Responses of Aphis fabae Scop (Hom., Aphididae) and its steno-phagous predator Cheilomenes propinque Muls. (Col., Coccinellidae) to neem seed water extract By Mohammed Abdalla Suliman Mohammed B.Sc. Agric. (Honours) Sci. (2002). University of Gezira A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of M. Sc. in Crop Protection Supervisor: Dr. Hamadttu Abdel Farag El Shafie Department of Crop Protection Faculty of Agriculture University of Khartoum September - 2005 1 DEDICATION To my father To my dear mother To my grand mother (Haja Sakina) To my brothers and sisters To my teachers and friends With love and respect `É{tÅÅxw Acknowledgements 2 First of all, I render my gratitude and praise to the Almighty “Allah”. I wish to express my sincere gratitude to Dr. Hamadttu Abdel Farag Elshafie, for his guidance continuous encouragement, and efforts to solve the problems arised during the accomplishment of this research. Iam deeply grateful to Dr. Abdelwahab Hassan for choosing a suitable design for the experiment and Dr. Salah Hamed Elturabi for his help with the statistical analysis. I would like to extended my thank to Dr. Moutassim Mohamed Khalfallah (Environment and Natural Research Institute) I acknowledge with thanks the help rendered to me by my colleagues and staff members of the Department of crop protection, Faculty of Agriculture, University of Khartoum. Finally, special thanks are extended to my family and my friends ABSTRACT 3 Two green house experiments were conducted at Shambat area to investigate the systemic and contact effect of neem seed kernel aqueous extracts on the bean aphid, Aphis fabae Scop. and their stenophagous Coccinellid predator Cheilomenes propinque (Muls.). In the first experiment, different concentrations of neem seed kernel aqueous extract (25g, 50g and 75g/liter of water), and insecticide Actara® (1g per 1.25 litter of water) were tested. Three methods of application: drenching in the soil, spraying on the foliage and topically on the aphid body were attempted. In the second experiment, the same concentrations of neem and the same insecticides were applied topically (direct spraying) on the different stages of C. propinque (Muls)(eggs, larva, adults). The results indicated that, Actara® was the most effective product to control aphids and kill their predator C. propinque (Muls.), compared with neem seed aqueous extracts. The results in all experiments indicated that the effect of neem seed kernel aqueous extract was effective in the first 3–4 days of the experiment. Also during these days, the high concentration of neem gave high mortality. The results have also shown that the neem products were less effective on the C. propinque (Muls.) than aphids. 4 ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ ﺨﻼﺼﺔ ﺍﻷﻁﺭﻭﺤﺔ ﺃﺠﺭﻴﺕ ﺘﺠﺭﺒﺘﺎﻥ ﺩﺍﺨل ﺍﻟﺼﻭﺒﺔ ﺍﻟﻤﺤﻤﻴﺔ ﺒﻤﻨﻁﻘـﺔ ﺸـﻤﺒﺎﺕ ﻟﺩﺭﺍﺴـﺔ ﺍﻟﻔﻌـل ﺍﻟﺠﻬـﺎﺯﻱ ﻭﺒﺎﻟﻤﻼﻤﺴﺔ ﻟﻤﺴﺘﺨﻠﺼﺎﺕ ﺒﺫﻭﺭ ﺍﻟﻨﻴﻡ ﻋﻠﻰ ﺤﺸﺭﺓ ﻤـﻥ ﺍﻟﻔـﻭل ﺍﻟﻤـﺼﺭﻱ Aphis fabae Scop ﻭﻤﻔﺘﺭﺴﻬﺎ ﺨﻨﻔﺴﺎﺀ ﺃﺒﻭ ﺍﻟﻌﻴﺩ ).Cheilomenes propinque (Muls ﻓﻲ ﺍﻟﺘﺠﺭﺒﺔ ﺍﻷﻭﻟﻰ ﺃﺴﺘﺨﺩﻤﺕ ﺜﻼﺜﺔ ﺘﺭﻜﻴﺯﺍﺕ ﻤﻥ ﻤﺴﺘﺨﻠﺹ ﺒﺫﺭﺓ ﺍﻟﻨﻴﻡ ) 25ﺠـﺭﺍﻡ50 ، ﺠﺭﺍﻡ ،ﻭ 75ﺠﺭﺍﻡ ﻟﻜل ﻟﺘﺭ/ﻤﺎﺀ( ﻭﻤﺒﻴﺩ ﺃﻜﺘﺎﺭﺍ ® Actaraﺍﻟﺤﺸﺭﻱ ﺒﺘﺭﻜﻴﺯ ) 1ﺠﺭﺍﻡ ﻟﻜـل 1.25 ﻟﺘﺭ ﻤﺎﺀ( ﻭﻗﺩ ﺃﺴﺘﺨﺩﻤﺕ ﺜﻼﺜﺔ ﻁﺭﻕ ﻟﻤﻌﺎﻤﻠﺔ ﺤﺸﺭﺓ ﺍﻟﻤﻥ :ﺍﻷﻭﻟﻰ ﺍﻟﺭﺵ ﻋﻠﻰ ﺍﻟﺘﺭﺒﺔ ﻭﺍﻟﺜﺎﻨﻴﺔ ﺍﻟﺭﺵ ﺍﻟﻤﺒﺎﺸﺭ ﻋﻠﻰ ﺍﻟﺠﺯﺀ ﺍﻟﺨﻀﺭﻱ ﻤﻥ ﺍﻟﻨﺒﺎﺕ ﻭﺍﻟﺜﺎﻟﺜﺔ ﺍﻟﺭﺵ ﺍﻟﻤﺒﺎﺸﺭ ﻋﻠﻰ ﺠﺴﻡ ﺍﻟﺤﺸﺭﺓ. ﻓﻲ ﺍﻟﺘﺠﺭﺒﺔ ﺍﻟﺜﺎﻨﻴﺔ ﺃﺴﺘﺨﺩﻤﺕ ﻨﻔﺱ ﺍﻟﺘﺭﻜﻴﺯﺍﺕ ﺍﻟﻤﺨﺘﻠﻔﺔ ﻟﻠﻨﻴﻡ ﻭﻨﻔﺱ ﺍﻟﻤﺒﻴﺩ ﺍﻟﺤﺸﺭﻱ ﺒﻁﺭﻴﻘﺔ ﺍﻟﺭﺵ ﺍﻟﻤﺒﺎﺸﺭﺓ ﻋﻠﻰ ﺃﻁﻭﺍﺭ ﺨﻨﻔﺴﺎﺀ ﺃﺒﻭ ﺍﻟﻌﻴﺩ ﺍﻟﻤﺨﺘﻠﻔﺔ )ﺍﻟﺒﻴﻀﺔ ،ﺍﻟﻴﺭﻗﺔ ،ﺍﻟﺤﺸﺭﺓ ﺍﻟﻜﺎﻤﻠﺔ(. ﺃﻭﻀﺤﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻥ ﻤﺒﻴﺩ ﺃﻜﺘﺎﺭﺍ ﺍﻟﺤﺸﺭﻱ ﺃﻜﺜﺭ ﻓﻌﺎﻟﻴﺔ ﻤﻘﺎﺭﻨﺔ ﺒﻤﺴﺘﺨﻠـﺼﺎﺕ ﺍﻟﻨـﻴﻡ ﻓـﻲ ﻤﻜﺎﻓﺤﺔ ﺤﺸﺭﺓ ﺍﻟﻤﻥ ﻭﺍﻟﻘﻀﺎﺀ ﻋﻠﻰ ﻤﻔﺘﺭﺴﻬﺎ ﺃﺒﻭ ﺍﻟﻌﻴﺩ .ﻜﺫﻟﻙ ﺃﻭﻀﺤﺕ ﺍﻟﻨﺘﺎﺌﺞ ﻓﻲ ﻜل ﺍﻟﺘﺠﺭﺒـﺔ ﺃﻥ ﺘﺄﺜﻴﺭ ﻤﺴﺘﺨﻠﺼﺎﺕ ﺍﻟﻨﻴﻡ ﺒﻜل ﺘﺭﻜﻴﺯﺍﺘﻬﺎ ﺇﻨﺤﺼﺭﺕ ﻓﻲ ﺍﻟﺜﻼﺜﺔ ﺇﻟﻰ ﺃﺭﺒﻌﺔ ﺃﻴﺎﻡ ﺍﻷﻭﻟﻲ .ﻜﻤﺎ ﺃﻭﻀﺤﺕ 5 ﺍﻟﻨﺘﺎﺌﺞ ﺨﻼل ﺍﻟﻴﻭﻡ ﺍﻟﻭﺍﺤﺩ ﺃﻥ ﺍﻟﺘﺭﻜﻴﺯ ﺍﻟﻌﺎﻟﻲ ﻤﻥ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻨﻴﻡ ﻜﺎﻥ ﻫﻭ ﺍﻷﻓﻀل ﺒﻴﻥ ﻤﻌـﺎﻤﻼﺕ ﺍﻟﻨﻴﻡ ﺤﻴﺙ ﺃﻋﻁﻲ ﻤﻌﺩل ﻤﻭﺕ ﻋﺎﻟﻲ. ﻜﺫﻟﻙ ﺃﻭﻀﺤﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻥ ﺘﺄﺜﻴﺭ ﻤﺴﺘﺨﻠﺹ ﺍﻟﻨﻴﻡ ﻜﺎﻥ ﻀﻌﻴﻔﹰﺎ ﻋﻠﻰ ﺍﻟﻤﻔﺘـﺭﺱ ﺃﺒـﻭ ﺍﻟﻌﻴـﺩ ﺒﺎﻟﻤﻘﺎﺭﻨﺔ ﺒﺤﺸﺭﺓ ﺍﻟﻤﻥ. 6 LIST OF CONTENTS Page Dedication………………………………….…………………………………….…… i Acknowledgements ……………………………….………………………….…… ii Abstract …………………………...……….…………………………………….…… iii Arabic Abstract ……………………………………………………………….…… iv List of Contents ……………………………………………………………….…… v List of Tables and Figures ….….…….…………………………………….…… ix List of Plates ………………………………...……………………………….…… x List of Abbreviations……………………………………………..………….…… xi CHAPTER ONE: INTRODUCTION …….………………..…………….… 1 CHAPTER TWO: LITERATURE REVIEW…….……..…………….… 3 2-1-1 Classification of aphids…………..…………………..……………….… 3 2-1-2 Nature of damage………………………………………………………..… 3 2. The control measures adopted against aphids…….……………….… 4 2.1. Conventional method….………………….…………………………….… 4 2.1.1. Chemical control….…………………………..……………………….… 4 2.1.2. Biological control…….………………...……………………………….… 4 7 2.2. The use of botanical (Neem tree) in control of aphids……...… 5 2.2.1. Chemical constituents of neem…….……………………...……….… 5 2.2.2. The mode of action of azadirachtin…….……………………….… 5 2.2.3. The side effect of neem kernel Aqu- Ext on the aphids….… 6 2.3 The Coccinellids…….………………………...…………………………….… 8 2.3.1. Classification of C. propinque (Muls)…….………………….… 8 2.3.2. Population dynamic of C. propinque (Muls) …….…………… 8 2.3.3. The efficiency of coccinellids in checking aphid popula-tion …….…………….…………….………………….………………………………… 9 2.3.4. Side–effect of neem seed Aqu-Ext on the Coccinellids 10 CHAPTER THREE: MATERIALS AND METHODS…….… 13 3.1. Aphids culture….……………………….………………………………….… 13 3.2. Collection and rearing of Cheilomenes propinque (Muls) ……. 13 3.3. Preparation of test materials……..…………………………………….… 15 3.3.1. Neem seeds kernel powder (NSKP) …….………………………… 15 3.3.2. Preparation the aqueous- extract…….……………………………… 15 3.4. Synthetic insecticide…….………………..……………………………….… 15 3.5. Equipment and spraying technique…….……………..…………….… 16 8 3.6. The effect of neem aqueous-eztract on the Aphis fabae (Scop) …….………………………………………………………..…………….… 16 3.6.1 Soil drenching experiment…….…………………………………….… 16 3.6.2. Foliar application experiment…….……………………………….… 20 3.6.3. Topical application experiment …….…………………………….… 20 3.7. The efficacy of neem aqueous extract on the Cheilomenes propinque (muls) …….…………………………………………..……………….… 21 3.7.1. Egg stage…….……………………………………….…………………….… 21 3.7.2. Larval stage…….………………………………………………………….… 22 3.7.3. Adult stage…….………………………………….……………………….… 22 3.7.4. Feeding of C. propinque (Muls) on aphids treated topically…… 22 CHAPTER FOUR: RESULTS……………….…………………………… 25 4.1.1. The effect of NSK Aqu-Ext, applied as soil drenching, on aphids (drenching experiment) …….………………………………….… 25 4.1.2. The effect of NSK Aqu-Ext sprayed on the faba bean plants…….…………………………………………………….…………………….… 27 4.1.3. The effect of NSK Aqu-Ext sparyed topically on the aphids…….………………………………………………………...………………….… 27 4.2.1. The effect of NSK Aqu-Ext on the Egg hatchability of C. propinque (Muls) 48 hours after treatment .…………………. ……….… 9 30 4.2.2. The effect of NSK Aqu-Ext on the Aduts C. propinque Muls. 30 4.2.3. The effect of NSK Aqu-Ext on the 3rd, larval instar…….… 30 4.2.4. The feeding of C. propinque (Muls) on aphids treated topically with NSK Au- Ext………………………………………………….… 32 CHAPTER FIVE: DISCUSSION …….……………………………….… 36 5.1. General…….………………………………………………..………………….… 36 5.2. The effects of NSK Aqu-Ext on aphids…….………………….… 36 5.3. The effects of NSK Aqu-Ext and Actara® on the different stages of the Coccinellid Cheilomenes propinque (Muls) …….… 38 5.4. Conclusions and recommendations……………………………….… 39 REFERENCES…….……………...………………..…………………………….… 41 APPENDICES …….…………………………...……………………………….… 51 10 LIST OF TABLES & FIGURES Table Title 1. Page Survival and corrected mortality (CM) of A. fabae (20 insects) fed on seedling of faba bean treated with NSWE (soil application)…………………………………………………………… 2. 26 Survival and corrected mortality (CM) of A. fabae (20 insects) after foliar application of NSWE on faba bean (post infestation). ………………………….……………………………………… 3. Survival and corrected mortality (CM) of A. fabae (20 insects) topically sprayed with NSWE…………………………… 4. 29 The effect of topical application of NSK Aqu-Ext on the egg hatchability of (C. propinque Muls) 48 hours after treatment 5. 28 31 Survival and corrected mortality (C.M) of C. propinque adults (10 insects) after direct spraying with, neem seed water extract (NSWE)…….……………………………………………… 6. 33 Survival and corrected mortality (C.M) of 3rd larvae (10 insects) of C. propinque after direct spraying with, neem seed water extract (NSWE)…………………………………………… 7. 34 Survival and corrected mortality (C.M) of C. propinque adults (10 insects) fed on aphids (A. fabae) treated topically with, neem seed water extract (NSWE)………………….………… 11 35 LIST OF PLATES Plate Title Page 1. Aphid and Coccinellids rearing culture………………………… 14 2. The sprayer used in experiments……….………………………… 17 3. The experimental unit…………………..….………………………… 19 4. The test stages of C. propinque …………………………...……… 24 12 LIST OF ABBREVIATIONS ANOVA Analysis of variance ARC Agricultural Research Center AZA Azadirachtin CM Corrected mortality cm Centimeter D.f. Degree of freedom Ec Emulsifiable concentrate Fcal The ratio of ms of treatment to ms of experimental error Fig. Figure IPM Integrated pest management L Liter NSO Neem seed oil NSK Neem seed kernel NSK Aqu-Ext Neem seed kernel aqueous extracts NSKP Neem seed kernel powder NSWE Neem seed water extract Ss Sum of square 13 CHAPTER ONE INTRODUCTION Broad bean (Vicia faba L.) has many names but the name faba bean has recently been widely used. It is considered as good source of protein, carbohydrates and fiber (El Tinay et al., 1993.). Faba bean is subject to attack by several insect pests and disease from the early development phase to the post harvest time. The main pest that attacks faba bean in the early development phase is aphid. Aphids are pests of world-wide importance and they cause heavy crops losses by feeding on plant sap and transmitting viruses as vectors. In addition, they also secrete copious amount of honey dew which attracts sooty mould. Although aphids are readily killed by various insecticides, yet they are not easily controlled because they multiply rapidly and many species feed on the lower side of the foliage and therefore not easily reached by sprays or dusts (Frohlch and Rode Wald, 1963). Aphid, are also known to develop resistance against insecticides rapidly. Therefore, it is necessary to find alternative methods of control preferably with agents non- toxic to humans and less hazardous to the environment. The natural enemy of aphids [C. propinque (Muls)], as an example taken in this study plays a fundamental role in integrated pest management program, Coocinellids were found to be the common predators attacking cereal aphids in spring cereal fields (Dean, 1974, Jones, 1972). They are also important predators of aphids in several field crops (Nevenschewander et al. 1976, Kring et al. 1985. Rice and Wide 1988). 14 Obvious pollution problems in the environment, and the toxic effects of synthetic chemicals on non-target organisms have prompted investigation on exploiting pesticides of plant origin. Natural plant products and their analogues are important source of new agricultural chemicals used in the control of insect pest (Cardllina 1988, Emosairue and Ukeh 1996 Gulter, 1998, Schmutterer and Huber, 2005). Recent studies have shown the importance of natural chemicals as possible source of non- phytotoxic, systemic, and easily biodegradable alternative pesticides (Singh, 1994; Qasem and Abou-Blan, 1996, Mason and Mathew, 1996). Furthermore, pesticides of plant origin are cheap, readily available and cost effective in developing countries. Many pesticides of plant origin have been used as pest control agents. About 2000 plant species were reported to posses pest control properties, out of which very little are being used for insect control purposes. Products from the neem tree (Azadirchata indica A Juss) proved to have promising effects against pests. Mortality, antifeedent, repellency development and oviposition inhibitions are some of the impacts of the neem on insects. This studied was taken under the green house conditions with the main following objectives. To evaluate the contact and systemic action of the extracts from the neem seed kernel (NSKWE) on faba been aphid Aphis faba (Scop). To elucidate the negative impact of neem, if any on the developmental stages of the steno-phagous aphid, on predator [Cheilomenes propingue (Muls)]. 15 CHAPTER TWO LITERATURE REVIEW 2-1-1 Classification of aphids: Aphids belong to the order: Hemiptera, Sub order: Homoptera, super family: Aphidoidea and family Aphididea (Imms, 1957). 2-1-2 Nature of damage. Aphids are pests of world-wide importance and they cause heavy crop losses by feeding on plant sap and transmitting viruses as vectors. Inaddition, they also secrete copious of honey dew which attracts sooty moulds. Many aphids species are vectors of viral disease and they can cause serious damage at low densities. Some species of aphids such as the green peach aphid Myzus persicae (Sluzer) which is vector of many viral disease has developed resistance to insecticides (Fernhurst et al., 1989). The virus of bean yellow mosaic (BYMV) is transmissible by sap inoculation and by aphids in a non- persistant manner. The well known aphid vectors include Aphis craccivora, Aphis fabae, A. Pisum and Myzus persicae (Quantz, 1954; Nitzany and Cohen, 1962; Evans 1973). Black bean aphids (Aphis fabae Scop) is commonly recognized as pest of number of crop in Europe. In Poland this species has been recorded as a harmful pest of broad bean, field bean and sugarbeet (Goszczyhski et al., 1992, Hurej, 1991). In field bean Vicia faba, yield losses due to this species were found to depend on the timing and intensity of colonization 16 (Hins et al, 1981). In the Sudan, a number of aphid species were reported from agricultural research stations. They include, The wheat aphids Schizaphis graminum (Rondani), Rhopalosiphum maidis (Fitcg), the cotton Aphid gossypii (Glover), the cowpea aphid Aphis craccivora Koch, the sorghum aphid Aphis sorghi (Theo); the milk weed or the oliander aphid, Aphis nerii Fonscolombe; and Aphis lutescents Monell. 2. Control measures adopted against aphids: 2.1. Conventional methods: The conventional methods of insect pests control used in Sudan are applicable also for aphid control viz. chemical control use of resistant varieties, good crop husbandry…etc. 2.1.1. Chemical control: Mudathir (1971) found that various insecticides gave a good control and effectively reduced aphid population. These were mostly organo phosphorus compounds namely Folimat®, 80% Primor® 25%, vamifen® 25%, Thimulion®, Anthio ® 25%, Ekatin® 25%, Dimethoate® 32%, cytrolane® 50%, Metasytox® 50%, Lannate ® 90%, Cp5 11%, Trihion 4E, Tamarn® 50 % and Bidrin® 24%. 2.1.2. Biological control: Cereal aphids as example are attacked by wide range of parasitoids, predators and fungal pathogens. Bacteria and viruses have not been reported (Elamin, 1975). Coccinellids were found to be the common predators attacking cereal aphids in spring cereal fields (Dean, 1974; Jones, 1972). 17 In the Sudan Gezira, the following aphid predators were reported and found to play an important role in aphid control: the beetle Coccinella undecimpunctata. (L), the green lace wing (GLW) Chrysoperla (=chrysopa) zastrowi Ebson-petrson, C. paudica Naras, the lady bird Cheilomenes propinque propinque (Mulsant)[=Cydonia propinque (Mulsant)], the ladybird Hippodamia variegata (Goeze), the syrphid Ischiodon aegyptium Wieden mann, and the lady bird Hyperapsis spp. ( Munir et al. 1992). 2.2. The use of botanical (Neem tree) in the control of aphids: Neem is a forest plant in Sudan .It had been introduced from India. It is now extensively grown throughout the country as shade tree in public parks, street and residential areas. It is also used in shelter belts in dry area. The origin of neem tree is south-east Asia, where it has been used in traditional medicine, production of insecticides, lamping oil, soap from manufacturing the margosa oil pressed from its seed kernels which contain up to 40% oil (Siddig, 1991). 2.2.1. Chemical constituents of neem: All of the well characterized compounds identified in Neem tree belong to the class triterpenoids. These include Azadirachtin, Salannin, Salannol, Salannol acetate, 3-deacetyl-salannin, nimbin and nimbidin and nimbolid ( Schmutterer and Zebitz, 1984). 2.2.2. The mode of action of azadirachtin: Within azadirachtin molecule, the declain fragment is responsible for the insect growth regulation and development effects observed, while the 18 hydroxylfuran fragment causes the anti-feedant effects more widely observed among target species (Aldhous, 1992). The IGR and anti-feedant effects of azadirachtin are independent of each other, but both remain relative to concentration (Koul & Isman 1991). Schmutterer (1990) suggested that azadirachtin modifies the developmental programs of insects by influencing hormonal systems, especially that of ecdysone. The effects of azadirachtin are both dose and time dependent, prevent both ecdysis and apolysis, and can cause insect death. 2.2.3. The side effect of neem kernel Aqu- Ext on the aphids: Neem seeds and leaves extract gave better control of Aphis gossypii on cotton and potato, compared to the standard insecticides Sevin + Anthio and Endosulfan (Venkatrtan et al., 1987 and Sidding, 1991). Elshafie (2001) investigated the effect of three different neem formulations including NeemAzal T/S on Aphis gossypii, Jacobiasca lybica and Bemisia tabaci damaging potato and egg plants. He reported that neem preparation, particularly NeemAzal gave results which were comparable with Sumicidin- Neem seed water extracts gave good result against the flea beetle, Podagrica spp., white fly Bemisia tabaci Genn, cotton jassid Jacobiasca lybica Pruthi, Aphis spp. and spying boll worm Earias insulana Boisd on okra crop in the Sudan (Siddig, 1991). In the Sudan, neem seed and leaves water extracts at 1kg /40L of water,repelled foliage pest of potato i.e Bemisia tabaci, Jacobiasca lybica and Aphis gossypii increased yield by 0.5 tones/ha. Therefor, a pest 19 management program should include neem treatments for combating potato pests in Sudan (Siddig, 1987). Tertiary–methyl–butyl ether extract and methonolic extract of neem seed kernel (MENSK) were toxic to the first instar of Aphis pisum and Aphis fabae (Schauer, 1984). Dimetry and Schmidt (1992) counted more apetrous females of the bean aphid, Aphis fabae on plants in a choice test where as “NeemAzals” especially after application of higher concentrations (0.1 – 2%) deterred the aphids. A mixture of plant extract including neem at (1%, 4%, 10%) was highly repellent to the bean aphids, Acyrthosiphon pisum (Hunter and Ullman, 1992). Molting failure was observed in young aphids, Aphis pisum, exposed to bean plants sprayed with NSKEs (Schauer, 1984). Persistence of toxic neem oil to aphids was limited to a bout 3 to 7 days outdoors and it remained active for longer periods on plant in green house ( lowery , 1992). Lowery (1992) observed that the third instar of Myzus persica and Nasonovia ribisnigri exposed to 5% neem oil molted successfully to adults, but they were generally much smaller in size than those in the control. Many homopterous and aphids are highly sensitive to neem products (Elshafie, 200l). Neem based insecticides can be effective in controlling aphid (Schauer 1984, schmutterer, 1990 and Nisbet et al., 1994). 2.3 The Coccinellids: 2.3.1. The Classification of Cheilomenes propinque (Muls): 20 The Coccinellid Cheilomenes propinque (Muls) belong to the order Coleoptera, suborder: polyphaga, superfamily Cucujoidea and the family Coccinellidae Imms, 1957) . C. propinque is a polyphagous Coccinelld with a wide range of prey acceptance. It has been reported by many workers to prey on different aphid species and other agronomic and horticultural pests in Africa. In the sudan this predator was previously reported to feed on Melaphis sacchari (Zehnter) and Myzus utricae (Kalt) Cited by Bashir (1968). Pea cock (1913) and Lamborn (1914), also reported that A. gossypii infesting cotton in south Nigeria was kept well in check by C. propinque (Ibrahim, 1988). 2.3.2. Population dynamic of C. propinque (Muls). Adults and larvae of C. propinque were common in lucerne fields during April to October. They seemed to migrate from by nearly sorghum fields, they where already present in large numbers during January-April and when M. sacchari started to decline (Basher, 1968). Similar observations were made by Mohamadein (1979), but he added that C. propinque was the least prevalent in lucerne fields. Bashir (1968), showed that this predator is of less important in the natural control of A. craccivora. The sensitivity of C. propinque to pesticides was reported by Mohamadein (1979). The two species almost disappeared from the sprayed fields until the end of September under natural conditions, the efficiency of C. propinque as a predator of the cowpea aphid may be affected by inter and intra specific competition ….etc. However, the laboratory data are supportive of considering C. propinque as an important biotic control agent of the cowpea aphid (Ofuya, 1986). 21 2.3.3. The efficiency of Coccinellids in checking aphid population: The predators (Coccinellids) play an important role in the control and reduction of aphids internationally. The following reports are of pertinent. Kenchaiah and Porte (1989) reported that field surveys were conducted in several districts of Kanataka in India during Kharif (autumn) and rabi (spring) seasons of 1984 – 1989 to determine the major insect pests of groundnut. The average incidence of aphids during the Kharif and rabi seasons was moderate (16-35 %), and low (1–5%), respectively. The maximum incidence of aphid population on groundnut was recorded between July and September, 1987–1988. Increases in the number of aphids were controlled by the Coccinellid predators Coccinellia sp, and Menochilus sexmoculatus Fabricius (Cheilomenes sexmoculata F.). In the Harihar and Chitradurga districts, 5–10 larvae of aphid populations during mild weather. The predation efficiency of the Coccinellds Coccinella transversalis Fab, and M. sexmaculatus against 3 species of aphids (Aphid faba Scop., A. Gossypii, and A. nerrii) were discussed in relation to prey preference, and bioenergeties of the predators. C. sexmeculata tended to exhibit lower predatory efficiency than C. transsversalis. Feeding preference, longevity and reproductive potential, beside predatory efficiency of these predators showed significant correlation with the aphid prey quality (Babu and Ananthakmishan, 1993). The relationship between A. craccivora on groundnut and its predators was studied in Gujarat, India, 1981. The predators collected were 22 identified as H. variegata, C. Septempunctata, and M. sexnaculatus. An infestation index was calculated by dividing the number of plants showing various extents of damage by the total number of plant examined. A positive correlation was observed between aphid index and population of active states of the predators during March, while in April the aphid population decreased with increased abundance of the Coccinellids. The predators population declined by the 1st week of may, most of them having migrated to other areas, due to decrease in prey abundance. It was concluded that application of insecticides is unnecessary, if the Coccinellds are present in the groundnut crop in Summer (Butani and bharodia, 1984). 2.3.4. Side–effects of neem seed Aqu-Ext on the Coccinellids. The side effects on beneficials and other non- target organisms is important aspect concerning the use of neem products in integrated production and organic farming. Neem products can cause some mild effects on non- target insects and therefore, recommended for pest control especially in IPM (Schmutterer, 1995). Experiments with adults of the Coccinellid beetle (Coccinella septempunctata) kept on neem oil did not show increased mortality or reduction of fecundity when compared with untreated control, but the metamorphosis of the larvae was interrupted (Schmutterer, 1981). Lowery and Isman (1995), reported that the number of larvae of predacious Cecidoryiid’ was reduced in the field after application of neem seed extract and neem oil as compared with the control. 23 Neem was found to be harmless or slightly harmful to natural enemies like predators and parasites. Predtors spider. L pseudonulata was not affected even by 50g neem oil per individual (Saxena, et al., 1984). Lowery and Isman (1995) reported that the edosion of adult Coccinellids (Coccinella undecimpunctata) and the syrphid (Eupeodes fumipennis) was negatively affected. El shafie (2000) reported that the application of neem water extract, neem oil and NeemAzal did not reduce the population of the aphid predators Scymnus spp. He observed that the reduction in the population of the Coccinellid was correlated to the reduction of the prey and was not due to the dirct effect of neem preparation. Neem research has led to the assumption that fewer side-effects on beneficial organisms could be expected when neem is compared with the numerous pesticides with strong contact and neuro –toxic mode of action against the active Coccinellids. The application of neem formulation in the laboratory, when used with the same concentration as in the filed, lead to a strong side-effect on beneficial organisms than the field, where, they are often not hit by sprays and where relatively rapid degradation of active ingredients takes place. Spraying of neem oils against the sorghum aphid Melanoaphis sacchari in the field trials at concentration of 0.1, 0.2, 0.5, and 1.0, did not harm the larvae of Coccinellids or the larvae of syrphids (Schmutterer, 1995). Green house experiments were conducted at Shambat area to investigate the systemic and contact effects of different neem formulations on the Coccinellid Hippodamia variegata (Goeze). The results indicated 24 that Sumicidin® 20% Ec (0.5mL/L) was the most effective on the different developmental stages of the Coccinellids specially, the larvae compared to the neem products, while neem seed water extract (2.5%) showed the least effect compared to celatlor® (4mL/L). It is found that feeding of Coccinellid on aphid treated topically was the most effective treatments compared to direct spraying and aphid treated systemically. Also feeding of Coccinellid on aphid treated systemically gave weak effect as compared with other treatments (Hamad, 2003). 25 CHAPTER THREE MATERIALS AND METHOD 3.1. Aphids culture: Four plastic cups, 30 cm in diameter, were sown with faba bean (Vicia faba L.) cultivar Hudieba 93 and every cup was kept inside small cages (62.5×45×45cm) made of wooden frames and wire-mesh at the sides and covered with muslin cloth at the top (plate 1). Adults of the Black bean aphid [Aphis faba (Scop)] were obtained from the Demonstration farm of the Faculty of Agriculture, University of Khartoum in Shambat from untreated faba bean plants. The insects were collected in Petri-dishes with the aid of fine camel hair brush and were used to infest the faba bean plants. Insects were left to reproduce and develop for many generation before being used in the different experiment. 3.2. Collection and rearing of Cheilomenes propinque(Muls): Stock culture of C.vcina (Muls) established from pupal stages collected from the medicag sativa grown in the farm of the Faculty of Agriculture, University of Khartoum in Shambat. The collected pupae were put in Petri-dishes. Cages (62.5×45×45cm) were used to rear the Coccinellid in the green house (plate 1). The Adult insects were f ad libitum on aphid reared on faba bean plants. They were left to reproduce and kept as test insects until the end of all experiment. 26 Plate 1. Aphid and Coccinellids rearing cage 27 3.3. Preparation of test materials: The collection of neem seeds, the preparation of powder, the extraction and methods of storage were carried out according to those described by Dreyer (1984), Ermal et al (1984), Schmutterer (1988) and Siddig (1990). 3.3.1. Neem seeds kernel powder (NSKP): Mature fallen neem fruit berries were collected in Shambat area. The seeds were washed and crushed lightly by stone to break the seed coat which were then separated from the broken seed kernel by winnowing, Pestle and motor were then used to mill the kernels into fine powder. 3.3.2. Preparation the aqueous- extract: The neem seeds kernel powder prepared as above, was put in tap water in plastic bucket and stirred thoroughly with a piece of wood for 10 min, and allowed to stand overnight. After 24 hours the mixture was reagitated for 10 min and filtered, using a light cloth, to prevent blocking of the sprayer’s nozzle. 3.4. Synthetic insecticide: Actara® neonicotinoid was used at recommended dose (1g/1.25 liter water). This was to allow the comparison of its side–effects with that of aqueous-extract of neem seeds kernel. This insecticide was chosen because it has been proved as effective agent against 160 pest species in 115 crops. Moreover, it has a broad action against a wide spectrum of sucking and leaf –feeding pests. 28 3.5. Equipment and spraying technique: A plastic hand sprayer (0.5 liter) was used to apply the test chemicals in all experiment. The pressure was maintained by hand continuously during spraying (plate 2) . This sprayer gives very small droplets and hence a good surface coverage. The sprayer was washed thoroughly with water after each concentration used. The preparations were poured into the sprayer using a glass flask and filtered through piece of fine cloth. 3.6. The efficiency of neem aqueous-extract on the Aphis fabae (Scop): Three experiments were carried out in the Green house of the Faculty of Agriculture, University of Khartoum, Shambat “15°. 40 °North latitude, longitude 32°. 32´ and 280 meters above sea level” for the purpose of assessing the systemic and contact efficacy of neem seed kernel and time of application in controlling the black bean aphid Aphis fabae (Scop). The experiments were carried out during the period from November to December 2004. 3.6.1. Experiment–I (Soil drenching): This experiment was carried out when the faba bean (Vicia faba L) seedling reached the four leaves stage in winter season to study the systemic effect of the aqueous extract of NSKP on the faba bean aphid when applied by drenching the soil before infestation (pre- infestation application). 29 Plate 2. The sprayer used in the experiments 30 Twenty plastic pots 10 cm wide at the top and half-filled with sterile soil, were planted with faba bean (Vicia faba L) Cultivar (Hudieba 93). Three seeds were put in each pot and after germination, the seedlings were thinned to two plants per pot. Each pot was then covered with mulslin cloth (transparent cloth) to allow the access of plants to sun light and to prevent contamination of test plants with any other insect (plate 3). The pots were treated with the different treatments before aphids were transferred on them. Each treatment was replicated four times. The infestation was carried out by placing 20 aphids on the plants in each pot. The aphids transfer was carried out 24 hours after treatments (post-infestation application). The soil drenching of each pot was carried out by spraying the soil by 20 ml. The control pots (untreated) were drenched with water only. The treatments were as follows: 1- 25 g NSKP/Liter water. 2- 50 g NSKP/Liter water. 3- 75 g of NSKP/Liter water. 4- Actara® neconicotinoid used at recommended Rate (1g/1.25Liter water). 5- Control (water only). The number of live insects (Aphids) were counted in each pot every 24 hours for 7 days. 31 B4 Plate 3. The experimental unit 32 3.6.2. Experiment II (Foliar application): This experiment was performed during the same period when seedlings reached the four leave stage in the winter season. The specific objective of the experiment was to study the effect of the NSK aqueous extract on faba bean aphids when applied by spraying the plant before infestation. Twenty plastic pots (10 cm) at the top and each pot semi-filled with sterile soil, were planted with faba bean (Vicia faba) Cultivar “Hudieba 93” three seeds per pot.The seedlings were thinned to two plant per pot. Each pot was coverd with muslin cloth, the pots were treated with the different treatments before they were infested. Each treatment was replicated four times. The infestation was carried out by placing (20) aphids on the plants in each pot. After 24 hours, the aphids were placed on the plants (post infestation application). A plastic hand sprayer as described above was used. The foliar parts of the plants on each pot was sprayed carefully to ensure good coverage of the shoot part of the plants. The control plots (untreated) were sprayed with water.The same treatments as mentioned in the first were used. The number of live insects (aphids) were counted and recorded in each pot every 24 hours for 7 days. 3.6.3. Experiment – III (Topical application): This experiment was performed consonantly with the above two experiments, in the winter season to study the effect of NSK aqueous extract when applied by direct spraying (Topical application). Twenty Petridishes were arranged in a completely randomized design (CRD), twenty aphids were placed in every petri-dishes and were then treated with different treatments. The insect was fed with a parts of faba bean plants 33 daily. Each treatment was replicated four times. The same treatments, as mentioned in the above experiments were used. The number of live insect (aphids) were counted every 24 hours for 7 days. 3.7. The side-effects of neem aqueous extract on the Cheilomenses propinque (muls): Four experiments were carried out, for the purpose of assessing the effect of the neem seed kernel applied topically on the natural enemies C. propinque (Muls). All experiments were carried out during the period from the November to December 2004. The different stages of the C. propinque (Muls)(Plate 4) were sprayer with neem seed water extract (NSWE) and the Actara® insecticide. 3.7.1. Egg stage: Ten eggs in a petri-dishes containing filter papers were sprayed to determine the side effect on hatchability. Acompletely randomized design (CRD)was used toarrange the experimental units. Each treatment was replicated four times, the data was taken 48 hours after treatment. 3.7.2. Larval stage: Ten larvae (3rd Instar) in a petri-dishes containing filter papers were sprayed to determine the mortality percentage. The each treatment was replicated four times. 3.7.3. Adult stage: Ten adult insects of C. propinque (Muls) in a petri-dish containing filter papers were treated topically with the different treatments to study the 34 mortality percentage. Each treatment was replicated four times. The treatments used in this experiments were as follows: 1. 25 g of NSKP per liter water. 2. 50 g of NSKP per liter water. 3. 75 g of NSKP per liter water. 4. Actara® used at recommended rate of (1 gram bar 1.25 liter of water). 5. Control (water only). 3.7.4. Feeding of C. propinque (Muls) on aphids treated topically: This experiment was carried out to determine the effect of treated aphids on the feeding of the adult of C. propinque (Muls). The aphids Aphis fabae (Scop) were sprayed with neem seed water extract (NSWE), Actara and water (control). They were offered to adult insect of C. propinque (Muls) to feed freely on them. The aphids were kept in Petridishes, and then the mortality percentage was calculated. Each treatment was replicated four times. The treatments were as follows: 1. 25 g NSKP per liter water. 2. 50 g NSLP per liter water. 3. 75 g NSKP per liter water. 4. Actara® neonicotinoid used at recommended Rate (1gram bar 1.25 liter of water). 35 5. Control (water only). The number of live insects (Coccinellids) were counted in each dish every 24 hours for three days. Statistical analysis: The collected data was statistically analyzed using ANOVA. The means were separated by DMRT. 36 Eggs Larvae 3 37 Adult Plate 4. The test stages of C. propinque. 38 CHAPTER FOUR RESULTS Generally, the synthetic insecticide Actara® Nenicotionid active ingredient thiomethoxam used with a recommended dose of (560g/ha) was significantly better in reducing the number of Aphis faba (Scop) on faba bean. The same insecticide resulted in a 100% mortality of Coccinellid C. propinque (Muls). The test neem products proved to be less harmful to the predator compared with Actara. 4.1.1. The effect of NSK Aqu-Ext, applied in the soil as drenching on aphids (drenching experiment). The result of this experiment illustrated that all treatments on the 1st, 2nd, 3rd, and 5th days after treatment showed highly significant difference (P<0.01) when compared with the control. However, on the 4th, 6th, 7th days there was no significant difference between NSK Aqu-Ext and control as whereas there was a highly significant difference between Actara insecticide and control. The NSK Aqu-Ext (75g) gave high mortality on aphids (A. fabae Scop) followed by (50g). There was no significantly different mortality between the concentration (25g) and the untreated control (Table 1, and Appendix A). 39 Table 1. Survival and corrected mortality (CM) of A. fabae (20 insects) fed on seedling of faba bean treated with NSWE (soil application). Days 1st Treatments 2nd 3rd 4th 5th 6th 7th Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Control 89.43a - 86.34a - 89.43a - 81.88a - 89.43a - 86.34a - 86.34a - 25g NSKP/l 80.49a 5 74.94b 8 78.79b 5 72.94a 5.19 86.34a 1.25 73.59a 3.80 75.90a 3.80 50g NSKP/l 65.19b 19 69.25b 8 61.72d 22 70.09a 13.90 77.70b 6.25 82.38a 2.03 77.41a 5.06 75g NSKP/l 45.06c 50 73.23b 8 69.82c 12 68.60a 13.24 89.43a 0.00 80.17a 2.28 86.34a 0.00 Actara® 0.57d 100 0.57c 100 0.57e 100 0.57b 100 0.57c 100 0.57b 100 0.57b 100 C.V% SE± 12.53% 11.25% 7.27% 15.46% 7.16% 14.02% 11.73% 3.52 3.42 2.18 4.55 2.46 4.53 3.83 Figures followed by different letters are significantly different at P = 0.05 (DMRT). 40 4.1.2. The effect of NSK Aqu-Ext sprayed on the faba bean plants. The statistical analysis of the results showed that there was highly significant difference (P< 0.01) between all treatments and control on the 1st, 2nd , 3rd, and 4th days after treatment, however, on 5th, 6th, and 7th days there was no significant difference between NSK Aqu-Ext and control (Table 2, and Appendix B). The three concentrations of neem seed water extract showed the same effect on the first day after treatment. There was a significant difference between the neem treatment and the synthetic insecticide Actara® which gave a corrected mortality of 100%. No significant difference between the control and all treatments was obtained on 5th, 6th, and 7th, days after treatment indicating that this method of applying neem products was ineffective. 4.1.3. The effect of NSK Aqu-Ext sparyed topically on the aphids. Table 3and Appendix C summarized the effect of NSK Aqu-Ext, applied topiclly on the faba bean aphids. A highly significant difference between all treatments and the control was obtained on the 1st, 2nd, 3rd, and 4th days after treatment. However, there was no significant difference between the NSK Aqu-Ext and control. On the 1st day after treatment, all concentrations of NSK Aqu-Ext gave the same results. The neem treatments were however, significantly different from the control. On the 4th, 5th, 6th, and 7th, days after treatment, all concentrations of neem seeds kernel extracts showed no significant effect on aphid compared with the control. 41 Table 2. Survival and corrected mortality (CM) of A. fabae(20 insects) after foliar application of NSWE on faba bean (post infestation). Days 1st Treatments 2nd 3rd 4th 5th 6th 7th Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Control 89.43a - 89.43a - 86.34a - 89.43a - 81.88a - 81.88a - 83.26a - 25g NSKP/l 72.49b 12.5 65.55b 17.5 65.56b 16.46 78.46ab 7.5 86.34a 2.60 84.96a 1.3 80.17a 1.28 50g NSKP/l 74.77ab 12.5 57.72c 28.75 61.74b 21.52 68.02b 15 83.26a 1.30 84.96a 1.30 78.79a 2.56 75g NSKP/l 59.55b 26.25 70.76b 11.25 59.78b 25.32 68.02b 15 84.96a 1.30 77.41a 2.60 86.34a 1.28 0.57c 100 0.57d 100 0.57c 100 0.57c 100 0.57b 100 0.57b 100 0.57b 100 Actara® C.V% SE± 17.32% 6.58% 9.76% 12.53% 10.49% 11.97% 9.22% 5.14 1.87 2.67 3.82 3.54 3.95 3.03 Figures followed by different letters are significantly different at P = 0.05 (DMRT). 42 Table 3. Survival and corrected mortality (CM) of A. fabae( 20 insects) topically sprayed with NSWE. Days 1st Treatments 2nd 3rd 4th 5th 6th 7th Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Surviving C.M% Control 89.43a - 89.43a - 86.34a - 83.26a - 84.96a - 80.49a - 81.88a - 25g NSKP/l 56.86b 30 84.96a 2.5 72.76b 10.13 80.17a 1.28 89.43a 2.56 83.26a 2.63 77.41a 2.30 50g NSKP/l 53.86b 35 69.82b 12.5 50.85c 39.24 83.26a 0.00 86.34a 1.28 86.34a 3.95 86.34a 2.60 75g NSKP/l 51.61b 38.75 61.10b 25 58.83c 27.85 86.34a 1.28 86.34a 1.28 86.34a 3.95 86.34a 2.60 Actara® 0.57c 100 0.57c 100 0.57d 100 0.57b 100 0.57b 100 0.57b 100 0.57b 100 C.V% 7.97 11.08 15.32 8.94 8.04 10.14 10.16 SE± 2.01 3.39 4.13 2.98 2.79 3.42 3.38 Figures followed by different letters are significantly different at P = 0.05 (DMRT). 43 4.2.1. The effect of NSK Aqu-Ext on the Egg hatchability of C. propinque. In this experiment all treatments resulted in highly significant difference (P < 0.01), 48 hours after application when compared with the control. The best hatchability was obtained in the treatment of 25g NSK, followed by 50g and 75g respectively. The synthetic insecticide Actara® gave zero hatchability in contrast to the control (Fig1and Appendix D). 4.2.2. The effect of NSK Aqu-Ext on the 3rd, larva instar. There was no significant difference (P> 0.01) between the NSK (25g) , (50g) and the control on the 1st day after treatment. However, the concentration (75g) gave a significantly different mortality compared with the control. The same trend was observed on the 2nd day after treatment concerning the effect of NSK–Aqu- Ext All neem treatments were significantly different from the control. On the three days following the treatment, Actara resulted in a significant different mortality of 100% (Table 4 and Appendix F) 4.2.3. The effect of NSK Aqu-Ext on the Aduts of C. propinque (Muls). Table 5 and Appendix E showed that there was a significant difference (P<0.01) between all treatments and control. The mortality caused by Actara was 100% on all days after the treatment. The NSK-AquExt concentration gave the same results on the 1st day after treatment and the same trend was observed on the 2nd ,3rd, days after treatment. 44 . 90 80 Hatchability % 70 60 50 40 30 20 10 0 Control 25 gNSKE 50 gNSKE 75 gNSKE Treatment Fig. 1. The effect of topical application of NSK Aqu-Ext on the egg hatchability of (C. vicina "Muls") 48 hours after treatment 45 Table 4. Survival and corrected mortality (CM) of C. propinque adults (10 insects) after direct spraying with neem seed water extract. Days 1st Treatments 2nd 3rd Surviving C.M% Surviving C.M% Surviving C.M% Control 89.43a - 89.43a - 84.96a - 25g NSKP/l 67.50b 15 78.46ab 7.5 78.46ab 5.13 50g NSKP/l 59.41b 27.5 84.96a 2.5 65.47b 15.38 75g NSKP/l 67.94b 20 69.08b 17.5 71.97ab 10.26 Actara 0.57c 100 0.57c 100 0.57c 100 C.V% 14.38 14.51 15.16 SE± 4.10 4.68 4.57 Figures followed by different letters are significantly different at P = 0.05 (DMRT). 46 Table 5. Survival and corrected mortality (CM) of 3rd larvae (10 insects) of C. propinque after direct spraying with neem seed water extract. Days 1st Treatments 2nd 3rd Surviving C.M% Surviving C.M% Surviving C.M% Control 82.93a - 89.43a - 84.96a - 25g NSKP/l 70.25a 13.16 60.72b 30 84.96a 0.00 50g NSKP/l 75.30a 7.89 56.25b 35 78.32ab 10.26 75g NSKP/l 48.75b 42.11 66.77b 20 62.58b 20.51 Actara 0.57c 100 0.57c 100 0.57c 100 C.V% SE± 22.46% 22.55% 18.39% 6.24 6.17 5.73 Figures followed by different letters are significantly different at P = 0.05 (DMRT). 47 4.2.4. The feeding of C. propinque (Muls) on aphids treated topically with NSK Au- Ext. On the frist day after treatment, there was no significant difference (P> 0.01) between concentration 25g NSK Aqu-Ext and the control, however, there was a highly significant difference among the other treatments (Table 6 and Appendix G). On the 3rd day after treatment, the results showed highly significant difference (p<0.01) between 25g of NSK –Aqu- Ext,75g NSK Aqu-Ext concentration and the control, but there was no significant difference between the 50 g NSK Aqu- Ext concentration and control. 48 Table 6. Survival and corrected mortality (CM) of C. propinque adults (10 insects) fed on aphids treated topically with neem seed water extractNSWE. Days 1st Treatments 2nd 3rd Surviving C.M% Surviving C.M% Surviving C.M% Control 89.43a - 80.49a - 76.03a - 25g NSKP/l 76.43a 10 75.57ab 7.89 60.27b 18.92 50g NSKP/l 54.53b 35 74.00ab 5.26 74.00a 2.70 75g NSKP/l 55.39b 35 59.13b 23.68 64.33ab 13.51 0.57c 100 0.57c 100 0.57c 100 Actara C.V% SE± 18.59% 18.73% 14.91% 5.14 5.43 4.10 Figures followed by different letters are significantly different at P = 0.05 (DMRT). 49 CHAPTER FIVE DISCUSSION 5.1. General: In the last decade, the neem tree Azadirachta indica has come under close scientific scrutiny as a natural source of noval pest control materials. Neem derivatives were found to have systemic and contact effects against many sucking and chewing insect pests. As Homopteran plant insect pests generally feed by sucking plant sap, neem derivatives may affect them systemically. The natural enemies play a fundamental role in integrated pest management programs (Michel et al., 1997) and the study of the side effect of botanicals on them is very essential before being incorporated in any control program. In the Sudan, Coccinellids are important predators in several field crops specially in winter. A part from some limited laboratory experiments, there is no extensive field research in Sudan to determine the deleterious impact of neem products on natural enemies and other beneficial insects (Osman and Bradley, 1993). Schmutterer, (1992) stated that only little work has been generally carried our on the effects of neem on parasitoids or predators. 5.2. The effects of NSK Aqu-Ext on Aphis fabae : As shown in table 1. in the soil drenching experiment, the chemical insecticide Actara® showed the best results in reducing the aphids numbers. Mudathir (2000) found similar results, by using Sumicidin® in the field to control aphids on okra. The effectiveness of NSK Aqu-Ext increased with 50 time up to the first three days after application and decreased in the following last three days after treatment, this might be due to the high concentrations of neem extracts in the plant tissue in first 3 days. Heyde et al. (1984) found that the mortality of aphids increased up to (8 days) when he used NSK powder. He went to say that the residual effect of bioactive ingredients will be prolonged if a systemic effect (translocation of active material) takes place, directing the uptake of material by the treated plant. In foliar application – experiment, as shown in Table 2. Actara® NSK Aqu-Ext were significantly effective in reducing aphids numbers compared to the control, the Actara® gave highly mortality than NSK AquExt. This could be explained by the fact that neem products have no knockdown effect compared with synthetic pesticides. The NSWE treatment (Post infestation) was significantly different (P < 0.01) on the first 4 days following application. Lowery and Isman (1993) reported that neem seed oil, 48 hrs following aphid infestation (post-infestation) were not significantly different (P > 0.05) from the control. This insignificant effect might be due to the slow absorption of neem oil by plants. As shown in the Table 3, (the topical experiment), Actara® gave highly mortality than NSK Aqu-Ext concentrations. Lowery et al. (1993) found in a field experiment, that foliar applications of NSO resulted significantly better control of M. persicae on pepper. The difference between his results of the findings of present investigation could be due to different preparations of neem and the different conditions under which the experiments were undertaken. 51 5.3. The effects of NSK Aqu-Ext and Actara® on the different stages of the Coccinellid Cheilomenes propinque (Muls): As shown in Fig. 4, the Actara® pesticide gave zero hatchability while the hatchability was slightly affected by NSWE. The same results was obtained by other authors on different species of Coccinelld particularly Coccinella septempunctata (Schmutterer, 1981 and Kaethner, 1990). The two authors used AZT-VR-K (1000 ppm) and combined it with neem oil (250 – 30000 ppm). As shown in table 5, the insecticide Actara® spryed on Adults C. propinque (Muls) topically gave 100% mortality. The same results were obtained by (Sharma and Adlakha, 1981) who found that “Feniforthion” was very toxic to other species of Coccinellid C. septempunctata whereas “endosulfan” did not appear to be harmful. The effect of NSWE on adults is harmless as compared with synthetic pesticide Actara®. When development and growth are concerned, neem products can affect hormonal balance and hence development and growth of young stages and later fertility of adults. The same results were obtained by Hoelmer et al., (1990) who reported mortality of the coccinellid beetles Delphastus pusillus and Scymnus sp reared on leaf discs dipped in Morgosan-O and kept in sealed Petri-dishes and glass vials for two weeks. According to Schmutterer (1990) neem products have relatively weak contact toxicity NSWE applied in the field at rate of 50g/l water proved to be safe for the Coccinellid Scymnus sp. (Elshafie, 2001). The topical treatment of (3rd instar) the larvae of Coccinellid C. propinque (Muls), with neem concentrations of 25g and 50g gave no 52 significant difference (P < 0.01) on the first day after treatment compared with the control. However, significant mortality occurred during the following days. Ossiewatsch (2000) reported the same results on the C. septempunctata. He also reported that high mortality of larvae occurred in laboratory when the insects were dipped in NSWE. As shown by the results presented in Table 7 adults of Coccinellid fed on aphid treated topically with NSWE, showed an increase in mortality with increase concentrations of neem extract on the first day. These results are supported by the findings of Hoelmer et al. (1990) who reported that larvae and females of Coccinellids Delphastus pusills and Scymuns sp. fed on eggs of Bemisia tabaci treated with Morgosan-O for several days showed no mortality and females continued egg laying. 5.4. Conclusions and recommendations: Neem seed kernel aqueous extract (NSKWE) represent a relatively safe, and inexpensive, source of materials for integrated pest control in agroecosystem exosystem as compared with synthetic insecticides. The application of neem into the soil could possibly decrease the photochemical degradation and increase the possibility of controlling some soil pests like nematodes. The neem products applied systemically could be considered safe for the natural enemies. However, the effect of neem on predators 53 and prasitoids of aphids should be studied more fully before any practical application is carried out. Neem seed kernel water extract proved to be less harmfull to the aphidophagous Coccinellids compared with the chemical insecticide Actara®. Therefore, it is recommended for use against agricultural pests within the framework of IPM and organic farming. 54 REFERENCES Aldhous, P. (1992). Neem chemical: the pieces fall into place science 258: 893. Babu, A. and Ananthakrishan, T. N. (1993). Predatory efficiency, reproductive potential and bioenergetics of Coccinella transversalis L. and Menochilus sexmaculatus F. 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ANOVA: Analysis of variance tables: 1st day:source D.F Sum of Squr Mean squr F. cal F. table Treatments 4 19975.931 4993.983 100.825 0.000 Error 15 742.968 49.531 Total 19 20718.90 C. V = 12.53% SE= ± 3.52 2nd day: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 18823.668 4705.917 100.431 0.000 Error 15 702.861 46.857 Total 19 19526.529 C.V. 11.25% SE = ± 3.42 66 3rd - day:source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 19401.752 4850.438 254.024 0.000 Error 15 286.416 19.094 Total 19 19688.168 CV = 7.27% SE = ± 2.18 4th day: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 17386.103 4346.526 52.576 0.000 Error 15 1240.060 82.6761 Total 19 18626.163 C .v = 15.46% SE = ± 4.55 5th –days source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 23573.682 5893.420 243.668 0.000 Error 15 362.794 24.186 Total 19 23936.475 C.V = 7.16% SE = ± 2.46 67 6th – days: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 20847.134 5211.748 63.563 0.000 Error 15 1229.913 18.994 Total 19 22077.047 C. V = 14.02% SE = ± 4.53 7th – day:source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 21337.778 5334.444 90.897 0.000 Error 15 880.474 58.698 Total 19 22218.251 C. V = 11.73% SE = ± 3.83 68 APPENDIX (B) The analysis of variance tables at the effect of (NSKE) on the aphid (A. fabae Scop) treated as foliar on the plants. 1st day : source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 19081.530 4770.382 45.119 0.000 Error 15 1585.928 105.729 Total 19 20667.458 C.V =17.32% SE = ± 5.14 2nd- day:- source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 17996.050 4499.013 321.857 0.000 Error 15 209.674 13.978 Total 19 18205.725 C.V = 6.58% SE = ± 1.87 3rd – day:- source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 16497.332 4124.333 144.178 0.000 Error 15 429.089 28. 606 Total 19 16926.421 C.V = 9.76% SE = ± 2.67 69 4th –day :source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 19454.351 4863.588 83.498 0.000 Error 15 873.716 58.248 Total 19 20328.067 C.V = 12.53% SE = ± 3.82 5th – day:- source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 22377.661 5594.415 111.894 0.000 Error 15 749.964 49.998 Total 19 23127.626 C.V =10.49% SE = ± 3.54 6th day: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 21529.498 5382.374 86.294 0.000 Error 15 935.587 62.372 Total 19 22465.085 C.V = 11.97% SE = ± 3.95 70 7th day: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 21426.886 5356.722 145.395 0.000 Error 15 552.637 36.842 Total 19 21979.523 C. V =9.22% SE= ± 3.03 71 Appendix ( C) The analysis of variance tables of the effects of (NSKE) on aphid (A. fabae Scop) treated as topically on the aphid. 1st day: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 16246.272 4061.568 251.366 0.000 Error 15 242.370 16.158 Total 19 16488.642 C.V = 7.97% SE = ± 2.01 2nd day:- source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 20447.766 5111.941 111.196 0.000 Error 15 689.584 45.972 Total 19 21137.350 C.V = 11.08% SE = ± 3.39 3rd day: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 17144.346 4286.087 62.943 0.000 Error 15 1021.415 68.094 Total 19 18165.762 C. V = 15 .32% SE = ± 4.13 72 4th day :source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 21954.051 5488.513 154.221 0.000 Error 15 533.829 53.589 Total 19 22487.880 C.V= 8.94% SE = ± 2.93 5th day :source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 23819.896 5954.974 190.748 0.000 Error 15 468.286 31.219 Total 19 24288.183 C.V = 8.04% SE = ± 2.79 6th day:- source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 22426.982 5606.746 120.034 0.000 Error 15 700.643 46.710 Total 19 23127.626 C. V = 10.14% SE = ± 3.42 73 7th day: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 21958.314 5489.578 120.146 0.000 Error 15 685.365 45.691 Total 19 22643.679 C.V = 10.16% SE= ± 3.38 74 APPENDIX (D) The analysis of variance of tables of the effects of (NSKE) on egg hatchability of the C propinque (Muls) after 48 houre. source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 9407.170 2351.792 360.170 0.000 Error 15 975.305 65.020 Total 19 10382.475 C.V = 13.81% SE = ± 4.03 75 APPENDIX (E) The analysis of variance table of effects of (NSKE) on the adults predation C propinque (Muls) treated as topically. 1st – day :source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 17887.197 4471.799 66.664 0.000 Error 15 1006.191 67.079 Total 19 18893.388 C.V. = 14.38% SE = ± 4.10 2nd day:- source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 21372.545 5343.136 61.002 0.000 Error 15 1313.851 87.590 Total 19 22686.396 C.V = 14.51% SE = ± 4.68 3rd day :- source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 18474.950 4668.738 55.873 0.000 Error 15 1253.387 83.559 Total 19 19928.337 C.V = 15.16% SE = ± 4.57 76 APPENDIX (F) The analysis of variance tables of the effect of(NSKE) on the larvae of & ( 3rd in star ) of (C propinque Muls) treated or spraying topically. 1st day : Source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 17699.286 4424.821 28.405 0.000 Error 15 2336.682 155.779 Total 19 20035.968 C. V = 22.46% SE= ± 6.24 2nd day: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 17282.065 4320.516 28.346 0.000 Error 15 2286.303 152.420 Total 19 19568.368 C.V= 22.55% SE = ± 6.17 3rd day: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 20378.270 5094.567 38.823 0.000 Error 15 1968.399 131.227 Total 19 22346.669 C.V = 18.39% SE= ± 5.73 77 APPENDIX (G): The analysis of variance tables of the effects all the adult predation Coccinellids (C propinque Muls) with the aphids (A fabae Scop) treated tropically with (NSKE). 1st day :source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 18430.089 4607.522 43.653 0.000 Error 15 1583.222 105.548 Total 19 20013.311 C.V = 18.59% SE = ± 5.14 2nd day:- source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 17481.233 4370.308 37.090 0.000 Error 15 1767.464 117.831 Total 19 19248.697 C.V= 18.73% SE= ± 5.43 3rd day: source D.F Sum of Squr Mean squ F. cal F. table Treatments 4 15522.289 3886.572 57.597 0.000 Error 15 1010.617 67.374 Total 19 16532.906 C.V = 14.91% SE = ± 4.1 78 79
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