STUDIES ON THE INSECTICIDAL PROPERTIES OF EXTRACTS FROM ROOTS OF PRURIENS () AGAINST , LOCUSTA MIGRATORIA AND , SCHISTOCERCA GREGARIA

by

Abdalla Mohamed Abdalla B.Sc. ( Agric. ), Hon., 1987, University of Khartoum & M.Sc.( Agric.) 1993, University of Khartoum

A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in the University of Khartoum, Sudan

Supervisor: Prof. S. El-Bashir, University of Khartoum, Sudan Co-supervisors: Prof. M. Lecoq & Dr. M. H. Luong-Skovmand, CIRAD-Prifas, Montpellier, France

Department of Crop Protection Faculty of Agriculture University of Khartoum

June 2004

I

DEDICATION

To My: mother (Bit Omer Nour El-Zain), father, sisters and brothers with love and respect

II ABSTRACT

Locusts and their control have attracted a plethora of attention especially in the context of eco-toxicological studies and development of environmentally friendly control alternatives. products have proved to be suitable candidates that fit reasonably well in locust management programs. This manuscript, contains results of laboratory trials undertaken, in CIRAD- Prifas in Montpellier, France, during the period extending from 2000 to 2003 mainly to evaluate some botanical extracts against the Migratory locust, Locusta migratoria (Linné 1758) (: ) and to further investigate the bio-activity of Mucuna pruriens (Fabaceae) as locusticide against the foregoing species and the Desert locust, Schistocerca gregaria (Forskål 1775) (Orthoptera: Acrididae). A field survey was conducted in Western Sudan where 104 respondents were interviewed for their experience with Mucuna as pest control agent and about their indigenous knowledge in the field.

In this thesis, the importance of these locusts, their control and the role of plant materials as locusticides are reviewed. The varying views on locust control strategies are also discussed. Materials from some plant species namely: Mucuna pruriens, Adenium obesum, Azadirachta indica and Calotropis procera were collected from four different sites in the Sudan and processed to perform extraction and laboratory tests in France. Plant extracts were prepared using water or water/ethanol as solvents. These plant extracts were screened for their locusticidal properties and mode of action (contact and stomach) against the Migratory locust. The knockdown effect, mortality and time to death were recorded to evaluate the efficacy. Root extracts from Mucuna pruriens (Fabaceae) were used against the Migratory and the Desert locusts to determine the contact and stomach modes of action. Extracts of neem seed kernels were used as standard botanical insecticide and Deltametherin as standard synthetic locusticide. Mucuna mixed with reduced concentrations of Deltametherin was evaluated for compatibility, additive effect and/ or the possible synergism. Knockdown effect on locust was recorded one hour after treatment while mortality was recorded on 24h. basis.

Mucuna extracts were evaluated through a series of bioassay tests by applying concentrations of the extracts (by direct spraying, topical application and dipping of the

III locust in the tested plant materials) to investigate their efficacy as contact locusticides. As stomach poison the effect of the extracts was manifested through provision of treated wheat seedlings, cabbage leaf-discs and impregnated filter paper offered to the locusts. Mortality and faecal output was recorded in each test. Data were statistically analyzed using M-statc software statistical package, representative data for each test are presented, the most significant results are discussed and some recommendations are given.

The bio-tests have shown that Mucuna extracts act both as contact and stomach poisons. As contact locusticide mortality of up to 98% and 99% was achieved by direct spaying of water and water/ethanol extracts of Mucuna at 50g/l on the Migratory locust respectively. In topical application tests high concentration of Mucuna at 200g/l is required to cause mortality of 90% and relatively low knockdown effect was detected but it was statistically higher than that of the control treatment. Dipping of the in Mucuna extracts caused knockdown effect and mortality which were significantly higher than those obtained in untreated locust under untreated control. Mortality of up to 99% were obtained when locusts was fed on wheat seedlings treated with Mucuna at 50g/l (water/ethanol extract) indicating high stomach action. Mucuna extracts also appeared to act faster on locust than neem extracts. The effect given by Mucuna extracts was statistically similar to that of the standard botanical insecticide "neem" and the standard chemical insecticide Deltametherin "Decis". The toxic effect of low doses of Decis was enhanced when the insecticide was applied in combination with Mucuna root extracts. The Mucuna plant materials could be stored for a period of up to three years without serious losses in efficacy.

The study concluded that: Mucuna being a cheep natural product that can easily be obtained and processed in a simple way, may be of practical importance in future crop protection activities. For the upcoming studies further laboratory investigations on the biochemical properties and eco-toxicological aspects of the active molecules are recommended. Moreover, semi-field and field studies should be undertaken before recommending the root extracts for use as a pest control product.

IV ARABIC ABSTRACT

V

VI ACKNOWLEDGEMEMTS

I wish to express my sincere thanks for my supervisor Prof. S. EL-Bashir of the Dept. of Crop Protection, Faculty of Agriculture, University of Khartoum who provided guidance throughout all the stages of this work..

It is a pleasure to acknowledge with gratitude and sincere thanks the continuous help and guidance I received from my co-supervisors Prof. Dr. M. Lecoq and Dr. M. H. Luong- Skovmand of CIRAD-Prifas in Montpellier-France where most of this work was conducted. The kind supervision, the fruitful suggestions, the constructive criticism and the family atmosphere created and provided by each of them throughout the course of this study are highly appreciated.

The information provided by Dr. Abu El-Gasim Abdalla, University of Zalingai, and Dr. J. Duranton, CIRAD-Prifas, regarding Mucuna identification is very much appreciated. Provision of locust as starter for mass rearing by Dr. M. H. Luong-Skovmand (CIRAD- Prifas, Montpellier-France), Prof. Dr. M. O. Bashir (ICIPE, Port-Sudan), and Mr. M. A. Ould Baba (Centre de lutte antiacridienne de Mauritania CLAA,) is acknowledge with gratitude.

Special thanks and appreciations are reserved to Mr. T. Rachadi, Mr. A. Foucart, Mr. Emanuel and Mrs. L. .Schmidt of the laboratory of CIRAD-Prifas for their help throughout the study period in France. Thanks are extended to Dr. Abdel-Mohsin, University of Zalingai and My colleague Wad Hamid from University of Kordofan for the help offered during the field survey in Western Sudan. One should not forget to acknowledge the kind assistance of Mr. Idriss from ARC, El-Obeid and Mr. El-sarry El- Sheikh from Gezira University in the statistical analysis of the data presented in this thesis. The indispensable statistical consultancy provided by Dr. M. Kheir. (University of Kordofan) and Prof. F. El-Haj (ARC, El-Obeid) are very much appreciated. The printed materials I received, respectively, from Dr. H. A. El-Shafie ( Department of Crop Protection, Faculty of Agriculture, University of Khartoum) and Prof. R. Peevling (

VII Institute of Environmental Sciences, University of Basle, Switzerland) in the area of Neem effect and eco-toxicology were of a great help.

I would like to thank the Staff in the Dept. of Crop Protection, Faculty of Agriculture, University of Khartoum, especially Prof. El-Khidir and Dr. El-Taigani El-Tahir.

The fund provided by The French Government and The University of Kordofan is very much appreciated and acknowledged. Without such a generous financial support it would have been difficult to conduct this work properly.

Last but not least a very special recognition and thanks with indebtedness are reserved to my family especially my brother Mansour for the moral sport provided and for the many prayers which paved my way forward.

VIII

TABLE OF CONTENTS

DEDICATION...... II ABSTRACT ...... III ARABIC ABSTRACT ...... V ACKNOWLEDGEMEMTS...... VII TABLE OF CONTENTS...... IX TABLE OF ILLUSTRATIONS...... XIII LIST OF FIGURES ...... XIII LIST OF TABLES ...... XIV

1 INTRODUCTION...... 17

2 REVIEW OF LITERATURE ...... 23

2.1 Locust control...... 23 2.1.1 Importance of Locust and Locust control...... 23 2.1.2 Locust control strategies...... 25 2.1.3 Environmental impact of Locust control ...... 26 2.1.4 IPM of Locusts and ...... 26

2.2 in Locust control...... 27 2.2.1 Background on the history of plant materials in pest and disease control.. 27 2.2.2 Potentiality of Plant derived compounds as pest control agents ...... 28 2.2.3 Selection of plant materials for bio-test...... 30 2.2.4 Role of plant materials in pest control...... 30 2.2.4.1 Role of Neem (Azadirachta indica) in pest control ...... 30 2.2.4.2 Role of Neem in Locust and grasshoppers control ...... 31 2.2.4.3 Role of other Meliaceae in Locust and grasshoppers control ...... 33 2.2.4.4 Role of plants compounds other than Meliaceae ...... 33 2.2.4.5 Role of plant materials in integrated pest management ...... 34 2.2.4.6. Eco-toxicological risk assessment and the side effects of botanical insecticides ...... 35

2.3 Mucuna pruriens...... 35 2.3.1 Literature on Mucuna pruriens...... 35 2.3.2 General information on Mucuna pruriens ...... 36 2.3.2.1 and synonymy of Mucuna...... 36 2.3.2.2 Botanical description of Mucuna ...... 37 2.3.2.3 Geographical distribution of Mucuna ...... 39 2.3.2.4 Historical overview on Mucuna ...... 39 2.3.2.5 Mucuna in Folklores...... 39 2.3.3 Uses and chemistry of Mucuna ...... 40 2.3.3.1 General uses of Mucuna...... 40 2.3.3.2 Agronomic uses of Mucuna ...... 40 2.3.3.3 Chemistry of Mucuna...... 42 2.3.4 Mucuna as insecticide...... 43 2.3.4.1 Why Mucuna pruriens is selected ?...... 43

IX 2.3.4.2 Insecticidal properties of Mucuna ...... 43

2.4 Bioassay methods for plant materials ...... 44 2.4.1 Contact bioassay ...... 44 2.4.2 Topical bioassay ...... 45 2.4.3 Dipping bioassay ...... 45 2.4.4 Testing for stomach action ...... 45 2.4.5 Filter paper test ...... 46 2.4.6 Bioassay test of botanical compounds under choice/no-choice state ...... 48 2.4.7 Effect of time of exposure on activity of botanical treatments...... 48 2.4.8 Dose-response relationship...... 48 2.4.9 Insecticides mixtures and synergism ...... 49

3 MATERIALS AND METHODS...... 50

3.1 Field survey...... 50

3.2 Collection and preparation of the plant materials for extractions...... 51

3.3 Extraction of the plant materials...... 55

3.4 Locust rearing ...... 55 3.4.1 Rearing of Migratory Locust ...... 56 3.4.2. Rearing of the Migratory and Desert Locust ...... 57

3.5 Screening for insecticide properties of plant materials ...... 58 3.5.1 Objectives of screening ...... 58 3.5.2 Bioassays of the plant materials ...... 58 3.5.2.1 Contact action of the plant extracts ...... 59 3.5.3.2 Stomach action of the plant extracts ...... 60

3.6 Evaluation of biological activities and the mode of action of M. pruriens extracts ...... 60 3.6.1. Contact efficacy of Mucuna pruriens extracts ...... 61 3.6.1.1 Spraying Mucuna extracts...... 61 3.6.1.1.1 Efficacy on the 2nd nymphal instars of the Desert Locust ...... 61 3.6.1.1.2 Efficacy of Mucuna, Neem and Decis on the adult of the Desert Locust ...... 63 3.6.1.1.3 Efficacy on the 2nd nymphal instar of the Migratory Locust...... 63 3.6.1.2 Topical application of Mucuna extracts ...... 63 3.6.1.3 Dipping bio-assay with Mucuna extracts ...... 64 3.6.2 Stomach action of Mucuna ...... 65 3.6.2.1 Feeding Locusts, wheat seedlings treated with Mucuna extracts...... 65 3.6.2.2 Provision of treated wheat seedlings for limited feeding time...... 66 3.6.3 Feeding deterrence of Mucuna extracts...... 66 3.6.2.3.1 Cabbage leaf disc bioassay under no choice condition ...... 66 3.6.2.3.2 Cabbage leaf-disc bioassay under choice condition...... 66 3.6.2.3.3. Individual tests with filter paper...... 68

X 3.7 Persistence of bio-efficacy of Mucuna extracts from roots collected in different years...... 69

3.8 Effect of Mucuna mixed with Deltametherin (Decis)...... 69

3.9 Statistical analysis of the data...... 70

4 RESULTS...... 71

4.1 Field survey...... 71 umb4.1.1 N er of interviewees in each of the surveyed state according to their ethnic group...... 71 4.1.2 Level of education of the interviewees with regard to their ages...... 71 4.1.3 Educational levels and occupation...... 72 4.1.4 Local names of Mucuna pruriens...... 72 4.1.5 Methods of Mucuna extraction...... 73 4.1.6 Accessibility of Mucuna pruriens ...... 74 4.1.7 The latest application of Mucuna ...... 75 4.1.8 Years of experience with Mucuna pruriens in different interviewee groups in the study...... 76 4.1.9 Palatability of Mucuna to the grazing ...... 76

4.2 Screening for the insecticide properties of Mucuna pruriens and other plant extracts ...... 77 4.2.1 Efficacy of water/ethanol extracts of the plants materials based on directly sprayed on locust...... 78 4.2.1.1 Knockdown ...... 78 4.2.1.2 Mortality...... 79 4.2.1.3 Time to death...... 80 4.2.2 Efficacy of water extracts of the plant materials sprayed directly on locusts.81 4.2.2.1 Mortality...... 81 4.2.3 Efficacy of orally administered water/ethanol extracts of plants materials. 82 4.2.3.1 Mortality...... 82 4.2.4 Efficacy of orally administered water extracts of plants materials ...... 83 4.2.4.1 Mortality...... 83 4.2.4.2 Time to death...... 84

4.3 Evaluation of biological activities and mode of action of M. pruriens extracts ...... 85 4.3.1 Efficacy of Mucuna pruriens on the Desert locust by direct spraying...... 85 4.3.1.1 Knockdown effect on the 2nd nymphal instars...... 85 4.3.1.2 Mortality the of 2nd nymphal instars ...... 86 4.3.1.3 Knockdown effect on the adult of the Desert Locust by direct spraying87 4.3.1.4 Mortality of the adult of the Desert Locust by direct spraying ...... 88 4.3. 1.5 Time to death...... 88 4.3.2 Efficacy of Mucuna pruriens on the Migratory locust by direct spraying 89 4.3.2.1 Knockdown effect on the 2nd nymphal instar of the Migratory Locust89 4.3.2.2 Mortality of the 2nd nymphal instar of the Migratory Locust ...... 90 4.3.2.3 Time to death of the 2nd nymphal instar of the Migratory Locust ...... 91 4.3.2.4 Knockdown effect on the adult Migratory locust...... 92

XI 4.3.2.5 Mortality of the adult Migratory locust...... 93 4.3.2.6 Time to death of adult Migratory locust...... 94 4.3.4 Efficacy of Mucuna extracts against nymphs of Locusta treated by topical application ...... 95 4.3.4.1 Knockdown effect ...... 95 4.3.4.2 Mortality...... 96 4.3.5 Efficacy of Mucuna extracts against adults of Locusta treated by dipping 97 4.3.5.1 knockdown effects...... 97 4.3.5.2 Mortality...... 98 4.3.6 Effect of Mucuna as stomach poison...... 99 4.3.6.1 Mortality...... 99 4.3.6.2 Faecal output ...... 100 4.3.6.3 Effect of feeding period on wheat seedlings treated with Mucuna extracts on locust nymphs...... 101 4.3.6.3.1 Mortality ...... 101 4.3.6.3.2 Time to death ...... 102 4.3.6.4 Evaluation of extracts of Mucuna as locust antifeedant...... 103 4.3.6.4.1 Feeding on cabbaged leaf-disc under no choice condition ...... 103 4.3.6.5 Frass of Locust fed on cabbage leaf-disc under no choice condition.. 104 4.3.6.6 Cabbage leaf-disc bioassay under choice condition...... 105 4.3.6.7 Antifeedant effect of filter paper impregnated with Mucuna extracts offered to adults ...... 105

4.4 Effect of storage period on efficacy of Mucuna root materials...... 106

4.5 Effect from mixing extracts of Mucuna at 25g/l with reduced concentrations of Decis against the Desert Locust...... 108

5 DISCUSSION ...... 110

Recommendations...... 114

6 SUMMARY AND CONCLUSIONS...... 116

7 REFERENCES...... 118

APPENDEX ...... 140

Appendix 1 Key informant questionnaire : investigation on the uses of Mucuna pruriens as pest control agent in Darfur Western Sudan. Case of Kass and Zanlingi Areas...... 140

XII

TABLE OF ILLUSTRATIONS

LIST OF FIGURES Figure 1. Leaves of Mucuna pruriens...... 38 Figure 2. Pods and seeds of Mucuna pruriens...... 38 Figure 3. Map of Sudan showing areas where native people were interviewed for their experience with Mucuna pruriens (Fabaceae) and other botanicals used in pest control...... 51 Figure 4. Areas in Sudan where plant materials were collected for study...... 53 Figure 5. Digging of roots of Mucuna pruriens...... 54 Figure 6. Roots of Mucuna pruriens...... 54 Figure 7. Plexiglas cages for mass rearing of Locusts...... 57 Figure 8. 5l. plastic bottles used as cages...... 60 Figure 9. Application of Mucuna extract with a micro syringe...... 64 Figure 10. Dipping an adult of Migratory Locust in a Mucuna solution...... 65 Figure 11. Cabbage leaf-disk used in feeding deterrent experiments under free choice conditions...... 68 Figure 12. Single Migratory locust per tube in feeding trials for individual tests...69 Figure 13. The local manes given to the Mucuna pruriens in the surveyed areas in Western Sudan in the year 2001...... 73 Figure 14. Percentages of Traditional methods of Mucuna extraction according to the respondents in the surveyed areas in Western Sudan (2001) ...... 74 Figure 15. Availability of Mucuna pruriens to users in the surveyed areas in Western Sudan (2001)...... 75 Figure 16. % Palatability of Mucuna leaves and stems by the grazing animals in the surveyed areas in Western Sudan (2001)...... 77 Figure 17. Mean knockdown effect for different concentrations of Mucuna pruriens (Fabaceae) root extracts against 2nd instars nymphs of Locusta migratoria (Orthoptera, Acrididae) in laboratory(Temperature 25-28±1 º C. and RH. 60±5 % ).90 Figure 18. Mean mortality for different concentrations of Mucuna pruriens (Fabaceae) root extracts against 2nd instars nymphs of Locusta migratoria (Orthoptera, Acrididae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % )...... 91 Figure 19. Mean percentage knockdown of the adult Migratory Locust, Locusta migratoria (Orthoptera: Acrididae) Linné directly sprayed with water/ethanol (v: v ) extracts of roots of Mucuna pruriens (Fabaceae) under laboratory condition(Temperature 25-28±1 º C. and RH. 60±5 % )...... 93 Figure 20. Mean percentage mortality of the adult Migratory Locust, Locusta migratoria (Orthoptera: Acrididae) Linné directly sprayed with water/ethanol (v: v ) extracts of roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25- 28±1 º C. and RH. 60±5 % )...... 94 Figure 21. Mean time to death in days in adults of Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) directly sprayed with water/ethanol ( v: v ) extracts of roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25- 28±1 º C. and RH. 60±5 % )...... 95 Figure 22. Mean mortality of the 2nd instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) fed on wheat seedling( for different periods of times) treated with water/ethanol (v: v) extracts from roots of Mucuna pruriens

XIII (Fabaceae) at 50g/l. in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % )...... 102

LIST OF TABLES

Table 1. Distribution of interviewees in each of the surveyed state according to their ethnic group...... 71 Table 2. Level of education of the interviewees and their ages ...... 72 Table 3. Levels of education and occupation...... 72 Table 4. Year of latest application of Mucuna plant materials as pest control agent...... 75 Table 5. Years of experience with Mucuna pruriens of different interviewee groups in the study...... 76 Table 6. Mean knockdown effect on the 2nd. Instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) treated by direct spraying with water/ethanol (v: v) extracts of different plant materials in the laboratory (Temperature 25± º C. and % RH. 50-60)...... 78 Table 7. Mean mortality of the 2nd. Instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) treated with water/ethanol (v: v) extracts of different plants materials in the laboratory (Temperature 25± º C. and % RH. 50-60)...... 79 Table 8. Mean time to death in days of the 2nd. Instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) treated by direct spraying with water/ethanol (v: v) extracts from different plants materials in the laboratory (Temperature 25± º C. and % RH. 50-60)...... 80 Table 9. Mean mortality of the 2nd. Instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) treated by direct spray with water (v: v) extracts from different plants materials in the laboratory (Temperature 25± º C. and % RH. 50-60)...... 81 Table 10. Mean mortality of the 2nd. Instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) fed on wheat seedling treated with water/ethanol (v: v) extracts) of different plant materials in the laboratory (Temperature 25± º C. and % RH. 50-60)...... 82 Table 11. Mean mortality of the 2nd instar nymphs of The Migratory locust, Locusta migratoria Linné (Orthoptera: Acrididae ) fed on wheat seedlings treated with water extracts from roots of Mucuna pruriens ( Fabaceae ) and other plant extracts in the laboratory (Temperature 25± º C. and % RH. 50-60 )...... 83 Table 12. Mean time to death of the 2nd Instar nymphs of the Migratory Locust Locusta migratoria Linne( Orthoptera: Acrididae) fed on wheat seedlings treated with water extracts from different plants in the laboratory (Temperature 25± º C. and % RH. 50- 60 )...... 84 Table 13. Mean knockdown effect (%) on the 2nd nymphal instar of the Desert Locust, Schistocerca gregaria Forskål directly sprayed with 50g/l water/ethanol extracts from roots of Mucuna pruriens (Fabaceae), Neem Seed kernels and Decis (Deltametherin) in laboratory (Temperature 25± º C. and % RH. 50-60 )...... 85 Table 14. Mean mortality in the 2nd nymphal instar of the Desert Locust, Schistocerca gregaria Forskål directly sprayed with 50g/l water/ethanol extracts from roots of

XIV Mucuna pruriens (Fabaceae), Azadirachta seed kernels and Decis (Deltametherin) in the laboratory (Temperature 25± º C. and % RH. 50-60 )...... 86 Table 15. Mean(%) knockdown effect Mucuna water/ethanol extract ( v: v) sprayed on 7±1 day old adult Desert Locust, Schistocerca in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % )...... 87 Table 16. Mean(%) mortality of 7±1 day old adult Desert Locust, Schistocerca gregaria sprayed directly with water/ethanol (v: v) extracts of Mucuna pruriens (Fabaceae) in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % )...... 88 Table 17. Mean time to death in 7±1 day old adult Desert Locust, Schistocerca gregaria sprayed directly with water/ethanol (v: v) extracts of Mucuna pruriens (Fabaceae) in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % )...... 89 Table 18. Mean time to death resulting from different concentrations of Mucuna pruriens (Fabaceae) root extracts applied against 2nd instars nymphs of Locusta migratoria (Orthoptera, Acrididae) in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % )...... 92 Table 19. Mean knockdown effect on the 5th. instars nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) topically treated with Mucuna water/ethanol (v: v) extracts in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % )...... 96 Table 20. Mean mortality of the 5th. instars nymphs of the Migratory Locust, Locusta migratoria Linné of topically treated with of water/ethanol (v: v) extracts from roots of Mucuna pruriens (Fabaceae) in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % )...... 97 Table 21. Mean knockdown effect on 7±1day old adults of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) dipped for 1.5 sec. in different concentrations of extracts from roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % )...... 98 Table 22. Mean mortality in 7±1day old adults of the Migratory Locust, Locusta migratoria Linné dipped for 1.5 sec. in different concentration of extracts from roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % )...... 99 Table 23. Mean mortality of the 2nd instar nymphs of The Migratory Locust, Locusta migratoria Linné fed on wheat seedlings treated with different concentrations of water/ethanol extracts from roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % )...... 100 Table 24. Mean weight of frass collected after 24h from the 2nd instar nymphs of the Migratory Locust, Locusta migratoria Linné fed on wheat seedling treated with different concentration of water/ethanol extracts from roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % )...... 101 Table 25. Mean time to death in the 2nd instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) fed for different periods on wheat seedling treated with water/ethanol (v: v) extracts from roots of Mucuna pruriens (Fabaceae) (Temperature 25-28±1 º C. and RH. 60±5 % )...... 102 Table 26. Average percentage of cabbage leaf-disc (in weight) eaten by 7±1 days old adults of the Desert Locust, Schistocerca gregaria Forskål (Orthoptera: Acrididae) in a three hours feeding period on cabbage leaf-disc treated with extracts from roots of Mucuna pruriens (Fabaceae) under no-choice laboratory bioassay test(Temperature 25-28±1 º C. and RH. 60±5 % )...... 103

XV Table 27. Mean weight of frass of 7±1 days old adults of The Desert locust Schistocerca gregaria Forskål (Orthoptera: Acrididae) fed on cabbage leaf-disc treated with extracts from roots of Mucuna pruriens (Fabaceae) for a three hours period under no-choice laboratory bioassay test(Temperature 25-28±1 º C. and RH. 60±5 % ) ...104 Table 28. Mean percentage weight of Mucuna-treated cabbage leaf-disc consumed by 7±1 days old adults of the Desert Locust, Schistocerca gregaria Forskål (Orthoptera: Acrididae) in 3h under choice bioassay test(Temperature 25-28±1 º C. and RH. 60±5 % )...... 105 Table 29. Percent of Locust rejecting feeding on filter paper impregnated with extracts of Mucuna pruriens( Fabaceae) (Temperature 25-28±1 º C. and RH. 60±5 % )...... 106 Table 30. Mean percentage knockdown in the 2nd. Instar nymphs of the Migratory locust, Locusta migratoria Linné, sprayed with extracts of Mucuna pruriens roots collected at different years (2002/2001/2000) (Temperature 25-28±1 º C. and RH. 60±5 % )...... 107 Table 31. Mean Mortality of the 2nd instar nymphs of the Migratory locust, Locusta migratoria Linné, sprayed Mucuna pruriens extracts of roots collected at different years (2002/2001/2000) (Temperature 25-28±1 º C. and RH. 60±5 % )...... 108 Table 32. Mean mortality on the 2nd. Instar nymphs of the Desert Locust, Schistocerca gregaria Forskål (Orthoptera: Acrididae) treated with Decis/Mucuna and Decis or Mucuna extracts separately in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % )...... 109

XVI

1 INTRODUCTION

Under plague conditions, the Desert Locust, Schistocerca gregaria (Forskål 1775) (Orthoptera: Acrididae) is considered as a threat that endangers over 65 countries in Africa and Asia. The Locust under such conditions ravages agricultural crops and the natural pastures in an area estimated at 20% of the surface area of the earth (Joffe, 1995). Managing a pest with such capacity could only be achieved if the national, regional and international efforts are brought together in one tie. The African Migratory Locust, Locusta migratoria (Linné 1758) (Orthoptera: Acrididae) on the other hand, despite large distribution area, over the world, in comparison to the foregoing Locust species has a rather less invasive capacity (COPR, 1982). Locusta populations remained calm in the main land of Africa, since the last huge plague of the years 1928-1934, except for few and very sporadic upsurges (Balança et al, 1999).

Despite the on going debate, mainly, on which of the control strategies we should rely to combat Locust ravages, many acridologists and locust experts view locusts and grasshoppers as serious pests, especially in Africa, that worth active control operations or otherwise some sort of managements (Krall et al, 1997; Lomer et al, 1999 and 2001; Lomer and Langewald 2001; Lecoq 2000 and 2001; Lockwood et al 2000 and 2001). The controversy over the locust question may be in part due to the fact that most of the locust experts in the past time, were not from Locust-affected countries. Such experts may not have enough background on the social, political and geographical aspects of the problem. Some donors may also contribute to the development of such controversy through provision of their own products, equipment and personnel (Lecoq, p.c.). On the other hand it may be due to the ecology and behavior of the locust that may create a unique challenge in selection of management strategies. Moreover, locust control in Africa is always run under a crisis mode with just a little room of flexibility (Lomer and Langewald, 2001).

Under plague conditions, locusts are considered as a disaster that does not differ very much from the other natural disasters such as drought, floods and earth quakes. Hence, under such condition this pest generates a great pressure from social and political communities that elicit technically and monetary emergency responses at national and international levels (Peveling, 2001). The importance of these insects stems from the fact that under plague condition each locust can eat its weight (approximately 2g) per day and the swarms may include several millions and millions of locusts which travel up to 1.000 km in one week (U.S. Congress, 1990). In the 10 years preceding 1995, the sum of US$ 383 million had been offered by the international community to be invested by the Desert Locust-affected countries to meet the control operations (Joffe 1995). In the Sudan, however, the two species of locust co-exist in the Red Sea coastal area, Tokar Delta and some parts of Kassala State in the east where they endanger field and horticultural crops in the area. The former area is considered as the most important breeding site in which Locust breeding seasons may sometimes overlap. Acridids, in general, are very notorious pests in Sudan and the damage inflicted by the Desert locust alone was estimated at 55.000 metric tons of grains, valued at US$ 7.335 Million in the year 1954 and 2% of the country cereals which is equivalent to US$ 13.67 Million in 1988 (Joffe 1995). Sudan is considered as a watershed for the Desert Locust with a vast recession area that is approximately half the country in size ( about: 1.25 million km2.). This area includes both summer and winter breeding sites( El-Tom 1993). .

Comprehensive studies targeting the economy of locust control strategies are scanty, although a number of activities were planned within EMPRES to evaluate control strategies. Joffe (1998) addressed the issue of economy and policy in locust management. He gave a cost/benefit analysis of Locust control under different scenarios and found that control is generally very successful at reducing damage (from average of US$ 179 millions to a range between US$ 1-8.5 million with control under favorable scenario). Moreover, the results of cost/benefit simulation, based on 5 years period of preventive control, suggested that the cost of Locust control is around US$ 231 million under favorable conditions while under less favorable conditions it can increase up to US$ 288 millions.

Control of locusts in most of the Locust-affected countries is the responsibility of the state, with very little participation of the farming communities. Chemical pesticides are currently the only tools for combating the pest. Though, most of these chemicals are obtained through donations from rich countries and from international organizations, this situation cannot be sustained because the donor agencies are

18 reluctant to continue supporting such an expensive endeavor and also because of the environmental risks associated with the use of synthetic pesticides. The concern over the environmental risk of chemical control of locust has increased recently. It arose primarily from heavy reliance on chemical pesticides which pose many on the ecosystems and human health problems ( Isman 1990; McDonald, 1965; Ritchie & Dobson, 1995; Peveling, 2000, 2001; Peveling & Nagel, 2001). In the past there were very little data concerning assessment of eco-toxicological risks of chemical insecticides (van Der Valk, 1997). However, Balança & Visscher (1997) and Peveling et al (1999a, b) provided some data on the effect of chemical control of Locusts and grasshoppers on non-target fauna.

The current problems of chemical control of Locust including the high cost and the environmental hazards, have substantiated the need to investigate new products for acridid control. Accordingly, the trend now is toward overcoming the drawbacks of chemical pesticides by generating interest in the development of ecologically sound and economically justified technologies. Development of such technology is of great value especially in the case of subsistent farming systems. In this context a lot of discussion is going on with regard to the role and the place of the biological control in locust management. Lomer et al (2001) and Lomer and Langewald (2001) emphasized the importance of the entomo-pathogenic fungus, Metarhizium anisopliae in locusts and grasshoppers integrated pest management programs. However, other scientists doubted the visibility of using the slow action fungus such as M. anisopliae against a highly mobile insect like the Desert locust.

There is, therefore, a pressing need for alternative strategies and tactics for managing locusts is emerged regardless of the debate on management strategies. In this respect, there is a common view that IPM should be enforced and applied for acridid management in the future ( Lecoq et al, 1997; El Bashir, 1997; Peveling et al, 1999a; Lomer et al, 2001). Interesting research avenues in the context of alternative strategies that fit very well in IPM programs are plants that produce vast array of chemicals with insecticidal and semio-chemical properties. Plant species in the world are estimated at 250.000 different species and the figure could reach up to 500.000 species. Only 10% of these plants are chemically examined which reflects how wide is the window of the scope for further work with plant materials (Benner, 1993).

19 Moreover, due to the likelihood of bio-degradability of plant-derived chemicals, botanical insecticides have presented themselves as major tools for a novel approach on pest control, especially as on-crop active control measures. Positive results have been achieved by some scientists working with plant extracts as control agents e.g. Islam (1983) and Langewald and Schmuterer (1991). However, sufficient published recommendations are not available (Dales, 1996). In the last 30 years a plethora of research works targeting Meliaceae, especially the Neem tree Azadirachta indica and Melia volkonski, were conducted in different parts of the world. Schmutterer and Ascher (1983 , 1986); Schmutterer (1995) and Kleeberg and Zebitz (1997) compiled and edited hundreds of papers dealing with Neem bio-activities, mode of action, ways of extraction and preparation, in addition to the Neem chemistry. They also included some works on other tropical plants. The foregoing references included some articles that dealt with bio-activity of Neem product or extracts from other Meliaceae against some Orthoptera mainly the Desert or Migratory Locusts ( Iqbal and Strang 1997; Kämper 1997; Breuer and De Loof 1997). However, work in this field is still considered very meager with regard to the bio-activity of other tropical plants against acridid pests.

The present investigation accesses the locusticide properties of Mucuna pruriens Linné (Fabaceae) a pan tropical leguminous plant commonly known as “cowitch”, “magic ” or “velvet bean” and as “Irg El-Ghamoul” locally in western Sudan. Morris (1999) studying genetic resources with novel effect listed some as source of bio-active products. The list includes M. pruriens as a sources of bio-active substances like Bufotenine and Serotonin which are cholinesterase inhibitors. Mucuna is widely used in folklore medication, hunting and pest control in many African countries (Neuwinger, 1996). Root extracts of this plant are widely used by the cattle herders (Baggara tribes) in western Sudan to combat ecto-parasites on cattle. To the best of my knowledge there is no published work in the context of M. pruriens as locusticide before the year 2000. Therefore, this plant has been the target of the laboratory investigations since October 2000 in CIRAD-Prifas in Montpellier, France.

The work described in this study involves assaying extracts from roots of M. pruriens in comparison with three other plants extracts namely: Calotropis procera

20 (Asclepiadaceae), Adenium obesum (Apocynaceae) and Azadirachta indica (Meliaceae). The plant materials were assayed against the Migratory locust in the year 2000. It is of value to mention that: the candidate plants materials were screened for contact and stomach efficacy against locust. Extracts of Neem seed kernel were considered as the standard botanical insecticide. The main purpose of screening is to find out the most efficient bio-active plant material to be subjected to further bio- tests. The tests were designed to further investigate the bio-efficacy of the selected material in terms of knockdown and the killing capacity on the Migratory locust and the Desert locust. Investigations also targeted the mode of action of the plant materials. Moreover, different adapted concentrations of the tested materials were examined for the same purpose. The work reported here also aimed at mixing the candidate plant extracts with a standard chemical locusticide in order to explore the possible additive or synergistic effect. In the field survey, natives were interviewed for their experience with M. pruriens. Investigations to isolate the active molecule and field-testing of the plant materials are recommended to be undertaken in future studies. The output of the conducted experiments in screening of plant materials, studying the bioactivity and persistence of Mucuna extracts and the efficacy of Mucuna extracts when mixed with other materials are presented and the most significant results are discussed. The scope here is not to design an area wide management program for locust, it is rather an endeavor to find a material that can reduce acridids damage to crops, to avail a low cost pest control agent and to safeguard the environment.

The specific objectives of the current work could be summarized in the following points:

1- To investigate the insecticidal properties, the mode of action of the studied plant materials and to explore the possibility of their use in insect pest control programs.

2- To avail an effective, environmentally friendly, easily prepared, low cost and locally available pest control agent of plant origin to be used by resource-poor farmers.

3- To explore the compatibility of M. pruriens when mixed with chemical insecticides (Decis) and to investigate the possible synergistic effect of the plant extracts.

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22 2 REVIEW OF LITERATURE

2.1 Locust control

2.1.1 Importance of Locust and Locust control

With Locust plagues and some grasshoppers outbreaks in arid and semiarid zones of Africa, food and feed resources are particularly at risk (Peveling 2001). The Desert Locust, Schistocerca gregaria (Forskål 1775) (Orthoptera: Acrididae) is considered as a threat because it has the capacity to invade over 65 countries in Africa and Asia. Under plague conditions an estimated surface area of one fifth of our planet is prone to the Locust invasion (Magor 1994; Joffe 1995 and Anon. 1998 ). The African Migratory Locust, Locusta migratoria (Linné 1758) (Orthoptera: Acrididae) on the other hand, poses a comparatively less threat due to its limited invasion area (COPR 1982). The latter species reflected low tendency of upsurge and remained calm in its main recession area in Africa since the last plague of the years 1928-1934. However, heavy outbreaks were recently, reported in some areas of Chad and Cameroon, in West Africa, where the situation necessitated active intervention for crop protection (Balança et al 1999). In Madagascar, the local sub-species (L. migratoria capito Sauss.) invaded the whole island from 1996 to 2000 (Lecoq 2001).

There is increasing literature and information in recent years on the economic importance of Locust and its control (FAO 1993, 1999 ; Ritchie and Dobson 1995; Joffe 1998; Krall and Herok 1997; Symmons 1997). The literatures reflects a broad spectrum of views which indicate some controversy as far as the Locust triumvirate (socio-economic and environmental aspects) is concerned. In the last upsurge of the Desert Locust, there were those who claimed that, chemicals should deserve full credit as they were merely the factor that stopped the Locust plague at that time. Yet, others view weather factors to have the over-riding contribution and that they were the main cause of the Locust plague decline (U.S. Congress 1990). Based on his experience with the Migratory Locust in Madagascar and Africa Peveling (2001) emphasized the role of the ecological factors and the type of control strategy in Locust management. Moreover, he elucidated and compared different views, attitudes and

23 empirical evidences and finally a discussion was given on how the gap between control-oriented Locust operations and the environmental locust management could be bridged. However, the current situation is still paradoxical despite the tremendous improvement in knowledge and method of Locust control and detection (Lecoq 2001). The of latter author quoted the case of the last upsurge of the Desert Locust, Schistocerca gregaria, in the main land of Africa and the Migratory Locust, Locusta migratoria capito in Madagascar to highlight the controversy. He gave an overview for the Locust question with emphasis on the past and the current Locust control strategies integrating the socio-economic, institutional and political components.

Different views have been expressed with respect to control strategies and the economy of Locust control ( van Huis 1997; El-Bashir 1997; Lockwood et al 2001; Lomer et al 2001). In this respect, the last Locust plague period in the late eighties of the 20th. century is considered as the main event that triggered this controversy (Peveling 2001). In that period Africa experienced the largest simultaneous upsurges of several Locusts and grasshoppers species known in the last 50 years. Another small Locust upsurge was reported in Africa in 1992, only three years after the major one which ended in 1989, a period considered very short in comparison with a roughly three decades of recession period which preceded the 1986-89 plague. This phenomenon substantiates the need for definition of a global Locust control strategy (Chara 1997). From 1986 until the first half of 1989 donors invested about US$ 275 million to contain this pest in 23 Locust-affected countries in Africa which also allocated similar funds and resources to face the threat of Locust invasion (U.S. Congress 1990). This sum of money is within the range given by Symmons (1997) to which the cost of 15 million liters of pesticide concentrates provided by donors can be added. However, despite all these costs no one knows, precisely, what the over all achievements were (Ibid).

Regardless of the on going discussion, on the success and failure of the different control strategies, many Orthopterist's and Locust experts view Locusts and to some extent grasshoppers as the most feared insect pests of crops in Locust-affected zones in Africa, especially during plague periods (Krall et al 1997; Lomer et al 1999, 2001 ; Lomer and Langewald 2001; Lecoq 2000, 2001; Lockwood et al 2000, 2001).

24

2.1.2 Locust control strategies

Krall et al (1997) edited a book that reflected the research conducted in the recent years in different aspects of Locust control. The book provided a valuable overview of the research situation with special emphasis on the recent development in the control strategies including the potentiality of some botanical Locusticides, namely Melia volkensii. Historical background of Locust control was given by van Huis (1997) who cited some references about historical records of Locust appearance as pest; he evaluated the three control strategies in relation to: outbreaks, upsurges and plagues and summarized the constraints of upsurge prevention into the following: - Lack of specific criteria of control - Necessity for excessive implementation of resources for monitoring and control - Detection of gregarization at the start of the upsurge - Decreased persistence and accumulation of insecticides in the insect body - Lack of readiness and backup capacity of immediate response to the upsurge once developed.

Showler (1997) discussed the preventive, proactive and reactive approaches of Locust control. He identified pro-action as the most suitable modality of Locust control but he pointed out that : dependence on one single strategy is not advisable. Meinzingen (1997) gave an overview of Locust and grasshoppers new control agents and emphasized the need for implementing new control strategies especially with regard to the control of Locust in the recession areas. He reviewed the previous work with Neem extracts (A. indica) against the Desert Locust and indicated that mass production and registration of the Neem product and registration were still the obstacles that should be removed if Neem pesticides are to be used widely. El Bashir (1997) proposed alternative Locust management tactics and strategy. The proposed strategy should be preventive in nature, environmentally soft and sustainable. It advocated an integrated pest management tactics with ultimate objectives of preventing the gregarious tendency in Locust through the use of semio-chemicals. However, he reflected the need for field data on the performance of the Locust semio- chemicals in addition to the need for involving rural community, Locust-affected countries, regional and international organizations and donor countries in Locust

25 management. In the same context, Ferenz (1990) concluded that the Locust semio- chemicals, which are very specific and with less damage to the environment, could be used in an IPM program. Lecoq (2001) reviewed the current Locust control strategies and concluded that, despite many debates during the last 15 years, the preventive strategy is still considered the best approach and it is the only rational solution to the Desert Locust problem.

2.1.3 Environmental impact of Locust control

Due to the heavy reliance on synthetic Locusticides and the fact that vast areas will have to be sprayed with these chemicals, many of non-target biota are prone to have some sort of exposure to the insecticides either directly or indirectly. Due to the recent concern with regard to the environmental impact of the use of pesticides, some data on risk assessment and environmental impacts of the chemical Locusticide were made available (Peveling et al 1999). However, there is no economic scale to measure at what environmental cost the benefits of control are achieved (FAO. 1995; Balança and Visscher 1997; Peveling et al 1999ab; Peveling 2000, 2001). The risk of applying Locust and tsetse fly control agents to wildlife was evaluated in birds, reptiles and small mammals in Africa. An overview was given which confirms the impacts on the environment as a result of the wide application of pest control agents against these two pests in Africa (Peveling and Nagel 2001).

2.1.4 IPM of Locusts and grasshoppers

Carson (1962) in her fascinating book, “Silent Spring”, described spraying with synthetic chemical pesticides as the amazing rain of death. The book was written in such a style that paved the way for the wide acceptance of the integrated pest management concept (van Emden and Peakall 1996). Accordingly, the environmental awareness increased a great deal, especially with respect to the chemical control of Locust. More recently the increase of the environmental awareness has been motivated by some data reflecting the negative impact of the broad spectrum synthetic chemicals, a situation which emphasized the need for the adoption of an integrated pest management program (see Ritchie and Dobson 1995; El Bashir 1997; Lecoq et al 1997; Lomer et al 2001; Peveling 2001). Lomer et al (2001), advocating the use of

26 Metarhizium as bio-pesticides against Locust and grasshoppers, identified three steps that are essential in developing an IPM program: 1- Exploration of novel and environmentally friendly Locust control methods. 2- Evaluation of novel and existing control technologies for their efficacy and impact on the Acridids environment. 3- Implementation of the evaluated novel technology with improved monitoring and better forecast in an integrated manner.

2.2 Plants in Locust control

2.2.1 Background on the history of plant materials in pest and disease control

There are numerous examples of natural products from plants that attracted agrochemical interests due to their allelo-chemical properties. The most important example is the case of pyrethrum obtained from Chrysanthemum cinerariaefolium. Later synthetic pyrethroids have largely superseded the natural pyrethrum. Rotenone and were used in pest control (Benner, 1996). Historically, people used plant materials for various aspects of biological activities and there are many examples of plants that had places in tribal and ethnic folks. In China, flowering plants have been used against insects for more than 2000 years and a famous book, “The Quintessence of the Qi Dynasty”, was published in 540 A.D. A detailed description of botanical insecticides from Chinese plants was given in “Encyclopedia of Agriculture Management”, published in 1639. In 1959, a “Handbook of Chinese Native Pesticides’’ was published which includes 220 plant species actually in use in china (Shin-Foon 1985 and Er-Cae 1992) cited in Shang (1996). In an ethno-botanical study targeting plants that are traditionally used as native medicine, Singh et al (2002) extrapolated that in Sonbhadra district alone, in , 157 plants from 57 different families were known to be used for their medicinal values against many diseases. In East Africa, simple preparations of Neem (A. indica) fruits or bark are used against malaria and yellow fever (Meinzingen 1997). Different parts of Mucuna pruriens are traditionally used in many African countries against some diseases and pests or as an aphrodisiac and nerve tonic (Neuwinger 1996). As a result of a comprehensive working strategy in the development of botanical pest control agent, a complete

27 protocol has been detailed by Sharma (1984). The protocol included the method of selection, collection, preparation and conduction of bio-tests on plant materials. He concluded that “The socio-economic compulsions in the developing countries may make the proposition of the crude extracts of plant materials or enriched one against pest of special attractiveness”.

2.2.2 Potentiality of Plant derived compounds as pest control agents

Mankind has a continuous battle to protect crops against pests. Most of the earliest pest control materials were of plant origin. Tiuterev et al (1997) from their studies of the Russian flora as bioactive sources against plant pests concluded that indigenous plants are rich sources of compounds that have negative impacts on other organisms. With time several plants were widely exploited as sources of commercial pesticides. However, on the advent of the discovery of DDT and the other synthetic chemical insecticides in early Forties of the past century onward, these synthetic chemical insecticides have largely replaced traditional botanicals as key commercial pest control agents. Now it is evident that plant materials still have enormous potentiality to inspire and influence modern agrochemicals research activities in the world (Benner, 1993, 1996). Stoll (1988) compiled and edited a book about plants that are used in crop protection in tropical zones. He gave comprehensive information on different plants and their parts or products which are used in pest control. For each plant he gave a brief information about the botany of the plant, the bioactive properties of the plant, list of the sensitive pests and the practical utilization of the active ingredient of the plant materials. Ahmed et al (1983b) provided some relevant information about 26 promising plant species in the field of crop protection and listed the insects which are reported to show a response to these plant materials. In the U.S.A. and in the framework of an on going multi-years and a multi-disciplinary international program of coordinated research, a research was conducted to study the potentiality of using plant materials for pest control. The studies implied biological investigations to verify the efficacy of the selected plant materials, chemical studies to explore alternative procedure of preparation, formulation and the possibility of adoption by small-scale farmers, socio-economic survey to assess the reaction of farmers and cottage industry to the plant materials used and finally policy studies to determine the possible implication of using botanicals( Ahmed et al 1983a).

28 Therefore, plants, without any doubt, have great potentialities to provide vast array of bioactive compounds that could be used against pests.

29 2.2.3 Selection of plant materials for bio-test

The most obvious way to select a certain plant as a candidate to undergo test for the biological activities is its reputation in folks and literature (Sharma 1984; Benner 1993). Recently, a lot of research interests were generated in the area of botanical insecticides as suitable bio-rationale alternatives for the notorious synthetic chemicals that inflict direct hazard and toxicity to man and his animals and indirectly influence the biotic and the a biotic environmental components (a phenomenon lead to the residue in produce, pest resurgence, secondary outbreak and pest resistance). Plant materials which are readily broken down in the environment and have repellent and anti-feedants action, qualify as the most suitable pest control tools, especially within a framework of integrated pest management.

2.2.4 Role of plant materials in pest control

Higher plants are rich sources of bioactive secondary metabolites and contain over 80 % of alkaloids, phenols, terpenoids and other bioactive metabolites (Robinson 1980 cited in Tiuterev et al 1997). More than 2000 plant species reportedly possess anti- pest properties. However, only few species have been exploited for pest control (Ahmed et al 1983; Benner 1993). Roots of some tropical Leguminosae especially species of Derris have been used as insecticides. The bioactivity of this plant material is attributed to rotenone (Dividson 1930). Parts of Adenium obesum ( Apocynaceae) a shrub distributed in hilly areas in the sahelian belt of Africa are used as pesticides, poisons and medicines. Root extracts of Adenium are used against lice. However, the latex from this plant species is extremely poisonous compound to the cardiac muscles (Vogt, 1995).

2.2.4.1 Role of Neem (Azadirachta indica) in pest control

Some plants species have got an anti-insect action and could be used against insect pests. In this regard the Neem tree, Azadirachta indica (A. Juss), which is widely grown in the tropics, is rediscovered as a source of natural pesticides. The tree has a cocktail of bioactive compounds of limonoids. The highest concentration of these

30 bioactive materials was reported in the seed kernels. The most important active molecules in Neem are azadirachtin, meliantriol and salannin which deter insect feeding or interfere with insect development and metamorphosis (Ascher 1983; Haasler 1983; Sharma et al 1984; Jacobson 1986; Ascher 1993; Schmutterer 1990; Mordue and Blackwell 1993). Stefens and Schmutterer (1982) studying the effect of crude methanolic extracts of Neem seed kernels on the Mediterranean fruit fly, Ceratitis capitata Wied. reported the growth regulatory effects of Neem and that larval mortality increased as a result of exposure to the Neem treated diet. The effect on the larval growth appeared as a prolongation of the larval period. The same results were obtained by Courdriet et al (1985) who applied Neem seed extracts against whitefly, Bemisia tabaci on cotton. They found that Neem extracts prolonged nymphal development, reduced insect fecundity and induced mortality. The insect growth regulation as a result of treatment with Neem products was documented on various insects by several authors (Sieber and Rembold 1983; Sharma et al 1984; Haasler 1983 and Ascher et al 1983). Such effect is caused by influencing and blocking of the ecdyson path.

Accordingly, the Neem tree, Azadirachta indica (A. Juss) has been a subject for intensive research in the last four decades and literature is very rich with regard to the bio-activity of this plant (Jacobson et al 1983; Jotwani and Srivastava 1983; Schmutterer 1983; Kleeberg and Zebitz 1997). The research in Neem was conducted with the objective of demonstrating the bioactivity, mode of action of this potential plant as an anti-insect agent with the aim of exploring the possibility of using Neem products as crop protection agents. In this connection many advances were reported and Neem products and their analogous compounds are now available as commercial botanical crop protection agents (Schmutterer el al 1981; Schumtterer and Ascher 1983 and Schmutterer 1986). In the way of introducing the Neem products as easily prepared, highly effective anti-insect agents some scientists investigated and screened for the efficacy of the Neem crude extracts. The crude extracts of Neem were found superior to the refined extracts as observed by Jotwani and Srivastava (1983) due to the removal of some anti-feedant materials in the refining process.

2.2.4.2 Role of Neem in Locust and grasshoppers control

31 The Neem tree, Azadirachta indica (A. Juss.), produces many bio-active limonoids which are reported to have anti-insect activity against 314 species in 16 different insect orders including the notorious Orthoptera (Schmutterer and Singh 1995). Schmutterer et al (1993), from their laboratory and semi-field trials on the effect of Neem products on three plague Locusts and one in Africa, concluded that Neem could be used by subsistent farmers to protect their crops from acridid pests ravages. This can be achieved by spraying crops with Neem oil or water extract of seed kernels which provide protection against the pest for up to 10 days. Neem seed kernels and other Neem products are widely used as crop protection agents. In this respect Langewald et al (1995) evaluated the efficacy of Neem materials against the , Nomadacris septemfasciata (Serv.) when maize fields were sprayed with the Neem extract. Significant differences were noted between the treated plots and the untreated control. The results reflect that Neem aqueous solution, prepared in a very simple way, could be easily applied by the local farmers against Locust to give at least one week protection for the maize crop. However, the approach was considered unrealistic in the Malagasian context because of the very large quantity of water needed in a countryside which is already suffering water shortage. Olaifa et al (1991) found that Neem can offer 100% protection to crops for a period of two weeks against five acridid species of grasshoppers infesting cassava and maize. Neem also proved to be very effective against the rangeland grasshoppers. According to Shah et al (1993) and Shah (1995) Neem seed kernel extract at 2% concentration was the most effective treatment against grasshoppers followed by Neem leaf extract at 20% concentration. Nasiruddin and Mordue (1994) found that the Desert Locust, S. gregaria was most sensitive to azadirachtin compared to other products. They concluded that “for optimal crop protection in the field a compound must be relatively of high anti-feeding effect to give immediate protection to the crops and it must show sufficient toxicity to kill the pest”. Hence sound laboratory screening of potential pesticide candidates is a prerequisite for the field trials in the future. Locally produced plant materials of biological activity, if exploited in a sound way to supplement use of synthetic insecticides, will increase the cost-effectiveness of control measures and safeguard the environment (Obeng-Ofori et al 1996). Studies in the efficacy of Neem product, evaluated for its direct killing effect, against nymphs of S. gregaria confirmed that Neem product could be used to control the Locust ( Nasseh et al. 1992). Various Neem products applied against resting and flying Desert Locust

32 indicated a remarkable reduction in fitness and fight activities of the flying adults( Wips et al 1992).

2.2.4.3 Role of other Meliaceae in Locust and grasshoppers control

Most of research conducted on Meliaceae as potential botanical bioactive substances was carried out on Neem (A. indica). However, some research had been conducted on other members of Meliaceae, such as Melia volkensii, which is widely distributed in East Africa. Mwangi (1982), Rembold (1997), Mwangi et al (1997) and Diop and Wilps (1997) demonstrated the potentiality of using fruit extracts of this tree as anti- Locust products. They concluded that the product of this tree could offer an environmentally-friendly Locusticide that would reduce the cost of control. However, the latter authors had the following reservations towards the use of botanicals: 1- with the exception of Indian case, there is a lack of standardized product in adequate amount; 2- botanicals are relatively slow in action.

In an overview of Locust control agents, Meinzingen (1997) quoting from Nasseh et al (1993) mentioned that Melia volkensii had similar characteristics to those of Neem and it could be used against nymphs and adults of the Desert Locust. Breuer and De Loof (1997) in their investigation of the efficacy of an enriched fruit extracts of M. azadirach, observed the strong antifeedant and high mortality in the 2nd instar nymphs of S. gregaria of the extracts at high concentrations.

2.2.4.4 Role of plants compounds other than Meliaceae

The research on bio-testing of plant materials has mainly focused on the Neem tree products and to some extent on other meliaceae mostly Melia azadirach and Melia volkensii. Very little work is devoted to the other plant families. In this respect, Proksch and Rodriguez (1983) reviewed the recent findings on distribution, biosynthesis, isolation chemistry and the biological significance of chromenes and benzofurans of the family Asteraceae. Isman and Proksch (1985) bio-assayed chromenes and bezofurans from Encelia(Asteraceae) for their feeding deterrence and

33 toxicity to some insect pests. The authors isolated more than 120 bio-active substances were from Artemisia monosperma, a plant species wildly grown in the Egyptian Desert and elsewhere. Saleh (1984) obtained crude extracts and steam distillates which were subjected to bio-assay tests against a susceptible strain of the housefly, Musca domestica and the 4th instar larvae of the cotton leaf worm, Spodoptera littorallis using topical application methods. The result of the bioassay tests revealed that the steam distillate of A. monosperma was the most toxic fraction against the test insects. Ping et al (2001) in their assay of plant materials from Stellara chamaejasme L. (Thymelaeaceace) against Aphis gossypii and Schizaphis graminum reported the importance of botanical insecticides in pest control. Beside Neem there are so many other plants known for their antifeedancy and growth regulatory effects on insects in the laboratory. These plant products include leaf extracts of Annona squamosa, Lantana camara var aculeata, Calotropis procera and Ipomea camea. The mentioned plant materials provided 50-100 % protection against some forest insects ravaging trees (Kulkarnii and Joshi 1998). Morsy et al (2001) tested the latex of C. procera against the 3rd instar of the housefly Musca domestica in Egypt and reported the bioactivity of this plant latex. Generally, plant materials other than Meliaceae received little attention, in terms of studies on the bio-activity, in the past time despite their potentialities. In Sudan a laboratory bio-test conducted by Ali (1999) demonstrated the potentiality of using Neem and Usher, Calotropis procera, against the Desert Locust, S. gregaria.

2.2.4.5 Role of plant materials in integrated pest management

Neem extracts do not show quick knockdown effect as do chemical insecticides but they exhibit ant-feeding properties, act as growth regulators, show ovicidal action and cause sterility. For these qualities they fit into integrated pest management programs (Venkateswarlu et al 1997). The role of Neem products as integral components of the integrated pest management program, especially for small-scale farm holders and subsistent farming could be extrapolated from the study of Verkerk and Wright (1993) who investigated the biological activity of Neem seed kernel extracts against the cabbage moth (Plutella xylostella). Their field part of the study revealed that Neem seed kernel water extracts were more effective than the methanolic extracts. Hence

34 they considered the water-based Neem materials as the most suitable for small-scale vegeTable growers in the developing countries as the materials are locally available and can be easily prepared by farmers in the field.

2.2.4.6. Eco-toxicological risk assessment and the side effects of botanical insecticides

In the period between 1990-1992 Peveling et al ( 1994) carried out field studies on side effects of six botanicals for Locust control among them were Neem oil and methanolic extracts of M. volkensii. on epigeal non-target in Sudan, Niger, Mauritania and Cape Verde Island. The study evaluated and assessed the environmental risk from using some non chemical locusticides. Results of the study revealed no severe side effect on the tested fauna

2.3 Mucuna pruriens

2.3.1 Literature on Mucuna pruriens

Literature on the role of Mucuna as a botanical pesticide is scanty although it is very rich with regard to its seed chemistry. There is some confusion as far as the taxonomy of the plant is concerned (St-Laurent et al 2000; Szabo and Tebbett 2002). As information on the uses of Mucuna root extract in pest management is not available, a field survey has been conducted on the traditional uses of Mucuna root material as pesticide in Sudan. A key informant questionnaire was designed (APPENDEX 1.) and 104 persons in two different areas in Western Sudan were interviewed on how they prepare and apply Mucuna root material. The interview includes questions concerning the experience of the respondent with Mucuna as well as with other botanical material. In the survey all the interviewees indicated that they used Mucuna root extracts as pesticide and the way of preparation of Mucuna for extraction and the subsequent application was more or less the same.

35 2.3.2 General information on Mucuna pruriens

2.3.2.1 Taxonomy and synonymy of Mucuna

Despite the fact that the genus Mucuna is very well known (Neuwinger 1996), the taxonomy at species level is confusing as the genus Mucuna includes many different varieties which are poorly known. Around 100 species of wild and cultivated Mucuna may be present in the tropics and subtropics of the world (Wilmot-Dear 1991, cited in Lorenzetti et al 1998). The genus Mucuna, with such a large number of species ( > 100 species) about 5 species or more of them are domesticated plants has not been subjected to modern molecular techniques or phyto-chemical markers to aid in its classification. This could be a reason for confusion in the taxonomy (Lorenzetti et al 1998). Recently, the extensive review of the literature by St-Laurent et al (2000) provided a tool of description for 137 species, subspecies and varieties under the genus Mucuna. This is why part of this taxonomic confusion may probably arise from the enormous variability in seed phenotype and the wide range of duration to maturity (Giller 2001). The taxonomy of Mucuna becomes more complex when cultivars or annual species are considered as many of these cultivars are cultivated as fodder or green manure in southern USA and elsewhere in the world which led to the proliferation of names. The situation substantiates an urgent need to clearly identify the different cultivars by using the gene marking technology (St-Laurent et al, 2000). The limited knowledge on the taxonomy is now regarded as one of the main impediment of using Mucuna as food and feed (Capo-Chichi et al 2003, Kiome and Hartmann 2003). A recent study of the genetic diversity of Mucuna attempted the classification of the plant at accession levels using molecular markers (Capo-Chichi et al 2003a) because classification of the various accessions of Mucuna is uncertain (Capo-Chichi et al 2003b). From an internet search in Legume web (2002) under Mucuna pruriens as key words the following scientific names were reported as synonyms for Mucuna in the International Legume Database Information Service (ILDIS) web page: - Dolichos pruriens - Mucuna atropurpurea - Mucuna cochinchinensis (Lour) A. Chev - Mucuna esquirolli A. leveille

36 - Mucuna nivea (Roxb.) DC. - Mucuna prurita Hook. - Mucuna prurita (L.) Hook. - Stizolobium pruritum (Wight) Piper.

From the same sources the tribe of Mucuna appeared to be as Phaseoleae and the Genus is Mucuna. However, the sources included a list for the vernacular names of Mucuna : - Buffalo bean - Cow- - Cowage Velvet bean - Velvet bean - Itchy bean - Hill fire bean

2.3.2.2 Botanical description of Mucuna

Mucuna was described as a semi-woody twiner or climber with more or less hairy furrowed stems. The plant is highly variable and with many varieties which are poorly known. The pods are very conspicuous, S-shape covered with urticating hairs; leaf is trifoliate the two lateral leaves are obliquely ovate but asymmetrical. Mucuna is a large raceme with dark purple color. The genus Mucuna includes both perennial and annual species (Broun and Massey 1929, Wilmot-Dear, 1984 and Neuwinger 1996). The annual species had a rather erect or crawling growth habits and phenology (Wilmot-Dear, 1984) (Figures 1 and 2).

37

Figure 1. Leaves of Mucuna pruriens.

Figure 2. Pods and seeds of Mucuna pruriens.

38

2.3.2.3 Geographical distribution of Mucuna

The Genus Mucuna is a pan tropical plant widely distributed in tropical Africa mainly in savannah forest edges and gallery forests. In Sudan it grows across the savannah belt mainly, around water streams and at the foot of hills. Before 1900 the seeds were sometimes considered as “calabar ” (Neuwinger 1996). In Sudan Broun and Massey (1929) reported the plant to be distributed in Bahr El-Ghazal province (in Southern Sudan). However, the survey conducted by the author in Western Sudan revealed that the plant is widely known and used by some nomads in that region.

2.3.2.4 Historical overview on Mucuna

Buckles (1995) provided one of the earliest articles in the history of Mucuna plant. Further overviews on the historical background of Mucuna was recently made available (Carsky et al 1998; Elittä and Sollenberger 2002; Elittä et al 2002; and Elittä and Carsky 2003). They reviewed research conducted in the plant since 1900, with emphasis on the research efforts during the era of the eighties and the nineties of the past century. The mentioned era marked the renewal of interest in Mucuna as a crop that fitted in multidisciplinary approaches and projects in agroecosystems.

2.3.2.5 Mucuna in Folklores

Mucuna is widely used in native medicine. In many African countries various parts of the plant are used. In Nigeria intestinal worms are treated with Mucuna fruits hairs mixed with honey. In India where Mucuna has largely been used as native medication for hundreds of years, seed extracts are still in use as a nerve tonic and aphrodisiac drinks. Water extracts of leaves are used in treatment of snakebites in traditional African medicine. Roots and fruit extracts have the effect of reducing blood pressure (Dahr et al 1968 cited in Neuwinger 1996). Roots are used for curing the diseases of the nervous system, elephantiasis and kidney problems while the leaves are applied to treat ulcers ( Aruna et al. 1998). In western Sudan the tribal ethno-botanist used

39 special preparation of Mucuna root to combat ravages of some ecto-parasites on cattle in the area (Abdalla et al 2001a).

2.3.3 Uses and chemistry of Mucuna

2.3.3.1 General uses of Mucuna

Seed extracts of Mucuna inhibit the effect of the venom of poisonous snakes in experimental , rats (Aguiyi et al 2001). Mucuna is used as food and drinks, medicine, toxins, forage and as a . M. pruriens has irritant hairs on pods and calyx (Legume web 2002). In Malawi, this species is the only annual legume that produced more than 2000kg/ha of seeds. If the seeds of Mucuna are to be eaten, they should be carefully processed as they contain considerable amounts of the L-Dopa, a toxic material used as a medicine against the Parkinson disease (Gilbert R., 2000).

The different species of the genus Mucuna are traditionally used in many ways: as a dye (Standley and Steyermark 1946), as a native medication to treat pains in joints and for the irregular menstruation (Ding et al 1991) and as food (Wilmot-Dear, 1984 and Versteeg et al 1998). Recently there are some endeavors for incorporating Mucuna seed flour with wheat flour for production of high protein biscuits. The over all acceptability of the Mucuna biscuit was appreciable (Ezeagu et al, 2001). Mucuna is used as hunger-food in Malawi where 2ton/ha of seed could be produced without any fertilizer, where maize yields less than 1 ton of seeds/ha (Gilbert 1998 cited in Giller 2001).

2.3.3.2 Agronomic uses of Mucuna

Kiome and Hartmann (2003) introduced Mucuna as a promising multipurpose crop to the resources-poor or smallholder farmers in tropical Asia, Mesoamerica and Africa. This is mainly due to its favorable impact on the main crop in the rotation, effective weed and pest control and finally due to the high potentiality of being as food and feed crop. Mucuna has been a subject of many tests as green manure for intensive maize production in West Africa (Hauser S. Nolte 2002) as well as a cover crop in

40 many African countries. The seeds could be used as sources of edible flour after soaking and heat processing, manual de-hulling, dried and ground (Ahenkora et al 2001). Mucuna pruriens which is used in Sudan savannah of West Africa as a cover fallow is a promising way to protect soil and suppress weeds (Carsky et al 1998 and Vissoh et al 1998). Some 36 cultivars of Mucuna were examined for their L-Dopa content. A range of 1.8-7.6% of the Mucuna seed dry matter was reported as L-dopa in the tested cultivars. In several areas of the world Mucuna is used as rotational crop that adds to the soil fertility. For instance, Mucuna inclusion in crop rotation with rice and corn resulted in a yield increase of these crops (Berhe 2000). Historical background of the ample uses of the plant was given in the southern U.S. in the period from 1900-1950 by Eilita (2000). During that period of time Mucuna was viewed as an excellent multipurpose crop in the U.S. agriculture that fitted very well in the cropping pattern of the area as it required little labor, enhanced corn and maize production and helped in the integration of the animals in the cropping systems.

41 2.3.3.3 Chemistry of Mucuna

Recently, the chemistry of Mucuna has received more attention and attracted a plethora of research work. The works are mainly focused on the chemical analysis of seeds as potential sources of food and feed as well as sources of many medicinal, toxic and anti-feedant substances (Siddh Uraju et al 1996; Lorenzetti et al 1998; Flores et al 2002; Diallo and Berhe 2003; Egounlety 2003; Ezeagu et al 2003; Gurumoorthi et al 2003; Matenga et al 2003; Teixeira et al 2003 and Szabo 2003). The chemistry of Mucuna was also extensively studied by some scientists (see Lorenzetti et al 1998, Szabo 2002) and emphasis was on L-Dopa, a toxic substance in the seed that limits its value as human food and amimal feed. Moreover, Szabo (2002) reviewed and summarized, the pharmacological aspects of L-Dopa which is also used as an anti-Parkinson disease remedy. She stated that Vitamin B-6 could be used as an antidote to the L-Dopa in Mucuna. Other secondary compounds like Tryptamines and Bufotenine and others were also investigated. Tryptamines, bufotenine and N,N-Dimethyltryptamine are naturally occurring compounds that are structurally related to Serotonin (5-hydroxytryptamine). These compounds have potent effect on the brain. However many plants and insects produce serotonin ( eg. Pine apple and banana from plants and stinging nettles ( Urtica sp.) and some wasps from insects (Feldman and Lee 1985 cited in Szabo and Tebbett 2002 ). Pods of some Mucuna varieties are famous for causing a severe stinging but it is not yet confirmed whether this sting is from contact with serotonin, due to contact with Mucunain a protein present in some Mucuna varieties or due to some other unknown substances in Mucuna plant materials (Fairbrothers et al 1985, quoted by Szabo 2002). Ayala et al (2003) presented data on the chemical composition of Mucuna seed and the husk of the seed pots as a supplement feed for ruminants. For the digestibility they found that grains are more available in the rumen than the husk and hence grains could be an excellent source of fermenTable nitrogen and energy to the rumen micro-flora which aid further digestion. Some alkaloids like Mucunine, Mucunadine in addition to the highly toxic nicotine are found in small amount in the seed (Neuwinger 1996 quotation from Majumdar 1944). M. pruriens (L.) DC. produces the toxic principle L-Dopa more over it has been reported to contain a hallucinogenic compound relevant to N, N-Dimethyltryptamine (Lorenzetti et al 1998). In alteration of the toxic

42 substances in Mucuna, the plant resistance to pests and the allelopathy should be considered (Lorenzetti et al 1998).

Studies on Mucuna root chemistry are very rare. Aruna et al (1998) conducted phyto- chemical studies on the roots of M. pruriens Baker which revealed the presence of sterols, triterpenes and flavonoids, the latter were isolated for the first time from Mucuna roots.

2.3.4 Mucuna as insecticide

2.3.4.1 Why Mucuna pruriens is selected ?.

It is possible to apply folklore to help in targeting a certain plant material for its bio- activity. In this folklore, if a plant is reported to have biological activity against insects, or kills the ecto-parasite of both man and animal, this may represent a good avenue of research (Benner 1993). Mucuna seeds are mainly free of insect problem unlike other legumes seeds which are attacked by seed borers, mainly bruchids (Lorenzetti et al 1998) For M. pruriens, that it's extensively used in a traditional way by some ethno-botanist in Africa and elsewhere in the world against a panorama of health and pest problems. In western Sudan the M. pruriens root extracts are widely used to control ravages of the animal lice in the young calves of cattle. This is why Mucuna was selected to undertake these investigations.

2.3.4.2 Insecticidal properties of Mucuna

Although the phyto-chemical properties of Mucuna were intensively targeted, with special emphasis on L-Dopa in the plant seeds, testing the bioactivity of Mucuna products is rare. Lorenzetti et al (1998) observed that seeds of Mucuna are mainly free of insect problem unlike other legume seeds, which are attacked by seed borers mainly bruchids. Investigations of pesticidal properties are even more rare especially with regard to the root products. Abdalla et al (2001ab) provided the only available reference to date on the Locusticidal properties of root extracts of Mucuna. The study was conducted as a laboratory bioassay test for the efficacy of Mucuna in comparison

43 with other plant materials indigenous to Sudan. The contact and stomach efficacy of the candidate materials were evaluated against the 2nd nymphs of the Migratory Locust L. migratoria Linné. Mucuna root extracts caused some poisoning by contact and exhibited knockdown effect on the Locust larvae. Water soluble and ethanol/water soluble extracts of roots were found similar to the standard extracts of Neem seed kernel in the same solvents and mortality rates of 100%, 98.8% respectively were recorded, while the Neem extracts yielded 93.3%, 91.7% mortality, respecting the orders of the solvents (Abdalla et al a, 2001).

2.4 Bioassay methods for plant materials

Bioassay tests of botanicals is mostly concentrated in testing Neem products and some other Meliaceae on various types of insects and with different mode of action (Schmutterer and Ascher 1983; Kleeberg and Zebitz 1997). For the other plant materials, few experiments were conducted.

2.4.1 Contact bioassay

Wilps et al (1992) reported up to 75% mortality in S. gregaria flying adults sprayed with Neem oil compared to mortality not higher than 8% when non flying adults were sprayed. Methanolic Neem seed kernel extracts had no effect when were applied to the resting adults of S. gregaria at a dose of 1ml./m2. The effect appears to be a 50% decrease in the flight ability of the Locust compared to the untreated control. When the same product of Neem is applied to the flying adults of the Desert Locust mortality was increased up to 65% in comparison with the untreated control. With palm oil, the mortality rate remained less than 10%. Flight activity of the Neem treated Locust was reduced by more than 60% and these Locusts failed to sustain flight (Wilps et al 1993). For the contact efficacy of Neem product on the larvae of the Desert Locust, S. gregaria, Nicol (1993) reported the highest mortality coupled with greatest disturbance in morphogensis of the Locust. In these trials Neem also exhibited morphogenic effect when it was applied on the inter-segmental membrane. Ping et al (2001) investigated contact and stomach efficacy of Stellara chamaejasme L. (Thymelaeaceace) a plant known for its traditional medicinal value in China. They

44 conducted bioassay trials with root extracts of this plant against Aphis gossypii and Schizaphis graminum. Strong contact activity was reported against the test subjects.

2.4.2 Topical bioassay

Mwangi (1982) applied 10µl of 2% kernel extract of Melia volkensii topically every other day on the neck membrane of the Desert Locust and concluded that topical application with this extract lead to reduced food intake probably through physiological disturbances. From Artemisia monosperma, a herb wildly grown in the Egyptian Desert and elsewhere, Saleh (1984) obtained a crude extract and steam distillate which were subjected to bio-assay test against a susceptible strain of the housefly, Musca domestica and the 4th instar larvae of the cotton leaf worm, Spodoptera littorallis. The plant extracts were applied topically to the tested insects.

2.4.3 Dipping bioassay

Studies in insect dipping bioassay are generally very rare or not at hand, especially for orthopteroid insects. However, dipping methods of bioassay was used by Sombatsiri and Tigvattanont (1983) who dipped, momentarily, the 5th instar larvae of tobacco cutworms in 1% Neem seed extracts. Ping et al (2001) used insect dipping bioassay technique with aphids to evaluate some botanical materials as aphicides.

2.4.4 Testing for stomach action

The laboratory and field efficacy of Melia toosendan was studied together with other botanical extracts including Neem. The active principles in this Melia product is the triterpenoid toosendanin which showed high potential as anti-feedant against the larvae of Spodoptera litura. But toosendanin showed no activity as growth regulator, which is the case for Azadirachtin (Shin-Foon 1983). Ping et al (2001), in their antifeedant assay of Stellara chamaejasme L. (Thymelaeaceace) against Aphis gossypii and Schizaphis graminum, reported a very hight anti-feedant activity of the target plant materials. Ould Ahmedou et al (2001) demonstrated the potential of plant materials from Glinus lotoides as toxic and antifeedant against the Desert Locust. They reported 100% mortality of the 4th nymphal instar fed on G. lotoides within 10

45 days. Moreover, a loss in weight was noticed which indicates the feeding deterrence on Locust fed on this plant. They attributed this antifeedant effect to some plant allelochemicals which might be the cause of Locust avoidance to this plant in the field. The feeding deterrence of Neem products to Locust and grasshoppers has been demonstrated by many workers (Mordue and Evans 1987, Freisewinkel 1993, Mordue et al 1998, Nasseh et al 1993, Schmutterer et al 1993, Baumgart 1994, Nasiruddin and Mordue 1994, Langewald et al 1995). Butterworth and Morgan, (1971) who used Neem products against the Desert Locust reported that feeding was completely inhibited in the 5th instar nymphs when a filter paper impregnated with Azadirachtin was provided for the Locust feeding. For the grasshoppers, Passerini and Hill (1993) in their laboratory and field trials in Mali found that Neem had a great antifeedant effect on the sahelian grasshopper, angulifera (Acrididae, ). In their bioassay trials on Neem extracts they sprayed millet plants until complete coverage was achieved then the grasshoppers were introduced in cages and allowed to feed. They noted that, application of aqueous Neem extracts at 0.5% and 1% concentrations effectively reduced feeding of grasshoppers. The antifeedant effect reported by Passerini and Hill (1993) confirmed the results of Radcliff et al (1991) who worked with Neem extracts against the same species of grasshoppers in Niger. Most of the Neem anti-feedant activity was investigated for the seed kernel extracts, however, leaf extract also exhibited an anti-feedant effects on Orthoptera (Joshi and Lockwood, 2000). Schmutterer et al (1993) found that Schistocerca was more vulnerable to the phagorepellent effect of Neem oil than Locusta. Freisewinkel (1993) found that the mortality on the 3rd instar nymphs of Locusta fed on maize leaves treated with Neem oil was much higher than in those directly sprayed with Neem oil and he reported a dose-response relationship.

2.4.5 Filter paper test

Plant materials were sometime assayed for their bio-activity by impregnating filter paper with plant extracts. Then the paper is given to Locust for feeding. Butterworth and Morgan (1971) investigating the Locust feeding inhibition of Neem seed extracts reported complete feeding inhibition in Locust with test solution even if they decrease the test concentration up to 40µg/l. Using the same method of filter paper test,

46 Mwangi (1982) reported a substantial feeding inhibition in the S. gregaria provided with filter paper impregnated with seed kernel extract of M. volkensii.

47 2.4.6 Bioassay test of botanical compounds under choice/no-choice state

Nasseh et al (1993) investigated the effect of Neem oil treatment against adults L. m. migratorioides. Neem oil was applied to one plant of Schouwia thebaica Webb (Brassicaceae), while another plant was untreated. Then 120 adult Locusts were introduced. They found that the untreated plant was first eaten (100%), then the adult started feeding on the treated one. On a second trial the Locusts were offered only Neem treated S. thebaica plants. The result is that 80% of the Locust died within 10 days. The rest of Locusts started feeding on the treated plant.

2.4.7 Effect of time of exposure on activity of botanical treatments

Mortality in assays was found to increase with time of exposure to the tested materials. Some studies were conducted to assess the effect of time of exposure of target insect to certain bio-active plant materials or synthetic analogues. Verkerk and Wright (1993) investigated this effect by exposing Plutella xylostella larvae to Azadirachtin-treated cabbage leaf-disc for the different time: 6h, 24h, 48h and 120h. They reported increase in mortality of the larvae with increase of exposure time: 27%, 47%, 61%, and 68% for 6h, 24h, 48h and 120h respectively.

2.4.8 Dose-response relationship

The dose-response relations in insects treated with Neem product was investigated by Verkerk and Mordue et al (1998) for the anti-feedant response of S. gregaria and L. migratoria to different treatments of azadirachtin or its analogues. They found that L. migratoria was less responsive towards the change in the dose than S. gregaria. They showed that Azadirachtin was an extremely effective anti-feedant agent against polyphagous insects such as S. gregaria whereas oligophagous ones like L. migratoria were less responsive. The exact reasons for such differences in are unknown. They further investigated the dose-response relationships on some Lepidoptera which showed a wide range of responses over different concentrations. Ping et al (2001) assaying plant products extracted from roots of Stellera chamaejasme (Thymelaeaceae ) against aphids reported for the first time the

48 aphidicidal activity of the tested plant material which also exhibited dose-dependant relationships against aphids

2.4.9 Insecticides mixtures and synergism

Olaifa and Akingbohungbe ( 1986) studying the antifeedant and insecticidal properties of some botanical mixtures against the variegated grasshopper in Nigeria reported a substantial enhancement in the performance of Neem extracts when mixed with other botanical extracts. Mixing and subsequently applying pesticide mixtures can enhance toxicity of pesticides, a phenomenon known as synergism, which can be of value in pesticide formulation (Godson et al 1999). Application of Locusticide mixtures was discussed by the FAO Pesticide Referee Group. The data available to the Group were for the mixtures of pyrethroid/organophosphate and organgophosphate/carbamate only. The mixture will reduce the level of individual active ingredient in the environment. Mixtures of pyrethroid/organophosphate were extensively tested against the Desert Locust in Mauritania to study the possible synergism and to reduce the amount of each insecticide. The results are still insufficient for a concrete recommendation on a certain mixture (FAO 1999). However, Abdalla et al. (2003) provided some evidence for the possible synergism resulting from mixing Decis (Deltametherin) with root extracts of M. pruriens. They studied the effect of low concentrations of Decis mixed with fixed concentrations of root extracts of M. pruriens against The Desert Locust. Increases in the knockdown and killing effects of the mixtures were reported when compared with the effect from Decis or M. pruriens applied alone.

49

3 MATERIALS AND METHODS

The work presented in this thesis was carried out during the period extending from August 2000 to Oct. 2003 and was mainly conducted in the laboratory of CIRAD- Prifas at Montpellier, France. However, a field survey was conducted in the Sudan on the traditional use of plant materials in pest control in western Sudan. The survey was conducted with special emphasis on Mucuna pruriens( Fabaceae), a plant widely used by some nomadic tribes and cattle raising settlers of that area.

3.1 Field survey

A key informant questionnaire (appendix 1.) was designed to gather some data and to document the indigenous knowledge on the use of plant materials to combat pests in the area. The survey covered two provinces; Zalingi in Western Darfur state and Kas in Southern Darfur state (Figure 3). In the survey 104 respondents from the surveyed areas were interviewed for their experience with M. pruriens and other plant materials. The survey included some aspects pertaining to the social life of respondents, local names given to Mucuna in the area, the part of the plant used, methods of extraction and the ways of application, availability of Mucuna and the years of experience of respondent in using Mucuna. A group discussion with key informant groups was held in each of the surveyed provinces with the objective of cross checking and reaching common understanding on the issues raised.

50

Figure 3. Map of Sudan showing areas where native people were interviewed for their experience with Mucuna pruriens (Fabaceae) and other botanicals used in pest control.

3.2 Collection and preparation of the plant materials for extractions Plant materials from Adenium obesum ( im Tree), Calotropis procera (Usher shrub),, Azadirachta indica (Neem Tree) Mucuna pruriens ( a twisting climber locally known as Erig El-Ghamoal) were collected from different areas in the Sudan as indicated in the map (Figure 4) to undergo bio-test. Selection of the plant materials followed the criteria described by Benner (1993). These plant materials are traditionally used by Sudanese as pest and/or disease control agents. The collected materials were then prepared, to perform extraction, as follow:

51

‰ Roots of the Sim Tree, Adenium obesum : Roots of Adenium obesum an Apocynaceous shrub locally known as the Sim Tree were collected from Dilling town (800 km Southwest Khartoum) in August season 1999/2000. They were chopped into small pieces and allowed to be dried under the shade. The root materials were then ground into powder by a laboratory electrical grinder.

‰ Leaves of Usher, Calotropis procera : Leaves of an Asclepiadaceous shrub known locally as AUsher shrub, Calotropis procera were collected from Khartoum area and from El-Obeid (600 km Southwest Khartoum) in September 2000, dried under shade condition, crushed and subsequently ground in the same way as described earlier.

‰ Seed Kernels of Neem Tree Azadirachta indica: Dry fruits of Neem tree, Azadirachta indica( Meliaceae) were collected from the vicinity of El-Obeid (600 km Southwest Khartoum) in June/July 1999/2000, 2000/2001 and 2001/2002. Collected fruits were then soaked in water for 6 hours to depulp them to release the seeds, which were shade- dried. The kernels were obtained by decorticating the seeds in a wooden mortar with pestle. As most of the available literature on botanical insecticides is from Neem products (Azadirachta indica), mainly the seed kernels, Neem was selected as the standard botanical insecticide throughout the course of this study.

‰ Roots of Mucuna pruriens : Roots of a plant locally known as Erig El-Ghamoal ( identified by Abu El-Gasim A., eco-botanist in The University of Zalighi in western Sudan, as Mucuna pruriens, Fabaceae ex. Paplionaceae). were dug out from the fringes of Zalinghi at the vicinity of Marra Mountain of western Sudan in September season 1999/2000 (Figures 4, 5 and 6). To provide this material for the rest of the experimental work, Mucuna roots were collected in September from the same area in the subsequent years( i.e. 1999/2001 and 2001/2002 seasons) Roots were chopped in slices and dried under

52 shade and then ground using the same laboratory grinder as in the case of Usher and Sim Tree materials

Figure 4. Areas in Sudan where plant materials were collected for study.

53

Figure 5. Digging of roots of Mucuna pruriens.

Figure 6. Roots of Mucuna pruriens.

54 3.3 Extraction of the plant materials

The extraction process was performed according to the protocol developed in collaboration with Prof. Andary from Faculty of Pharmacy, University of Montpellier I. 50gm of each of the plant materials was separately mixed with one liter of solvent which was either distilled water alone or in mixture with ethanol (v: v).. The obtained mixtures were first blended for 3min. in a laboratory blender (8010 E Model 38BL40 400W, Waring Commercial, Torrington, Connecticut, USA). Then put on a magnetic agitator( Model CB162, 550W from Bibby Sterlin Ltd., Slone, Stofforshire, UK).at 500r/min and 40°C for 20min and finally put in an ultra-sonic device (Sonication 110W J.P. Selecta S. A. Barcelona, Spain) for 15min to enhance and accelerate extraction. Each of the four solutions was filtered through a fine muslin cloth and used as crude extract against the test insects. A similar method of extraction was described by Ascher et al (1983) who worked with Neem seed kernel against Spodoptera littoralis (Lepidoptera). To reduce the surface tension and to insure an even spray and good coverage of the targets, 3 droplets of a chemical surfactant (Alkylphenol Oxyethylene ) were added to each extract before application. The above mentioned extraction procedures is a modification of the extraction procedures applied earlier by Naidu and Ganapaty ( 1998) in their phytochemical studies on the roots of M. pruriens. They used different solvents such as chloroform, methanol and hexane. The former two solvents were not used in this study due to their toxicity and hazard during handling.

3.4 Locust rearing

The Migratory Locust, Locusta migratoria (Linné 1758) and the Desert Locust, Schistocerca gregaria (Forskål 1775) were reared as described hereunder and used for the tests of plant materials throughout the course of the study. Migratory Locust was reared separately under semi-control conditions in the year 2000, while the two species were reared together under control conditions in the subsequent years of the study. The two Locust species were used in the bioassay tests during the course of this study. The first species is oligophagous while the other is highly polyphagous.

55 3.4.1 Rearing of Migratory Locust

The starter culture was provided by Dr. Skovmand who collected the Locust from Sumatra Island, in June 2000. Mass rearing of Locusta migratoria Linné was performed according to the rearing techniques in the laboratory of CIRAD- PRIFAS, France in the year 2000. Locusts were reared in clear plexiglas cages (40 x 40 x 50cm) with a removable floor that supported four egg laying tubes (6.5cm in diameter and 10 cm in height), while the top and the front sides were of plexiglas with a flappable slit that allowed for handling, cleaning and feeding of the Locust (Figure 7). The three sides of the rearing cage were covered with a mosquito screen to provide ventilation and to allow for light. A 40-Watts lamp was placed against one screened side of the cage to provide light and some local heating for 12h a day. The general room temperature is kept at 25°C ; the relative humidity in the rearing room ranged between 50-60 %. Pots of 10-15 days old wheat seedlings with some wheat bran were provided daily for feeding. Some grasses and bamboo leaves were added from time to time to enrich the Locust diet. The 2nd nymphal instars, which were selected for the bioassay were from the 3rd generation reared under the described laboratory conditions. This method of rearing is like that described earlier by Ishikawa and Kanke, (2000) who reared L. migratoria Linné on wheat seedling. The cages they used they are some what smaller( 30x30x30cm). Light/darkness periods used was as well different( 16/8h.). Quesada and Santiago-Álvarez, (2001) studying the Moroccan Locust Dociostaurus maroccanus used a similar method of rearing with minor modifications.

56

Figure 7. Plexiglas cages for mass rearing of Locusts.

3.4.2. Rearing of the Migratory and Desert Locust

To provide Locust materials for the different sets of the bioassay experiments during the years 2001 and 2002 the Desert Locust, Schistocerca gregaria, and the Migratory Locust, Locusta migratoria, were separately reared in the laboratory of CIRAD- Prifas. The two Locust species were mass reared in controlled chambers calibrated at 28 ± 1°C during day time and 25 ± 1°C at night; relative humidity was 60% ± 5% and Light/ Dark time regime of 12/12h. The Desert Locust culture was started by three Locust lots obtained as egg pods from different sources. The first lot was provided by Professor M. O Bashir, from The International Centre for Insect physiology and Ecology (ICIPE) in Port-Sudan, Sudan. The second was provided by M. A. Ould Babah from the Mauritanian anti-Locust center (Centre de Lutte Antiacridienne of Mauritania, CLAA) while the third lot was bought as sexually mature adults from a private company in France. For Locusta, the offsprings of the Locust reared at CIRAD in the year 2000 were used. Males and females were reared together throughout the rearing period. Primarily they were fed on wheat seedlings and wheat

57 bran but from time to time Locusta was provided with bamboo leaves, while cabbage leaves and some plants of Lactuca serriola L. (Asteraceae) were added to insure the diet diversity of S. gregaria. The Locusts were reared in clear plexiglas cages (40 x 40 x 50cm) with a removable floor that supported four egg laying tubes (6.5cm in diameter and 10 cm in height), while the top and the front sides were of plexiglas with a flappable slit that allowed for handling, cleaning and feeding of the Locust. After eggs laying, egg tubes were transferred to an incubator at temperature 28±1ċ. The newly emerged nymphs were then transferred into the cages and reared until adulthood. The daily routine work regarding rearing condition, sanitation of culture, culture manipulation and handlings of Locusts are similar to the procedures followed/described by Hunter-Jones (1956), Harvey (1990), Ochieng et al (1991) and Hinks and Erlandson (1994).

3.5 Screening for insecticide properties of plant materials

3.5.1 Objectives of screening

All experiments in this part were done with the aim of screening for Locusticidal properties of root extracts of M. pruriens, an indigenous plant in Sudan, with three other plant materials that are traditionally used by some natives in the Sudan to combat insect pest of different types. These plant materials were obtained from roots of Sim Tree Adenium obesum (Apocynacae), leaves of Usher Calotropis procera (Asclepiadaceae) and seed kernels of Neem Azadirachta indica (Meliaceae). The latter one is mainly used as standard botanical insecticide. The four plant materials were tested against the second nymphal instar of the Migratory Locust L. migratoria. The plants material is prepared for application as crude extract made as described earlier in this chapter.

3.5.2 Bioassays of the plant materials

The efficacy of plant extracts was determined the second nymphal instar of L. migratoria reared under the mentioned conditions. These nymphs were kept in cages and deprived of food for 4-5h before the test in case of stomach action. However,

58 water was provided, ad libitum, in a piece of wet cotton in a Petri dish. This nymphal stage was chosen for its relatively short duration that allow for testing a large number of insects in a relatively standard period of time compared to other developmental stages of locust. The of modes of action of the plant extracts investigated included contact and stomach. The results were assessed either as knockdown, death and time to death.. All experiments were conducted under room condition where temperature fluctuated between 25-26 °C and 12/12h light/dark time regime.

3.5.2.1 Contact action of the plant extracts

The plants extracts were applied by direct spraying on Locust nymphs using a small hand sprayer (400ml.) at an application rate of 21l/ha(approximately). For each of the water extracts and ethanol/ water extracts of the candidate plant materials, ten second instar were sprayed in a separate cage (5 liter plastic bottle). to the point of the runoff, transferred into a new cage of the same type and supplied with wheat seedlings for feeding. Cages were covered by a piece of muslin cloth fixed with a rubber band and kept under the described controlled laboratory conditions (Figure 8). For the untreated control, nymphs were either sprayed with distilled water in the case of water extract, or with distilled water/ ethanol (95%) at a ratio of 1:1 (v:v). Three droplets of Alkylphenol Oxethylene were added to the plant extracts and to the control solution to act as a surfactant.

The treatments were replicated six times each. Knockdown effect was recorded for each treatment one hour after the start of the experiment. The mortality caused by the candidate plant extracts was assessed after 24h and then on daily basis. The number of living insects, number nymphs completed development and numbers of dead individuals per cage and per treatment were determined separately. The tests continued until 100% normal moulting or 100% mortality was observed. Nymphs that successfully moulted into third instars were picked out of the cage. At the end of the trials percentage mortality, mean duration of the second nymphal instar, time to death and the cumulative faecal output were taken for each treatment. Collected feaces was desiccated at 40 °C for 24h and then weighted.

59

Figure 8. 5l. plastic bottles used as cages.

3.5.3.2 Stomach action of the plant extracts

The previously prepared plant extracts as described in paragraph 3.1.1.2. were applied to wheat seedlings. One week old wheat seedlings of 10-15 cm length grown in small plastic pots (20 seedlings/pot) were dipped for one minute in the one of plant extracts (i.e. ethanol/water or water extracts of the plant materials). The treated wheat seedlings were allowed to dry under room conditions for one hour and then transferred into plastic cages. Ten nymphs, previously starved for 4-5 hours, were introduced in each cage and allowed to feed on the seedlings. For the untreated control, seedlings were dipped in a solution of ethanol/water (v:v) or water only and allowed to dry for one hour under room conditions and exposed to the starved nymphs.

3.6 Evaluation of biological activities and the mode of action of M. pruriens extracts

60 After screening for the Locusticidal properties of the extracts of M. pruriens in relation to extracts from A. obesum, A. indica and C. procera which are traditionally used for pest control, extracts of M. pruriens were compared with extracts of A. indica as standard botanical pest control agent and Decis as standard chemical pesticide. For evaluation of the contact and stomach action, these three materials were applied to the Migratory and the Desert Locust in the same way as described before.

The Mucuna extracts were evaluated for their contact bio-efficacy by direct spraying on Locust using hand sprayers of 400ml capacity. A series of concentrations ranging from 100g/l to 0g/l were applied on lots of 10 individuals each of the Desert Locust and the Migratory Locust kept in 5l capacity plastic bottle. For both Locusts, treatments were applied to the individuals of the 2nd instar nymphs and fledglings (one week after the final moult) to observe the knockdown effect and mortality. In these sets of experiments, knockdown was observed one hour after treatments while, mortality was recorded on hourly basis for the 1st 6h and then every 24h until the end of the experiments. For the 2nd instar nymphs, the experiment will end either at 100% mortality or 100% moulting into 3rd instars nymphs, while, for the adults observations continued for 7 days after treatment. Unless otherwise indicated, a minimum of six replicates were conducted for each treatment.

3.6.1. Contact efficacy of Mucuna pruriens extracts The extracted Mucuna root materials were examined for their efficacy as contact botanical materials against the candidate Locust as:

3.6.1.1 Spraying Mucuna extracts The Mucuna extracts were directly sprayed on the assays. Knockdown and mortality on the test Locust were observed.

3.6.1.1.1 Efficacy on the 2nd nymphal instars of the Desert Locust

Investigations carried out in the context of the contact effect were performed through direct spray of the treatments on the 2nd instar nymphs of the Desert Locust. Control

61 group, was sprayed with water/ethanol only. Four replicates were made. Observation on knockdown effect, mortality and moulting to the third nymphal instars were kept.

62 3.6.1.1.2 Efficacy of Mucuna, Neem and Decis on the adult of the Desert Locust

These trials were conducted on young Desert Locust adults (7±1 day). Six replicates were made for the extracts of Mucuna, Neem and four replicates only for Decis were applied. Data on knockdown was reported one hour after spraying, mortality was recorded daily for ten days. Faecal out put was daily collection of faeces from the available insects continued for seven days. Faeces were oven dried at 40 °C to constant weight. The faeces from each treatment was weighed separately for comparison.

3.6.1.1.3 Efficacy on the 2nd nymphal instar of the Migratory Locust

The mentioned concentrations in paragraph 3.1 were applied here. Ten insects were sprayed in each treatment and data on knockdown were reported once 1hr from spraying. For mortality data were collected on a 24h basis. Five replicates were conducted.

3.6.1.2 Topical application of Mucuna extracts

Mucuna extracts were topically applied to the 5th nymphal instars of the Migratory Locust, adopting a modified version of the topical application method used earlier by Verkerk and Wright (1993) on larvae of Plutella xylostella using an Arnold micro- applicator fitted with an Agla glass syringe. In this study, however, 20µl of ethanol/water extracts of M. pruriens were applied topically, 10 droplets of 2µl each, on the neck membrane and at the base of wings using a calibrated glass micro syringe( 1ml.in capacity ) fitted with the above mentioned device (Figure 9). The following series of concentrations of Mucuna extracts were applied against the nymphs : 200g/l., 150g/l., 100g/l. and 0g/l. The last concentration was a control treatment of water/ethanol (v: v ) mixture. Knockdown and mortality were recored. The test was concluded when there was complete mortality or complete moulting into the adult stage. Six replicates were made (5 replicates only for 150g/l.). Kämper,

63 (1997) studying the responses of Locusta migratoria topically treated with Neem used the same nymphal stage and he applied neen product topically on the contra- lateral wing bud.

Figure 9. Application of Mucuna extract with a micro syringe.

3.6.1.3 Dipping bio-assay with Mucuna extracts

This experiment was conducted on one week old adults of the Migratory Locusts. The Locusts were dipped for 1.5 sec. in Mucuna solution of the following concentrations: 100g/l, 50g/l, 25g/l and 0g/l (i.e. with solvent only). Knockdown and mortality were recorded in each treatment in the same manner as mentioned earlier (Figure 10).

64

Figure 10. Dipping an adult of Migratory Locust in a Mucuna solution.

3.6.2 Stomach action of Mucuna

To study the efficacy of M. pruriens extracts as the stomach poison on Desert and Migratory Locusts, the following trials were made.

3.6.2.1 Feeding Locusts, wheat seedlings treated with Mucuna extracts

Wheat seedlings were dipped for one minute in Mucuna extracts and left to dry under room condition, and then ten of the 2nd instars nymphs of each of the studied Locusts were allowed to feed on them. The extracts tested were Mucuna 100g/l, 50g/l, 25g/l and 0g/l ( control). Six replicates were made for each concentration. Observations on mortality were kept throughout while, the faecal output was collected after 24h, oven dried at 40 °C overnight and weighted.

Some trials were conducted on Locusta and Schistocerca imagoes (one week old) using Mucuna 100g/l to observe the impacts on adult Locusts in term of knockdown and mortality. Six replicates were made.

65 3.6.2.2 Provision of treated wheat seedlings for limited feeding time

This experiment aimed at exploring the effect of time on feeding activity of Locust on seedlings treated with Mucuna extracts. Wheat seedlings, previously dipped in Mucuna extracts (100g/l), were provided to 2nd instar nymphs of the Migratory Locust. The nymphs had access to the treated seedling for 6h, 24h and 48h. After each specified time period, the treated wheat seedlings were removed and replaced by fresh untreated ones. Observation on mortality was kept throughout while the feacal output was collected 24h after treatment, oven dried at 40°C to a constant weight.

3.6.3 Feeding deterrence of Mucuna extracts Anti-feedant effects of Mucuna were evaluated against locusts through various bioassay methods as follows:

3.6.2.3.1 Cabbage leaf disc bioassay under no choice condition

Extracts of M. pruriens were assessed as anti-feedants against 7±1 day old imagoes of the Desert Locust, Schistocerca gregaria. The Locusts were offered discs of cabbage leaves of known weight under no choice condition. The discs were dipped in Mucuna extracts (100g. /l.) for 1 min., allowed to dry under room condition for one hour and then offered to the insects. Five starved Locusts were allowed to feed for three hours before the discs were weighed again. After the removal of the disc, the Locusts were left without food for 24h. , the faecal output was collected, oven dried and weighted. For the control, the same number of Locusts was offered a cabbage leaf disc that was dipped for the same period of time in a solution of water/ethanol (v:v). Twenty replicates were made for the treatments and for the control as well. The percent loss in weight of the treated cabbage leaf discs and the untreated control was calculated. The difference in weight of the frass was also calculated.

3.6.2.3.2 Cabbage leaf-disc bioassay under choice condition

Another set of experiments was designed to investigate the feeding deterrence resulting from application of the Mucuna extracts to cabbage leaf discs. In this experiment a cabbage leaf was cut in the form and size of a round disc (9 cm in

66 diameter). The cabbage leaf was then divided into two identical halves. Verkerk and Wright (1993) used similar method to study the biological activity of Neem and synthetic azadirachtin against Plutella xylostella on cabbage leaf-disc. The two halves were weighed separately. One half was dipped for 1min in the Mucuna extracts, while the other was dipped for the same period of time in the solvent (water/ethanol=V: V) to serve as control. The two halves were then reassembled in the form of a circle and put in a plexiglas cage. Five individuals of the adult Locust(of 7±1day), previously deprived of food overnight, were introduced into the cage and allowed to feed for 3 hours. After feeding the remaining parts of each half (Figure 11) were weighed again. The test was replicated 30 times. The loss in weight of the treated and the untreated disc control was calculated by subtracting the weight of each half after feeding from the initial weight of the respective half. Then the following formula was applied to calculate the percent loss in weight in each case:

The %age loss in weight of cabbage = Wt.b.- Wt.a X 100 Wt.b.

Whereas: Wt.b.=Weight of cabbage before the feeding Wt.a= Weight of cabbage after feeding

.

67 Figure 11. Cabbage leaf-disk used in feeding deterrent experiments under free choice conditions.

3.6.2.3.3. Individual tests with filter paper

In this test Locusts were used to investigate the feeding inhibition action from extracts of Mucuna. The trials were conducted on the adult Desert and Migratory Locusts separately. A piece of filter paper 1x2 cm was submerged for 30 sec. in 10% sugar solution allowed to dry and then held on crocodile clip fixed in a test tube plastic stopper. Sugar was added to stimulate feeding. 20µl of Mucuna 100g/l were applied on the filter paper in the form of droplets by means of Arnold micro applicator fitted with glass micro syringe of 1ml capacity. The treated filter paper was dried under room conditions for one hour and introduced to individuals of adult Locust (7±1 day old) in a test tube 2.3 cm diameter and 15 cm length (Figure 12) . Data were collected on the acceptance or rejection of the filter paper by Locust in every hour for the first 6h then after 24h. Thirty replicates of one Locust each were conducted for the Migratory Locust and 33 replicates for the Desert Locust. Equivalent numbers of replicate were made for the respective Locusts to serve as control by applying 20µl of solvent on the filter paper. This method was devised by Butterworth and Morgan, (1971) which was modified so that 5th nymphal instars were used which were put in groups of four insect per kilner jar instead of a single insect per test. Moreover, in the present study the volume of the test material was known and the insects were allowed to feed only and not to have body contact with test materials.

68 Figure 12. Single Migratory locust per tube in feeding trials for individual insect tests.

3.7 Persistence of bio-efficacy of Mucuna extracts from roots collected in different years

A trial was made to study the effect of time on rpersistance of the bio-efficacy of Mucuna roots materials. Extracts prepared from root materials collected in the years 2000, 2001 and 2002, stored under room condition, were applied by direct spraying on 2nd instar nymphs of Desert and Migratory Locusts to evaluate their biological activities with respect to year of collection. Knockdown and mortality were determined. Extracts of 100g/l and 50g/l were applied. Control series were sprayed with solvents only.

3.8 Effect of Mucuna mixed with Deltametherin (Decis)

The effect resulting from mixing Mucuna root extracts was manifested in the following set of tests:

Mucuna extracts were mixed with reduced concentrations of Deltametherin (Decis). The tests were executed to explore the possible synergism resulting from a combination of Mucuna extracts with Decis. The tests dealt with a series of mixtures of various Decis concentrations with a fixed concentration of Mucuna at 25g/l.

In this experiment a series of sub-lethal doses of Decis were mixed with a fixed concentration of water extracts from Mucuna pruriens to form the following mixtures: 25g Mucuna with Decis at 3.125g, 1.56g, 0.78g, 0.39g and 0.195g per litre. Correspondent concentrations, of Decis only, were kept to be used for comparison. These treatments were applied to sets of ten 2nd. instars nymphs of the Desert Locust by direct spraying. Another set of the same number were sprayed with water only to serve as a control group. The treatments were replicated four times at least. Mortality was recorded on hourly basis for the first six hours and then on each subsequent day. At the end ( Death of all nymphs or moulting into the 3rd instar nymphs), the total mortality was calculated for each treatment.

69 3.9 Statistical analysis of the data

In the cases where the untreated control series yielded a mean mortality, the correction formula as in Abbott (1925) was applied using the following equation:

Test Mortality - Control Mortality Corrected Mortality = ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ 100 % 100-Control Mortality

The data of each experiment were analyzed separately using Mstatc statistical soft package. A completely randomized design was applied to all laboratory experiments except in the case of cabbage leaf-disc bioassay where t-test was used for the analysis of the data. For the cabbage leaf-disc trials data were transformed using arcsine transformation. Means comparison was run in each case

70 4 RESULTS

4.1 Field survey The field survey questionnaire (Appendix 1.) exhibits the questions asked to 104 respondents on different aspects of their experience with Mucuna. Two group discussions were held in each of the surveyed provinces which reached common views that were generally similar, more or less, to the results of the analysis of the survey data. The results obtained from the statistical analysis of this field survey are can be summarized as follows:-

4.1.1 Number of interviewees in each of the surveyed state according to their ethnic group The distribution of the interviewees for their experience with insecticidal properties of Mucuna in the surveyed area in western Sudan is presented in Table (1).

Table 1. Distribution of interviewees in each of the surveyed state according to their ethnic group

Variable Number of interviewees in Total (Ethnic group) South Darfur Western Darfur 1-Buggara 29 59 88 2-Others 9 7 16 Total 38 66 104

4.1.2 Level of education of the interviewees with regard to their ages Individuals of different age groups with different educational background and experience with Mucuna products used against insect pests in the study area are given in Table (2 ).

71 Table 2. Level of education of the interviewees and their ages

Education level Age group Literate Khalwa Primary Intermediate Secondary Total in years 1-Adults 15 9 5 1 1 31 to<30yrs 2-30-50yrs 18 30 8 - - 56 3->50yrs 8 9 - - - 17

Total 41 48 13 1 1 104

4.1.3 Educational levels and occupation Level of education and the way of living are given in Table (3 ) for the respondents surveyed for their experience Mucuna, in western Sudan.

Table 3. Levels of education and occupation

occupation Literate Khalwa Primary Intermediate Secondary Total

1-Nomadic 9 9 - - 1 19 2-Settlers 32 38 13 1 - 84 3-Others - 1 - - - 1

Total 41 48 13 1 1 104

4.1.4 Local names of Mucuna pruriens In Figure (13) the results of the field survey for the local names of Mucuna are presented. 93% of the respondents call it Erg El-Ghamoul.

72 7% Others (Erg Elhagar)

Erg-Elgamul 93%

Figure 13. The local manes given to the Mucuna pruriens in the surveyed areas in Western Sudan in the year 2001.

4.1.5 Methods of Mucuna extraction Results indicate that root extraction in cold water is the preferred method in comparison to extraction in hot water (Figure 14).

73 28% hot water

cold water 72%

Figure 14. Percentages of Traditional methods of Mucuna extraction according to the respondents in the surveyed areas in Western Sudan (2001)

4.1.6 Accessibility of Mucuna pruriens Results indicate that Mucuna materials are quite accessible to all who wish to use for pest control (Figure 15).

74 10% others (foot of hills)

90% valleys

Figure 15. Availability of Mucuna pruriens to users in the surveyed areas in Western Sudan (2001)

4.1.7 The latest application of Mucuna Table (4) reflects years where respondents had their latest experience of application of Mucuna root material against pests of cattle.

Table 4. Year of latest application of Mucuna plant materials as pest control agent.

Variables Last date of Mucuna application Total

(Ethnic group)... Last y. Last 2y. Last 4y > 5y

1- Buggara 18 38 10 22 88 2-Others 2 11 1 2 16 3-Total 20 49 11 24 104

75 4.1.8 Years of experience with Mucuna pruriens in different interviewee groups in the study.

Years of experience in using Mucuna root materials to combat ecto-parasites on cattle as given by different groups of the respondents in the study area are shown in (Table 5)

Table 5. Years of experience with Mucuna pruriens of different interviewee groups in the study.

Variables Years of experience Total ( Ethnic group) <20 yrs 20-40yrs >40yrs 1-Buggara 27 34 27 88 2-Others 3 9 4 16 3-Total 30 43 31 104

4.1.9 Palatability of Mucuna to the grazing animals Respondent’s views about the palatability of Mucuna plant to grazing animals in the study area are given in (Figure 16).

76 10% palatable

90% not palatable

Figure 16. % Palatability of Mucuna leaves and stems by the grazing animals in the surveyed areas in Western Sudan (2001).

4.2 Screening for the insecticide properties of Mucuna pruriens and other plant extracts The results of the screening trials for the locusticide properties of extracts from roots of Mucuna pruriens (Fabaceae) and other plant materials namely: leaves of Calotropis procera (Usher shrub); roots and stems of Adenium obesum (Sim tree) and seed kernels of Azadirachta indica (Neem tree) against the 2nd Instar nymphs of the Migratory Locust, Locusta migratoria Linné, are presented under different mode of action in the following sections. The main purpose of the screening trials was to study the potentiality as bioactive materials against locust and grasshoppers and to demonstrate a simple way of their preparation to be used by farmers in some parts of Africa.

77 4.2.1 Efficacy of water/ethanol extracts of the plants materials based on directly spray on locust.

4.2.1.1 Knockdown

Table (6) shows the knockdown effect of water/ethanolic (v: v) extracts of the studied plants materials against the 2nd instar nymphs of Locusta. There are significant differences between control and any of the plants extracts. However, no significant difference is shown among the different plants extracts.

Table 6. Mean knockdown effect on the 2nd. Instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) treated by direct spraying with water/ethanol (v: v) extracts of different plant materials in the laboratory (Temperature 25± 1º C. and % RH. 50-60).

Treatments No. of Knockdown SD SE Insects/ % ± ± test

1-Control 60 01.67b 0.41 0.59 2-Calotropis 50/l 60 20.00a 1.26 0.59 3-Azadirachta 50g/l 60 20.00a 2.00 0.59 4-Adenium 50g/l 60 21.67a 1.60 0.59 5- Mucuna 50g/l 60 28.33a 1.47 0.59

NB.: Means followed by the same lower case letters are not statistically different at P< .05

78

4.2.1.2 Mortality Table (7) shows the results of the efficacy of the screened plants extracts in term of mortality. Mucuna water/ethanol extracts gave a mortality of 98.67±0.38% followed by Neem extracts where 97.17±0.38% and for The Sim tree and Usher extracts it was 29.33±0.38 and 14.50 respectively. There was no significant difference in mortality between Neem and Mucuna or between Usher and control. However, there was significant difference between Mucuna and Neem extracts compared to other treatments and to control.

Table 7. Mean mortality of the 2nd. Instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) treated with water/ethanol (v: v) extracts of different plants materials in the laboratory (Temperature 25±1º C. and % RH. 50-60).

Treatments No. of Mortality SD SE Insect/ % ± ± test

1-Control 60 20.50bc 1.16 0.38 2-Calotropis 50g/l 60 14.55c 1.12 0.38 3-Azadirchta 50g/l 60 97.17a 0.69 0.38 4-Adenium 50g.l 60 29.33b 1.10 0.38 5- Mucuna 50g/l 60 98.67a 0.33 0.38

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

79 4.2.1.3 Time to death The mean time to death, in days, as results as the result of direct spraying of the 2nd. Instar nymphs of the Migratory Locust, with water/ethanol (v: v) extracts from different plants materials is presented in (Table 8). It shows that Neem takes much longer time to kill as compared to the other plant extracts and it was followed by Calotropis extracts. However, Mucuna and the Adenium killed the nymphs much faster than the other plant extracts

Table 8. Mean time to death in days of the 2nd. Instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) treated by direct spraying with water/ethanol (v: v) extracts from different plants materials in the laboratory (Temperature 25±1 º C. and % RH. 50-60).

Treatments No. of Time to death SD SE Insect/ In days ± ± test

1-Control - - - - 2-Calotropis 50g/l 60 2.17ab 2.93 0.60 3-Azadirachta 50g/l 60 3.25a 1.26 0.60 4-Adenium 50g/l 60 1.20b 0.32 0.60 5- Mucuna 50g/l 60 1.00b 0.00 0.60

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

80 4.2.2 Efficacy of water extracts of the plant materials sprayed directly on locusts.

4.2.2.1 Mortality The mean percentage mortality as a result of direct spraying of aqueous extracts of plant materials on the 2nd nymphal instar of the Migratory Locust is presented in Table (9). Data in this table clearly indicate that Mucuna root extracts were more toxic than the other extracts followed by Azadirachta seed kernel. These two extracts are significantly different from one another but they are both highly significantly different from the other treatments. The water extracts of Calotropis and Adenium have extremely poor toxic effect on the nymphs of Locusta

Table 9. Mean mortality of the 2nd. Instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) treated by direct spray with water (v: v) extracts from different plants materials in the laboratory (Temperature 25± 1º C. and % RH. 50-60).

Treatments No of Mortality (%) SD SE Insects/ ± ± test

1-Control 60 0.00c 0.00 0.00 2-Mucuna 50g/l 60 98.33a 0.41 0.22 3-Adenium 50g/l 60 03.33c 0.52 0.22 4-Calotropis 50g/l 60 05.00c 0.84 0.22 5-Azadirachta 50g/l 60 96.67a 0.52 0.22

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

81 4.2.3 Efficacy of orally administered water/ethanol extracts of plants materials

4.2.3.1 Mortality

The highest mortality was recorded among nymphs treated with Mucuna water/ethanol extracts followed by extracts of Azadirachta seed kernels and the Calotropis and Adenium respectively. All the plant extracts showed significantly different toxicity from the control which exhibited no effect on the nymphs. The Mucuna extracts shows no significant difference (at P < 0.05) from Azadirachta which was used as standard botanical insecticide (Table 10).

Table 10. Mean mortality of the 2nd. Instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) fed on wheat seedling treated with water/ethanol (v: v) extracts) of different plant materials in the laboratory (Temperature 25± 1º C. and % RH. 50-60).

Treatments No. of Mortality SD SE Insect/ % ± ± test

1-Control 60 00.00c 0.00 1.05 2-Calotropis 50g/l 60 55.17b 4.91 1.05 3-Azadirachta 50g/l 60 93.33a 1.03 1.05 4-Adenium 50g/l 60 35.33b 3.00 1.05 5- Mucuna 50g/l 70 98.57a 0.38 0.97

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

82 4.2.4 Efficacy of orally administered water extracts of plants materials

4.2.4.1 Mortality

In trials where water extracts of the screened plants materials were applied Azadirachta extracts caused the highest mortality of nymphs followed by Calotropis extracts and then by Mucuna extracts. Adenium extracts were found nontoxic and so were the control treatment (Table 11).

Table 11. Mean mortality of the 2nd instar nymphs of The Migratory locust, Locusta migratoria Linné (Orthoptera: Acrididae ) fed on wheat seedlings treated with water extracts from roots of Mucuna pruriens ( Fabaceae ) and other plant extracts in the laboratory (Temperature 25± 1º C. and % RH. 50-60 ).

.

Treatments No. of Mean (%) SD SE Insect/ Mortality ± ± test

1-Control 60 00.00c 0.00 0.00 2-Calotropis 50g/l 60 50.08b 4.76 0.91 3-Adenium 50g/l 70 00.00c 0.00 0.84 4-Mucuna 50g/l 100 30.00b 2.11 0.70 5-Azadirachta 50g/l 70 98.57a 0.38 0.84

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

83 4.2.4.2 Time to death For the water extracts the highest records of time to death was reported in Calotropis (5.12±0.59 days). 3.94±0.45 and 3.20 ±0.54 days were recorded for Mucuna and Azadirachta respectively. Comparisons of the result reflect no significant difference in times to death between Neem and Mucuna. However, Calotropis showed a significant difference from Azadirachta treatments (Table 12).

Table 12. Mean time to death of the 2nd Instar nymphs of the Migratory Locust Locusta migratoria Linne( Orthoptera: Acrididae) fed on wheat seedlings treated with water extracts from different plants in the laboratory (Temperature 25±1 º C. and % RH. 50-60 ).

Treatments No of Mean SD SE Insects/ Time to death ± ± test

1-Control 60 - - - 2-Calotropis 50g/l 60 5.12a 2.14 0.59 3-Adenium 50g/l - - - - 4-Mucuna 50g/l 100 3.94ab 2.06 0.45 5-Azadirachta 50g/l 70 3.20b 0.61 0.54

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

84

4.3 Evaluation of biological activities and mode of action of M. pruriens extracts

4.3.1 Efficacy of Mucuna pruriens on the Desert locust by direct spraying

4.3.1.1 Knockdown effect on the 2nd nymphal instars

The results of evaluation of Mucuna roots extracts with Neem seed kernel and Decis( Deltametherin) as standard Locusticides against the 2nd. Instars nymphs of the Desert Locust, S. gregaria are given for the knockdown in Table(13). Decis, the standard synthetic Locusticide gave 100%±0.47 knockdown, Mucuna root extracts ranked second and gave 88±0.47% knockdown which is significantly different from the knockdown given by Neem seed kernels, the standard botanical insecticide, which gave only 15±0.47% knockdown on the nymphs. All the treatments showed significant differences from the untreated control sprayed with the solvents only.

Table 13. Mean knockdown effect (%) on the 2nd nymphal instar of the Desert Locust, Schistocerca gregaria Forskål directly sprayed with 50g/l water/ethanol extracts from roots of Mucuna pruriens (Fabaceae), Neem Seed kernels and Decis (Deltametherin) in laboratory (Temperature 25± 1º C. and % RH. 50-60 ).

Treatments No of Knockdown SD SE insect/ test % ± ±

1-Control 60 00.00d 0.00 0.47 2-Muc50g/l 60 88.00b 1.94 0.47 3-Neem50g/l 60 15.00c 1.22 0.47 4-Decis12.5g/l 60 100.00a 0.00 0.47

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

85 4.3.1.2 Mortality the of 2nd nymphal instars

Table (14) shows the results of the efficacy of the tested toxic agents against the 2nd instar nymphs of the Desert Locust, S. gregaria. The highest records of mortality were in nymphs treated with Decis( 100%) followed by Azadirachta and Mucuna extracts which gave mortality of 90.93% and 79.17 respectively. The Duncan multiple range tests run for the means indicating significant differences between the treatments.

Table 14. Mean mortality in the 2nd nymphal instar of the Desert Locust, Schistocerca gregaria Forskål directly sprayed with 50g/l water/ethanol extracts from roots of Mucuna pruriens (Fabaceae), Azadirachta seed kernels and Decis (Deltametherin) in the laboratory (Temperature 25± 1º C. and % RH. 50-60 ).

Treatments No of Mortality SD SE Insects/ test % ± ±

1-Control 60 10.33d 1.10 0.55 2-Muc50g/l 60 77.00c 1.69 0.55 3-Neem50g/l 60 90.00b 1.78 0.55 4-Decis12.5g/l 60 100.00a 0.00 0.55

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

86

4.3.1.3 Knockdown effect on the adult of the Desert Locust by direct spraying

Results of the experiments conducted to study the efficacy of Mucuna against the young adults of the Desert Locusts are given in Table(15) where Mucuna 100g/l gave the highest percent knockdown( 73.33±0.63%) Mucuna 50g/l gave only 1.67% .

Table 15. Mean(%) knockdown effect Mucuna water/ethanol extract ( v: v) sprayed on 7±1 day old adult Desert Locust, Schistocerca in the laboratory(Temperature 25- 28±1 º C. and RH. 60±5 % )..

Treatments No. of Knockdown SD SE Insects/ test % ± ±

1-Muc100g/l 60 73.33a 2.66 0.63 2-Muc50/l 60 01.67b 0.41 0.63 3-Control 60 00.00b 0.00 0.00

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

87

4.3.1.4 Mortality of the adult of the Desert Locust by direct spraying

Table ( 16 ) shows efficacy of two concentrations of water/ethanol( v: v) extracts of M. pruriens (Fabaceae) by direct spraying on 7±1 day old adult the Desert Locust, S. gregaria. Mortality of 91.67±0.67% was obtained in Locust treated with Mucuna 100g/l which was significantly higher than the mortality of locusts treated with Mucuna 50g/l. Control mortality was also rather high though it was significantly lower than mortality obtained in locust treated with Mucuna 50g/l.

Table 16. Mean(%) mortality of 7±1 day old adult Desert Locust, Schistocerca gregaria sprayed directly with water/ethanol (v: v) extracts of Mucuna pruriens (Fabaceae) in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No. of Mortality SD SE Insects/ test % ± ±

1-Muc100g/l 60 91.67a 1.17 0.67 2-Muc50/l 60 37.00b 2.32 0.67 3-Control 60 23.33c 1.21 0.67

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

4.3. 1.5 Time to death The mean time to death in days as a result of direct spraying of water/ethanol( v: v) extracts of M. pruriens on 7±1 day old adult Desert Locust, S. gregaria. Was much lower (1.50±0.46 day) at higher concentration, but relatively high at the lower dose of Mucuna and the untreated control ( Table 17).

88

Table 17. Mean time to death in 7±1 day old adult Desert Locust, Schistocerca gregaria sprayed directly with water/ethanol (v: v) extracts of Mucuna pruriens (Fabaceae) in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No. of Time to death SD SE Insects/ test In days ± ±

1-Muc100g/l 60 1.50b 0.64 0.46 2-Muc50/l 60 3.73a 0.78 0.46 3-Control 60 4.35a 1.68 0.46

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

4.3.2 Efficacy of Mucuna pruriens on the Migratory locust by direct spraying

4.3.2.1 Knockdown effect on the 2nd nymphal instar of the Migratory Locust

Figure (17) show mean percentage knockdown on the 2nd instar nymphs of Migratory locust treated with different concentrations of Mucuna when compared with Decis. No difference of statistical significance was noticed between Decis and Mucuna 50g/l. However, all the treatments gave significantly higher knockdown than the control.

89

100

a a 80

60 b b 40 Knockdown (%)

20 c

0 1-Decis 12.5 2-Muc 50g/l 3-Muc 25g/l 4-Muc12.5g/l 5-Control

Treatments

Figure 17. Mean knockdown effect for different concentrations of Mucuna pruriens (Fabaceae) root extracts against 2nd instars nymphs of Locusta migratoria (Orthoptera, Acrididae) in laboratory(Temperature 25-28±1 º C. and RH. 60±5 % ).

4.3.2.2 Mortality of the 2nd nymphal instar of the Migratory Locust

Mortality resulting from testing different concentrations of M. pruriens compared with Decis as standard locusticide against the 2nd nymphal instar of the Migratory locust is given in Figure (18). All the treatments cause significantly higher mortality than the untreated control. Mucuna at 50g/l was similar in effect the standard.

90

100 a a

80 ) 60

b 40 Mortality (%

b 20

c 0 1-Decis 12.5 2-Muc 50g/l 3-Muc 25g/l 4-Muc12.5g/l 5-Control

Treatments

Figure 18. Mean mortality for different concentrations of Mucuna pruriens (Fabaceae) root extracts against 2nd instars nymphs of Locusta migratoria (Orthoptera, Acrididae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % ).

4.3.2.3 Time to death of the 2nd nymphal instar of the Migratory Locust

Table(18) indicates that time to death resulting from application of Decis and that resulting from application of Mucuna 50g/l and 25/l were similar. However, there is significant difference between these treatments and the control.

91 Table 18. Mean time to death resulting from different concentrations of Mucuna pruriens (Fabaceae) root extracts applied against 2nd instars nymphs of Locusta migratoria (Orthoptera, Acrididae) in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No. of Time to death SD SE Insects/ test in days ± ±

1-Decis 12.5 60 1.00c 0.00 0.56 2-Muc 50g/l 60 1.02c 0.04 0.56 3-Muc 25g/l 90 1.31c 0.33 0.45 4-Muc 12.5g/l 60 2.55b 3.17 0.56 5-Control 60 - - -

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

4.3.2.4 Knockdown effect on the adult Migratory locust

The knockdown of the Migratory Locust L. migratoria directly sprayed with water/ethanol( v: v ) extracts of roots of M. pruriens under laboratory conditions in are given in Figure ( 19 ). The comparison reflected no significant differences when the knockdown from control is compared with knockdown in Locust treated with Mucuna 12.5g/l. However, Mucuna 50g/l and Mucuna 25g/l yielded a significantly higher knockdown effect when they are compared with Mucuna 12.5g/l and the control and they are a gave a significant difference when compared with each other.

92

100 a

80 ) 60 b

40 Knockdown (%

20 c c

0 1-Muc.50g/l 2-Muc.25g/l 3-Muc.12.5g/l 4-Control

Treatments

Figure 19. Mean percentage knockdown of the adult Migratory Locust, Locusta migratoria (Orthoptera: Acrididae) Linné directly sprayed with water/ethanol (v: v ) extracts of roots of Mucuna pruriens (Fabaceae) under laboratory condition(Temperature 25-28±1 º C. and RH. 60±5 % ).

4.3.2.5 Mortality of the adult Migratory locust

Data for the average percentage mortality of the adult Migratory Locust, L. migratoria directly sprayed with water/ethanol( v: v ) extracts of roots of M. pruriens under laboratory condition are presented in Figure ( 20 ) The comparisons suggested significant differences where all the treatments gave significantly higher mortality than the control. No significant difference was detected between mortality in the Locust nymphs as a result of applying Mucuna 50g/l when compared with mortality at Mucuna 25g/l. However, the latter two concentrations gave a significantly higher mortality when compared with mortality from applying Mucuna 12.5g/l.

93

100 a a 80 ) 60

b 40 Mortality (%

c 20

0 1-Muc.50g/l 2-Muc.25g/l 3-Muc.12.5g/l 4-Control

Treatments

Figure 20. Mean percentage mortality of the adult Migratory Locust, Locusta migratoria (Orthoptera: Acrididae) Linné directly sprayed with water/ethanol (v: v ) extracts of roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % ).

4.3.2.6 Time to death of adult Migratory locust Figure ( 21 ) Shows the results of time to death in adults Migratory locust, L. migratoria directly sprayed with water/ethanol( v: v ) extracts of roots of Mucuna pruriens( Fabaceae) under laboratory conditions. Locust nymphs treated with higher concentrations of Mucuna died in shorter time. Nymphs treated with Mucuna 50g/l died within 24h after treatment which is significantly shorter than the time to death given by Mucuna 25g/l and time to death from each of these two treatments is significantly shorter when compared with time given by Mucuna 12.5g/l and the control.. No significant difference was detected when time to death is compared in the last two sets (Mucuna12.5g/l and Control).

94 6

5 a a 4

3

b 2 Time to death (days) death to Time

c 1

0 1-Muc.50g/l 2-Muc.25g/l 3-Muc.12.5g/l 4-Control

Treatments

Figure 21. Mean time to death in days in adults of Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) directly sprayed with water/ethanol ( v: v ) extracts of roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % ).

4.3.4 Efficacy of Mucuna extracts against nymphs of Locusta treated by topical application

4.3.4.1 Knockdown effect Percentage knockdown resulting from topical application of 10µl of water/ethanol extracts from root of M. pruriens against the 5th nymphal instar of the Migratory locust is shown in Table (19 ) . In general there was insignificant knockdown effect even at the highest concentration of 200g/l of the extract. All the treatments are similar to one another but they are significantly different from the untreated control.

95 Table 19. Mean knockdown effect on the 5th. instars nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) topically treated with Mucuna water/ethanol (v: v) extracts in the laboratory (Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No of Mean(%) SD SE insects/test knockdown ± ±

1-Muc200g/l 60 16.67a 0.52 0.23 2-Muc150g/l 50 12.00a 0.84 0.25 3-Muc100g/l 60 10.00a 0.63 0.23 4-Control 60 0.00b 0.00 0.00

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

4.3.4.2 Mortality Table (20) shows the percentage mortality of the 5th. instar nymphs of the Migratory Locust L. migratoria Linne obtained after the topical application of various concentrations of water/ethanol extracts of Mucuna. The highest mortality of 90.00±0.58 was obtained in the nymphs treated with 200g/l. but there was no difference in response when nymphs were treated with 150g/l and 100g/l however all treatments were significantly different from the untreated control.

96 Table 20. Mean mortality of the 5th. instars nymphs of the Migratory Locust, Locusta migratoria Linné of topically treated with of water/ethanol (v: v) extracts from roots of Mucuna pruriens (Fabaceae) in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No. of Mean (%) SD SE insects/test mortality ± ±

1-Muc200g/l 60 90.00a 0.89 0.58 2-Muc150g/l 50 54.00b 2.07 0.63 3-Muc100g/l 60 55.00b 1.64 0.58 4-Control 60 06.67c 0.82 0.58

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

4.3.5 Efficacy of Mucuna extracts against adults of Locusta treated by dipping

4.3.5.1 knockdown effects The efficacy of different adapted concentrations of Mucuna water/ethanol extracts against the adult Migratory locust was determined by dipping (Table 21 ). The highest knockdown was seen in the insect dipped in Mucuna100g/l followed by Mucuna50, but Mucuna25g/l cause very low Knockdown which was similar to that of the untreated control.

97 Table 21. Mean knockdown effect on 7±1day old adults of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) dipped for 1.5 sec. in different concentrations of extracts from roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No. of Knockdown(%) SD SE insects/test ± ±

1-Control 60 21.67b 1.60 0.56 2-Muc25g/l 60 26.67b 1.03 0.56 3-Muc50g/l 50 78.00a 1.10 0.62 4-Muc100g/l 50 86.00a 1.67 0.62

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

4.3.5.2 Mortality Table (22) shows the mean percentage mortalities in the7±1day old adults of the Migratory Locust, Locusta migratoria Linne dipped for 1.5 sec. in different concentration of extracts from roots of Mucuna pruriens(Fabaceae) under laboratory condition. There was no significant difference between the effect of Mucuna 100g/l and Mucuna 50g/l but there was significant difference between the mortality caused by the two higher concentrations and that of Mucuna 25g/l and the control treatment. It must be noted that control mortality was rather high.

98 Table 22. Mean mortality in 7±1day old adults of the Migratory Locust, Locusta migratoria Linné dipped for 1.5 sec. in different concentration of extracts from roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No. of Mortality(%) SD SE Insect/ test ± ±

1-Control 60 31.67c 1.72 0.57 2-Muc25g/l 60 34.14b 0.84 0.57 3-Muc50g/l 50 80.00a 2.07 0.63 4Muc100g/l 50 100.00a 0.00 0.00

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

4.3.6 Effect of Mucuna as stomach poison.

4.3.6.1 Mortality

Table (23) shows the mean mortality of the 2nd instar nymphs of the Migratory Locust, L. migratoria fed on wheat seedlings treated with different concentrations of water/ethanol extracts from roots of Mucuna pruriens( Fabaceae) under laboratory conditions. The data show that Mucuna at 100g/l caused the highest mortality followed by Mucuna 50g/l and Mucuna at 25g/l. Control mortality was low. The Duncan multiple range test gave evidence that: there were statistical differences among the means of mortalities caused by the different treatments.

99

Table 23. Mean mortality of the 2nd instar nymphs of The Migratory Locust, Locusta migratoria Linné fed on wheat seedlings treated with different concentrations of water/ethanol extracts from roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No. of Mortality(%) SD SE Insect/test ± ±

1-Muc.100g/l 70 98.57a 0.38 0.46 2-Muc.50g/l 70 82.14b 1.63 0.46 3-Muc.25g/l 60 20.00c 1.67 0.49 4-Control 60 03.00d 0.52 0.49

NB.: Means followed by the same lower case letters are not significantly different at P. < 0.05

4.3.6.2 Faecal output The mean weight of frass collected from 10 nymphs after 24h. following the introduction of wheat seedlings for feeding is presented in Table. (24) The Locust nymphs fed on the control gave significantly higher weight of frass in comparison with nymphs fed on Mucuna treated wheat seedlings. Moreover, there are significant differences among the average weights of frass collected from nymphs under different treatments.

100 Table 24. Mean weight of frass collected after 24h from the 2nd instar nymphs of the Migratory Locust, Locusta migratoria Linné fed on wheat seedling treated with different concentration of water/ethanol extracts from roots of Mucuna pruriens (Fabaceae) under laboratory conditions(Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No. of insects Weight of frass SD SE tested (g/10 nymphs)

1-Muc.100g/l…. 70 11.43d 3.78 4.40 2-Muc.50g/l 70 18.57c 18.64 4.40 3-Muc.25g/l 60 31.67b 7.53 4.76 4-Muc.Control 60 43.33a 10.33 4.76

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

4.3.6.3 Effect of feeding period on wheat seedlings treated with Mucuna extracts on locust nymphs.

4.3.6.3.1 Mortality Figure ( 22 ) shows the mortality of 2nd instars nymphs of locust fed on wheat seedlings treated with Mucuna extracts for periods of 6, 24 and 48 hours. There was no significant difference between the effect of feeding for 24hours and 48 hours while the effect of feeding for 6 hours was significantly different.

101

100 a a 80 ) 60

b 40 Mortality (%

20

0 1-6 hours 2- 24 hours 3- 48 hours

Treatments

Figure 22. Mean mortality of the 2nd instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) fed on wheat seedling( for different periods of times) treated with water/ethanol (v: v) extracts from roots of Mucuna pruriens (Fabaceae) at 50g/l. in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % ).

4.3.6.3.2 Time to death The mean time to death taken by the 2nd. Instar nymphs of the Migratory locust fed on Mucuna treated wheat seedlings is given in Table (25).

Table 25. Mean time to death in the 2nd instar nymphs of the Migratory Locust, Locusta migratoria Linné (Orthoptera: Acrididae) fed for different periods on wheat

102 seedling treated with water/ethanol (v: v) extracts from roots of Mucuna pruriens (Fabaceae) (Temperature 25-28±1 º C. and RH. 60±5 % ).

Time allowed No. of Time to death SD SE for feedings insects/test in hours ± ±

1-6hours 60 73.60ab 36.77 9.78 2-24hours 60 80.40a 13.64 9.78 3-48hours 60 45.05b 13.55 9.78

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

4.3.6.4 Evaluation of extracts of Mucuna as locust antifeedant

4.3.6.4.1 Feeding on cabbaged leaf-disc under no choice condition

Table (26) shows the mean percent of Mucuna treated cabbage leaf-disc consumed by adult locust under no-choice test conditions. Differences in mean percentage weight between the treated and untreated cabbage leaf-disc consumed was less than 10% which rather low, though probability of t. at alpha 0.05 is 0.0000 which mean that there is a high significant difference between treated and untreated control. The confidence level is 9.20±3.94).

Table 26. Average percentage of cabbage leaf-disc (in weight) eaten by 7±1 days old adults of the Desert Locust, Schistocerca gregaria Forskål (Orthoptera: Acrididae) in a three hours feeding period on cabbage leaf-disc treated with extracts from roots of

103 Mucuna pruriens (Fabaceae) under no-choice laboratory bioassay test(Temperature 25-28±1 º C. and RH. 60±5 % ).

No. of Control Treated Probability of t. Confidence insects/test (%) (%) at P< 0.05

100 47.36±6.37 38.16±5.94 0.0000 9.20±3.94

4.3.6.5 Frass of Locust fed on cabbage leaf-disc under no choice condition Table (27) shows the mean weight of frass collected from group of 5 locusts each fed on Mucuna treated and untreated cabbage leaf-disc for a period of 3hrs. It is clear that there was significant difference between the weight of frass in the treated and the untreated disc

Table 27. Mean weight of frass of 7±1 days old adults of The Desert locust Schistocerca gregaria Forskål (Orthoptera: Acrididae) fed on cabbage leaf-disc treated with extracts from roots of Mucuna pruriens (Fabaceae) for a three hours period under no-choice laboratory bioassay test(Temperature 25-28±1 º C. and RH. 60±5 % )

No. of Weight in Weight in Probability of t Confidence insects/test Control Treated at P< 0.05 (g.) (g.)

100 178.5±106.2 91.7±30.8 0.0012 20±3.93

104 4.3.6.6 Cabbage leaf-disc bioassay under choice condition The results of choice bioassay of Mucuna water/ethanol( v: v) extracts on cabbage leaf-disc were given in Table ( 28 ) and they show that treatment with Mucuna extracts significantly decrease feeding ability of locusts as compared with the untreated control. Comparison between weight consumed in the treated leaf-disc and that of the untreated control indicates a highly significant difference at P. < 0.05.

Table 28. Mean percentage weight of Mucuna-treated cabbage leaf-disc consumed by 7±1 days old adults of the Desert Locust, Schistocerca gregaria Forskål (Orthoptera: Acrididae) in 3h under choice bioassay test(Temperature 25-28±1 º C. and RH. 60±5 % )

% weight of cabbage

No. of Consumed Consumed Probability Confidence insects/test cabbage in cabbage in of t. at 0.05 Control (%) Treated (%)

150 58.49±11.93 31.68±9.95 0.0000 26.81±4.81

4.3.6.7 Antifeedant effect of filter paper impregnated with Mucuna extracts offered to adults Results of Mucuna impregnated filter paper trials conducted on 7±1 day-old adults of The Migratory and The Desert locusts are presented in Table.( 29 ). These results indicate that Locusta was off food for the first five hours and only few individuals were tried feeding when compared with Schistocerca. For the two locust species the rejecting feeding is rather high in Mucuna impregnated filter papers when compared with that of the untreated control. These results suggest antifeedant effect from extracts of Mucuna

105

Table 29. Percent of Locust rejecting feeding on filter paper impregnated with extracts of Mucuna pruriens( Fabaceae) (Temperature 25-28±1 º C. and RH. 60±5 % )

Time of Locust rejecting feeding on filter paper observation Schistocerca gregaria Locusta migratoria Treated Untreated Treated Untreated

1st hour 100 81.8 100 73.3 2nd hour 81.8 78.8 100 36.3 3rd hour 87.9 66.7 100 65.7 4th hour 93.9 63.7 100 56.7 5th hour 90.9 60.6 100 56.7 6 hour 84.8 48.5 96.7 53.3 24th hour 78.8 36.4 93.3 33.3 48th hour 75 26.5 93.1 19.4

4.4 Effect of storage period on efficacy of Mucuna root materials

Results of trials on the effect of the storage period on the efficacy of the Mucuna root materials are given in Tables ( 30) and (31). The root materials were collected at different years namely: 2002, 2001 and 2000 stored under room condition, prepared and tested for the knockdown effect and mortality. It appears that the efficacy of the plant materials decreased with an increase of the storage period but the rate of loss was relatively slow. This suggests that the materials are probably stable under normal storage conditions.

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Table 30. Mean percentage knockdown in the 2nd. Instar nymphs of the Migratory locust, Locusta migratoria Linné, sprayed with extracts of Mucuna pruriens roots collected at different years (2002/2001/2000) (Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No of Knockdown SD SE insects/ test (%) ± ±

1-Muc.2002 60 82.86a 0.95 0.45 2-Muc.2001 60 70.00ab 1.26 0.48 3-Muc.2000 60 58.33bc 1.47 0.48 4-Control 60 46.67c 1.03 0.48

NB.: Means followed by the same lower case letters are not significantly different at P < 0.05

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Table 31. Mean Mortality of the 2nd instar nymphs of the Migratory locust, Locusta migratoria Linné, sprayed Mucuna pruriens extracts of roots collected at different years (2002/2001/2000) (Temperature 25-28±1 º C. and RH. 60±5 % )

Treatments No of Mortality SD SE insect/test (%) ± ±

1-Muc.2002 60 98.00a 0.49 0.65 2-Muc.2001 60 83.67ab 1.39 0.65 3-Muc.2000 60 75.33b 2.60 0.65 4-Control 60 18.33c 1.17 0.65

NB.: Means followed by the same lower case letters are not significantly different at P< 0.05

4.5 Effect from mixing extracts of Mucuna at 25g/l with reduced concentrations of Decis against the Desert Locust

Table (32) shows the mortality of the 2nd instar nymphs of the Desert locust treated with different concentrations of Decis alone and in combination with a fixed dose of Mucuna root extracts. These results indicate that the toxicity of the mixture of Decis and Mucuna was higher than that of either of the two compounds when applied alone. This tends to suggest that there was a form of interaction between the constituents of the two components which could well be a synergistic effect.

108 Table 32. Mean mortality on the 2nd. Instar nymphs of the Desert Locust, Schistocerca gregaria Forskål (Orthoptera: Acrididae) treated with Decis/Mucuna and Decis or Mucuna extracts separately in the laboratory(Temperature 25-28±1 º C. and RH. 60±5 % ).

Treatments No. of Mean(%) SD SE insects/test mortality ± ±

1-Dec3.12/Muc25g./l 50 100.00a 0.00 0.48 2-Dec1.56/Muc.25/l 50 92.00ab 0.45 0.48 3-Dec0.78/Muc.25g/l 40 90.00ab 0.00 0.54 4-Dec0.39/Muc25g/l 60 91.67ab 0.98 0.44 5-Dec0.195/Muc25g/l 70 62.29d 1.60 0.41 6-Decis3.12 50 86.00b 1.52 0.48 7-Decis1.56 60 80.00bc 1.10 0.44 8-Decis0.78 80 71.13cd 1.64 0.38 9-Decis0.39 50 46.00e 1.14 0.48 10-Decis0.195 70 8.57f 0.90 0.41 11-Mucuna25g./l 60 3.83f 0.41 0.44 12-Control 50 2.00f 0.45 0.48

NB: Means followed by the same lower case letter are not significantly different at P < 0.05.

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5 DISCUSSION

Locusts and grasshoppers are serious pests of crops in Africa and other arid zones of the world ( Krall et al 197; Lomer et al 1999 and 2000; Lecoq 2000 and 2001and Lockwood et al 2000 and 2001). Sudan is considered as watershed area for the Desert locust where 50% of the country is a recession area for this insect (El-Tom 1994 and Joffe 1995). The Desert locust has, therefore, been selected for this study because of its economic importance as a crop pest in this region. The highly polyphagous locust species, Schistocerca gregaria (Forskål 1775) was compared with an oligophagous one, Locusta migratoria (Linné 1758). Hence, the output of this study could be extrapolated to cover the cases of several economically important locusts and grasshoppers in the Sahelian countries.

It is generally held that the heavy reliance on chemical locusticides will not be sustained for long due to the increase in environmental awareness and the rapid escalation in prices of these synthetic chemical insecticides. Lecoq et al (1997); El Bashir (1997); Peveling et al (1999a) and Lomer et al (2001) emphasized the need for alternative control strategies to minimize the heavy reliance on synthetic chemical locusticides and to enforce integrated locust management programs. Plant materials, which are readily degradable in the environment could be a suitable substitute for the chemical pesticides and they may fit very well in future IPM approaches. The plant materials chosen for screening during the course of this study were selected on the basis of their reputation in the Sudanese folklore. They are already in use by some ethno-botanists as native medicines and as control agents against some household and livestock pests. Extracts of Mucuna are commonly used by cattle owners in Western Sudan against some ecto- parasites of cattle. Azadirachta (Neem), Calotropis ( Ushar) and Adenium ( Sim tree) are known as sources of bio-active compounds.

The presence of some bio-active substances of pesticidal properties in Mucuna plant materials was confirmed earlier by Neuwinger (1996) and Morris (1999). The toxic effect of M. pruriens extracts against locust was proved by Abdalla et al. (2001a, b and 2003).The present study demonstrates that extracts of Mucuna pruriens, can

110 easily be prepared and applied against locust. Moreover, the study manifested the compatibility of Mucuna extracts with the synthetic locusticide (Decis).

Results of the field survey confirm the availability of Mucuna in Western Sudan while earlier reports by Broun and Massey (1929) also pointed out its presence in Bahr El- Ghazal area. However, while most of the cattle owners of Western Sudan use this plant extracts for control of cattle ecto-parasites, little is known about similar use by cattle owners in Bahr El-Ghazal. It is rather interesting to note that to date up to 66% of the interviewed cattle owners still use Mucuna extracts for treatment of their cattle against ecto-parasite despite the availability of chemical pesticides.

Recent developments in natural products as pest control agents have been well documented especially for neem products (Stefens and Schmutterer 1982; Ascher 1983; Haasler 1983; Sharma et al 1984; Jacobson 1986; Ascher 1993; Schmutterer 1990; Mordue and Blackwell 1993). Many scientists object to the heavy reliance on synthetic chemical insecticides and warn from their eco-toxicological risks (Isman 1990; McDonald, 1965; Ritchie & Dobson, 1995; Balança & Visscher 1997; van Der Valk, 1997; Peveling et al 1999a, b; Peveling, 2000, 2001; Peveling & Nagel, 2001). Hence the search for effective botanical pesticides has become a priority in recent decades. Accordingly, the current investigation focused on four plant materials vis-à- vis: Mucuna pruriens ( Fabaceae ); Calotropis procera (Asclepiadaceae); Adenium obesum (Apocynaceae) and Azadirachta indica (Meliaceae). The main objective was to evaluate their efficacy as locusticides against the African Migratory locust. The plant materials were examined for their contact and stomach effects. The lethal toxic effect of Mucuna was comparable to that of neem, the well known botanical insecticide. Similar results on the bio-efficacy of Mucuna roots extracts were documented earlier by Abdalla et al. (2001a, b). The results obtained in the present investigations support earlier findings by Morris, (1999) who listed some leguminous plants as sources of bio-active molecules. The list includes Mucuna pruriens as a source of bio-active substances of pesticidal nature such as Bufotenine, Mucunain and Serotonin. Szabo, (2002) gave further evidence that seeds of Mucuna contain some bio-active materials like Mucunine, and Bufotenine, the latter is structurally related to Serotonin. However, most of previous work in the area of chemical analysis of Mucuna products focused on the seeds. This is mainly because Mucuna seeds are

111 considered as a potential source of L-dopa, a toxic compound used as an anti- Parkinson remedy (Siddh Uraju et al 1996; Lorenzetti et al 1998; Flores et al 2002; Diallo and Berhe 2003; Egounlety 2003; Ezeagu et al 2003; Gurumoorthi et al 2003; Matenga et al 2003; Teixeira et al 2003 and Szabo 2003). In the case of neem where bio-active compounds are also present in leaves, stems and roots, the seed kernels contain the highest concentration of biologically active molecules (Schmutterer 1990 and Verkerk and wright 1993 ). Beckstorm-Sternmerg and Duke (1994) indicated that Mucuna contained many useful phyto-chemicals. The roots were selected in the present bio-tests instead of the seeds and the other plant parts because Mucuna root extracts are widely used by cattle raisers, in Western Sudan, to combat pests of their animals, mainly lice. Moreover, the roots are available throughout the year in the required quantities. Also it may be due the fact that seed pods are covered with irritating hairs which render their handling by the rural communities a difficult task. Studies on Mucuna root chemistry are very rare. Aruna et al (1998) in his phyto-chemical studies on the roots of M. pruriens confirmed the presence of sterols, triterpenes and flavonoids as bio-active substances. Lorenzetti et al 1998 observed that seeds of Mucuna are mainly free of insect infestation, unlike other legume seeds which are attacked by seed borers. Extracts of different parts of M. pruriens, are extensively used in traditionally by some ethno-botanist in many tropical areas of the world against a panorama of health and pest problems.

Mucuna can act very well both as contact and as stomach poison. The roots extracts also cause knockdown when directly sprayed on tested insects. In the Migratory locust Mucuna at 50g/l gave significantly higher knockdown effect than the control and was similar to Decis in effect. In the Desert locust, however, knockdown effect was noted only in the case of nymphs where Mucuna at 50g/l gave an effect significantly higher than that of the control but significantly lower than the knockdown obtained in locust treated with Decis. The adult Desert locust showed only little response in term of knockdown at this level of concentration. However, increasing the concentration of Mucuna resulted in a substantial increase in the knockdown effect on the adults. For the Migratory locust the obtained knockdown at Mucuna 50g/l was similar to that obtained by applying the selected dose of Decis. Mortality resulting from application of Mucuna 50g/l against the Migratory locust was significantly higher than that of the control and similar to the mortality obtained

112 in locust treated by Decis. The high contact efficacy of the Mucuna root extracts on locust was confirmed earlier by Abdalla et al. (2001).

Mucuna roots materials collected and stored for up to three years and then used for treatment of nymphs of Migratory locust showed that: the bio-activity of the materials had decreased with time. This is expected since it is a crude unstable plant material which is subject to various degradation factors. However, it is note worthy that the loss in efficacy with time was low ( < 23 in 3 years ).

The apparent high contact efficacy of extracts of Mucuna, could be of help in future studies on the identification of the active molecules, mechanism of action and the possibility of using these extracts as control agents against some other insect pests.

The antifeedant properties of natural products from neem tree and other related plants were demonstrated by several workers (Schmutterer and Ascher 1986; Schmutterer 1990; Jacobson 1990; Kleeberg and Zebitz 1997). This antifeedant effect of plant materials may have some advantages over conventional insecticides. In the bioassays conducted during the course of this study Mucuna extracts were subjected to further investigations to evaluate their efficacy when orally taken by the Migratory locust. Stomach action was evident because all the treatments were significantly more toxic than the untreated control and caused high mortality. However, no knockdown effect was reported in any of the tests confirming the finding of Abdalla et al. ( 2001b). It should be noted that because of the antifeedant effect of Mucuna extracts, the insect will not consume large enough quantities to kill, however, there is need to increase intake of food by addition of feeding stimulant and attractants. Frass collected from locusts fed on Mucuna treated food gave evidence that extracts can work as anti- feedants. In this study locust provided with Mucuna treated wheat seedlings gave a significantly less weight of frass than the untreated control. For the Desert locust the tests for antifeedant activity of Mucuna were conducted by the provision of cabbage leaf-discs under choice and no choice conditions. Under no choice and choice feeding tests the locusts were found to eat significantly less weight of Mucuna treated cabbage leaf-discs when compared with feeding for the same period on the untreated discs. Significantly less frass was collected from locust fed on cabbage leaf-discs treated with Mucuna as compared with that collected from the untreated control under no

113 choice conditions. The results obtained in the present bioassay were in line with the findings presented by Abdalla et al. (2004). The feeding deterrence from Mucuna is also evaluated against the Desert and the Migratory locusts through filter paper feeding tests. Most of the locusts rejected feeding on filter papers treated with M. pruriens extracts. The antifeedant effect was more in the Migratory locust than the Desert locust.

Studies on pesticide mixtures are scanty but according to Godson et al. (1999) mixtures of pesticides formulations can enhance toxicity through some sort of additive or synergistic effect. When M. pruriens was mixed with a series of reduced concentrations of Decis and used against locusts there was increase in both knockdown and killing capacity of the mixture. The resulting effect of the mixture (Decis/ Mucuna) on Desert locust nymphs was significantly better than the effect obtained when each of the two compounds was applied separately. This finding was observed earlier by Abdalla et al. (2003). The practical implication of this result is that the amount of Decis in the mixture would be much less than the recommended dose thus there should be a decrease in the monetary and the environmental costs.

The need for implementing IPM in locust management programs and provision of novel locust control options is emphasized by many workers (Ritchie and Dobson 1995; El Bashir 1997; Lecoq et al 1997; Lomer et al 2001; Peveling 2001). Plant materials, which are efficacious and readily degradable in the environment have been the most suitable choice in integrated pest management programs. Of particular interest in this context is the potentiality of integrating Mucuna plant materials both as toxic plant materials and as products of recognized antifeedant effect.

Recommendations

The following are recommendations for future studies in this field:

• Mucuna root extracts show promising activities against locust under laboratory conditions. Further semi-field tests should be carried out in order to recommend its use by the resource-poor farmers under typical field conditions

114

• Mucuna root extracts show reasonable anti-feeding activity. This requires further investigation so that it can be incorporated in future integrated pest management programs.

• Mucuna products when mixed with synthetic insecticides gave additive and/ or synergistic effect, a phenomenon that could be utilized in pest management programs. However, further laboratory, semi-field and field studies are needed to determine the optimum combinations of Mucuna/ Decis in a mixture.

• There is need to investigate and characterize the active molecules responsible for the activity of Mucuna extracts. Studies on mode of action, biochemical properties and eco-toxicological impact should be initiated.

115

6 SUMMARY AND CONCLUSIONS

- The different locust control strategies are discussed and the varying views on their merits and shortcomings are highlighted. The role of materials of plant origin used in locust control and management is also reviewed and discussed.

- Indigenous knowledge of some local ethno-botanists on the extraction methods of the active materials and insecticidal properties of Mucuna pruriens is documented.

- Some plant materials, traditionally known for their activity against certain pests in Sudan, are screened as contact and stomach poisons against the Migratory locust, Locusta migratoria Linné. Extracts of Mucuna pruriens ( Fabaceae ) produced promising results as up to 98%mortality was recorded in locust treated with Mucuna extracts by direct spraying using water or water/ethanol extracts. The same results were obtained when the water/ethanol extracts were used as stomach poisons. The effect given by Mucuna extracts was statistically similar to that of the standard botanical insecticide "neem" and the standard chemical insecticide "Decis".

- The Desert locust appeared to be less susceptible to Mucuna extracts than the Migratory locust and that Desert locust adults were slightly more tolerant than the nymphal stages.

- Mucuna extracts also appeared to act faster on locust than neem extracts. This can be seen by comparing the results of knockdown effect and time to death of extracts on both locust species.

116

- Through topical application a high concentration of Mucuna extracts ( 200g/l). is needed to cause a mortality of 90% on the 5th nymphal instar of the Migratory locust. Topical application caused a relatively low knockdown effect though it was statistically higher than that of the control treatment.

- Dipping of the adult Migratory locust for 1.5 Sec. caused knockdown effect and mortality which were significantly higher than the effect of the control treatment.

- Extracts of Mucuna acted as stomach poisons and antifeedant against the Migratory and the Desert locust. These findings my be of practical in future crop protection activities.

- The bioactivity of substances from the root extracts of Mucuna pruriens decreased with time, but the material can be stored for a period of up to three years without serious losses in activity.

- The toxic effect of low doses of Deltametherin (Decis) was enhanced when the insecticide was applied in combination with Mucuna root extracts. The exact nature of the interaction between Decis and the extracts is not known but it could be due to potentiation or some form of synergism.

- Possibility of integrating the plant materials in locust’s management programs was discussed.

- M. pruriens root extracts may have potential use against both the Migratory and Desert locusts. This is mainly because the material is a cheep natural product that can easily be obtained and processed.

117

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139 APPENDEX

Appendix 1 Key informant questionnaire : investigation on the uses of Mucuna pruriens as pest control agent in Darfur Western Sudan. Case of Kass and Zanlingi Areas.

Surveyor………………………………………………………Date…......

1. respondents name………………………………………………………………. 2. age………………3. occupation…………..4. tribe…………………………… 5. village……………6. locality……………..7. province…………8. state…….. 9. Way of living: i. Nomadic ii. Settlers iii. Others (specify)………………. 10. educational level: i. Khalwa ii. Primary iii. Intermediate iv. Secondary 11. do you use plants for pest control or as native medicine? i. Yes ii. No 12. if yes, specify these plants?...... 13. do you use Mucuna spp (irg elghamol)? i. Yes ii. No 14. if yes, please specify other local names……………………………………….. 15. if yes, specify the uses: i. control of animal pests ii. Crop protection iii. Human health 16. please specify the treated organism and the pest(s)……………………………. 17. which part of the Mucuna spp do you use for the above purpose? i. whole of the plant ii. Leaves iii. Roots iv. Leaves and roots v. young shoots vi. Fruits/seeds explain………………………………………………………………………………… 18. in which form do you prepare Mucuna spp? i. fresh (1. chopped in slices 2. squeezed 3. others (specify)………) ii. dry (1. powder 2. weTable 3. slices 4. others (specify……….) iii. with other solvents (specify the way of extracting the active ingredients………….. ………………………………………………………………………………………….. 19. do you mix Mucuna spp. With other additives? i. Yes ii. No 20. if yes, specify………………………………………………………………………..

140 21. for how long do you store Mucuna spp and how?...... 22. how do you get access to Mucuna spp? i. valleys and water streams ii. Hilly areas iii. Open forest iv. On farm 23. is access constrained by land ownership (land tenure)? i. Yes ii. No explain………………………………………………………………………………….. 24. is Mucuna spp easily available in areas? i. Yes ii. No 25. if yes, where does the plant abundantly available?...... 26. in which time do you collect Mucuna spp and why? i. autumn ii. Summer iii. Winter (early, mid, late)………………………... 27. for how long have you been used Mucuna spp? i. less than 20 years ii. 20-40 years iii. More than 40 years 28. do you sell/ buy Mucuna spp? i. Yes ii. No 29. if yes, specify the way of market supply…………………………………………… 30. could Mucuna spp be considered as a range species? i. Yes ii. No 31. if yes, is it palaTable? i. Yes ii. No 32. other possible uses of Mucuna spp…………………………………………………. 33. traditional orals (proverbs) and lidends……………………………………………. 34. other comments and suggestions…………………………………………………..

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