidolomia binotalis Zeller (Lepidoptera: Pyralidae). Bull. Plant. Pests and Dis. 7:
50-60.
Berenbaum, M. 1985. Brementown revisited: interactions among allelochemicals in

plants. Rec. Adv. Phytochem. 19: 39-169.
Berenbaum, M. R., Nitao, J . K., and Zangerl, A. R. 1991. Adaptive significance of

furanocoumarin diversity in Pastinaca sativa. J. Chem. Ecol. 17: 207-215.
Bernays, E. A. and Chapman, R. F. 1994. Host-plant selection by pythophagous

insects. Chapman and Hall. New York. 312 pp.
Blaney, W. M., Simmonds, M. S. J . , Ley, S. V., Anderson, J . C. and Toogood, P.

L. 1990. Antifeedant effects of azadirachtin and structurally related compounds on
lepidoptereous larvae. Entomol. Exp. Appl. 55: 149-160.
Bomford, M.K. and Isman, M.B. 1996. Desensitisation of fifth instar Spodoptera
litura to azadirachtin and neem. Ent. Exp. Appl. 81: 301-313.
121
Brunner, J . F., Dunley, J . E., Doerr, M. D., and Beers, E. H. 2001. Effect of
pesticides on Colpoclypeus florus (Hymenoptera: Eulophidae) and Trichogramma
platneri (Hymenoptera: Trichogrammatidae) parasitoids of leafrollers in Washington.
J. Econ. Entomol. 94: 1075-1084.
Capinera, J . L. 2001. Handbook of Vegetable Pests. Academic Press, San Diego.
729 pp.
Champagne, D. E., Isman, M. B. and Towers, G. H. N., 1989. Insecticidal activity

of phytochemicals and extracts of the Meliaceae. In: Insecticides of Plant Origin.
J.T. Arnason, B.J.R. Philogene and P. Morand (Eds.). American Chemical Society
Symposium Series 387. Washington D.C. pp. 95-109.
Champagne, D. E., Koul, O., Isman, M. B., Scudder, G. G. E. and Towers, G. H.

N., 1992. Biological Activity of limonoids from the Rutales. Phythochemistry 31: 377-
394.
Champagne, D. E., Isman, M. B., Downum, K. R. and Towers, G. H. N., 1993.

Insecticidal and growth-reducing activity of foliar extracts from Meliaceae.
Chemoecology 4: 165-173.
Chen, W., Isman, M. B., and Chiu S.-F. 1995. Antifeedant and growth inhibitory
effects of the limonoid toosendanin and Melia toosendan extracts on the variegated
cutworm, Peridroma saucia (Lep., Noctuidae). J. Appl. Entomol. 119: 367-370.
Chiu, S-F.1987. Experiments on the practical application of chinaberry, Melia

azedarach, and other naturally occurring insecticides in China. In: Natural Pesticides
From the Neem Tree (Azadirachta indica A. Juss) and Other Tropical Plants.
Proceedings of the Third International Neem Conference, Nairobi. H. Schmutterer,
and K.R.S. Ascher (Eds). German Agency for Technical Cooperation, Eschborn,
Germany, pp. 661-668.
Chiu, S-F. 1989. Recent advances in research on botanical insecticides in China. In:
Insecticides of Plant Origin. J.T. Arnason, B.J.R. Philogene and P. Morand (Eds.).
American Chemical Society Symposium Series 387. Washington D.C. pp. 69-77.
Cole, M. D. 1994. Key antifungal, antibacterial and anti-insect assays- a critical

review. Biochem. System. Ecol. 22: 837-856.
122
Corrigan, J . E., and Laing, J . E. 1991. An improved method for producing small,
consistent samples of hosts for presenting to the egg parasitoid, Trichogramma
minutum. Proc. Entomol. Soc. Ont. 122: 103-104.
Cronquist, A., 1981. An Integrated System of Classification of Flowering Plants,

Columbia University Press, New York. 1262pp.
Crosby, D. G. 1971. Minor insecticides of plant origin, In: Naturally Occurring

Insecticides. M. Jacobson and D.G. Crosby (Eds.). Marcel Dekker Inc., New York,
pp. 171-239.
Dreyer, M. 1987. Field and laboratory trials with simple neem products as
protectants against pests of vegetable and field crops in Togo. In: Natural Pesticides
From the Neem Tree (Azadirachta indica A. Juss) and Other Tropical Plants.
Proceedings of the Third International Neem Conference, Nairobi. H. Schmutterer,
and K.R.S. Ascher (Eds). German Agency for Technical Cooperation, Eschborn,
Germany, pp. 431-447.
Eisemen, F and M. 1988. Fruits of Bali. Periplus Editions (Hk) Ltd., Singapore. 60
pp.
Elzen, G.W. 2001. Lethal and sublethal effects of insecticide residues on Orius
insidiosus (Hemiptera: Anthocoridae) and Geocoris punctipes (Hemiptera:
Lygaeidae). J. Econ. Entomol. 94: 55-59.
Ermel, K., Pahlich, E., Schmutterer, H. 1987. Azadirachtin content of neem kernels
from different geographical locations, and its dependence on temperature, relative
humidity, and light. In: Natural Pesticides From the Neem Tree (Azadirachta indica
A. Juss) and Other Tropical Plants. Proceedings of the Third International Neem
Conference, Nairobi. H. Schmutterer, and K.R.S. Ascher (Eds). German Agency for
Technical Cooperation, Eschborn, Germany, pp. 71-184.
Escoubas, P., Lajide, L. and Mizutani, J . 1994. Insecticidal and antifeedant

activities of plant compounds potential leads for novel pesticides. In: Natural and
Engineered Pest Management Agents. P.A. Hedin, J.J. Menn, and R.M.
Hollingworth (Eds). American Chemical Society Symposium Series 551,
Washinghiton, D.C. pp.162-179.
123
Ewete, F. K., Arnason, J . T., Larson, J . and Philogene, J . R. 1996. Biological
activities of extracts from traditionally used Nigerian plants against the European
corn borer, Ostrinia nubilalis. Entomol. Exp. Appl. 80:531-537.
Fagoonee, I. 1987. Use of neem in vegetable crop protection in Mauritius. In:

Natural Pesticides From the Neem Tree (Azadirachta indica A. Juss) and Other
Tropical Plants. Proceedings of the Third International Neem Conference, Nairobi,
Kenya. H. Schmutterer, and K.R.S. Ascher (Eds). German Agency for Technical
Cooperation, Eschborn, Germany, pp. 419-429.
FAO, 1998. IPM-trained farmers in Indonesis escape pest outbreaks. News and
highlights. http://www.fao.org/NEWS/1998/981104-e.htm.
Fang, X. P., Rieser, M. J . , Gu, G. X., and McLaughlin, J . L., 1993. Annonaceous
acetogenins: an updated review. Phytochem. Anal. 4: 27-49.
Feng, R., and Isman, M.B. 1995. Selection for resistance to azadirachtin in the
green peach aphid, Myzus persicae. Experientia 51: 831-833.
Finney, D. J . 1971. Probit Analysis. Cambridge University Press. 333 pp.
Fujimoto, Y., Eguchi, T., Kakinuma, K., Ikekawa, N., Sahai, M., Gupta, Y. K.
1988. A new bis-tetrahydrofuran containing acetogenins from Annona squamosa.
Chem. Pharm. Bull. 36 : 4802-4806.
Fujimoto, Y., Murasaki, C , Shimada, H., Nishioka, H., Kakinuma, K., Sing, S.,
Gupta, Y.K., and Sahai, M. 1994. Annonaceous acetogenins from the seeds of
Annona squamosa. Non-adjacent bis-tetrahydrofuranic acetogenins. Chem. Pharm.
Bull. 42: 1175-1184.
Gallagher, K. D. 1992. IPM development in the Indonesian national IPM program.

In: Pest management and the Environment in 2000. A.A.S.A. Kadir and H.S. Barlow
(Eds.). C.A.B. International, Wallingford, Oxon, UK. pp.82-89.
Gonzalez-Coloma, A., Valencia, F., Martin, N., Hoffmann, J.J., Hutter, L., Marco,
J. A., Reina, M. 2002. Silphinene sesquiterpenes as model insect antifeedants. J.
Chem. Ecol. 28: 117-129.
124
Gu, Z.-M., Zhao, G.-X., Oberlies, N. H., Zeng, L. and McLaughlin, J . L. 1995.
Annonaceous acetogenins: potent mitochondrial inhibitors with diverse applications,
In: Recent Advances in Phytochemistry. J.T. Arnason, R. Mata (Eds.). Plenum
Press, New York. pp. 249-310.
Guandano, A., Gutierrez, C , de la Pena, E., Cortes, D. and Gonzales-Coloma,

A. 2000. Insecticidal and mutagenic evaluation of two Annonaceaous acetogenins.
J. Nat. Prod. 63: 773-776.
Hama, H. 1992. Insecticide resistance characteristics of the diamondback moth. In:

Diamondback Moth and Other Ccrucifer Pests. Proceeding of the Second
International Workshop, Tainan, Taiwan. N.S.Talekar (Ed). Asian Vegetable
Research and Development Center, Taipei, pp. 455-463.
Hamilton, G. C , and Lashomb, J . H. 1997. Effect of insecticides on two predators

of the Colorado potato beetle (Coleoptera: Chrysomelidae). Flor. Entomol. 80: 10-
23.
Hammig, M., Rauf, A., and Carner, G. 1997. Integrated pest management in non-
rice food crops in Indonesia: opportunities to reduce chemical pesticide use. In:
Agricultural Production and Nutrition. Proceedings of an International Conference,
Boston, Massachusetts. W. Lockeretz (Ed.) School of Nutrition and Policy, Tufts
University, Medford, M.A. pp.131-139.
He, K., Zeng, L., Ye, Q., Shi, G., Oberlies, N. H., Zhao, G.-X., Njoku, C.J., and
McLauhglin, J . L. 1997. Comparative SAR evaluation of Annonaceous acetogenins
for pesticidal activity. Pestic. Sci. 49: 372-378
Henn, T., and Weinzierl, R. 1990. Alternatives in insect pest management.

Beneficial insects and mites. University of Illinois. Circular 1298. 24 pp.
Hoffmann, M. P., and Frodsham, A. C. 1993. Natural Enemies of Vegetable Insect
Pets. Cooperative Extension, Cornell University, Ithaca, NY. 63 pp.
Hollingworth, R. M., Ahmmadsahib, K. L., Gadelhak, G., and McLaughlin, J . L.

1994. Inhibitors of complex I in the mitochondrial electron transport chain with
activities as pesticides. Biochem. Soc. Trans. 22. 230-233.
125
Hui, Y,-H., Rupprecht, J . K., Liu, Y. M. and Anderson, J . E. 1989. Bullatacin and
bullatacinone: Two highly potent bioactivity acetogenins from Annona bullata. J. Nat.
Prod. 52: 463-477.
Isman, M. B., 1993. Growth inhibitory and antifeedant effects of azadirachtin on six
noctuids of regional economic importance. Pestic. Sci. 38: 57-63.
Isman, M.B. 1994. Botanical Insecticides and Antifeedants: New Sources and
Perspectives. Pest. Res. J. 61: 11-19.
Isman, M. B., 1995. Leads and prospects for the development of new botanical
insecticides. In: Reviews in Pesticide Toxicology, Vol.3. R. M. Roe and R. J. Kuhr
(Eds.) Toxicology Communications Inc, Raleigh, NC, pp.1-20.
Isman, M. B. 1997. Barriers to the commercialization of new botanical insecticides.

Phytoparasitica 25: 339-344.
Isman, M. B., and Rodriguez, E., 1983. Larval Growth inhibitors from species of
Parthenium (Asteraceae). Phytochemistry 22: 2709-2713.
Isman, M. B., Koul, O., Luczynski, A., and Kaminski, J . 1990. Insecticidal and
antifeedant bioactivities of neem oils and their relationship to Azadirachtin content. J.
Agric. Food Chem. 38: 1406-1411.
Isman, M. B., Koul, O., Arnason, J . T., Stewart, J . and Salloum, G. S. 1991.
Developing a neem-based insecticide for Canada. Mem. Ent. Soc. Can. 159: 39-47.
Isman, M. B., Arnason, J . T. and Towers, G. H. N. 1995. Chemistry and biological

activity of ingredients of other species of Meliaceae. In: The Neem Tree Azadirachta
indica A. Juss. And Other Meliaceous Plants: Sources of Unique Natural Products
For Integrated Pest Management, Medicine, Industry and Other Purposes. H.
Schmutterer (Ed.). VCH, Weinheim. pp. 652-666.
Isman, M. B., Matsuura, H., MacKinnon, S., Durst, T., Towers, G. H. N. and
Arnason, J . T. 1996. Phytochemistry of the Meliaceae: So many terpenoids, so few
insecticides. Recent Advances in Phytochemistry 30: 155-178
126
Iwuala, M. O. E., Osisiogu, I. U. W. and Agbbakwuru, E. O. P. 1981. Dennettia oil,
a potential new insecticide: test with adults and nymphs of Periplaneta Americana
and Zonocerus variegates. J.Econ. Entomol. 74: 249-252.
Jacobson, M., 1989. Botanical pesticides. Past, present and future. In: Insecticides
of Plant Origin.J.T. Arnason, B.J.R. Philogene and P. Morand (Eds.). American
Chemical Society Symposium Series 387. Washington D.C. pp. 1-10.
Jacobson, D. A., and Kring, T. J . 1995. Efficacy of predator attacking Helicoverpa

zea (Boddie) (Lepidoptera: Noctuidae) eggs on grain sorghum in the field. J.
Entomol. Sci. 30: 251-257.
Janprasert, J . , Satasook, C , Sukumalanand, P., Champagne, D.E., Isman,

M.M., Wiriyachitra, P. and Towers, G.H.N. 1993. Rocaglamide, a natural
benzofuran insecticides from Aglaia odorata. Phytochemistry 32: 67-69.
Johnson, D. R. 1953. Plutella maculipennis resistance to DDT in Java. J. Econ.

Entomol. 46: 176.
Johnson, H, A., Oberlies, N. H., Alali, F. Q., and McLaughlin, J . L., 2000.
Thwarting Resistance: Annonaceous Acetogenins as New Pesticidal and Antitumor
Agents. In: Biologically Active Natural Products: Pharmaceuticals. S.J. Cutler and
H.G. Cutler (Eds.). CRC Press, Washington, D.C. pp. 173-183.
Johnson, H. A., Gordon, J . , and Mc Laughlin, J . L., 1996. Monthly variations in

biological activity of Asimina triloba. In: Progress in New Crops: Proceedings of the
Third National New Crops Symposium Indianapolis. V.J. Janick (Ed.) American
Society of Horticulture Science, Alexandria, V.A, pp. 609-613.
Jolad, S. D., Hoffman, J . J . , Schram, K .H., Cole, J . R., Tempesta, M. S., Kriek,
G. R., and Bates, R. B. 1982. Uvaricin, a new antitumor agent from Uvaria
accuminata (Annonaceae). J. Org. Chem. 47: 3151-3153.
Kawazu, K., Alcantara, J . P. and Kobayashi, A. 1989. Isolation and structure of

neoannonin a novel insecticidal compound from the seeds of Annona squamosa.
2719-2722.
Agric. Biol. Chem. 53:
127
Kirsch, K., and Schmutterer, H. 1988. Low efficacy of a Bacilus thuringiensis
(Berl.) formulation in controlling the diamondback moth, Plutella xylostella (L.) in the
Philippines. J. Appl. Entomol. 105: 249-255.
Klemm, U., and Schmutterer, H. 1993. Effects of neem preparation on Plutella

xylostella L. and its natural enemies of the genus Trichogramma. J. Plant Dis.
Protec. 100: 113-128
Klocke, J.A. and Kubo, I. 1982. Citrus limonoid by-products as insect control
agents. Entomol. Exp. Appl. 32: 299-301.
Kohyama, Y. 1986. Insecticidal Activity of MK-139 (CME 134) against diamondback

moth. In: Diamondback Moth Management. Proceedings of the first international
workshop. Talekar, N.S and Griggs, T. D. (Eds.). Asian Vegetable Research and
Development Center, pp. 265-269.
Landolt, J . L., Ahammadsahib, K. I., Hollingworth, R. M., Barr, R., Crane, F. L.,
Buerck, G. P., McCabe, G. P., and McLaughlin, J . L., 1995. Determination of
stucture activity relationships of Annonaceous acetogenins by inhibition of oxygen
uptake in rat liver mitochondria. Chemico. Biol. Interact. 98: 1-13.
Leboeuf, M., Cave, A., Bhaumik, P. K., Mukherjee, B. and Mukherjee, R. 1982.
The Phytochemistry ofthe Annonaceae. Phytochemistry 21: 2783-2813.
Lee, S. M., Klocke, J . A., Barnaby, M. A., Yamasaki, R. B. and Balandrin, M. F.

1989. Insecticidal constituents of Azadirachta indica and Melia azedarach
(Meliaceae). In: Insecticides of Plant Origin. J.T. Arnason, B.J.R. Philogene, and P.
Morand (Eds.). American Chemical Society Symposium Series 387, Washington,
D.C. pp. 293-304.
Lewis, A. C , and van Emden, H. F. 1986. Assays for insect feeding. In: Insect-
Plant Interactions. J.R. Miller and T.A. Miller (Eds.). Springer Verlag, New York. pp.
95-119.
Lewis, M. A., Arnason, J . T., Philogene, J . R., Rupprecht, J . K., and

McLaughlin, J . L., 1993. Inhibition of respiration at site I by asimicin, an insecticidal
acetogenin ofthe paw paw, Asimina triloba (Annonaceae). Pestic. Biochem. Physiol.
45: 15-23.
128
Londershausen, M., Leicht, W., Lieb, F., and Moesschler, H., 1991. Molecular
mode of action of annonins. Pestic. Sci. 33: 427-433.
MacKinnon, S., Chauret, D., Wang, M., Mata, R., Pereda-Miranda, R., Jiminez,
A., Bernard, C.B., Krishnamurty, H.G., Poveda, L.J., Sanches-Vindas, P.E.,
Arnason, J.T. and Durst, T. 1997. Botanicals from the Piperaceae and Meliaceae
of the American neotropics: Phytochemistry. In: Phytochemicals for Pest Control. P.
A. Hedin, R. M. Hollingworth, E. P. Masler, J . Miyamoto and D. G.Thompson (Eds.).
American Chemical Society Symposium Series 658, Washington, D.C. pp. 49-57.
Mariapan, V., and Saxena, R. C. 1983. Effect of custard-apple oil and neem oil on
survival of Nephotettix virescens (Homoptera: Cicadellidae) and on rice tungro virus
transmission. J. Econ. Entomol. 76: 573-576.
Mariapan, V., and Saxena, R. C. 1984. Effect of mixtures of custard-apple oil and
neem oil on survival of Nephotettix virescens (Homoptera: Cicadellidae) and on rice
tungro virus transmission. J. Econ. Entomol. 77: 519-521.
Mariapan, V., Jayaraj, S. and Saxena, R.C. 1988. Effect of nonedible seed oils on
survival of Nephotettix virescens (Homoptera: Cidadellidae) and on transmission of
rice tungro virus. J. Econ. Entomol. 81: 1369-1373,
McLaughlin, J . L., Zeng, L., Oberlies, N. H., Alfonso, D., Johnson, H. A., and
Cummings, B., 1997. Annonaceous Acetogenins as New Natural Pesticides:
Recent Progress. In: Phytochemicals for Pest Control. P.A. Hedin, R. M.
Hollingworth, E. P. Masler, J. Miyamoto and D. G.Thompson (Eds.). American
Chemical Society Symposium Series 658, Washington, D.C. pp. 117-133.
Mikolajczak, K. L., and Reed, D. K., 1987. Extractives of seeds of the Meliaceae:
Effects on Spodoptera frugiperda (J.E.Smith), Acalymma vittatum (F) and Artemia
salina Leach. J. Chem.Ecol. 13: 99-111.
Mikolajczak, K. L., McLaughlin, J . L. and Rupprecht, J . K. 1988. Control of pests

with annonaceous acetogenins. U.S. Patent No. 4721727.
Mikolajczak, K. L., Zilkowski, B. W., and Bartlet, R.J., 1989a. Effects of

Meliaceous seed extracts on growth and survival of Spodoptera frugiperda (J. E.
Smith). J. Chem. Ecol. 15: 121-128.
129
Mikolajczak, K. L , McLaughlin, J . L. and Rupprecht, J . K. 1989b. Control of
pests with annonaceous acetogenins. U.S. Patent No. 4855319.
Mitsui, T., Atsusawa, S., Ohsawa, K., Yamamoto, I., Miyake, T. and Umehara, T.
1991. Search for insect growth regulators. In: Pesticides and the Future.
Toxicological E. Hodgson, R. M. Roe and N.
Studies of Risks and Benefits.
Motoyama (Eds.). North Carolina State University, Raleigh. Rev.Pestic.Toxicol. 1:
239-247.
Miyakado, M., Nakayama, I. And Ohno, N. 1989. Insecticidal unsaturated

isobutylamides: from natural products to agrochemical leads. In: Insecticides of Plant
Origin. J.T. Arnason, B.J.R. Philogene, and P. Morand (Eds.). American Chemical
Society Symposium Series 387, Washington, D.C. pp. 173-187.
Miyakado, M. and Watanabe, K. 1992. In search for insecticidal leads from natural
sources. Proc. '92 Agric. Biotech.Symp. on New BioPesticides, Suwon, Korea.
Moeschlar, H. F., Pfuger, W., and Wendlisch, D. 1987. Pure Annonin and a
process for the preparation thereof, U.S. Patent No. 4689323.
Mohan, M., and Gujar, G. T. 2000. Toxicity of Bacillus thuringiensis strains and
commercial formulations to the diamondback moth, Plutella xylostella (L.). Crop
Protection 20: 311-316.
Morre, D.J., Cabo, R.D., Farley, C , Oberlies, N.H., and McLaughlin, J.L. 1995.
Mode of action of bullatacin, a potent new antitumor acetogenin: inhibition of NADH
oxidase activity of HELA and HL-60, but not liver plasma membranes. Life Scie. 56:
343.
Morton, J. F., 1987. Fruits of Warm Climates. Media, Inc, Greensboro, N. C ,
Murray, K.D., Groden, E., Drummond, F.A., Alford, A.R., Conely, S., Storch,
R.H. and Bentley, M.D. 1995. Citrus limonoid effects on Colorado potato beetle
(Coleoptera: Chrysomelidae). Env. Entomol. 24: 1275-1283.
Murty, U. S., Sirram, K. and Jamil, K. 1997. Effect of leaf extract of Polyalthia
longifolia (Family: Annonaceae) on mosquito larvae and pupae of Culex
130
quinquefasciarus (Diptera: Culicidae) Say of different habitats. Inter. Pest Control 39:
52-53
Nagakartti, S., and Nagaraja, H. 1977. Biosystematics of Trichogramma and

Trichogrammatoidea species. Ann. Rev. Entomol. 22: 157-176.
Nakasone, H. Y., and Paull, R. E. 1998. Tropical Fruits. Crop Production Science in
Horticulture 7. CAB International, Wallington, Oxon, UK. 454 pp.
Nawrot, J . , Koul, O., Isman, M. B. and Harmatha, K. 1991. Naturally occurring

antifeedants: effects on two polyphagous lepidopterans. J. Appl. Entomol. 112: 194-
201.
Oberlies, N. H., Chang, C. J . , and McLaughlin, J . L., 1997. Structure -activity

relationships of diverse Annonaceous acetogenins against multidrug resistant
human mamary adenocarcinoma (MCF-7/Adr). J. Med. Chem. 40: 2102-2106.
Perera, D. R., Armstrong, G., and Senanayake, N. 2000. Effect of antifeedants on

the diamondback moth {Plutella xylostella) and its parasitoid Cotesia plutellae. Pest
Manag. Scie. 56: 486-490.
Philogene, B. J . R., Arnason, J . T., Berg, C. W., Duval, F. and Morand, P. 1986.
Efficacy of the plant phototoxin a-terthienyl against Aedes intrudens and effects on
nontarget organisms. J. Chem. Ecol. 12: 893-898.
Piper, J. M., 1989. Fruits of South-east Asia, facts and folkore. Oxford Univ. Press
Ltd., New York. 94 pp.
Pree, D. J., Archibald, D. E., Morrison, R.K. 1989. Resistance to insecticide ofthe
common green lacewing (Chrysoperla carnea (neuroptera: Chrysopidae) in southern
Ontario. J. Econ. Entomol. 82: 29-34.
Prijono, D., and Hassan, E. 1993. Laboratory and field efficacy of neem
(azadirachta indica A. Juss) extracts against two broccoli pests. J. Plant Dis. Prot.
100: 354-370.
Prijono, D., and Hindayana, D. 1993. Insecticidal activity of pond-apple (Annona

glabra L.) and neem (Azadirachta indica A. Jussieu) seed extrcats against
Phaedonia inclusa Stal (Coleoptera: Chrysomelidae). Proced. Seminar on Botanical
Pesticides, Bogor, Indonesia. Abstracts.
131
Prijono, D., Gani, M. S., and Syahputra, E., 1997. Insecticidal activity of
Annonaceous seed extracts against Crocidolomia binotalis Zeller (Lepidoptera:
Pyralidae). Bull. Plant Pests and Dis. 9: 1-6.
Prijono, D., Manuwoto, S., and Soemawinata, R.A.T. 1994. Insecticidal activity of
sugar apple (Annona squamosa L.) and pond apple (A. glabra L.) seed extracts
against rice brown planthopper, Nilaparvata lugens (Stal.). Proceedings Unesco
National Seminar, Depok, Indonesia, pp. 335-341.
Ratnayake, S., Rupprecht, J . K., Potter, W. M., and McLaughlin, J . L., 1992.
Evaluation of various parts of the paw paw tree, Asimina triloba (Annonaceae) as
commercial sources of the pesticidal annonaceous acetogenins. J. Econ. Entomol.
85: 2353-2356.
Reddy, G.V.P. and Urs, K.C.D. 1988. Effect of plant extracts on brown planthopper
(BPH) oviposition. International Rice Research Newsletter^: 42.
Ridgway, R. L., Abies, J . R., Goodpasture, C , and Hartstack, A. W. 1981.

Trichogramma and its utilization for crop protection in the U.S.A In: Proceedings of
the Joint American-Soviet Conference on Use of Beneficial organisms in the control
of crop pests. Washington, D.C. J.R. Coulson (Ed.). The Entomological Society of
America, College Park, M.D. pp. 41-48.
Rieser, M. J . , Fang, X. P., Rupprecht, J . K., Hui, Y. H., Smith, D. L. and

McLaughlin, J . L., 1993. Bioactive single-ring acetogenins from seed extracts of
Annona muricata. Planta Med. 59: 91-92..
Rieser, M. J . 1996. Five novel mono-tetrahydrofuran ring acetogenins from the

seeds of Annona muricata. J. Nat. Prod. 59: 100-108
Rumpf, S., Frampton, C , and Chapman, B. 1997. Acute toxicity of Insecticides to

Micromus tasmaniae (Neuroptera: Hemerobiidae) and Chrysoperla carnea
(Neuroptera: Chrysopidae): LC o and LCgo estimates for various test duration. J.
5
Econ. Entomol. 90: 1493-1997.
Rupprecht, J . K., Hui, Y.H., and McLaughlin, J . L., 1990. Annonaceous

acetogenins: a review. J. Nat. Prod. 53: 237-276.
132
Sahai, M., Singh, S., Singh, M., Gupta, Y. K., Akashi, S., Yuji, R., Hirayama, K.,
Asaki, H., Araya, H., Hara, H., Eguchi, T., Kakinuma, K., and Fujimoto, Y., 1994.
Annonaceous acetogenins from the seeds of Annona squamosa: adjacent bis-
tetrahydrofuranic acetogenins. Chem. Pharm. Bull. 42: 1163-1174.
Satasook, C , Isman, M. B. and Wiriyachitra, P. 1992. Activity of rocaglamide, an

insecticidal natural product, against the variegated cutworm, Peridroma saucia
(Lepidoptera: Noctuidae). Pestc. Sci. 36: 53-58.
Satasook, C , Isman, M. B., Ishibashi, F., Medbury, S., Wiryachitra, P. and

Towers, G. H. N. 1994. Insecticidal bioactivity of crude extracts of Aglaia species
(Meliaceae). Biochem. System. Ecol. 22: 121-127.
Saxena, A. Harshan, V and Saxena, R. C. 1992. Mosquito larvacidal activity of

Annona squamosa extract. Z. ang. Tool. 79: 185-191.
Schluter, U., Bidmon, H. J., and Grewe, S. 1985. Azadirachtin affects growth and
endocrine events in larvae of the tobacco hornworm, Manduca sexta. J. Insect
Physiol. 31: 773-777.
Schmutterer, H., 1990. Properties and potential natural pesticides from the neem
tree. Ann. Rev. Entomol. 35: 271-297.
Schmutterer, H., 1992a. Higher plants as sources of novel pesticides. In:
Insecticides: Mechanisms of Action and Resistance. D. Otto and B. Weber. (Eds.)
Intercept Ltd., Andover. pp. 3-15.
Schmutterer, H., 1992b. Control of diamondback moth by application of neem

extracts. In: Diamondback Moth and Other Ccrucifer Pests. Proceeding of the
Second International Workshop, Tainan, Taiwan. N.S.Talekar (Ed). Asian Vegetable
Research and Development Center, Taipei, pp. 325-332.
Schoonhoven, L. M. 1982. Biological aspects of antifeedants. Entomol. Exp. Appl.

31:57-69.
Secoy, D. M., and Smith, A. E., 1983. Use of plants in control of agricultural and
domestic pests. Econ. Bot. 37: 28-57.
133
Shelton, A. M., and Wyman, J . A. 1992. Insecticide resistance of diamondback
moth in North America. In: Diamondback Moth and Other Ccrucifer Pests.
Proceeding of the Second International Workshop, Tainan, Taiwan. N.S.Talekar
(Ed). Asian Vegetable Research and Development Center, Taipei, pp. 447-454.
Shelton, A. M., Vanderberg, J . D., Ramos, M., and Wllsey, W. T. 1998. Efficacy
and persistence of Beauveria bassiana and other fungi for control of diamondback
moth (Lepidoptera: Plutellidae) on cabbage seedlings. J. Entomol. Sci. 33: 142-151.
Singh, R. P. 1987. Comparison of antifeedant efficacy and extract yields from

different parts and ecotypes of neem (Azadirachta indica A. Juss) tree. In: Natural
Pesticides From the Neem Tree (Azadirachta indica A. Juss) and Other Tropical
Plants. Proceedings of the Third International Neem Conference, Nairobi. H.
Schmutterer, and K.R.S. Ascher (Eds). German Agency for Technical Cooperation,
Eschborn, Germany, pp. 185-194.
Snedecor, G. W. and Cochran, W. G. 1989. Statistical Methods.Iowa States

University Press, Ames, Iowa. 503 pp.
Soares, G. G. and Quick, T. C. 1992. MVP, a novel bioinsectide for the control of
diamondback moth. In : Diamondback Moth and Other crucifer Pests. Proceedings
of the Second International Workshoop, Tainan, Taiwan. N. S. Talekar (Ed.). Asian
Vegetable Research and Development Center. Taipei, pp. 129-137.
Sombatsiri, K., and Temboonkeat, K. 1987. Efficacy of an improved neem kernel
extract in the control of Spodoptera litura and Plutella xylostella under laboratory
conditions and in field trials. In: Natural Pesticides From the Neem Tree
(Azadirachta indica A. Juss) and Other Tropical Plants. Proceedings of the Third
International Neem Conference, Nairobi, Kenya. H. Schmutterer, and K.R.S. Ascher
(Eds). German Agency for Technical Cooperation, Eschborn, Germany, pp. 195-
200.
Sookvanichsilp, N., Gritsanapan, W., Somanabandhu, A. O., Leckcharoen, K.,

and Tiankrop, P. 1994. Toxicity testing of organic solvent extracts from Annona
squamosa: effects on rabbit eyes and ear skin. Phytother. Res. 8: 365-368.
Sparks, T. C , Thompson, G. D., Kirst, H.A., Hertlein, M. B., Larson, L. L.,

Worden, T. V., and Thibault, S. T. 1998. Biological activity of the Spinosyns, new
fermentation derived insect control agents, on tobacco budworm (Lepidoptera:
Noctuidae) larvae. J. Econ. Entomol. 91: 1277-1283.
134
Srivastava, S.D. 1986. Limonoids from the seeds of Melia azedarach. J. Nat. Prod.
49:56-61.
Steibl, M., Nawrot, J . and Herout, V. 1983. Feeding deterrent activity of

enantiomeric isoalantones. Biochem. System. Ecol. 11: 381-382.
Tabshnik, B. E., Cushing, N. L , Finson, N., and Johnson, M. W. 1990. Field

development of resistance to Bacillus thuringiensis in diamondback moth
(Lepidoptera: Plutellidae). J. Econ. Entomol. 83: 1671-1676.
Tabashnik, B. E., Schwartz, J . M., Finson, N., and Johnson, M. W. 1992.

Inheritance of resistance to Bacillus thuringiensis in diamondback moth
(Lepidoptera: Plutellidae). J. Econ.Entomol. 85: 1046-1055.
Talekar, N. S., and Shelton, A. M. 1993. Biology, ecology and management ofthe
diamondback moth. Annu. Rev. Entomol. 38: 275-301.
Venkataramireddy, P., Chitra, K. C , and Rao, P. K. 1990. Efficacy of the plant

extracts in the control of brinjal spotted leaf beetle, Henosepilachna
vigintioctopunctata F. In: Botanical Pesticides in Integrated Pest Management.
Proceedings of National Symposium on Problems and Prospects of Botanical
Pesticides in IPM, Rajahmundry, India, pp. 225-227.
Weaver, D. K., Wells, C. D., Dunkel, F. V., Bertsch, W., Sing, S. E. and Sriharan,
S. 1994. Insecticidal activity of floral, foliar and root extracts of Tagetes minuta
(Asterales : Asteraceae) against adult Mexican bean weevils (Coleoptera:
Bruchidae). J. Econ. Entomol. 87: 1718-1725.
Wheeler, D. A., 1999. The effects of Trichilia americana (Meliaceae) crude extract
on the growth, development and behavior of the Asian armyworm, Spodoptera litura
(Lepidoptera: Noctuidae). PhD Thesis. University of British Columbia, Vancouver,
Canada. 167pp.
Wiriyachitra, P. 1991. Botanical Pesticides. International Development Research

Center (Canada) Rpt. 3-P-89-1012-02.
135
Wolvetang, E. J . , Johnson, K. L , Krauer, K., Ralph, S. J . and Linnane, A. W.
1994. Mitochondrial respiratory chain inhibitors induce apoptosis. FEBS Lett. 339
40-44.
Zafra-Polo, M. C , Gonzales, M. C , Estornell, E., Sahpaz, S., and Cortes, D.,

1996. Acetogenins from Annonaceae: inhibitors of mitochondrial complex I.
Phytochemistry 42: 253-271.
Zeng, L , Ye, Q., Oberlies, N.H., Shi, G., Gu, Z.-M, He, K., and McLaughlin, J.L.
1996. Recent advances in Annonaceous acetogenins. Nat. Prod. Rep. 13: 275-306.
Zhang, X., Wang, X.-L and Chiu, S.-F. 1992. Studies on the bioactivities and
applications of Chinese botanical insecticide-toosendanin. Proc. XIX Intl. Congr.
Entomol. Abstracts.
136

Abe 2

Enviado por

Dados do documento

Título original

Direitos autorais

Formatos disponíveis

Compartilhar este documento

Compartilhar ou incorporar documento

Opções de compartilhamento

Você considera este documento útil?

Este conteúdo é inapropriado?

Direitos autorais:

Formatos disponíveis

Abe 2

Enviado por

Direitos autorais:

Formatos disponíveis

DEVELOPMENT OF A BOTANICAL INSECTICIDE FROM AMBON AND

SURROUNDING AREAS (INDONESIA) FOR LOCAL USE

Johanna Audrey Leatemia

B.Sc. (Honors), Bogor Agricultural University, 1987

M . S o , University of Guelph, 1994

THE FACULTY OF GRADUATE STUDIES

Faculty of Agricultural Sciences

W e accept this thesis as conforming

©Johanna Audrey Leatemia, 2003

Department of pfa^ bteiu faculty c\ N

Intensive use of synthetic insecticides to control insect pests had lead to

many problems such as pest resistance and resurgence, effects on non-target

organisms, human exposure and environmental impacts. These negative effects

been shown to possess promising insecticidal properties. The general objectives of

Crude ethanolic seed extracts of Annona muricata L , A. squamosa L.

(Annonaceae), Lansium domesticum Corr., and Sandoricum koetjape (Burm. F.)

geographic as well as annual differences among the extracts of both species.

Extracts of L. domesticum and S. koetjape did not show sufficient bioactivity to be

showed toxic as well as antifeedant effects against both species.

Greenhouse trials were conducted to assess the efficacy of the extracts

against some commercially available biocontrol agents. An aqueous emulsion of an

than 1 % Rotenone, a commercial insecticide. Crude aqueous seed extracts showed

efficacy comparable to pyrethrum (0.1% a.i), a widely used botanical insecticide.

followed by Orius insidiosus (Say.) adults, while Trichogramma brassicae (Bezd)

adults were the most susceptible.

A preliminary economic analysis was conducted to evaluate the feasibility of

slightly more economical than using synthetic insecticide.

Good efficacy of crude seed extracts of A. squamosa both in the laboratory

make this species a promising candidate for development as a simple botanical

insecticide for local use in Ambon (Indonesia).

CHAPTER 1. GENERAL INTRODUCTION AND LITERATURE

1.2.4. Ecology and Botany of Annona spp, Lansium

1.3. Thesis objectives 33

CHAPTER 2. SCREENING FOR INSECTICIDAL ACTIVITY OF

CHAPTER 3. LABORATORY EVALUATION OF CRUDE SEED

CHAPTER 5. FEASIBILITY OF PRODUCING CRUDE AQUEOUS

CHAPTER 6. SUMMARY OF CONCLUSIONS 115

Table 2.1. Growth inhibitory effect of crude ethanolic seed extracts

Table 2.2. Growth inhibitory effect of crude ethanolic seed extracts

Table 2.3. Growth inhibitory effect of crude ethanolic seed extracts

Table 2.4. Growth inhibitory effect of crude ethanolic seed extracts

Table 2.5. Effect of crude ethanolic seed extracts of A squamosa

Table 3.1. Range of concentrations of aqueous extracts (AE) and

Table 3.2. Efficacy of crude aqueous seed extracts of A. squamosa

Table 3.3. Efficacy of crude aqueous seed extracts of A. squamosa

Table 3.4. Efficacy of crude aqueous seed extracts of A. squamosa

Table 3.5. Efficacy of aqueous emulsions of ethanolic seed extracts

Table 4.1. Mortality of diamondback moth (P. xylostella) larvae sprayed

Table 4.2. Mortality of diamondback moth (P. xylostella) larvae

Table 4.3. Mortality of 1 -instar C. carnea after 24 h when exposed

to crude aqueous seed extracts of A. squamosa in a direct spray test ... 92

Table 4.4. Cumulative mortality of 1 -instar C. carnea exposed to crude

aqueous seed extracts of A. squamosa in a residual contact test 93

Table 4 . 5 . . Toxicity of crude aqueous seed extracts of A. squamosa

Table 4.6. Mortality of adult T. brassicae after 24 ht when exposed to

Table 5.1. Comparison of costs of using synthetic insecticide with

Table 5.2. Comparison of costs of using synthetic insecticide with

Figure 1.1. Chemical structures of promising botanical insecticides 12

Figure 1.2. Chemical structures of Annonaceous acetogenins with

promising insecticidal properties 21

Figure 1.3. Tree, fruits and seeds of A., muricata 28

Figure 1.4. Tree, fruits and seeds of A. squamosa 30

Figure 2.1. Maps of Indonesia and Ambon 39