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Development of a botanical insecticide from Ambon and surrounding areas (Indonesia) for local use Leatemia, Johanna Audrey 2003

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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.So , University of Guelph, 1994 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQIUREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Faculty of Agricultural Sciences We accept this thesis as conforming to the required standards THE UNIVERSITY OF BRITISH COLUMBIA 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. p f a ^ bteiu faculty c\ N Department of 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 FIGURES ix A C K N O W L E D G E M E N T S 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 3.1. Introduction 55 3.2. Materials and Methods 58 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 3.2.8. Data Analysis 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 4.1. Introduction 801 4.2. Materials and Methods 82 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 4.2.3. Data Analysis 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 5.1. Introduction 102 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 Table 2.2. Growth inhibitory effect of crude ethanolic seed extracts of A. muricata (soursop) from different locations and years of collection on neonate S. litura (5000 ppm = 0.5% fwt, n = 20) 47 Table 2.3. Growth inhibitory effect of crude ethanolic seed extracts of L. domesticum from different locations on neonate S. litura (5000 ppm = 0.5% fwt, n = 20) 49 Table 2.4. Growth inhibitory effect of crude ethanolic seed extracts 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 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 67 Table 3.4. Efficacy of crude aqueous seed extracts of A. squamosa 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 1st-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 1st-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 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 r d and 4 t h -instar P. xylostella on leaf dip bioassay with aqueous extracts of A. squamosa 69 Figure 3.3. Feeding deterrency of crude aqueous seed extracts of A.squamosa on 4 t h - instar P. xylostella overtime in the leaf disc choice test .... 70 Figure 3.4. Residual effect of aqueous seed extracts of A. squamosa on 3 rd-instar P. xylostella 71 ix Figure 3.5. Residual effect of aqueous emulsions of ethanolic seed extracts of A. squamosa on 3 rd-instar P. xylostella 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 LD5o of 12.5 mg/kg) but commercial sabadilla contains only 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 (CH2)4-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 LD 5 0 of this compound in larvae of the diamondback moth, Plutella xylostella was found to be 20 ug/cm2, but one-tenth of 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 As 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 s t - and 2n d-instars; 50, 100, 250, 500, 750 ppm for 3 rd-instar and 250, 500, 750, 1000, 2000 ppm for 4 t h - and 5 th-instar larvae. Control diets were treted with carrier solvent (ethanol) alone. Bioassay was performed with neonate larvae as described above. Bioassays with other larval instars (2 n d, 3 r d , 4 t h and 5 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 5th/early 6 t h instars (this is the stage reached after 10 days, starting as neonate larvae). For 2 n d instars, this equated to 7 days, 3 r d instar 6 days, 4 t h instars 4 days and 5 t h instars 3 days. Insects were weighed after this time and larval weights compare to larvae fed on control diets. The E C 5 0 (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 ± SE 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). Location Larval growth (% relative to control) (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 Significant Difference (LSD) test. 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) Location Larval growth (% relative to control) (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 *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) 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) Location Larval growth (% relative to control) (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 *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) 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 5 0s of 192 and 202 ppm, respectively). The 3 r d and 4 t h instars were much less sensitive (EC50S of 533 and 705 ppm, respectively) and the 5 t h instar was relatively insensitive (EC 5 0 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 s t 191.7 0.87 202.0 0.94 3rd 533.1 0.98 4 t h 704.9 0.70 5 t h 1707.5 0.94 degression lines were calculated from five points, n=20 for each point. b EC 5 0 = effective concentration to reduce larval growth by 50% relative to the control 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 r d and 4 t h instars were much less sensitive and the 5 t h 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 th-instar larvae of the diamondback moth obtained from the 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 rd-instar or five 4 th-instar larvae were 60 placed in each cup. Five and 10 replicates were used for each treatment for 3 r d and 4 t h 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 (pooled) Drench T. n/(1st) P. xylostella (1st) (2nd) 1 - 1 6 0.125 -2 0 .25 -4 Leaf-dip P. xylostella (3rd) (4th) 0 .25 -4 0.625- 10 All (pooled) Drench T.ni (1st) P. xylostella (1 s) (2nd) 1.25-20 0.0625 - 1 0 .125-2 Leaf-dip P. xylostella (3rd) (4th) 1.25-20 2 .5 , -40 AS All (pooled) Drench T.n/(1st) P. xylostella (1st) 2 n d) 0 .05-0.8 0.00625-0.1 0.0125-2 Leaf-dip P. xylostella (3rd) (4th) 0.0625 - 1 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 th-instar 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 9 co 5 o > —, J5 != to _ •— o « •o oo © = -5 2 © « 2 0 . 2 * 5 CO £ 3 =5 8 z § 5 f U 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 © u o c J= co ° -3 U CO CO 3 '•5 o" •s © u © CO -J © • CO © © «-!= T3 .SP =• u- O CD £ oi ** *-c • T3 CD © "D © O © J= © © >-© » © 5 > o « .© "Z "° « JC 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 rd-instar P. 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 rd-instar larvae were placed in each cup and five 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 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). LC 5 0 (lethal concentration causing 50% mortality) in drench and leaf dip bioassays, was calculated using probit analysis (Finney, 1971). Estimated LC 5 0 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 2n d-instar larvae when tested 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 5 0s of 0.02 and 0.03%) (Table 3.5) However, 2n d-instar larvae were significantly (p<0.05) 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). Table 3.2. Efficacy of crude aqueous seed extracts of A. squamosa from Namlea ('96, '98 and '99) in drench bioassays Insect (larval instar) LC50 (95 % CI)* Control mortality ± SE (% w/v) (%) T.n/(1st) 6.64 (4.09- 10.78 5.63 1.42 P. xylostella (1st) 0.53 (0.33-0.84) 12.38 ±2.57 (2nd) 0.97 (0.75- 1.26) 13.60 + 2.23 * LC 5 0 values differ significantly where 95% Cis do not overlap 66 3.3.2. Leaf dip bioassay Leaf dip bioassays showed that mortality of 3 r d and 4 th-instar P. xylostella fed 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 5o between the 3 r d and 4 t h instars (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 5 0s of 12.96 and 35.20) (Table 3.4). However, LC 5 0 values of 4 th-instar larvae at 48 and 72 h were significantly (p<0.05) higher than those of 3 rd-instar (Table 3.3). 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 LC5o (95 % CI)* Control Mortality ± SE time (hours) (% w/v) (%) 3 r d 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 4m 24 h 8.65 (6.64- 11.27) 0 48 h 4.20 (3.49 - 5.07) 1.25 ± 1.25 72 h 2.03 (1.72-2.40) 5.00 ± 2.04 * LC 5 0 values differ significantly where 95% Cis do not overlap 67 Fourth instar larvae P. xylsotella was significantly (p<0.05) less susceptible than 3 r d -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 5o (95 % CI)* Control Mortality ± SE (larval instar) (% w/v) ' (%) r.A?/(1st) Drench 5.19 (4.26-6.31) 2.00 ±0 .88 P. xylostella (1st) Drench 0.18(0.14-0.25) 9.17 ±1.99 (2nd) Drench 0.57 (0.45-0.73) 5.00 ±1.58 (3rd) Leaf-dip 12.96(7.71-21.80) 5.00±2.24 (4th) Leaf-dip 35.20(16.7-73.79) 2.50 ±1.06 * LC 5 0 values differ significantly where 95% Cis do not overlap 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 5 0 (95% CI)* Control Mortality ± SE (larval instar) (% w/v) (%) T.n/'(1st) Drench 0.27 (0.19-0.38) 3.85 ± 1.25 P. xylostella (1st) Drench 0.02 (0.01 - 0.03) 11.25 ± 2.51 (2nd) Drench 0.03(0.02-0.03) 5.33 ±1.42 (3rd) Leaf-dip 0.39(0.33-0.46) 3.00 ±1.53 (4th) Leaf-dip 0.67(0.57-0.78) ' 1.00 ±1.00 * LC 5 0 values differ significantly where 95% Cis do not overlap 68 Figure 3.2. Mortality of 3rd and 4th-instar P. xylostella 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 4th- 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 rd-instar P. xylostella. There were no significant differences in the 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 Concentration (% w/v) 30 Figure 3.4. Residual effect of aqueous seed extracts of A. squamosa on 3rd-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 rd-instar larvae (Figure 3.5). However, when larvae fed on leaf discs 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 3rd-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. 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 th-instar P. xylostella. This 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 r d and 4 th-instar larvae (Table 3.3), suggesting slow-acting toxicity of the extracts. A 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 th-instar larvae of the Colorado potato beetle, Leptinotarsa decemlineata. The highest mortality of the beetles was observed after 7 days. I obtained LC 5 0 s for aqueous seed extracts of A. squamosa on 3 rd-instar P. 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 5 0 s for aqueous seed extracts of A. squamosa on 3 rd-instar C. binotalis at 2 and 3 d of 0.29 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 2n d-instar DBM of 0.17 - 2.56%. They concluded that 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 2n d-instar DBM were 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 5 0 s of these metabolites against 3 rd-instar DBM of 3 ppm using a leaf dip assay and 150 - 980 ppm in a topical assay. I obtained LC 5 0 values for aqueous emulsions of ethanolic extracts of 0.02-0.67% on neonate to 4 t h-instar DBM (Table 3.5). These values are much lower 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/m3 mixture). Plants were 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 CN o o GO LO CO CM o 00 co t E 1 E CM CO O o CO E o LO o o CO LO E o o LO LO CN 'E o o E LO c t/5 o _ ^ : C O o o . Q • o ° d) CO N ® E 2> O <l> = 1 _ CD re ^ c u — c re g> CO O) re c d> E |5 re a) IS (A (0 c c a) re | CL 8 w Q. X C o> E S * o £ c </> 22 re o o - g ^ ° 9 c - a > ro CN _ l O • U ^ O H <* S 3 £ .2 = •=2 B ~ o u . 5 o 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 3rd-instar larvae. A pre-spraying count was made one day after infestation and 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 o 3 O « co .2 JZ CJ) CO CO CD CO o £ E "TT CO ° 3 O SI 2 CD * C i CD CO o •= c 'co CD O OO C 03 co ^ CD" -CO O E 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 1st-instar 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 1st-instar larvae (< 24 h-old) were placed inside each vial and 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 o f A. squamosa to adult 7. brassicae. Glass vials (7 ml) were coated with 50 ul of 0, 5, 7.5, o r 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) f o r 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 DAT3 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 1st-instar C. carnea after 24 h when exposed to crude aqueous seed extracts of A. squamosa in a direct spray test Concentration (%w/v) Corrected mortality (%) ± SE 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 1st-instar C. carnea exposed to crude 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 (day) LC 5 0 (95% CI) (%w/v) Control mortality ± SE (%) 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 *LC5 0 values differ significantly where 95% Cis do not overlap 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 (% w/v) 1 3 7 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. a Control 1 = water + 0.05% powdered laundry detergent.b Control 2 = water 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 5 0 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. 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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 1 1 1 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. 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