EFFECTS OF ESSENTIAL OILS OF Lantana camara AND TWO OCIMUM SPECIES ON BEAN WEEVIL (Acanthoscelides obtectus) AND THEIR CHEMICAL COMPOSITIONS

Transcription

1 EFFECTS OF ESSENTIAL OILS OF Lantana camara AND TWO OCIMUM SPECIES ON BEAN WEEVIL (Acanthoscelides obtectus) AND THEIR CHEMICAL COMPOSITIONS BY MUINDE ESTHER SYOMBUA REG. NO. I56/22556/2011 A thesis submitted in partial fulfillment for the requirements for the award of the Degree of Master of Science in Chemistry in the School of Pure and Applied Sciences, Kenyatta University APRIL 2015

2 DECLARATION I hereby declare that this thesis is my original work and has not been presented in any other institution for the award of a degree or any other award. MUINDE ESTHER SYOMBUA CHEMISTRY DEPARTMENT KENYATTA UNIVERSITY Signature.. Date.... We confirm that the work reported in this thesis was carried out by the candidate under our supervision PROFESSOR CAROLINE THORUWA CHEMISTRY DEPARTMENT KENYATTA UNIVERSITY Signature Date PROFESSOR AHMED HASSANALI CHEMISTRY DEPARTMENT KENYATTA UNIVERSITY Signature Date. ii

3 DEDICATION This research work is lovingly dedicated to my husband Benjamin and my children Joseph and Faith for their encouragement, moral and spiritual support as I travelled through the research journey. iii

4 ACKNOWLEDGEMENTS I thank my research advisors Prof. Caroline Thoruwa and Prof. Ahmed Hassanali for their tireless support, guidance and for being instrumental in molding me into a keen researcher. I am highly indebted to research advisors at ICIPE Dr. Wilber Lwande and John Bwire for the opportunities they gave me to gain practical laboratory skills, handling essential equipment and critical laboratory procedures. I would also thank my research colleagues, Alex and Davis for their support and encouraging words during difficult moments and precious research suggestions they offered. I would also thank my husband Benjamin Manyala and our children Joseph and Faith for their loving concern and encouragement I am grateful to Dennis Osoro for assisting me to analyse my data. I appreciate the support of my employer, Teachers Service Commission, for granting me leave to attend to my postgraduate studies. Finally, I thank the National Commission for Science Technology and Innovation (NACOSTI) for the financial support to conduct this research. God bless you abundantly. iv

5 TABLE OF CONTENTS Title page......i DECLARATION... ii DEDICATION... iii ACKNOWLEDGEMENTS iv TABLE OF CONTENTS.v LIST OF TABLES...ix LIST OF FIGURES...x LIST OF PLATES...xi ABBREVIATIONS AND ACRONYMS.. xii ABSTRACT...xiii CHAPTER ONE Introduction Background to the study Statement of the Problem Justification of the study General Hypotheses General objective Specific objectives Scope and limitation of the study Significance of the study Conceptual Framework...5 v

6 CHAPTER TWO Literature Review Bean weevils Callosobruchus maculatus Life cycle Habitat Behaviour Acanthoscelides obtectus Life cycle Economic importance of Acanthoscelides obtectus Sources of infestation Control measures Ocimum gratissimum L Chemical constituents of essential oil of Ocimum gratissimum Ocimum americanum L Chemical constituents of essential oil of Ocimum americanum Lantana camara L Chemical constituents of essential oil of Lantana camara.. 17 CHAPTER THREE Methodology Location of the study Mass rearing of bean weevils Plant materials...18 vi

7 3.4 Cleaning of Glassware Extraction of the oils Chemical identification Essential oils composition Toxicity bioassay with ground powder Repellency Bioassay Choice Bioassay Toxicity test of essential oils Adult mortality tests Subtractive bioassays Toxicity of individual constituents of essential oils and their blends on A. obtectus Data analyses...23 CHAPTER FOUR Results and discussion Introduction Chemical identification of oils Composition of essential oil of Ocimum americanum (Kitui) Composition of essential oil of Ocimum americanum (Machakos) Composition of essential oil of Ocimum gratissimum (Kitui) Composition of essential oil of Ocimum gratissimum (Machakos) Composition of essential oil of Lantana camara (Kitui) Composition of essential oil of Lantana camara (Machakos) Effect of powdered materials of the plant on mortality of A. obtectus...31 vii

8 4.2 Repellency bioassays Mortality bioassays Toxicity of individual constituents of the three essential oils and their blends.40 CHAPTER FIVE Conclusions and Recommendations Conclusions Recommendations Recommendations from the study Recommendations for the study.. 48 REFERENCES...50 APPENDICES...54 viii

9 LIST OF TABLES Table 1: Effects of powdered plant materials from Kitui region on the mortality of A. obtectus Table 2: Effects of powdered plant materials from Machakos region on the mortality of A. obtectus Table 3: Percentage repellency of A. obtectus adults exposed to essential oils of the three plants from Kitui region Table 4: Percentage repellency of A. obtectus adults exposed to essential oils of the three plants from Machakos region Table 5: Percentage mortality of A. obtectus caused by the essential oils from O. americanum, O. gratissimum and L. camara from Kitui region Table 6: Percentage mortality of A. obtectus caused by the essential oils from O. americanum, O. gratissimum and L. camara from Machakos region. 39 Table 7: Percentage mortality of A. obtectus adults exposed to O. americanum (Kitui) constituents, individually and in blends equivalent to the relative proportion of each compound in 100% lethal dose of the essential oil Table 8: Percentage mortality of A. obtectus adults exposed to L. camara (Kitui) constituents, individually and in blends equivalent to the relative proportion of each compound in 58% lethal dose of the essential oil Table 9: Percentage mortality of A. obtectus adults exposed to O. gratissimum (Kitui) constituents, individually and in blends equivalent to the relative proportion of each compound in 59% lethal dose of the essential oil ix

10 LIST OF FIGURES Figure 1: GC-MS chromatogram of O. americanum (Kitui) essential oil Figure 2: GC-MS chromatogram of O. americanum (Machakos) essential oil Figure 3: GC-MS chromatogram of O. gratissimum (Kitui) essential oil Figure 4: GC-MS chromatogram of O. gratissimum (Machakos) essential oil Figure 5: GC-MS chromatogram of L. camara (Kitui) essential oil Figure 6: GC-MS chromatogram of L. camara (Machakos) essential oil Figure 7: Percentage repellency of A. obtectus exposed to essential oils from three plants from Kitui region Figure 8: Percentage repellency of A. obtectus exposed to essential oils from three plants from Machakos region Figure 9: Percentage mortality of A. obtectus caused by essential oils of O. americanum, O. gratissimum and L. camara from Kitui region Figure 10: Percentage mortality of A. obtectus caused by essential oils of O. americanum, O. gratissimum and L. camara from Machakos region x

11 LIST OF PLATES Plate 1: Callosobruchus maculatus.. 6 Plate 2: Acanthoscelides obtectus....9 Plate 3: Ocimum gratissimum 13 Plate 4: Ocimum americanum...15 Plate 5: Lantana camara 16 Plate 6: Clevenger apparatus.. 19 Plate 7: Choice bioassay system...21 Plate 8: Mortality bioassay xi

12 ABBREVIATIONS AND ACRONYMS ANOVA EI FAO GC GC- MS ICIPE LD N C N T PR RD SE SNK WPI Analysis of Variance Electron Impact Food and Agricultural Organization Gas Chromatography Gas Chromatography-Mass Spectrometry International Centre of Insect Physiology and Ecology Lethal Dose Number of insect in the control jar Number of insect in the treated jar Percentage Repellency Repellency Dose Standard Error Student-Newman-Keuls Test Weevil Perforation Index xii

13 ABSTRACT Crude plant products have been widely used by different communities in Africa for pest control. Synthetic insecticides have a number of drawbacks, such as development of genetic resistance, eco-toxicity, negative health effects to users and high cost of application. However, not enough scientific research has been conducted on the efficacy of traditional methods in weevil control, the chemical profiles of plant products and their safety to the users. The bioactivities of the powedered products and essential oils of three plants, namely Ocimum gratissimum, Ocimum americanum and Lantana camara, growing in Eastern Kenya against common bean weevil (Acanthoscelides obtectus) were determined. Actellic Super and untreated bean grains were used as positive and negative control, respectively. The powdered plant materials (dried leaves) induced relatively low mortality, with the most effective O. americanum (Kitui) causing 52% mortality at dose level of 30% w/w after 28 days. The essential oils of all the three plants were found to have significant repellency to the bruchid. The most repellent essential oil was that of L. camara (Kitui) (RD µl per cm 2 ), followed by O. gratissimum (Kitui) (RD µl per cm 2 ), then O. americanum (Kitui) (RD µl per cm 2 ). However, the repellencies of the essential oils of the same species of plants from Machakos region were lower [L. camara (Machakos) RD µl per cm 2 ; O. americanum (Machakos) RD µl per cm 2 and O. gratissimum (Machakos) RD µl per cm 2 ]. The essential oils of the three plants also showed significant mortality, with LD 50 of that of O. americanum (Kitui) being 5.28µl per cm 2, that of O. gratissimum (Kitui) being 5.39 µl per cm 2 and that of L. camara (Kitui), µl per cm 2. The essential oils of the plants from Machakos region gave the following mortalities: O. americanum (Machakos) LD µl per cm 2, O. gratissimum (Machakos) LD µl per cm 2 and L. camara (Machakos) LD µl per cm 2. The oil from O. americanum (Kitui) gave the best mortality. The essential oils were analysed by gas chromatography (GC) and gas chromatography-linked mass spectrometry (GC-MS) and the major constituent compounds identified by comparison with MS database and GC co-injections with authentic samples. The relative amounts of different constituents of oils of the plants from the two regions varied sharply. Subtractive bioassays were carried out to characterize the constituents in the essential oil blends responsible for mortality. The most effective essential oil was O. americanum whose constituent compounds were 1,8-cineole with mortality of 51%, α-terpineol 39% and lastly linalool 28%. The results provide a baseline for the use of O. americanum and L. camara essential oils as bio-organic post-harvest pest control agents. xiii

14 CHAPTER ONE INTRODUCTION 1.1 Background to the study In most developing countries, small scale bean production represents a significant part of rural economies. Small scale farmers are faced by serious post-harvest problems from bruchids especially in storage of grains. Research has shown that development stages of bruchid (A. obtectus) under field and laboratory conditions include: incubation period, duration of the evolution of larvae stage, pupae duration and longevity of new adults. The whole lifecycle takes optimum 27 days at 30 C and 80% humidity (Koridinium, 2009). Eggs are laid in cracks in seed coat, larvae bores into the seed making translucent window in seed before pupation. Larvae cause damage by eating the inner side of the bean, and this makes the beans useless for consumption and inappropriate for sowing (Maldonado et al., 1996). The adult bruchid, then emerges through the window leaving a neat round hole. When beans are attacked by bruchids they become less marketable and this causes economic loss to the producers and quality loss to the consumers (Upadhyay et al., 2011). As a result of these problems, many small scale farmers prefer to sell most of their crop immediately after harvesting to avoid making losses from the infestation of the bruchids. Farmers who want to keep their produce from bruchids infestation result to the use of synthetic pesticides if they can afford them. T h e misuse of these chemicals frequently occurs since majority of small scale farmers in many African countries are illiterate. The chemicals contaminate stored food commodity, leaving behind harmful residue, especially when 1

15 application dosages are not properly followed (Maribet and Aurea, 2008). Synthetic pesticides are not only expensive but may also have harmful effects in the health of the consumers. Moreover, some pests have developed some resistance to some of the synthetic pesticides; hence, it is necessary to find alternative natural solutions to the problem of bruchids in beans. This project investigated hydro-distillates (essential oils) from selected Ocimum species and Lantana camara growing in the eastern region of Kenya that may be used as natural protection against the common bean weevil Acanthoscelides obtectus. 1.2 Statement of the problem Previous observations have shown that use of some synthetic insecticides has serious drawbacks such as development of genetic resistance, toxic residues, worker safety and increasing cost of application (Jembere et al., 1995). Because of these problems there is a need to search for safer bio-organic post-harvest pest control agents. Traditionally, farmers in Machakos and Kitui counties have been protecting grains from pests by mixing them with powdered plant materials including those of Lantana camara, Ocimum gratissimum and Ocimum americanum. However, limited scientific studies have been undertaken to establish their efficacy, identify the active constituents, and establish the dosage for effective protection. Beans being a major source of proteins affordable by most of the people in the rural and urban areas need to be protected since t h e y are damaged by bruchids. Due to this risk most farmers do not grow large quantities of beans, and if they do, they sell them immediately after 2

16 harvesting to avoid post-harvest storage losses (Ishimoto & Chrispeels, 1996). Bio-organic insect control agents will enable farmers to keep beans safe for consumption for long and this will ensure food security. They also help the farmers to store the beans and sell when the market prices are good. 1.3 Justification of the study Controlling stored grains from pests using synthetic pesticides is not demanding, since it requires high skills to mix with the two in the right proportion, and is also very expensive. The chemicals need to be handled with great care since they are toxic to the user. Once the grains have been treated they need to be left for particular duration before they are safe for consumption. The synthetic pesticides are not environmentally friendly because they kill untargeted organisms. Therefore, cheap and locally available pest control agent is required for both small scale and large scale farmers to keep their grains safe from post-harvest pests. Plant extracts and powders have gained popular ground in the world of traditional agriculture as an alternative to synthetic insecticides. Not enough scientific research has been conducted on the efficacy, the chemical profiles and safety of O. gratissimum, O. americanum and L. camara plant products used in Kitui and Machakos counties. 1.4 Hypotheses i. Products derived from O. gratissimum L, O. americanum L. and L. camara L. have deleterious effects on the bean post-harvest pest A. obtectus. ii. The deleterious effects are largely due to specific blends of the essential oils of the plants. 3

17 51.5 General objective The purpose of this study is to investigate the effectiveness of two Ocimum species (O. americanum and O. gratissimum) and L. camara essential oils against a bruchid (A. obtectus) in beans Specific objectives i. To determine the repellence and toxicity of O. gratissimum L, O. americanum L. and L. camara L. essential oils and their powders against A. obtectus in beans. ii. To determine the major constituents present in the two Ocimum species and L. camara from different geographical locations using GC-MS and GC co-injections with authentic standards. iii. To determine the compounds responsible for bioactivities of the essential oils using subtraction bioassays. iv. To determine the dosage range of the essential oils required for effective protection against the common bean weevil. 1.7 Scope and limitation of the study The study was limited to two Ocimum species (O. gratissimum and O. americanum) and L. camara found growing in Kitui and Machakos counties in eastern region of Kenya. The plants were collected at their floral stage. Further, the study was also limited to A. obtectus bruchid species in kidney beans (Phaseolus vulgaris). 1.8 Significance of the study The goal of the study was to come up with bio-organic pest control agents which can be used to protect beans from post-harvest weevils. The farmers from these regions and beyond will be able 4

18 to use Ocimum and L. camara essential oils to fight against bruchids in beans. This will help increase food security and promote good health to the local communities. In addition, the usefulness of these plants growing in the area will be appreciated and this may enhance their conservation. 1.9 Conceptual framework The conceptual framework of this research was to evaluate natural plant products as protectants against post harvest weevils. Crude Ocimum and L. camara plant products have been used traditionally as protectants against weevils in maize (Sitophilus zeamais). However, their efficacy was found to be limited. Previously, the essential oils of some plants have been found to have repellence and toxic effect on the maize weevils (Jembere et al., 1995). The same concept has been applied in this study to test the efficacy of O. americanum, O. gratissimum and L. camara essential oils on A. obtectus in beans 5

19 CHAPTER TWO LITERATURE REVIEW 2.1 Bean weevils There are two common bean weevils, namely Callosobruchus maculatus and Acanthoscelides obtectus Callosobruchus maculatus Plate 1: Callosobruchus maculatus (http//en.wikipedia.org/wiki/file:callosobruchus_maculatus.jpg) It is commonly known as cowpeas weevil. It is not a true weevil but instead a species of bruchid beetle of family Chrysomelidae. C. maculatus lacks distinctive snout of weevils. It can be distinguished from the other bruchid beetles by its more elongated body and red-brown coloration as an adult. The beetle elytra is short in comparison to the rest of its body and ends leaving the last segment of the abdomen exposed. The average body length of an adult beetle is 4-6mm and average body mass is 4-6mg with female beetles generally slightly larger than the males. The beetles are sexually dimorphic, i.e. their sex can be easily determined by the naked 6

20 eye. Males tend to be smaller and possess a more rotund shape than the females. Females have dark stripes on each side of the dorsal abdomen. The eggs of C. maculatus are translucent while the larvae are yet to hatch and are 0.6mm length. The eggs are small, flat and off white once the larvae have hatched from the eggs. The larva of C. maculatus is curved white and has a small head (Fox & Cope, 2003) Life cycle A female C. maculatus beetle can lay over a hundred eggs during their adult life and most of them hatch. The eggs are laid on the surface of the bean (the type of the bean is variable) where they remain. The larvae hatch out of the egg and barrow straight into the bean about 4-8 days after oviposition. During maturation the larva chew near the surface of the bean leaving behind a window-like layer in the seed coat. The larva emerges from the window after days from the first barrowing into the bean. About 8-10 larvae can develop within the same bean. The larval crowding limits the amount of resources available to each individual. This results in longer development time, high larval mortality and smaller adult size. Once adult beetle emerge from the bean they mature within 36 hours. The adult have a mean lifespan of 7 days in the laboratory environment, but some are able to live up to 14 days. The adult do not need to feed but spend their time as adult mating and ovipositing (egg laying) (Fox, 1993). Callosobruchus maculatus can live in a range of humidity level and temperature which has made its invasion of multiple continents successful, but there are optimal conditions. Variation in development time can be affected by factors like humidity, environment temperature, type of legume, number of larva in the same bean and amount of inbreeding experienced by the family. A bean that is too dry will be difficult for the larva to bore into it and a bean that is too wet will have too much fungal growth for the larva to survive. A humidity range of 25-80% is acceptable for development but 7

21 each life stage has a different optimal humidity level. The most eggs hatch at 44% and 63% humidity. Adults live longer at higher levels of 80-90% humidity (Reed and Fox, 2010). The age of the mother affects the development and survival of the offspring Habitat C. maculatus lay eggs directly on the seed of the legume. This is done on nearly mature pod of the preferred legume and the next generation emerges after the harvest or complete maturation of the legume. C. maculatus infests a wide range of legume but in general it prefers members of Virgina genus (Fox and Cope, 2003) Behavior C. maculatus have fully functional wings. Both sedentary morph and dispersal morph can fly but sedentary tend to walk instead of flying when moving unless threatened. Females prefer to lay eggs on the smooth cheekˮ of the bean. They avoid wrinkled top of the bean and avoid legumes that do not have smooth surfaces. They have some unknown method to determine the mass of the bean they encounter. When the beetles are presented with a combination of bean sizes (small and large), they distribute their eggs so that each larva has access to roughly the same amount of nutrients. A single larva of C. maculatus diminishes the weight of cowpeas by 3% and multiple larvae will infest one cowpea in most storage infestation (Reed and Fox, 2010). 8

22 2.1.2 Acanthoscelides obtectus Plate 2: Acanthoscelides obtectus: (http// The weevil belongs to family bruchidae, genus acanthoscelides. The beetle body is ovoid, slightly convex, light and dark brown with yellow-green golden hairs and longitudinal spots from above. Its length varies from 2 to 5mm. Dark brown short elytra do not cover abdominal end. The insect has clavate antenna and yellow red legs (Karapetyan, 1983). Oblong egg is white, about mm in length. Young cylindrical larva has bristles and three pairs of legs. White pupa is about 4mm in length. Overwintering of the bean weevil takes place at the stages of beetle or larva usually in store houses. The beetles appear at the temperature 12.5ºC, but they are inactive at temperature below 16ºC (Darka et al., 2009; Berim, 2009) Life cycle In some days after coupling, female lay eggs into ripening pods by groups (5-20), piercing the pod sutures or growing holes. Fecundity of one female reaches 200 eggs. The eggs develop 9

23 during days. Larvae penetrate into the grain and eat the grain content completely. Several larvae can develop in one grain. Larval period lasts weeks. Pupation also takes place in the grain 9-29 days. Life cycle of one generation takes days. The pest arrives in the store houses with grain where it develops (Sapunaru, 2006). The analysis showed that the bean pest causes great damages during the grain storing. A. obtectus affects all varieties of beans and most affected ones are kidney beans, chickpea and soya bean (Fox, 1993). 2.2 Economic importance of Acanthoscelides obtectus After harvest, farmers store their products to have supply throughout the year, and sell when prices of products increase for high profit. During storage the grains are liable to pest infestation leading to losses. FAO estimates worldwide annual losses in stored produce to be up to 10% of all stored grains (Solomon & Evans, 2008). About 35% of crops all over the world are destroyed by insect pests (Shan, 2000). It is reported that 60-70% of all grains produced in the tropics are stored at farm level by small scale farmers (Golop et al., 2009). The bean weevil (A. obtectus) has a field generation and 2-3 generations in store houses where it finds a proper temperature for its development. It hibernates at the adult stage in shivered grains which remain in the field after harvesting and 90% in stored grain (Sapunaru, 2006). In a bean grain 1-28 larvae develop eating the entire grain content. The weevil infests bean crop during stage of floral bottom formation. Egg laying time coincides with pod formation. The bean weevil causes significant damage to bean. Larva usually eats the pod content completely decreasing the yield by 50-60%. Partially damaged grains lose their germinating power and taste quality. For controlling bean weevil from store houses it is recommended to treat the grain before storage. In the field treatments should be done during flowering (Odagiu and Porca, 1996). 10

24 2.3 Sources of infestation Beans are harvested twice in a year in Machakos and Kitui regions. Those grown in October November of previous year are harvested February-March of the following year and those grown in March-April are harvested August September. By the end of the year the farmer has bulk of food products to store. Proper storage management of harvested cereals is required to ensure quality of stored products. 2.4 Control measures Chemical substances have been used as repellents to protect the grain weevils. Repellents like N,N-diethyl-m-toluamide (DEET) and Actellic Super are widely used but have negative health effects like swelling, itching and eye irritation, brain swelling in children, hypotension and even death (Chris and Coats, 2001). Due to these problems, safer pest control agents are required to replace these synthetic pesticides. Additional methods of protection against crop pest and diseases make use of plants products. This may be partly because of the high cost of most synthetic protective pesticides which makes farmers unable to afford them for use against crop pests. Small-scale farmers use plant materials for post-harvest pest control because they are easily available and not as expensive as synthetic pesticides. The bulk of African grain production comes from small scale farm holdings and the traditional farmers use different kinds of plants for pest control. The protection of stored grains involves mixing them with protectants made from plant materials. In Kitui and Machakos counties most small scale farmers use powdered plant materials of L. camara and O. americanum and O. gratissimum as protectants against post-harvest pest. 11

25 In a research by Jembere and others, the efficacy of Ocimum kilimandscharicum for protection against weevils was determined (Jambere et al., 1995). Thus, small-scale farmers can make use of available natural based solutions. Their research was limited to maize and sorghum. Extracted phytochemicals of O. kilimandscharicum and Ocimum kenyense were e v a l u a t e d for pest and microbial control. This targeted Sitophilus zeamais and Rhyzopertha dominica weevils but did not address bruchids in bean (Bekele and Hassanali, 2001). Vernonia amygdalina essential oil has been reported to be potential protectant against maize weevil (Aswalam and Hassanali, 2006). Artemisia annua has been tested for use against Callosobruchus maculatus in cowpeas. Other plants like Azadirachta indica and O. gratissimum have been tested against the same pest (Keita et al., 2002). The control set up was use of convention Actellic Super 2% dust. The research reported that irrespective of the concentrations tested all three plant materials significantly (P < 0.05) increased mortality rate of adult insects earlier than the control. Higher concentrations of the botanical pesticides equally resulted in an increased reduction in the number of surviving bruchids and reduction to seed damage through a lower number of eggs laid and weevil perforation index (WPI) after 90 days (Brisbe et al., 2011). Artemisia eriopoda has been reported to have some insecticidal activities against the maize weevil, Sitophilus zeamais. The main components of the essential oil were germacrene and eucalyptol which were responsible for contact toxicity (Jiang et al., 2009). Essential oils, derived from the leaves of O. gratissimum against Sitophilus zeamais were found to be moderately repellent to the maize weevil and induced high mortality in the weevils (Asawalam et al., 2008). Ocimum suave and Eugenia caryophyllata have been reported to be good weevil repellents. The two have eugenol as the major constituent compound and is a potential repellent of maize weevils (Hassanali et al., 1990). Further, these researchers 12

26 observed that gas chromatography-linked mass spectrometry (GC-MS) and GC co-injections with authentic samples showed the following major constituents: thymol (32.7%), paracymene (25.4%), γ-terpinene (10.8%), β-selinene (4.5%), phellandrene (3.9%) and β-myrcene (3.1%) (Asawalam et al., 2008). The outcome provided a scientific rationale for the use of plant in postharvest protection. The above outline reveals that although oils from different natural products have been evaluated for repellency and insecticidal activities, little has been done to test the activities of O. gratissimum, O. americanum and L. camara against the common bean weevil, A. obtectus. Therefore, this study will establish the repellency and insecticidal activities of the essential oils of O. gratissimum, O. americanum and L. camara by studying the major constituents in order to find out their suitability as post-harvest protection against the common bean weevil, A. obtectus. 2.5 Ocimum gratissimum L Plate 3: Ocimum gratissimum (http//en.wikipedia.org/wiki/binomial_nomenclature) 13

27 Familiar name is holy Basil. It is an aromatic herb and shrub indigenous to the tropical region. It is wildly cultivated in India. It has been used as medicine and well known sacred plant to the Indian sub-continent (Joshi, 2009). Leading phytochemical compounds are eugenol (volatile oil) which has antifugal activity, ursolic acid (triterphenoid) and rosmarinic acid (phenylpropanoid). Other active compounds are caryophyllene and oleanolic acid (Mbata, 2000). It has nutritional components like vitamin A and C, minerals like calcium, iron and zinc (Maimes, 2004). In medicine it has been used in treatments of disorders in cardiovascular-circulatory system, digestive system (esophagus, stomach, intestines, liver and pancreases), endocrine system immune system, skin system, nervous system, reproductive system and excretory system (Maimes, 2004). The recommended dosage is mg of dried leaves daily for preventive therapy and mg for corrective therapy. The essential oils have insecticidal activity (Keita et al., 2002) Chemical constituents of essential oil of O. gratissimum The major constituents of essential oil of O. gratissimum identified by GC-MS analysis are: eugenol, linalool, limonene, methyl eugenol, β-caryophyllene, farnesene, α-terpineol, β salinene, methyl isoeugeneol, geraniol, α-copaene, bisabolol, α-pinene, p-cymene, fenchone, cubenene, camphene, T-cadinol, ϒ-eudesmol, sabinene, myrcene, β-bisoboline, α-humelene and β-elemene. The quantities of the constituents varied considerably, e.g. eugenol yield at am was 98% and at 5.00 pm was 11%. The results show influence of the solar light on eugenol production. This is useful in determining the optimal time for the plant collection (Prabhu et al., 2009). 14

28 2.6 Ocimum americanum L. Plate 4: Ocimum americanum ( Common names are lime, hairy, hoary basil or americanum basil in English and Zitronan basilicum in Germany. The useful parts are mostly the leaves in medicinal purpose especially intestinal worms (Sathish, 2011). O. americanum is often found growing on roadsides, in fields, in thick forests and in open waste places close to settlements (Vieira et al., 2003). Chemical analysis done in Brazil showed O. americanum contains high methyl (E)-cinnamate (90%) (Robert et al., 2000). It is also used as mosquito repellent in Lake Victoria regions (Seyoum et al., 2009) Chemical constituents of essential oil of Ocimum americanum Aerial parts yield essential oil which contains camphor and linalool as major constituents. Other constituent include citronellal, methyl cinnamate, citronellic acid, eugenol, cetronellol, methyl 15

29 heptenone, limonene, pinene, sabinene, camphene and caryophyllene (Shaikh, 2011). 2.7 Lantana camara L. Plate 5: Lantana camara L. ( The plant L. camara is also known as Spanish flag or West India Lantana. It is mainly used as an ornamental plant in most parts of the world. It has distinctive pungent odor which is similar to that of tomato greens. In Kenya it is found along footpaths, deserted fields and farms. The berries are edible when ripe. Ingestion of L. camara (including unripe berries) is not associated with significant human toxicity. In India, some house furniture such as tables and chairs are made from the stalks of the plant. The small branches are bundled together to make brooms. Methanolic extracts of L. camara leaves have been used to treat gastric ulcers. The extracts of fresh leaves are antibacterial and traditionally used in Brazil as an antipyretic, a carminative, and in the treatment of respiratory system infections (Sathish, 2011). 16

30 2.7.1 Chemical constituents of essential oil of Lantana camara Lantana camara is wide spread plant species mostly native to subtropical and tropical regions of the world. The major constituent of essential oil of L. camara identified by GC- MS analysis were α-humulene, cis-caryophyllene, germacrene-d, bicyclogermacrene, aromadendrene, and β- curcumine (Nooshin et al., 2012). The quantities and quality of the constituent compounds vary due to genetic, climate, geographical, and seasonal variations (Saikia and Sahoo, 2011). 17

31 CHAPTER THREE MATERIALS AND METHODS 3.1 Location of the study The experiments were conducted at the ICIPE laboratories. 3.2 Mass rearing of bean weevils Adult bean weevils (A. obtectus) were isolated from already infested beans. The weevils were collected from a local farmer in Machakos county and identified at International Centre of Insect Physiology and Ecology (ICIPE). Materials such as kidney beans (Phaseolus vugaris) used to culture the weevils were thoroughly cleaned and exposed in an oven at a temperature of 40 o C to ensure the absence of insect, mites or disease-causing micro-organisms (Bekele and Hassanali, 2001). The treated beans were put inside plastic containers previously washed, sterilized and dried. The bean weevils were then introduced into these containers. The plastic containers were then covered with a net fastened tightly with rubber bands. The rearing of the insect was done in the laboratory to adapt them to the laboratory condition. The rearing was given enough time until new adults insect emerged; these were then used for the experiments. 3.3 Plant materials The aerial parts (leaves) of O. americanum, O. gratissimum and L. camara were collected from Machakos and Kitui counties in Eastern province of Kenya. The plants are widely used in these regions for post-harvest protection against weevils. The collected plants were identified by plant taxonomist from National Museum Herbarium Botany Department and voucher specimen deposited there (voucher numbers NMK/BOT/CTX/1/2, NMK/BOT/CTX/2/2) and NMK/BOT/CTX/3/2. The leaves were dried under shade for one week before extracting the oils. 18

32 3.4 Cleaning of Glassware The glassware were cleaned with 6M HNO 3 acid and then rinsed with distilled water and acetone. They were dried in an oven. The glasswares for storing the extracts were rinsed with hexane and dried in an oven before use. 3.5 Extraction of the oils Plate 6: Clevenger apparatus Various quantities ( g) of leaves were subjected to steam distillation for 4 hours to obtain the essential oils. The distillation process was carried out using Clevenger apparatus. The condensing oil dissolved in hexane. The hexane solution was filtered through a filter paper containing anhydrous sodium sulphate to remove traces of moisture. Hexane was then removed by distillation at 60 C (significantly lower than boiling points of volatile monoterpenoid 19

33 constituents) the residual oil collected and weighed. 3.6 Chemical identification Essential oils composition GC-MS analysis was carried out using Hewlett Packard; HP 8060 series II Gas Chromatograph coupled to VG platform II mass spectrometer in order to identify the constituents of the essential oils. The MS was operated in the electron impact mode (EI) at 70 ev and an emission current of 200µA. The temperature of the source was held at 180 C and multiplier voltage at 300 V. The pressure of the source and MS detector were held at 9.4x10-6 mbar. The MS had a scan cycle of 1.5 seconds (scan duration of second and inter-scan delay, 0.5 seconds). The mass and scan range was set at m/z and , respectively. The standard used was heptacosaflourotributyl amine [CF 3 C(CF 2 ) 3 ] 3 N (Apollo Scientific Ltd., UK). The analysis was made in the splitless mode with helium as the carrier gas. The identification of the constituents was based on computer matching components of mass spectra data against the standard Wiley and NIST library spectra, constituted from spectra of pure substances and components of the essential oils, and literature MS data. They were confirmed by their GC retention time comparison with those of reference compounds. 3.7 Toxicity bioassay with ground powder Kidney beans (Phaseolus vulgaris) (50g) were mixed with 15g (30% w/w) or 5g (10% w/w) ground powders of L. camara, O. americanum and O. gratissimum in separate plastic jars, each in four replicates. Bean grains were also treated with Actellic Super 2% dust at 0.02% w/w as positive control (Epidi and Esther, 2008). Untreated beans were used as negative control. 20 adult A. obtectus were introduced into each of the jars. The top of each jar was covered with nylon 20

34 mesh held tightly with elastic bands. The insects were monitored every day for the first ten days, then on the 14 th day and 21 st day. The percentage mortality (%) was calculated by expressing the dead as a percentage of the total number of adult insects introduced into the jars at the start of the experiment. 3.8 Repellency bioassay Choice bioassay Plate 7: Choice bioassay system The repellence of each essential oil against the bruchids was assessed in a choice bioassay system consisting of two 1 litre glass jars connected together at their rims by means of a 30 cm x 21

35 10 cm nylon mesh tube. A 5.0 cm diameter circular hole was cut in the middle of the mesh for the introduction of the bruchids. 50g of beans were put into each of the two glass jars. The grains in one of the jars were treated with the essential oil, each time at a particular dose (2µl, 4µl, 6µl, 8µl, 10µl, 12µl, 14µl and 16µl). 20 adult A. obtectus of mixed sex and age were introduced into the nylon mesh tube through the circular hole by means of a 5 cm diameter funnel. After 1 hour the number of the insects in the control jar (Nc) and treated jar (Nt) were counted. After each test the glass jars were thoroughly cleaned and dried at 100 C. The assay for each dose of the oils was replicated five times. A control setup of untreated grains in both jars was also done. The percentage repellency (PR) values were computed using the formula: [Nc-Nt/Nc+Nt] x Toxicity test of essential oils Adult mortality tests Plate 8: Mortality bioassay These were conducted in pyrex glass vials. A sample of each essential oil was dissolved in 1 ml hexane and delivered to a Whatman filter paper in the glass vials. Appropriate dose ranges of 22

36 2µl-16µl for each essential oil (O. americanum, O. gratissimum and L. camara) was used after initial preliminary assessment to determine the dose which caused some mortality. Hexane treated filter papers were used as controls. The solvent in both treated and control Petri dishes were allowed to evaporate off (20 mins) prior to introduction of the bruchids. 20 bean bruchids were then introduced and the glass vials were kept for 24 hours in the laboratory at normal room temperature, after which the number of dead insects was counted Subtractive bioassays Toxicity of individual constituents of essential oils and their blends on A. obtectus Percentage mortality of A. obtectus was obtained by exposing them to commercially available constituents of the essential oils at their natural proportions (GC) in the oils and amounts present in the minimum lethal dose of the oils. Each component was absent (subtracted) from the blend in turn to determine its relative contribution to the overall toxicity of the natural oil (Bekele and Hassanali, 2001) Data analyses The percentage repellency of each replicate experiment was calculated using the formula R = [Nc - Nt/Nc + Nt] x100 where Nc and Nt represent the number of weevil in the control and test arm respectively and averages calculated. The data for mortality was subjected to analysis of variance (ANOVA). Treatment means showing significant difference (p 0.05) were separated using Student-Newman-Keuls test (SNK). 23

37 CHAPTER FOUR RESULTS AND DISCUSSION 4.1 Introduction In this chapter, the results of the study are provided and discussed. Identification of the various constituent compounds in the oils was represented by chromatograms. The identities of the compounds, their retention times and p ercentage amounts are shown in the chromatograms (Fig. 1-6). The mean repellency and mortality on the bean weevil by the various oils and ground plant materials from Kitui and Machakos regions are summarized in Tables and Bar graphs. 24

38 4.1 Chemical identification of oils Essential oil composition of O. americanum (Kitui) Figure 1 GC-MS chromatogram of O. americanum (Kitui) essential oil (1) 1,8-Cineole (21%); (2) Camphene (3%); (3) Humulene (2.5%); (4) Linalool (22%) (5) α-terpenol (17%); (6) Terpene-4-ol (2%); (7) Pinene (5%); (8) Caryophylene (6%) The highest constituent was linalool (22%) followed by 1,8-cineole (21%), then α-terpenol (17%). 25

39 4.1.2 Essential oil composition of O. americanum (Machakos) Figure 2: GC-MS chromatogram of O. americanum (Machakos) essential oil (1) 1,8-Cineole (38%); (2) Camphene (0.2%); (3) Humulene (0.1%); (4) Linalool (3%); (5) α-terpenol (7%); (6) Terpenen-4-ol (8%); (7) Pinene (9%); (8) Caryophyllene (7%) The highest constituent was 1,8-cineole (38%) followed by pinene (9%) then terpenen-4- ol (8%). Apart from 1,8-cineole which was a major compound in both plants from the two region, the other compounds were significantly different. 26

40 4.1.3 Essential composition of O. gratissimum (Kitui) Figure 3: GC-MS chromatogram of O. gratissimum (Kitui) essential oil (1) Ocimene (0.5%); (2) Terpinen-4-ol (2%); (3) Eugenol (30%); (4) α-copaene (0.8%); (5) Caryophyllene (8%); (6) Germacrene (3%); (7) Methyl isoeugenol (10%); (8) Spirolepechinene (6%). The highest constituent was eugenol (30%) followed by methyl isoeugenol (10%), then caryophyllene (8%). 27

41 4.1.4 Composition of essential oil of O. gratissimum (Machakos) Figure 4: GC-MS chromatogram of O. gratissimum (Machakos) essential oil (1) Ocimene (5%); (2) Terpinen-4-ol (0.5%); (3) Eugenol (1%); (4) α-copaene (19%); (5) Caryophyllene (23%); (6) Germacrene (3%); (7) Methyl isoeugenol (17%); (8) Spirolepechinene (6%) The highest constituent was caryophyllene (23%) followed by α-copaene (19%) then methyl isoeugenol (17%). Eugenol was a major compound in O. gratissimum Kitui, but the one for Machakos it was minimal (1%). 28

42 4.1.5 Composition of L. camara (Kitui) essential oil Figure 5: GC-MS chromatogram of L. camara (Kitui) essential oil (1) Sabinene (10%); (2) 1,8-Cineole (11%); (3) Camphor (2%); (4) Eugenol (0.5%); (5) Caryophyllene (16%); (6) Guaiadiene (0.5%); (7) Byclogermacrene (4%); (8) Caryophyllene -14- hydroxy (4%) The highest constituent was caryophyllene (16%) followed by 1,8-cineole (11%), then sabinene (10%). 29

43 4.1.6 Essential composition of L. camara (Machakos) Figure 6: GC-MS chromatogram of L. camara (Machakos) essential oil (1) Sabinene (0.4%); (2) 1,8-Cineole (3%); (3) Camphor (2%); (4) Eugenol (4%); (5) Caryophyllene (20%); (6) Guaiadiene (11%); (7) Byclogermacrene (9%); (8) Caryophyllene -14- hydroxy (4%) The highest constituent was caryophyllene (20%) followed by guaiadiene (11%), then byclogermacrene (9%). The quantity of caryophyllene in the plants from the two regions did not vary significantly. The other constituents varied significantly. 30

44 4.2 Effect of powdered materials of the plant on mortality of A. obtectus The effect of powdered plant materials on the mortality of A. obtectus was very low and was felt after the 14 days. The tables shows the results obtained for various plants from the two regions. Table 1: Effects of powdered plant materials from Kitui region on the mortality of A. obtectus Dose level Percentage mortality ± SE Percentage mortality ± SE Percentage mortality ± SE Percentage mortality ± SE Days 10 th day 14 th day 21 st day 28 th day p-value O. americanum 10% 5.00 ± 0.32 Cc 8.80 ±.37 Cc ± 0.68 Bd ± 1.69 Ad < % ±1.58 Cb ±1.58 Cb ± 2.92 Bb ± 2.55 Ab <0.001 O. gratissimum 10% 2.00 ± 0.63 Cc 5.60 ± 0.60 Bc 9.20 ± 0.49 Bd ±1.38 Ae < % 5.00 ± 2.24 Dc ± 2.55 Cb ± 3.39 Bc ± 1.87 Ac <0.001 L. camara 10% 0.00 ± 0.00 Cd 0.00 ± 0.00 Cd 2.60 ± 0.87 Be 7.20 ± 0.49 Ae < % 2.00 ±1.22 Dc 6.00 ±1.00 Cc ±1.58 Bd ± 2.92 Ae <0.001 Actellic Super ± 0.00 a ± 0.00 a ± 0.00 a ± 0.00 a Untreated grain 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 e 0.00 ± 0.00 f p-value <0.001 <0.001 <0.001 <0.001 Mean mortality followed by the different small letter(s) within the same column are significantly different and (p <0.05, SNK); mean mortality followed by different capital letter(s) within a row are significantly different and (p <0.05, SNK). By the 10 th and 14 th day, the powdered plant materials showed very low mortality (< 12% for both doses). There was progressive increase in mortality with the period of exposure. O. americanum caused the highest mean mortality of 52% at dose level of 30% w/w, O. gratissimum 44% and L. camara 16%. Actellic Super caused 100% mortality after few hours of treatment. Another study on efficacy of L. camara and T. vogelii showed that there was significant increase in mortality with exposure time (Ogendo et al., 2005). 31

45 Table 2: Effects of Powdered plant materials from Machakos Region on mortality of A. obtectus Dose Percentage Percentage Percentage Percentage level mortality ± SE mortality ± SE mortality ± SE mortality ± SE Days 10 th day 14 th day 21 st day 28 th day p-value O. americanum 10% 0.80 ± 0.49 Cc 4.00 ± 0.63 Bc 7.60 ± 0.75 Ac ± 0.75 Ac < % 5.00 ±1.58 Cb 12.00±1.22 Bb ±1.87 Ab ± 2.24 Ab <0.001 O. gratissimum 10% 0.80 ± 0.49 Cc 4.00 ± 0.63 Bc 7.60 ± 0.75 Ac ± 0.75 Ac < % 1.00 ± 1.00 Dc 4.00 ±1.87 Cc 7.00 ± 1.22 Bc ± 2.00 Ac <0.001 L. camara 10% 0.00 ± 0.00 Bc 1.60 ± 0.75 Bd 4.80 ± 0.37 Ac 6.40 ± 0.68 Ad < % 1.00 ± 1.00 Cc 3.00 ± 1.22 Cc 8.00 ±1.22 Bc ± 1.87 Ac <0.001 Actellic Super ± 0.0 a ± 0.0 a ± 0.0 a ± 0.0 a Untreated grain 0.00 ± 0.00 c 0.00 ± 0.00 d 0.00 ± 0.00 d 0.00 ± 0.00 e p-value <0.001 <0.001 <0.001 <0.001 Mean mortality followed by the different small letter(s) within the same column are significantly different and (p < 0.05, SNK). Mean mortality followed by different capital letter(s) within a row are significantly different and (p < 0.05, SNK). The powdered plant materials from Machakos region caused very low mortality, with O. americanum giving the highest mortality of 25% after the 28 th day at dose level of 30%, O. gratissimum 13% and L. camara 14%. Comparing the effect of the plants from the two regions, the powdered plants materials from Kitui caused higher mortality than those of Machakos. There was no significant difference in mortality by L. camara powders from the two regions. The powders for the O. americanum and O. gratissimum from the two regions varied significantly. This could be due to chemotypic or genetic effect. Powdered plant materials of plants like Tephrosia vogelic have shown to be effective pest control (Katanga and Sileshi, 2012). 4.2 Repellency bioassays The effect of the essential oils from the various plants from the two regions on the repellency of 32

46 A. obtectus was high. The effect varied from one plant to another and the region. The results obtained are tabulated in tables 3 and 4, and also represented in bar graphs in figure 7 and 8. Table 3: Percentage repellency of A. obtectus adults exposed to essential oils of the three plants from Kitui region. O. L. camara O. americanum gratissimum Dose Mean ± SE Mean ± SE Mean ± SE p- value ± ± 6.19 b ± ± ± ± ± > ± ± >0.05 2µL ± b 6.34 b 4µL ± 4.79 ab 0.29 b 4.55 ab 6µL ± 3.43 a ± 0.00 a 4.03 ab 8µL ± 0.00 a 0.00 a 0.00 a 10µL ± 0.00 a 0.00 a 0.00 a RD RD RD p- value < Mean repellency followed by the different small letter(s) within the same column are significantly different (p < 0.05, SNK). The repellency of the essential oil of plants from Kitui region on A. obtectus was high. In a minimum dose of 10µl per cm 2 of filter paper, O. americanum essential oil caused 100% repellency of A. obtectus with RD 50 of 0.44µl per cm 2. For O. gratissimum, a minimum dose of 10µl per cm 2 caused 100% repellency with RD 50 of 0.33 µl per cm 2. That of L. camara, at a minimum dose of 10µl per cm 2 caused 100% repellency with RD 50 of 0.25 µl per cm 2. At dose of 8µL and 10µl, the essential oils from the three plants caused 100% repellency. This implies that grains can be treated with the essential oils to keep the safe from bruchids. 33

47 Figure 7: The percentage repellency of A. obtectus exposed to essential oils from the three plants from Kitui region 34

48 \ Table 4: Percentage repellency of A. obtectus adults exposed to essential oils of the three plant species from Machakos region O. americanu L. camara O. gratissimum p- value Dos m Mean ± SE Mean ± SE Mean ± SE e 2µL ±15.04 b ± b ± 3.15 c µL ± 4.42 a ± 5.40 ab ± 4.78 b µL ± 4.14 a ± 3.53 a ± 3.30 b µL ± 3.08 a ± 2.22 a ± 3.69 ab µL ± 0.00 a ± 0.00 a ± 0.00 a >0.05 RD RD RD p-value <0.001 Mean repellency followed by the different small letter(s) within the same column are significantly different (p < 0.05, SNK). The repellency of the essential oils of plants from Machakos region on A. obtectus was relatively low compared to those from Machakos. In a minimum dose of 10µl per cm 2 of filter paper, O. americanum essential oil caused 100% repellency of A. obtectus with RD 50 of 0.75µl per cm 2, O. gratissimum, 100% repellency with RD 50 of 0.92 µl per cm 2 and L. camara, 100% repellency with RD 50 of 0.62 µl per cm 2. The repellency of the essential oils of plants from Machakos region on A. obtectus was significant (RD 50 values 0.62, 0.75 and 0.92 for L. camara, O. americanum and O. gratissimum respectively but relatively lower compared to that of plants from Kitui region (RD 50 values 0.25, 0.44 and 0.33 for L. camara, O. americanum and O. gratissimum respectively. 35

49 Similar studies on volatile oils from Ocimum suave and Ocimum kenyense had also shown to have repellency activity against maize weevil (Hassanali et al., 1990, Bekele et al., 1997). Studies by Ogendo et al. (2004) found similar repellency against maize weevil using powders of Lantana camara and Tephrosia vogelii. Figure 8: The percentage repellency of A. obtectus exposed to essential oils from various plants from Machakos region 4.3 Mortality bioassays The effect of the essential oils from the various plants from the two regions on the mortality of A. obtectus was high compared to the powdered plant materials. The effect varied from one plant to another and the region. The results obtained are tabulated in tables 5 and 6, and also represented in bar graphs in figure 9 and

50 Table 5: Percentage mortality of A. obtectus caused by the essential oils from O. americanum, O. gratissimum and L. camara from Kitui region. Dose O. americanum O. gratissimum L. camara Hexane p-value Mean ± SE Mean ± SE Mean ± SE Mean ± SE 2µL 6.00 ± 1.87 Bd ± 2.74 Ab 4.32 ± 0.98 Bd 3.40 ±.24 Bb < µL ± 5.24 Bc ± 2.24 Ab 6.00 ± 0.78 Cd 3.80 ± 0.20 Cb < µL ± 7.97 Ab ± 1.22 Ab 7.11 ± 1.09 Bd 3.80 ± 0.20 Cb < µL ± 1.87 Aa ± 3.00 Ba ± 1.87 Cc 4.20 ± 0.20 Db < µL ± 1.00 Aa ± 2.92 Ba ± 2.24 Cc 6.00 ± 0.32 Da < µl 100 ± 0.00 Aa ± 2.00 Ba ± 2.24 Cb 6.00 ± 0.32 Da < µl ± 0.00 Aa ± 0.97 Ba ± 2.00 Ca 6.00 ± 0.32 Da < µl ±0.00 Aa ± 0.99 Ba ± 3.74 Ba 6.60 ± 0.24 Ca <0.001 LD ± ± ± 3.49 LD ± ± ± 5.78 LD ± ± ± p-value <0.001 <0.001 <0.001 <0.001 Mean mortality followed by the different small letter(s) within the same column are significantly different and (p < 0.05, SNK). % (mean ± SE) Mortality followed by different capital letter(s) within a row are significantly different and (p < 0.05, SNK). The influence of the essential oils of the three plants from Kitui region on the mortality of A. obtectus was significant. In a minimum dose of 16µl per cm 2 of filter paper, O. americanum essential oil caused 100% mortality of A. obtectus with LD 50 of 5.28µl per cm 2, O. gratissimum 60% mortality with LD 50 of 8.39µl per cm 2 and L. camara 58% with LD µl per cm 2. At LD 90, the values were as follows; O. americanum 9.31 µl per cm 2, O. gratissimum µl per 37

51 cm 2 and L. camara µl per cm 2. At dose of 12 µl to 16 µl, O. americanum gave 100% mortality, while O. gratissimum and L. camara at 16 µl caused lower mortality of 60% and 58% respectively. The difference could be attributed to the difference in the quantity of constituent compounds in the oils. At low dose of 2µL the activity of the essential oils was significantly low for O. americanum and L. camara. Figure 9: Percentage mortality of A. obtectus caused by the essential oils from O. americanum, O. gratissimum and L. camara from Kitui region. 38

52 Table 6: Percentage mortality of A. obtectus caused by the essential oils from O. americanum, O. gratissimum and L. camara from Machakos region. Dose O. americanum O. gratissimum L. camara Hexane p- Mean ± SE Mean ± SE Mean ± SE Mean ± SE value 2µL 6.00 ± 1.00 Ac 4.00 ±1.87 Ac 4.00±1.87 Ac 3.40 ± 0.24 Aa µL ± 4.00 Ab 9.00 ± 3.32 Bc 4.00 ± 0.32 Bc 3.80 ± 0.20 Ba µL ± 6.63 Ab 6.00 ± 1.87 Bc 5.00 ± 3.32 Bc 3.80 ± 0.20 Ba µL ± 2.55 Ab ± 3.00 Bb 5.50 ± 1.58 Cc 4.20 ± 0.20 Ca < µL ± 1.87 Aa ± 2.00 Ba ± 2.00 Bb 3.00 ± 0.32 Ca < µl ± 0.98 Aa ± 0.87 Ba ± 2.45 Bb 3.40 ± 0.32 Ca < µl ± 0.79 Aa ± 1.09 Ba ± 2.00 Aa 3.60 ± 0.24 Ca < µl ± 1.90 Aa ± 2.00 Ba ± 1.00 Aa 4.00 ± 0.32 Ca <0.001 LD ± ± ± LD ± ± ± LD ± ± ±53.26 p-value <0.001 <0.001 Mean mortality followed by the different small letter(s) within the same column are significantly different and (p < 0.05, SNK).Mean mortality followed by different capital letter(s) within a row are significantly different and (p < 0.05, SNK). The influence of the essential oils of the three plants from Machakos region on the mortality of A. obtectus was extremely low compared to that of Kitui region. In a minimum dose of 16µl per cm 2 of filter paper, O. americanum essential oil caused 28% mortality of A. obtectus with LD 50 of 34.20µl per cm 2, O. gratissimum, 20% mortality with LD 50 of 89.93µl per cm 2 and L. camara 34% mortality with LD 50 of µl per cm 2. At LD 90, the values were as follows; O. 39

53 americanum µl per cm 2, O. gratissimum µl per cm 2 and L. camara µl per cm 2. With respect to the three plants L. camara (Machakos) gave better results from the three plants. Despite the low values at lower doses O. americanum (Kitui) gave the best results at higher doses. Figure 10: Percentage mortality of A. obtectus caused by the essential oils from O. americanum, O. gratissimum and L. camara from Machakos region. 4.4 Toxicity of individual constituents of the three essential oils and their blends The above experiment on adult mortality tests was repeated with commercially available constituents of the essential oils and blends of these at their natural proportions (GC) in the oils and amounts present in the minimum 100% lethal dose of the oils, with each component absent (subtracted) from the blend in turn to determine its relative contribution to the overall toxicity of the natural oil. 40

54 Table 7: Percentage mortality of A. obtectus adults exposed to O. americanum (Kitui) constituents, individually and in blends equivalent to the relative proportion of each compound in 100% lethal dose of the essential oil. Treatments Compounds % Mortality Mean ± SE 1 Essential oil ± 0.00 a 2 1,8-Cineole ± 4.30 c 3 β-pinene 8.00 ± 1.22 e 4 α-terpineol ± 3.32 d 5 Caryophyllene 4.00 ± 1.87 e 6 Camphene 4.00 ± 1.87 e 7 Terpine-4-ol 8.00 ± 2.00 e 8 Linalool ± 2.55 d 9 Humulene 5.00±2.24 e combined ± 0.00 a without 1,8-Cineole ± 2.55 d without β-pinene ± 5.10 ab without α-terpineol ± 3.39 d without Caryophyllene ± 2.55 ab without Camphene ± 1.00 ab without Terpine-4-ol ± 3.00 ab without Linalool ± 1.93 d without Humulene ± 2.00 ab 19 Blend of 3 most effective and prominent constituents (2, 4 & 8) ± 3.39 b 20 1,8-Cineole and α-terpineol ± 3.87c 21 1,8-Cineole and Linalool ± 5.48c 22 α-terpineol and Linalool ± 2.92d Treatment means followed by the same letter are not significantly different from each other (P < 0.05, SNK). 41

55 The individual constituents of O. americanum (Kitui) at levels present in the lethal dose of the essential oil, 1,8-cineole caused highest individual (51%) mortality followed by α-terpineol 39% then linalool with 28%. The other constituents showed insignificant toxic effects against A. obtectus. 100% mortality of the insect was obtained when all the eight compounds were present. In subtraction assays, absence of 1,8-cineole caused largest drop in the activity of the resulting blend, showing that this compound contributes most to the activity of the essential oil. Subtraction of α-terpineol and linalool also caused significant drop in toxicity of the resulting blends. The other five components did not cause significant drop in the toxicity of the blends. A blend of three most effective and prominent constituents (1,8-cineole, α-terpineol and linalool ) gave a high mortality (82%), but not 100%, showing that the other less prominent components also contribute to the activity of the essential oil. Binary blends of 1,8-cineole and α-terpineol, 1,8-cineole and linalool, and linalool and α-terpineol, gave moderate mortality of 60%, 55% and 44%, respectively. The lethal toxicity to the insects is caused by combined effect of the constituents of the oil or major components in the oil (Asawalam et al., 2008). 42

56 Table 8: Percentage mortality of A. obtectus adults exposed to L. camara (Kitui) constituents, individually and in blends equivalent to the relative proportion of each compound in 58% lethal dose of the essential oil. Treatments Compounds % Mortality (± SE) 1 Essential oil ± 3.74 a 2 Caryophyllene 3.00 ± 2.00 b 3 Sabinene 2.00 ± 1.22 b 4 1,8-Cineole ± 2.92 a 5 Caryophyllene, Sabinene and 1,8-Cineole combined ± 2.74 a 6 Sabinene and 1,8-Cineole ± 4.30 a 7 Caryophyllene and 1,8-Cineole ± 3.54 a 8 Caryophyllene and Sabinene 3.00 ± 2.00 b Treatment means followed by the same letter are not significantly different from each other (P < 0.05, SNK). At levels present in the lethal dose of the essential oil, 1,8-cineole caused 51% mortality followed by caryophyllene 3% then guaiadiene with 2%. A minimum dose of 14µl per cm 2 of the filter paper was used and gave 58% mortality of A. obtectus. The blend of the three most abundant constituents gave mortality of 50%, showing that the other compound contribute to the mortality of the insects. Subtraction of 1,8-cineole gave a high drop in mortality, this implies that it is the most effective constituent. 43

57 Table 9: Percentage mortality of A. obtectus adults exposed to O. gratissimum (Kitui) constituents, individually and in blends equivalent to the relative proportion of each compound in 59% lethal dose of the essential oil. Treatments Compounds % mortality Mean ± SE 1 Essential oil ± 2.92 a 2 Eugenol ± 3.00 a 3 Methyl isoeugenol ± 2.24 c 4 Caryophyllene 2.00 ± 1.22 d 5 Eugenol, Methyl isoeugenol and Caryophyllene ± 5.24 a 6 Methyl isoeugenol and Caryophyllene ± 2.92 b 7 Eugenol and Caryophyllene ± 4.30 a 8 Eugenol and Methyl isoeugenol ± 3.32 a Treatment means followed by the same letter are not significantly different from each other (P < 0.05, SNK). At levels present in the lethal dose of the essential oil, eugenol caused 53% mortality followed by methyl isoeugenol 39% then caryophyllene with 2%. A minimum dose of 10µl per cm 2 of the filter paper was used and gave 59% mortality of A. obtectus. The blend of the three most abundant constituents gave mortality of 55%, showing that the other compounds contribute to the mortality of the insects. Subtraction of eugenol gave a significant drop from 59% to 46% in mortality; this implies that it is the most effective constituent. Eugenol is reported to be a more potent maize weevil repellent than the synthetic commercial compound, DEET (Hassanali et al., 1990). 44

58 CHAPTER FIVE CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions The study revealed that powdered plant materials had low toxicity to A. obtectus. The most effective powdered plant material was O. americanum from Kitui region with mean mortality rate of 52% after the 28 th day of treatment at the highest level of 30% w/w. O. gratissimum from Kitui region gave mean mortality rate of 44% and L. camara gave the lowest mean mortality of 16%. The low mortality of the aromatic plants as ground powders may be due to relatively low emission of the volatiles from these materials. The powdered plants materials from Kitui region had higher mortality of the bruchids compared with those from Machakos (O. americanum 25%, O. gratissimum 13% and L. camara 14%). The results shows that powdered plant material of O. americanum can be used for pest control. Powdered plant materials like, leaves of Tephrosia vogelii have also been used by mixing 100g of its powder with 100g of maize and beans. The treatment is reported to be effective upto three months, after which the process is repeated (Sileshi and Katanga, 2012). In repellency bioassays, all the essential oils were very effective, 2µl of oils of O. americanum, O. gratissimum and L. camara from Kitui region gave 74%, 80%, 81% repellency, respectively. At 8µl, repellency of all the three oils was 100%. The essential oils for the plants from Machakos region at dose level of 2µl were as follows: O. americanum 65%, O. gratissimum 54% and L. camara 62%. The effect at higher dose of 8µl was 97%, 91% and 98% respectively. At the highest dose level of 10µl all the three essential oils gave repellency of 100%. Thus, specific constituent(s) of the essential oils of the three plants appear to have potent repellent 45

59 activities individually and/or in blends against A. obtectus. Interestingly, the oils of the three plants contain α-terpineol, which has been reported to be most repellent compound against Rhipicephalus appendiculatus and Sitophilus zeamais (Asawalam and Hassanali, 2006). The high repellent activity of L. camara and O. gratissimum could also be attributed to the presence of eugenol. Earlier studies on the effects of the essential oils of Ocimum suave found eugenol as the most repellent against Sitophilus zeamias (Hassanali et al., 1990). In mortality bioassays, the effects of the essential oils of plants from Kitui region at 10 µl were as follows: O. americanum 99%, O. gratissimum 59% and L. camara 25%. At the same dose levels, the essential oils of plants from Machakos region gave the following mortalities: O. americanum 26%, O. gratissimum 18% and L. camara 12%. Just as the powdered plant materials, the mortalities caused by the oils from plants from Machakos were lower campared to those from Kitui. GC-MS analysis of the essential oils of O. americanum revealed the presence of compounds like, linalool, 1,8-cineole, α-terpineol, ᵦ-pinene, caryophyllene, camphene, terpinen-4-ol and humulene in high quantities. The more prominent constituents like 1,8-cineole, α-terpineol and linalool gave higher mean mortality of 51%, 39% and 26% respectively. Subtraction assays involving different combinations of the constituent compounds against A. obtectus indicated special potency of some of the mixtures. This shows that varying the composition can give a more active blend from less active constituents. This is illustrated by the high (82%) lethal activity of 1,8-cineole, α-terpineol and linalool when blended together in 46

60 proportion occurring in the essential oil. The lethal toxicity to the insect is caused by the combined effect of the major constituents in the oil. The findings of Asawalam et al., (2008) was that the major component of X. aetiopical, 1,8-cineole was found to be largely responsible for toxic action against S. zeamais, while the toxic action of V. amygdalina was due to the combined effects of its constituents. GC-MS analysis of the essential oil of O. gratissimum (Kitui) showed the presence of eugenol, methyl isoeugenol and caryophyllene as major constituents. The individual compounds gave mean mortality as follows: eugenol 53%, methyl isoeugenol 20% and caryophyllene 2%. When blend together in proportion present in the essential oil, the mean mortality was 55%, not significantly different from that of pure eugenol. GC-MS analysis of the essential oil of L. camara (Kitui) showed t h e p r es ence o f caryophyllene, α-guaiadiene and 1,8-cineole as the major compounds. Individual compounds gave mean mortalities as following: caryophyllene 3%, α-guaiadiene 2% and 1,8-cineole 51%. When blended together the mean mortality was 52% comparable to that of pure 1,8-cineole. GC-MS analysis of the essential oils of the plants from the two region showed that most compounds were common in all the plants. The compounds varied only in relative amounts. The difference could be due to genetic or ecological differences. In general, the essential oil of L. camara (Kitui) had high repellency to the bruchids (RD50, 0.25) than those of the other plants (O. americanum (Kitui) RD50, 0.44 and O. gratissimum (RD50, 47

61 0.33 but low mortality LD50, The essential oil of O. americanum had good combination of both repellency and mortality. 5.2 Recommendations Recommendations from the study i. Essential oil from O. americanum (Kitui) possesses a blend of compounds which is repellent and toxic to bruchids, and therefore it can be used by farmers to control the post- harvest pest. ii. Essential oil from L. camara possesses compounds which are repellent to the bruchids, therefore farmers can use it on uninfected grains to protect them from any invasion of the bruchids. iii. The results demonstrate a possible scientific rationale for the incorporation of the leaves of these plants into the grain protection practices by some farmers. iv. Potential users can be mobilized to transfer the essential oils-based post-harvest technology downstream (which could be a source of additional income to resourcepoor communities) Recommendations for further study From the findings of this research, it is recommended that; i. The bases of differences in quantities of the constituent compounds of each plant species from different regions should be determined to see if it is due to chemotypic (epigenetic) or genetic effect. 48

62 ii. Essential oils or extracts from other parts of the test plants like roots, stem or flowers need to be studied to establish if phytochemical constituents from these parts can enhance the activities of the essential oils. iii. Further research is needed on the time-course efficacy of essential oils in protecting grains from stored pests in traditional storage systems over longer period. iv. A survey of potential users needs to carried out to see if residues of essential oils in the grains are acceptable to the users 49

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67 APPENDICES STRUCTURES OF THE COMMON CONSTITUENTS OF ESSENTIAL OILS Appendix 1. Mass spectra of 1,8 - CINEOLE 54

68 Appendix 2. Mass spectra of CAMPHENE 54

69 Appendix 3. Mass spectra of HUMULENE 55

70 Appendix 4. Mass spectra of LINALOOL FORMATE 56

71 Appendix 5. Mass spectra of TERPINE-4-OL 57

72 Appendix 6. Mass spectra of α TERPINEOL 58

73 Appendix 7. Mass spectra of PINENE 59

74 Appendix 8. Mass spectra of CARYOPHYLLENE 60

75 Appendix 9. Mass spectra of OCIMENE 61

76 Appendix 10. Mass spectra of EUGENOL 62

77 Appendix 11. Mass spectra of α COPAENE 63

78 Appendix 12. Mass spectra of SABINENE 64

79 Appendix 13. Mass spectra of CAMPHOR 65

80 Appendix 14. Mass spectra of GUAIADIENE 66

81 Appendix 15. Mass spectra of GERMACRENE 67

82 Appendix 16. Mass spectra of METHYL ISOEUGENOL 68