*^*5 g n T rrTrr^ ^ U « * V f t t t l T V 0 9 O M A H A IdMAflV QK625.M74 G54 bite C.J G364060 T H E B A LM E LIBRARY 3 0 6 9 2 1 0 0 8 6 4 5 9 2 University of Ghana http://ugspace.ug.edu.gh THE POTENTIAL OF Beauveria bassiana FOR THE MANAGEMENT OF Cosmopolites sordidus (Germar, 1824) ON PLANTAIN {Musa, AAB) A THESIS SUBMITTED TO THE DEPARTMENT OF CROP SCIENCE, UNIVERSITY OF GHANA, LEGON BY IGNACE GODONOU IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OCTOBER 1999 University of Ghana http://ugspace.ug.edu.gh DECLARATION I do hereby declare that, except for references to works o f other researchers which have been duly cited, this work is the result o f my own original research and that this thesis either in whole or in part has not been presented for another degree elsewhere. I. GODONOU (STUDENT) , DR K. A. ODURO (SUPERVISOR/] AL PROF. K.UFREH-NUAM AH (SUPERVISOR) P- DR K. R. GREEN (SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENTS It is a real pleasure to express my heartfelt gratitude to my supervisors Dr K. A. Oduro and Professor K. Afreh-Nuamah both at the University of Ghana, Legon and Dr K. R. Green at the International Institute o f Tropical Agriculture (IITA) who guided me throughout the course o f this study. I am extremely thankful to them for all the time and care they devoted to this work. I would like to thank Professor E.V. Doku, for guiding me through my work and helping me to solve other problems at the University. I am very grateful to Professor J.N. Ayertey Head o f Crop Science Department, University of Ghana, Legon, for accepting me as student and for academic supervision. My thanks are extended to all the lecturers at Crop Science Department who took a keen interest in my work and who were always helpful to me. My thanks also go to the administrative staff o f the University o f Ghana for help that made me go through regulations at the University. I am greatly indebted to Drs C.J. Lomer, J. Langewald, C.S. Gold and P. Speijer o f IITA for their dedication to this research and for their useful suggestions and innovative help with the Methodology. I wish to thank Dr P. Neuenschwander, Director o f Plant Health Management Division (PHMD)/IITA and Dr. C.J Lomer, scientist/Leader at IITA/PHMD for releasing me from IITA and recommending me to undertake my course work at the University. My sincere thanks to Dr I. Ofori o f Crop Science Department o f the University o f Ghana and Mr S. Korie o f the International Institute o f Tropical Agriculture (PHMD) for their useful advice and guidance with data analysis. University of Ghana http://ugspace.ug.edu.gh iv Thanks to Drs J. K. Osei and K. G. Ofosu-Budu and Mr E. K. S. Ahiekpor at the University o f Ghana, ARS-Kade for their comments and suggestions throughout the course o f this study. Thanks to Eric Cornelius of the Crop Science Department for always being with me and guiding me from my course and research work to the end o f the thesis writing. Special thanks to my Technician, Mr. Aseidu Bismark for the nice job he did with me during my research work. To the staff o f the West African Plantain Project especially Messrs. Nsiah, Banfo, Brentu, Alex Eppi, Musa, Henry and Lambert, I say a big thank you for your support. Many thanks also go to all the LUBILOSA local staff in Cotonou; Pierre Affa, O. K. Douro-Kpindu, Gbongboui Comlan, Serge Attignon, Gabriel Heviefo, Olga Idohou, Leonard Nassara, Theotime Adouhoun, Romain Houenoussi, Etienne Dagbozounkou, N oelie Dossou, Yves Amoussougbo and Cooperi Gnanhoui for providing me technical assistance in IITA, Benin I wish to express my sincere thanks to Mr Sampson Nyampong for the useful advice and moral support he gave me during the course o f this work. Thanks to Dr Georgen and Mr. David both o f IITA/PHMD for identification o f some field collected insects I wish to express my thanks to Mr Ashian and his wife and Mr Ntri for their social assistance to me during my stay in Kade, Ghana. The project was funded by the German Federal Ministry for Economic Cooperation and Development (BMZ) and administered by the West African Plantain Project, IITA. The support o f these organisations is gratefully acknowledged. University of Ghana http://ugspace.ug.edu.gh LIST OF ABBREVIATIONS Anon. Anonymous ANOVA Analysis o f variance ARS Agricultural Research Station ARSEF Agricultural Research Service Collection o f Entomopathogenic Fungi BHC Benzene Hexachloride CABI CAB International CP Conidial powder CS-CP Clay soil-based formulation o f conidial powder DDT Dichloro Diphenyl Trichloroethane df Degrees o f freedom e.g. For example F Fisher’s test GDP Gross Domestic Product GLM General linear models GO-CP Groundnut oil-based formulation o f conidial powder GOK-CP Groundnut oil plus kerosene-based formulation o f conidial powder i.e. That is IITA International Institute o f Tropical Agriculture IMI International Mycological Institute IPM Integrated Pest Management LUBILOSA Lutte Biologique contre les Locustes et Sauteriaux MS Mean o f Square OPKC Oil palm kernel cake OPKC-C Oil palm kernel cake-based formulation o f conidia P Probability PDA Potato Dextrose Agar PHMD Plant Health Management Division SAS Statistical analysis system SDA Sabouraud Dextrose Agar University of Ghana http://ugspace.ug.edu.gh vii TABLE OF CONTENTS TITLE PAGE DECLARATION ' ' ACKNOWLEDGEMENTS ’>> LIST OF ABBREVIATIONS v TABLE OF CONTENTS v ii LIST OF TABLES x l > LIST OF FIGURES x v LIST OF PLATES x v i i ABSTRACT x v i i i CHAPTER 1 GENERAL INTRODUCTION 1 CHAPTER 2 LITERATURE REVIEW 5 2.1. Origin and distribution of plantain and banana 5 2.2. Botany of plantain and banana 5 2.3. Production and economic importance of plantain in Ghana 6 2.4. Constraints to plantain production 7 2.5. Origin and distribution of C. sordidus 9 2.6. Morphology and biology of C. sordidus 9 2.7. Life history of C. sordidus 10 2.8. Host range and damage caused by C. sordidus 11 2.9. Control of C. sordidus 12 2.9.1. Cultural control 12 2.9.2. Chemical control 14 2.9.3. Classical biological control of C. sordidus 15 2.9.4. Biological control using entomopathogens 16 2.9.4.1. Nematodes 1 7 2.9.4.2. Fungi 1 8 2-10. The entomopathogens Beauveria bassiana and Metarhizium anisopliae 1 9 2.10.1. Taxonomy 1 9 2.10.2. Infection processes by Beauveria and Metarhizium 20 University of Ghana http://ugspace.ug.edu.gh viii 2.10.3. Development o f m uscardine disease 22 2.10.4. Saprophytic developm ent o f muscardine 23 2.10.5. Mass production o f species o f Beauveria and Metarhizium 23 2.10.6. Formulation 24 2.10.7. Delivery systems 25 2.11. Practical and economic feasibility o f biocontrol using entom opathogenic fungi 26 CHAPTER 3 BIOLOGY AND SPATIAL DISTRIBUTION O F C. sordidus ON PLANTAIN IN GHANA 28 3.1. INTRODUCTION 28 3.2. MATERIALS AND METHODS 29 3.2.1. Duration o f different developm ental stages o f C. sordidus 29 3.2.2. Spatial distribution o f the different stages o f C. sordidus on plantain 32 3.3. RESULTS 35 3.3.1. Duration o f different developm ental stages o f C. sordidus 35 3.3.2. Spatial distribution o f the different stages o f C. sordidus on plantain 35 3.4. DISCUSSION 39 CHAPTER 4 SELECTION OF STRAIN OF B. bassiana FOR THE CON TRO L O F C. sordidus 41 4.1. INTRODUCTION 41 4.2. MATERIALS AND METHODS 1— 43 4.2.1. Strains ot B. bassiana used in experim ental work 43 4.2.2. Comparative virulence o f the strains A RSEF2624 and 194-907 to adults o f C. sordidus 44 4.2.3. Comparative potential for mass production and virulence o f the strains 194-907 and 1M I330194 o f B. bassiana to C. sordidus 46 University of Ghana http://ugspace.ug.edu.gh 47 55 58 58 61 64 71 73 73 76 76 77 82 83 83 86 86 Comparative potential for mass production of the strains 194-907 and IMI330194 of B. bassiana Comparative virulence of the strains 194-907 and IMI330194 of B. bassiana to C. sordidus RESULTS Comparative virulence of the strains ARSEF2624 and 194-907 to adults of C. sordidus Comparative potential for mass production of the strains 194-907 and IMI330194 of B. bassiana Comparative virulence of the strains 194-907 and IMI3 3 0194 of B. bassiana to C. sordidus DISCUSSION DEVELOPMENT AND EVALUATION O F DIFFERENT FORMULATIONS OF B. bassiana FOR THE CO N TRO L OF C. sordidus IN POT EXPERIMENTS INTRODUCTION MATERIALS AND METHODS Production of CP of B. bassiana strain IMI330194 on rice grains subjected to different treatments Effect of different liquid and solid-phase media on the production of B. bassiana strain IMI330194 Development of different formulations of B. bassiana strain IMI330194 Evaluation of the effect of different formulations of B. bassiana strain IMI330194 on adults of C. sordidus in pot experiments Effect of W-CP and GOK-CP of B. bassiana strain 1MI330194 on adults of C. sordidus Effect of GO-CP and CS-CP of B. bassiana strain 1MI330194 on adults of C. sordidus Effect ot OPKC-C of S. bassiana strain IM1330194 on adults of C. sordidus ix University of Ghana http://ugspace.ug.edu.gh 87 88 88 89 89 91 93 93 94 98 101 101 103 103 104 105 X Persistence in the soil of GO-CP and OPKC-C of B. bassiana strain IMI330194 against adults of C. sordidus RESULTS Production of CP of B. bassiana strain IMI330194 on rice grains subjected to different treatments Production of hyphae and blastospores of the strain IMI330194 in yeast plus sugar, cassava starch plus sugar and cassava starch suspensions Production of B. bassiana strain IMI330194 on OPKC Effect of W-CP and GOK-CP of B. bassiana strain IMI330194 on adults of C. sordidus Effect of GO-CP and CS-CP of B. bassiana strain IMI330194 on adults of C. sordidus Effect of OPKC-C of B. bassiana strain IMI330194 on adults of C. sordidus Persistence in the soil of GO-CP and OPKC-C of B. bassiana strain IMI330194 against adults of C. sordidus DISCUSSION FIELD EVALUATION OF SELECTED FORM ULATION OF B. bassiana STRAIN IM I330194 FOR TH E CONTROL OF C. sordidus INTRODUCTION MATERIALS AND METHODS Determination of the dose of conidia needed for field studies by comparing the effect of two doses of B. bassiana strain 1M1330194 conidia on C. sordidus adults in a field where weevils were artificially released Effect of OPKC-C of B. bassiana strain IMI330194 on adults of C. sordidus in a field where weevils were artificially released Effect of OPK.C-C of B. bassiana strain IM1330194 on adults of C. sordidus in a field naturally infested with weevils University of Ghana http://ugspace.ug.edu.gh 107 108 108 111 115 119 121 124 130 146 X I Evaluation of the spread of B. bassiana in a plantain field using adult weevils artificially infected with OPKC-C of B. bassiana strain IM I330194 RESULTS Determination of the dose of conidia needed for field studies by comparing the effect of two doses of B. bassiana strain IM I330194 conidia on C. sordidus adults in a field where weevils were artificially released Effect of OPKC-C of B. bassiana strain IM I330194 on adults of C. sordidus in a field where weevils were artificially released Effect of OPKC-C of B. bassiana strain IM I330194 on adults of C. sordidus in a field naturally infested with weevils Evaluation of the spread of B. bassiana in a plantain field using adult weevils artificially infected with OPKC-C of B. bassiana strain 1MI330194 DISCUSSION GENERAL DISCUSSION AND CONCLUSION University of Ghana http://ugspace.ug.edu.gh 6 20 36 36 36 43 59 59 66 66 79 90 90 x i i LIST OF TABLES Plantain production in different regions of Ghana. Outline classification o f some entomopathogenic fungi. Percentage viability o f eggs laid by adults o f C. sordidus on corm tissue under laboratory conditions. Mean duration o f different developmental stages o f C. sordidus under laboratory conditions. Parameter estimates from Generalized Linear Models (GENMOD) analysis of the number o f larvae at three levels on growing plantain (Figure 1.). B. bassiana strains used during trials. Mean percentage cumulative mortality of C. sordidus adults 7, 14, 21 days after exposure to pseudostem traps and pieces o f corm treated with inoculum o f B. bassiana strains ARSEF2624 or 194-907. Mean percentage cumulative dead weevil with signs o f mycosis 7, 14 and 21 days after exposure to pseudostem traps and pieces o f corm treated with inoculum o f B. bassiana strains ARSEF2624 or 194-907. Mean percentage cumulative mortality o f C. sordidus 7, 14 and 21 days after exposure to B. bassiana strains 194-907 and IMI330194. Mean percentage cumulative dead adults with signs o f mycosis 7, 14 and 21 days after exposure to B. bassiana strains 194-907 and IMI330194. Treatment combinations o f liquid substrate, solid substrate and water amendments used for production o f B. bassiana strain IMI330194. Mean weight of conidial powder, number of conidia and conidia viability of B. bassiana strain IMI330194 produced on rice subjected to different treatments. Production of blastospores and hyphae of B. bassiana strain IMI330194 in brewers yeast and cassava starch suspensions. University of Ghana http://ugspace.ug.edu.gh 90 92 92 95 95 96 96 109 109 113 113 X III Effect o f production method on production and viability o f conidia o f B. bassiana strain IMI330194. Mean percentage cumulative mortality o f C. sordidus at day 26 and 42 after exposure to W-CP and GOK-CP o f B. bassiana strain IM1330194. Mean percentage cumulative dead adults with signs of mycosis at day 26 and 42 after exposure to W-CP and GOK-CP o f B. bassiana strain IMI330194. Mean percentage cumulative mortality and percentage cumulative dead C. sordidus with signs of mycosis at day 18 after exposure to GO-CP and CS-CP o f B. bassiana strain IMI330194. Mean percentage cumulative mortality and percentage cumulative dead adults o f C. sordidus showing signs o f mycosis 18 days and 35 days after exposure to OPKC-C o f B. bassiana strain IMI330194. Mean percentage cumulative mortality o f C. sordidus after exposure to different formulations at day 0, 14. 28 and 42 after sucker treatment. Mean percentage dead adults of C. sordidus with signs o f mycosis when exposed to different formulations at day 0, 14, 28 and 42 after sucker treatment. Mean percentage mortality and dead adults o f C. sordidus with signs of B. bassiana mycosis, 28 days after exposure to two doses of B. bassiana conidia in a field. Mean percentage cumulative weevil mortality and percentage dead adults o f C. sordidus with signs of mycosis in planting holes, 28 days after exposure to two doses of B. bassiana conidia in a field. Mean percentage cumulative weevil mortality and percentage dead C. sordidus with signs of mycosis, 28 days after exposure to OPKC-C o f B. bassiana strain IMI330194 in a field. Mean percentage cumulative weevil mortality and percentage dead of C. sordidus with signs o f mycosis in planting holes, 28 days after exposure to OPKC-C o f B. bassiana strain IMI330194 in a field. University of Ghana http://ugspace.ug.edu.gh x iv T a b le 25 . Table 26. Table 27. Table 28. Table 29. Percentage mortality and percentage dead adults o f C. sordidus with signs of mycosis in the laboratory after collection from non-treated and treated suckers with OPKC-C of B. bassiana strain IMI330194 in a field. 116 Contrasts between OPKC-C o f IMI330194 and control treatments, for mortality o f C. sordidus collected from the experimental plots. 116 Total larvae of C. sordidus collected from plantain suckers treated with or without OPKC-C o f B. bassiana strain IMI330194, 60 days after planting. 117 Contrasts between OPKC-C o f B. bassiana strain IM I330194 and control treatments for number o f C. sordidus larvae collected from plantain suckers, 60 days after planting. 117 Percentage o f plantain suckers attacked and those killed by larvae o f C. sordidus 60 days after been exposed to OPKC-C o f B. bassiana strain IMI330194 in a field naturally infested with weevils. 117 University of Ghana http://ugspace.ug.edu.gh 34 37 37 37 38 38 38 60 62 62 62 63 63 68 XV LIST OF FIGURES Levels at which uprooted plantains were sampled for weevil stages. Duration o f C. sordidus egg incubation on corm pieces in the laboratory. Duration o f larval stage o f C. sordidus in corm pieces in the laboratory. Duration of pupal stage o f C. sordidus in corm pieces in the laboratory. Percentage of eggs o f C. sordidus at three sampling levels (Figure 1.) on plantain. Percentage of larvae o f C. sordidus at three sampling levels (Figure 1.) on plantain. Percentage o f pupae of C. sordidus at three sampling levels (Figure 1.) on plantain. Percentage mortality o f C. sordidus adults (a) and percentage dead adults with signs of mycosis (b) after exposure to pseudostem traps and pieces of corm treated with B. bassiana inoculum. Mean number o f conidia of B. bassiana strains per gram of rice grains. Mean weight o f conidial powder of B. bassiana strains per kilogram o f rice grains. Mean number of conidia o f B. bassiana strains per gram of conidial powder. Mean percent viability of conidia o f B. bassiana strains on rice grains. Mean percent viability o f the conidia of B. bassiana strains in powder extracted from rice. Percentage cumulative mortality of C. sordidus adults (a) and percentage dead adults o f C. sordidus with signs o f mycosis (b) after exposure to pseudostem traps treated with B. bassiana inoculum. University of Ghana http://ugspace.ug.edu.gh 69 69 70 70 97 110 110 114 114 120 120 121 x v i ®Mean percentage non-hatched egg of C. sordidus 10 days after exposure to 194-907 inoculum. '"’Mean percentage non-hatched eggs o f C. sordidus 10 days after exposure to IMI330194 inoculum. Mortality of larvae o f C. sordidus within pieces o f corm treated with B. bassiana strain 194-907 inoculum. Mortality of larvae o f C. sordidus within pieces o f corm treated with B. bassiana strain IMI330194 inoculum. Percentage mortality (top) and percentage dead weevil w ith signs of mycosis (bottom) when exposed to different formulation at different days. Percentage cumulative mortality o f C. sordidus at the soil surface 5-28 days after exposure to two doses of conidia o f the strain IMI330194 in a field. Percentage adults o f C. sordidus recaptured with pseudostem traps 19-28 days after exposure to two doses o f conidia of the strain IMI330194 in a field. Percentage cumulative mortality o f C. sordidus at the soil surface 5-28 days after exposure to OPKC-C o f B. bassiana strain IMI330194 in a field. Percentage adult o f C. sordidus recaptured with pseudostem traps 19-28 days after exposure to OPKC-C o f B. bassiana strain IMI330194 in a field. Regression fitted line for counts o f C. sordidus adults exposed or not exposed to OPKC-C of IMI330194 at different sampling distances recorded 2 to 63 days after release. Regression fitted line for counts of C. sordidus adults exposed or not exposed to OPKC-C o f IMI330194 recorded from 2 to 63 days after weevil release in a plantain field. Mortality and sporulation o f pot infected and non infected C. sordidus at different sampling distances 63 days after release in a plantain field. University of Ghana http://ugspace.ug.edu.gh xvii LIST OF PLATES Plate 1. Plastic bowls containing pieces o f corm on which C. sordidus eggs were incubated. Plate 2. Incubation o f B. bassiana conidia in liquid medium in Erlenmeyer Flasks (a) placed on a rotary shaker (b) for production o f hyphae and blastospores. Plate 3. Inoculation o f autoclaved rice grains in polypropylene bag with liquid medium containing B. bassiana hyphae and blastospores. Plate 4. Incubation and air drying o f rice grains inoculated with B. bassiana in plastic bowls. Plate 5. Extraction o f conidial powder o f B. bassiana from rice grains. Plate 6. Conidial powder of the B. bassiana strain IM I330194 obtained from rice grains in Petri dish. Plate 7. A dehumidifier (a) containing silica gel for drying conidial Powder placed in plastic containers (b). Plate 8. C. sordidus showing external growth o f B. bassiana at death after exposure to B. bassiana inoculum. Plate 9. Plate 5.2.1. Production o f oil palm kernel cake resulting from kernel oil extraction. Plate 10. B. bassiana strain IMI330194 growing on grounded oil palm kernel cake. Plate 11. Plastic buckets in which plantain suckers subjected to different treatments were planted and in which adult weevils were released. Plate 12. White growth o f B. bassiana strain IMI330194 on OPKC around a plantain sucker after application o f OPKC-C o f IMI330194. 31 49 50 51 52 53 53 65 80 81 85 118 University of Ghana http://ugspace.ug.edu.gh ABSTRACT The banana weevil, Cosmopolites sordidus (Germar) in association with other pests and diseases, represents a threat to the production of plantain (Musa spp., AAB), the preferred staple food in Ghana. Biological control of the banana weevil was considered the most promising management option for small-scale plantain production and studies were, therefore, undertaken to determine the efficacy o f the entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillemin in the management o f C. sordidus. The duration and spatial distribution o f the different developmental stages o f C. sordidus within plantain plants were determined to provide background information for evaluation o f B. bassiana against the banana weevil. The mean egg incubation period and the mean developmental period from larva to pupa and pupa to adult were 6.3 + 0.2, 28 ± 0.6 and 7.1 ± 0.3 days respectively. The developmental period from egg to adult ranged from 33 to 51 days with a mean o f 40.4 ± 0.7 days. Within the plantain plant, approximately 80% of the eggs were located in the rhizome, >80% of the larvae were found at the rhizome level and all o f the pupated larvae were located in the rhizome, suggesting that this is where a biocontrol agent should be targeted, rather than the pseudostem. Three strains o f B. bassiana were obtained and evaluated on the basis o f virulence tests and potential for mass production. From the results o f these tests, strain 1MI330194 o f B. bassiana was selected for subsequent studies. Laboratory studies using a water-based inoculum applied to corm pieces or pseudostem traps, showed that B. bassiana could control all stages o f C. sordidus, with up to 21.3%, 36.4% and 42.3% of eggs, larvae and adults respectively showing signs o f fungal disease. Pot experiments to compare different formulations o f the strain IMI330194 against adult weevils showed that the highest mortality (>60%) was obtained with groundnut oil plus kerosene-based formulation o f conidial powder (GOK-CP), groundnut oil-based formulation of conidial powder (GO-CP) and oil palm kernel cake-based formulation University of Ghana http://ugspace.ug.edu.gh xix o f conidia (OPKC-C). A persistence trial showed that OPKC-C o f IM I330194 still gave 61.0% weevil mortality one month after application, compared with only 12.3% for conidial powder (CP) o f IMI330194 and 3.9% for the control with no conidia. In field trials with artificial weevil release, mortality of adult weevils exposed to CP and OPKC-C o f IMI330194 ranged from 53.4 to 75.5%, compared to <8% in the control with no conidia. Under natural weevil infestation, 16.7% o f plantain suckers treated with CP o f IMI330194 and 19.4% of untreated suckers were killed by weevil attack. In contrast, none o f the suckers planted with OPKC-C of IMI330194 were killed. A study on the spread of fungal conidia using artificially infected and non-infected adult weevils showed a possible dissemination of B. bassiana conidia from infected weevils up to 18m from the release point. On the basis of results from the present study, the strain IMI330194 of B. bassiana could clearly play a key role in the management o f C. sordidus adults on plantain. University of Ghana http://ugspace.ug.edu.gh CHAPTER 1 GENERAL INTRODUCTION Plantain (Musa spp. AAB) is a particularly important crop for farmers in the humid forest agro-ecological zone o f West and Central Africa (Anon., 1990) where approximately 43% o f the world’s plantain is produced (Anon., 1993). Because o f its long history o f wide spread cultivation and distribution, the region has become a secondary centre of plantain diversity (Swennen and Vuylsteke, 1990). It is estimated that about 70 million people in West and Central Africa derive greater than one- quarter o f their food energy requirements from plantain, making it one o f the most important sources of carbohydrate throughout the African lowland humid forest zone (Swennen, 1990). Unlike sweet dessert bananas, plantain is a staple food which is fried, boiled and sometimes pounded or roasted and consumed alone or together with other food (Swennen, 1990; Afreh-Nuamah and Hemeng, 1993). In addition to being a staple food for rural and urban consumers, plantain provides an important source of income for resource-poor farmers (Anon., 1993). Plantain is a highly priced staple crop in Ghana. The crop is mainly grown by small-scale subsistence farmers who produce an average o f 7.1 tonnes per hectare (Anon., 1991a). In terms of national crop production, plantain is ranked fourth among the major crops in Ghana and accounts for 9% of agricultural Gross Domestic Product (GDP) (Anon., 1991a). As plantain is a major staple in Ghana, a shortfall in production results in scarcity on the general market and a corresponding price increase (Schill et a l , 1997) In the traditional cropping systems of Ghana, plantain is grown together with root crops, cereals and vegetables or as shade for tree crops, like cocoa (Afreh-Nuamah, 1993a). As a backyard crop, it also does well, coexisting easily with established farming systems. It can provide a continuous source o f food over the cropping year and counteracts degradation o f the soil through the prolific leaf mulch cover it produces. University of Ghana http://ugspace.ug.edu.gh 2 The yield o f banana and plantain in tropical and subtropical regions, where these crops are a major source o f food and revenue, is adversely affected by diseases and pest infestations (Simmonds, 1966). Out o f about two hundred insect pests recorded attacking Musa spp., the most important is the banana weevil, Cosmopolites sordidus Germar (Coleoptera: Curculionidae) (Purseglove, 1972). The damage done by the weevil is primarily the result o f destruction o f corm tissue, sometimes accentuated by secondary attacks by other insects and micro-organisms (Simmonds, 1966) leading to increased risk o f toppling. In Ghana, the combined effect o f nematode and banana weevil infestations on plantain can lead to yield losses o f 85% in the plant crop (Udzu, 1997). In Uganda, banana weevil outbreaks can cause up to 100% yield losses (Sengooba, 1986; Sebasigari and Stover, 1988). Limited control o f the banana weevil is achieved by use o f cultural, biological and chemical strategies. In the absence o f any better control strategy, many national programmes world-wide adopt and recommend chemical pesticides as the main control strategy to farmers (K. Afreh-Nuamah, personal communication, 1998). In the short term, chemical pesticides are expensive and provide only temporary relief. Moreover, reproductive and evolutionary capacities of the insects allow the pest to develop mechanisms of resistance to the chemicals (Metcalf, 1980). The detrimental effects o f pesticide application on human health, damage to non-target organisms and possible environmental pollution o f residential and agricultural lands, and o f ground water suggest that the use of chemicals has to be evaluated alongside other methods. For these and other reasons, biological control is now receiving considerable attention. Several natural enemies of C. sordidus, mainly in the orders Coleoptera and Diptera, have been identified and attempts to manage the population of C. sordidus using introduced predators and parasitoids have been made. The Histerid beetle (Plciesius javanus Erichson (Coleoptera: Curculionidae)), which is native to Java, has already been successfully introduced in many countries as a biological control agent o f C. sordidus, but in Africa this predator and many other predaceous beetles tested have not been effective (Nankinga, 1994). In Cuba, Tetramorium guineense Auct (= 71 University of Ghana http://ugspace.ug.edu.gh 3 bicarinatum Nylander) (Hymenoptera: Formicidae) has been used successfully to reduce the weevil population by 65% in heavily infested plantations and by 83.5% in less heavily infested ones (Roche and Abreu, 1983). Treverrow et al. (1991) reported that in Australia the entomopathogenic nematode Steinernema carpocapsae gave significant mortality of C. sordidus larvae in rhizomes (16-68% depending on the larval size and mode o f nematode application). In Brazil, the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metschnikoff) Sorokin have also shown good control against adults o f C. sordidus under laboratory conditions (Batista-Filho et al., 1987; Busoli et al., 1989). Laboratory infectivity tests conducted using different strains o f B. bassiana isolated from dead banana weevil and soil samples in East Africa (Nankinga, 1994) and in W est Africa (Traore, 1995) gave good results o f up to 100% weevil mortality. B. bassiana therefore, represents a promising alternative for the management o f C. sordidus in Africa. For sustainable management o f C. sordidus with an entomopathogenic fungus there is a need to know more about aspects o f its biology such as the developmental period o f its different stages, the spatial distribution o f the different stages within a standing plant and the movement of the adult in the field. Factors affecting the success o f any microbial agent for the control o f an insect pest include the selection o f an appropriate agent (i.e. most virulent strain), the potential o f the strain for mass-production, development o f an efficient and cost effective formulation and delivery system, and the feasibility o f the control measure in the small-scale farmer’s context. The present study therefore, aims to: (i) Determine the duration of the developmental period o f different stages o f C. sordidus in Ghana, (ii) Determine the spatial distribution o f the different stages of C. sordidus on plantain, University of Ghana http://ugspace.ug.edu.gh 4 (iii) Select virulent strain (s) o f B. bassiana with good production characteristics for the management of C. sordidus, (iv) Develop and evaluate different formulations o f B. bassiana against adults o f C. sordidus in pot experiments, (v) Evaluate the selected formulation of B. bassiana against adults o f C. sordidus in the field. University of Ghana http://ugspace.ug.edu.gh 5 CHAPTER 2 LITERATURE REVIEW 2.1. O rigin and distribution o f plantain and banana Plantain and banana are from the family Musaceae in the order Zingiberales (Purseglove, 1972). The family Musaceae has two genera, Musa and Ensete. Ensete is an old and declining genus, which probably originated in Asia and spread to Africa and is now equally divided between both continents (Purseglove, 1972). Musa is a rhizomatous herb which originated in South-eastern Asia and the Pacific, with its centre of diversity and probably o f origin in the Assam-Burma-Thailand area (Purseglove, 1972). Musa is the largest, most widely distributed and most diversified genus of the family, consisting o f up to 15 known species (Simmonds. 1982). Edible Musa cultivars are from two wild species, namely Musa acuminata Colla (A genome) and Musa balbisiana Colla (B genome) (Simmonds, 1982). Musa spp. are grown mainly in the lowland tropics (Purseglove, 1972). Simmonds (1982) believed that the dispersal o f AAA clones and hybrid cultivars (AAB and ABB) was from northern India by the way o f Saudi Arabia and the Horn o f Africa. The cultivation of plantain {Musa spp. type AAB), and highland banana (type AAA, AB, ABB) is limited to within 30° north and south o f the equator (Purseglove, 1972). Plantains are grown widely in Cote d ’Ivoire, Ghana, Nigeria, Cameroon, Congo and Zaire, while cooking bananas {Musa spp. type AAA-EA) and beer bananas {Musa spp. types AAA-EA, AB, ABB) are produced in Uganda, Rwanda, Burundi and northern Tanzania (Gold, 1993). 2.2. Botany o f plantain and banana Musa spp. are tree-like giant perennial herbs with an underground stem or corm, a pseudostem composed of leaf sheaths, and a terminal crown o f leaves through which the inflorescence emerges (Purseglove, 1972). The underground stem has extremely short internodes covered externally by closely packed leaf scars, which completely University of Ghana http://ugspace.ug.edu.gh 6 encircle the corm. The aerial part or pseudostem is composed o f leaf sheaths borne in the left-handed spiral with a phyllotaxy ol one-third on young suckers and a terminal crown (Purseglove, 1972). The pseudostem is functionally the trunk o f the plant, varying in height from 2 m to 8 m depending upon cultivar, and which later supports the fruit bunch. 2.3. Production and econom ic im portance of plantain in G hana Ghana is the fifth largest plantain and banana producer in West and Central Africa, Nigeria being the first followed by Zaire, Cameroon and Cote d ’Ivoire in that order (Anon., 1993). O f a total o f 7,960,000 tonnes produced by West and Central African countries, Ghana produced 8% with a per capita consumption o f plantain o f 60 kg a year (Anon., 1993). The total area cropped to plantain is about 129,000 ha (Anon., 1991a). Plantain is extensively grown throughout the forest zone o f the country and can be found in backyard gardens of most houses. Akomeah et al. (1995) gave the distribution o f plantain production in different regions of Ghana as follows: Table 1. Plantain production in different regions of Ghana. Region Land area Yield (hectares) (tonnes/hectare) Ashanti 50,940 6.8 Eastern 39,200 8.3 Western 31,000 6.2 Brong-Ahafo 24,700 5.5 Central 6,600 5.5 Volta 4,600 6.5 Source: Akomeah et al. (1995) University of Ghana http://ugspace.ug.edu.gh 7 In Ghana, plantain is the fourth most important starch-staple after grains, cassava, and yam (Cropley and Morriss, 1993). It is a highly priced crop, reflecting a strong consumer preference and huge demand (Schill et al., 1997). The bulk o f the crop is grown in the southern part of the country with rainfall o f about 1500 mm per annum and an annual water deficit below 400 mm (Ahiekpor, 1996). In the producing areas, any meal prepared without plantain, cocoyam, or yam is not considered as a full meal. In spite o f its cultural value, plantain production has declined during the past two decades (Akomeah et al., 1995). 2.4. C onstraints to plantain production A wide range of factors limits plantain and banana productivity. These factors include poor crop and soil management practices, inherent low soil fertility, reduced fallow periods (due to increasing population and food demand), inadequate supply o f good quality planting materials, low yield potential for most local varieties, high post­ harvest losses, and an extensive pest and disease complex (Karikari, 1970). The relative importance o f constraints varies within Africa. In Uganda for example, banana weevil is the key constraint followed by nematodes and black Sigatoka (a foliar disease caused by the fungal pathogen Mycosphaerella fijiensis Morelet) (Gold et al., 1993), while in Southern Cameroon, the major constraints in order o f importance are, black Sigatoka, nematodes (Radopholus similis (Cobb) Thome) and C. sordidus (Anon., 1997). Farmers in Ghana have identified several constraints to plantain production (Schill et al., 1997) among which nematodes, banana weevil, weeds and black Sigatoka are the most important. The major factor determining the productivity o f banana and plantain is the health of the root system, which is responsible for nutrient and water uptake (Swennen, 1986). According to Bridge et al. (1993), nematode species known to cause the most serious root damage to Musa spp. are R. similis, Pratylenchus coffeae (Zimmermann) Filipjev and Steckh., Pratylenchus goodeyi Sher and Allen, and Helicotylenchus multicinctus (Cobb) Golden. University of Ghana http://ugspace.ug.edu.gh In Ghana, Schill et al. (1996) reported that P. coffeae, H. multicinctus and Meloidogyne spp. are the most wide-spread nematodes species on plantain. The first two species occur at high densities while the latter occur at low density. The dominant nematode species in Ghana are all lesion-forming nematodes. Damage to the roots infected by plant parasitic nematodes and the resulting severe root-rot caused by secondary invasion o f fungal pathogens are considered to be responsible for a major part o f toppling o f banana and plantain and subsequent yield loss (Stover, 1966; Sikora and Schloesser, 1973). The feeding activity o f some plant parasitic nematodes on the root system causes root necrosis. Plants with necrotic roots are less able to take up water and nutrients resulting in stunted growth, delayed maturation time and reduced bunch size (Speijer et al., 1994). Udzu (1997) reported a yield reduction o f 63.2% due to nematodes. A combined infestation o f C. sordidus and nematodes reduced yield by more than 80% (Anon., 1997; Udzu, 1997). The larvae of C. sordidus bore into the corm and the pseudostem, causing mortality of suckers, snapping (Feakin, 1971; Koppenhofer, 1993) and toppling (Bosch et al., 1995; Pena et al., 1993; Rukazambuga, 1996) o f the pseudostem. Extensive tunnelling by the larvae interferes with root initiation as a result o f the destruction o f the corm ’s cortical tissue. This leads to the production o f a small number o f roots, which consequently affects the anchorage o f the plant (Wright, 1977) resulting in a general decrease in productivity (Pena et al., 1993). Damage is usually greater in ratoon crops (Mitchell, 1978; Taylor, 1991; Gold et a l , 1994; Pone, 1994). Heavily infested plants produce small bunches, and have reduced resistance to drought and strong winds may sweep many large or maturing plants down (Sikora et al., 1989). Such “blow-downs” or “toppling” can lead to crop losses ranging from 50 to 100% (Hord and Flippin, 1956; Stover, 1966). In heavily infested plantations, the suckers produced are weak (Froggatt, 1925). University of Ghana http://ugspace.ug.edu.gh 9 2.5. Origin and distribution o f C. sordidus C. sordidus is believed to have originated in Southeast Asia, probably the Malaya/Java/Borneo region, as it is there that the weevil’s natural predators were found (Kranz et al., 1977). The banana/plantain weevil has gradually spread with banana/plantain stalks to various parts o f the world (Harris, 1947). In 1900, the insect was recognised in the Far East, Australia and Brazil and during the following 20 years, was observed in Central Africa, Central America, the Pacific Islands, the Indian Ocean Islands and the Caribbean (Kranz et al., 1977). The banana weevil now has a pan-tropical distribution and is recognised as an important pest o f Musa spp. in most production areas o f plantain and banana (Hill, 1983; Neuenschwander, 1988). The banana/plantain weevil reached pest status between 1960s-1970s, more than 60 years after being introduced to Africa and is now widely disseminated (Anon., 1991b). The factors underlying changes in C. sordidus pest status remain undetermined. 2.6. M orphology and biology o f C. sordidus The adult weevil is soft and brown when newly emerged from the pupal case and later changes from dark-brown to black in colour. The adult weevil has a long slightly curved trunk in front o f the head and is approximately 12.5 mm long and 4 mm in width (Lescot, 1988). The mature adult lays its eggs in the standing stem o f the growing plant or the stump o f a recently harvested plant (Whalley, 1957). The female bites a small hole in the corm at the ground level and after preparing an incubation chamber, deposits a single egg (Kranz et a l , 1977). Oviposition takes place at night and occurs throughout the year (Woodruff, 1969) but is greatest during the rainy season. A single female can lay up to 100 eggs during its lifetime but usually the number does not exceed 50 (Kranz et al., 1977). The eggs o f C. sordidus are about 2 mm long, oval in shape and white in colour (Whalley, 1957, 1958). The duration of the various weevil stages varies widely according to season and locality (CuilltJ, 1950). For example, the duration of the egg incubation period is 5-7 days in America (Moznette, 1920), 6-7 days in Jamaica University of Ghana http://ugspace.ug.edu.gh 10 (Edwards, 1934), 5-8 days in Uganda (Bakyalire and Ogenga-Latigo, 1992), 3-7 days in Ghana (Afreh-Nuamah, 1993b) and 4-33 in Queensland (Froggatt, 1924). After emergence, the larvae tunnel into the corm for 20 to 100 days but after a series o f moults turn toward the periphery to pupate (Kranz et al., 1977). The larval stage is reported to last between 14 and 21 days (Hill, 1983), 30-40 days (Seshu Reddy, 1986), 55-65 days (Bakyalire and Ogenga-Latigo, 1992) or 11-16 days (Afreh-Nuamah, 1993b). The duration of the developmental period from larva to pupa decreases as temperature increases (Traore, 1995). The pupal stage is reported to last 5 to 8 days (Harris, 1947; Whalley, 1957; Woodruff, 1969), or 8 to 10 days (Bakyalire and Ogenga-Latigo, 1992; Afreh-Nuamah, 1993b). The mature adult often remains longer within the plant before biting the external sheath (Cuille, 1950). The life cycle o f the weevil has commonly been reported to last 30 to 50 days (Wolfenbarger, 1964; Woodruff, 1969; Hill, 1983) but studies by Bakyalire and Ogenga-Latigo (1992) in Uganda have shown a longer duration ranging between 53 and 72 days. 2.7. Life history o f C. sordidus The adult weevils live in the soil, feeding on rotten materials o f banana and plantain and visiting the growing plant for oviposition. They are sluggish and nocturnal in habit (Cuille, 1950) and are negatively phototactic being more active in hours of darkness and positively hydrotactic, preferring high humidity areas (Ittyeipe, 1986). The weevil is known to live up to two years or more (Whalley, 1957; Wolfenbarger, 1964; Hill, 1983; Waterhouse and Norris, 1987). It is also known to survive for long periods without food (Woodruff, 1969), and a maximum o f 180 days has been reported (Mitchell, 1980). The spread of the weevil within banana-growing areas generally happens in three ways; in planting material from infested plantations (most important), by crawling from adjacent infested plantations, or by rain. Since banana and plantain may be planted on steep slopes, flood water carries infested materials as well as adult weevils from high to low areas (Kranz et al., 1977). The adult weevils have functional wings but have rarely been observed to fly (Cuille, 1950). The banana weevil was observed University of Ghana http://ugspace.ug.edu.gh 11 to spread its functional wings without flight in the laboratory in dry conditions or when exposed to insecticides (Roth and Willis, 1963). Some authors have maintained that weevils never fly but Ostmark (1975) reported free-flight in Fiji on warm humid nights in the dry season, and it may also be a significant means o f spread. 2.8. H ost range and dam age caused by C. sordidus C. sordidus attacks only members o f the genus Musa (Froggatt, 1925). The banana weevil was recorded on sugarcane, Saccharum officinarum (L.) Lamk and yam (.Dioscorea spp.) but appears to be only a very minor pest on those crops, perhaps attacking them only when plantain and banana are not available (Woodruff, 1969). C. sordidus has also been reported on sweet potato tubers, Ipomoea batatas (L.) Lamk (Cuille, 1950). During dry weather conditions, when the plants are striving against adverse growing conditions, the effect o f the borer undermining the vitality o f the plants brings about a more or less complete breakdown o f the stools far more rapidly than would occur in a normal season (Froggatt, 1925). The larva is a voracious feeder, devouring an amount o f tissue equal to many times (5 to 10) its own body weight per day (Cuille, 1950). Plant losses due to the attack o f banana weevil depend on the stage o f the plant, and the level o f the weevil infestation in a field. According to Karamura and Gold (1996), the banana weevil damage has three main effects. Firstly, the infected plants are denied the ability to produce roots and normal suckers because o f the destruction of the cortical tissue of the co m . Thus the affected plants are unable to absorb sufficient nutrient and water and produce “water” suckers which rarely reach the flowering stage. Secondly, larval tunnels may destroy the central cylinder (with vascular bundles). When this happens, the physiological communication between the aerial shoot and the underground stem is cut and, depending on the growth stage, the plant may die or produce poor quality roots and reduced yield. Thirdly, weevil tunnels may affect the meristem region in which case growth will halt, leading to failure o f leaf production and death of the plant. It was reported that in a situation o f heavy University of Ghana http://ugspace.ug.edu.gh 12 infestation, mature plants may be killed or fail to flower while newly planted suckers within infested fields are readily destroyed almost immediately they are planted (Karamura and Gold, 1996). In Uganda for example 60% o f the suckers planted in heavily infested fields may die due to weevil attack (P. Speijer, personal communication, 1997). In West and Central Africa a range o f damage levels and yield losses have been reported. For example, in Cote d ’Ivoire, yield loss was correlated with intensity o f attack and reductions o f 30 to 60% were found to be common (Vilardebo, 1973). Lescot (1988) reported yield reductions o f 20 to 90% in Cameroon. In Ghana, Afreh-Nuamah (1993a), reported that one month after planting, percentage weevil infestation ranged from 0 to 82.5% depending on the origin o f planting material (i.e. nursery material or ratoon material), history o f land (cropped land or forest land) and cultivars. Also, Udzu (1997) reported a yield reduction o f 33.3% due to weevil infestation only and 86.1% yield reduction when the effect o f nematodes and weevil infestation was combined. 2.9. C ontrol o f C. sordidus Several methods are used to control the banana weevil. These include cultural, chemical and biological methods (Whalley, 1957; Simmonds, 1966; Hill, 1983). 2.9.1. C ultural control Cultural control involves destroying the sheltering and feeding places o f the adult weevils. Pseudostems from which bunches have been harvested are cut at the ground level, chopped up and scattered in the plantation, so that they dry o ff or rot as quickly as possible. The cut face of the old corm could also be covered with soil (Simmonds, 1966). Weed control is also important, especially in the immediate vicinity o f the banana stools. Since the adult weevils are sluggish and nocturnal in habit (Cuille, 1950), bare soil or soil with a low density o f weeds reduces their movement, thus resulting in a reduction in stool infestation. University of Ghana http://ugspace.ug.edu.gh 13 At planting, care should be taken not to use infested suckers. Suckers should be taken from a field known to be free o f the banana weevil. If this is not possible, the sucker should be closely inspected and, if necessary pared (by removal o f the outer tissue layer o f the rhizome) to remove the unhatched eggs and larvae (Harris, 1947; Whalley, 1957; Saouder, 1961; Feakin, 1971). When suckers have been dug they should be removed from the field at once and not left in heaps overnight. It has repeatedly been suggested that eggs and larvae in infested suckers can be killed by soaking the suckers in water for one to two days, however, it has been conclusively shown that only a very prolonged period o f soaking leads to disinfested planting material. In practice, therefore water soaking o f sucker is useless (Simmonds, 1966). Hot water treatment leads to less than 35 % larval mortality at 54°C for 20 min or 60°C for 15 min and more than 90% mortality at 43°C for 3 hours (C.S. Gold, personal communication., 1996). Good crop husbandry, involving such practices as clean weeding, desuckering, pruning, manuring and mulching produces vigorous plants that are more tolerant to weevil damage (Feakin, 1971). Good crop management (inter-cropping, soil amendment, sanitation) was found to reduce weevil infestation (Gold el al., 1997). For example, the same authors reported that banana fields inter-cropped with coffee coupled with good sanitation (i.e. weeding) and farms where crop residues were systematically destroyed, had a lower weevil population and lower weevil damage respectively than those having little or no crop sanitation. Trapping is also an important cultural method for controlling C. sordidus. The use of pseudostem and rhizome traps is common practice in the removal o f adult weevils from infested plantations. Pseudostem traps can either be a pseudostem split lengthways or a disc (Mitchell, 1978; Bujulu et al., 1983). Traps are placed at the base of plantain or banana plant stools and the weevils that are attracted to them are collected and destroyed (Hord and Flippin, 1956). The frequency of collection varies widely, ranging from daily to weekly but all authors seem to agree that collection should be carried out not less than once a week (Simmonds, 1966). It was shown that University of Ghana http://ugspace.ug.edu.gh 14 the younger the trap, the better the attraction, which falls markedly after one week (Hord and Flippin, 1956). It was found that rhizome traps were more attractive to the banana weevil than pseudostem traps (Hord and Flippin, 1956). Trapping is often of limited effectiveness against established infestation and is tedious and time consuming. Nevertheless trapping may be cheap and effective when used in combination with chemical or biological insecticides in an integrated weevil management programme (Whalley, 1957; Feakin, 1971; Kranz et al., 1977; Schmitt et al., 1992). 2.9.2. C hem ical control Chemicals (synthetic insecticides) can be used for the control o f banana weevil and are mostly used by commercial banana and plantain producers. They can be incorporated into traps or applied to soil (Simmonds, 1966). Insecticides, such as Paris green (alone or mixed with flour as bait), Benzene Hexachloride (BHC), Dichloro Diphenyl Trichloroethane (DDT) and others have been found to be repellent to the banana weevil and in any case, when applied in a field, do not reach the whole population (Simmonds, 1966). To improve the poison trapping technique, Cuille (1950) devised a mixture containing two insecticides (chlordane and parathion) with an attractive chemical earned in an inert dust. Ogenga-Latigo arid Mazanza (1996) reported population reduction o f C. sordidus by incorporating chemical insecticides to pseudostem traps. In general, although poison traps kill some borers, they appear to be relatively ineffective and severe infestation cannot be controlled by this means alone (Simmonds, 1966). Soil treatment with insecticides is based on the principle o f killing the egg-laying females on their way to plantain or banana plants. To achieve control by this means, the insecticide needs to be sufficiently persistent in the soil to avoid the need for frequent re-treatment (Simmonds, 1966). Persistent organochlorine, such as DDT and Dieldrin were until the 1970’s the standard recognised chemicals used against C. sordidus (Nankinga, 1994). Since then, however, environmental concerns such as pollution of agricultural lands and ground water and the development o f resistance by the weevils to the organochlorine chemicals in many countries (Mitchell, 1980), necessitated a change to organophosphate and/or carbamate insecticides, such University of Ghana http://ugspace.ug.edu.gh 15 as Primiphosethyl (Primicid) and carbofuran (Furadan) which are less persistent and less harmful to non-target organisms (Edge et al., 1974, Nankinga, 1994). The high cost and scarcity of synthetic insecticides have limited the farmers ability to acquire and use them. Farmers who can afford to buy chemicals tend to apply inadequate doses (under-dosing), a situation that renders them ineffective and enhances probability of pests developing resistance to chemicals (Nankinga, 1994). Chemical control methods are, therefore, environmentally and socio-economically unsound in the context o f the resource-poor smallholders who mainly grow the crop in Africa. 2.9.3. C lassical b iological control o f C. sordidus Attempts to control C. sordidus using introduced predators ana parasttoios nave oeen documented for a long time in different localities. The natural enemies o f C. sordidus include P. javanus, Hyposolenus laeviganus Marseul (Coleoptera: Histeridae), Hololepta quadridentata Fabricius (Coleoptera: Histeridae), Belonochus ferrugatus Erichson (Coleoptera: Staphylinidae), Priochirus unicolor L. (Coleoptera: Staphylinidae), Cathalus spp. (Coleoptera: Silvanidae) and Dactylostermim hydrophiloides Macleay (Coleoptera: Hydrophilidae). The known ’ diptera controlling the banana weevil is Chrysopilus ferruginosus Wiedemann (Diptera: Rhagionidae) (Neuenschwander, 1988). Some ants were recorded preying on the larvae of the banana weevil. These were Anochaetus sp. (Hymenoptera: Formicidae) (Seshu- Reddy, 1986) and T. guineense (Roche and Abreu, 1983). Previous efforts to control the banana weevil through the release o f natural enemies were inadequate and met with only limited success. For example P. javanus was imported from Java into Uganda in 1934, and released on Kibibi Island in Lake Victoria in 1935. The release site was visited in 1944 but no P. javanus was found, and it was presumed not to have established (Harris, 1947). P. javanus and H. quadridentata were released in Cameroon and were not able to establish (Greathead et al., 1971). P. javanus was introduced in the Pacific Region, Trinidad, Australia and University of Ghana http://ugspace.ug.edu.gh 16 most o f South America and was found to establish (Greathead et al., 1971). However, the natural enemies used were generalists, not specific to the banana weevil and releases were small and disregarded the possibility o f biotypes (W aterhouse and Norris, 1987; Neuenschwander, 1988; Greathead et al., 1971). In Africa, one o f the possible reasons for the failure o f the exotic natural enemies to establish may be their non-adaptability to the environment. The greatest potential for the biological control o f C. sordidus now seems to lie in the use o f endogenous or exotic pathogens (Nankinga, 1994; Traore, 1995). 2.9.4. B iological control using entom opathogens Pathogens used for insect and mites control include viruses, bacteria, fungi, protozoa and nematodes. It was estimated that about 650 viruses cause disease to insects (Cloutier and Cloutier, 1992). Bacteria are frequently found in association with insects (Poinar and Thomas, 1985) but only about 100 species are insect pathogens (Cloutier and Cloutier, 1992). Among fungal pathogens, about 700 species are found to cause insect diseases (Ferron, 1978; Hall and Papierok, 1982; Burges, 1981; Cloutier and Cloutier, 1992). The number o f nematodes species found in associations with insects was about 3000 (Poinar and Thomas, 1985; Cloutier and Cloutier, 1992). Tanada and Kaya (1993) stated that the use o f pathogens in the control o f an insect pest has the following advantages and disadvantages: The advantages are: 1. Specificity to target organisms or to a limited number of host species. 2. Harmless to invertebrates and plants. 3. No toxic residues. 4. Little or no environmental pollution. 5. Little or no development of resistance by the target organism. 6. No secondary pest outbreak. 7. Compatibility with many chemical pesticides, parasitoids, predators and other pathogens. University of Ghana http://ugspace.ug.edu.gh 17 8. Possibility of long-term control. 9. Ease of application o f pathogen with conventional spray equipment. 10. Mass production capability with facultative pathogens. 11. Adaptable to genetic modification through biotechnology. The disadvantages are: 1. Specificity only to the target organism. 2. Pathogen or its by-product(s) may be harmful to non-target organisms. 3. Strict timing of application is necessary for optimum effect. 4. Good coverage is essential for contact or ingestion o f pathogen by target organism. 5. Insect death does not occur immediately after infection 6. Susceptibility to inactivation by environmental conditions. 7. Loss o f virulence and pathogenicity by frequent sub-culturing. 8. Short selflife and/or requirements for special handling. 9. Obligate pathogens are difficult or expensive to mass produce. 10. Uneconomical except for high-value crops. 11. Fear of pathogens by the public. 12. Risk associated with genetically engineered pathogen (host range modification, gene exchange to other organisms and genetic stability) For the control o f C. sordidus, entomopathogenic nematodes (Treverrow et al., 1991) and entomopathogenic fungi (Batista-Filho et al., 1987; Busoli et al, 1989; Nankinga, 1994; Traore, 1995) are considered the most promising candidates. 2.9.4.1. N em atodes The entomopathogenic nematodes used in the control of C. sordidus include species o f Steinernema, Heterorhabditis and Neoaplectana. The effectiveness o f these nematodes against the banana weevil was demonstrated in Australia, the Caribbean, Florida and Brazil (Figuerda, 1990; Treverrow et al., 1991; Schmitt et a l , 1992). It was shown that entomopathogenic nematodes such as S. carpocapsae can cause high University of Ghana http://ugspace.ug.edu.gh 18 mortality (up to 85%) to the banana weevil (Treverrow, 1994). Evaluation of entomopathogenic nematodes for the control of C. sordidus showed significant mortality o f both the banana weevil larvae already in the rhizome and the adult weevil attracted to the treatment sites (Treverrow et al., 1991). The insect larvae size is a limiting factor in attempting control with steinernematid nematodes. The larvae o f C. sordidus may need to grow appreciably before steinernematid nematodes can penetrate them. This is because steinernematid nematodes unlike heterorhabditids, can enter their hosts only through mouth, anus and spiracles (Bedding and Molyneux, 1983) and these orifices are likely to be too small in newly-hatched banana weevils. In Brazil, Schmitt et al., (1992) sprayed S. carpocapsae suspended in water onto split pseudostem stumps which were used as a bait for the weevils. This method of application gave significantly greater control o f weevil than the application of nematodes to soil around banana plants. 2.9.4.2. Fungi The first micro-organisms found to cause diseases in insects were fungi because of their conspicuous growth on the surface o f their hosts (Tanada and Kaya, 1993). Various species and strains of entomopathogenic fungi have been found and isolated from C. sordidus and other insects hosts. The most important are species in the genera Beauveria, Metarhizium, Paecilomyces and Nomurea (Delattre and Jean-Bart, 1978; Batista-Filho et al., 1987; Busoli et a l , 1989; Allard and Rangi, 1991; Nankinga, 1994). Some o f these entomopathogenic fungi, such as isolates o f the fungi B. bassiana and M. anisopliae, have been found to be potential microbial agents against the banana weevil in Africa and elsewhere (Delattre and Jean-Bart, 1978; Mesquita, 1988; Nankinga, 1994; Traore, 1995). Batista-Filho et al. (1987), infected field- collected C. sordidus with B. bassiana and M. anisopliae cultured on rice and beans. The C. sordidus mortality recorded was above 85% for B. bassiana cultured on the different media, while the M. anisopliae cultured on beans caused only 56% mortality. The same authors also tested strains of B. bassiana isolated from Ligyrus sp. (Coleoptra: Scarabaeidae) and Diatreae saccharalis F. (Lepidoptera: Pyralidae), and M. anisopliae isolated from Ligyrus sp. and Deois flavopicta Stal (Hemiptera: University of Ghana http://ugspace.ug.edu.gh 19 Cercopidae) against the banana weevil. The weevil mortality rate obtained was high (94.7% to 98.6%). Delattre and Jean-Bart (1978) tested B. bassiana, Beauveria brongniartii (Sacc.) Petch (= B. tenella (Delac) Siemi), M. anisopliae and Nomuraea rileyi (Farlow) Samson (= Spicaria rileyi (Farlow) Charles) under field conditions. Weevil infection ranged from 64 to 100% using a dose of 10" spores/m2. M esquita (1988) used B. bassiana and M. anisopliae against C. sordidus and the weevil mortality was up to 100% and 64% respectively in 36 days for B. bassiana and M. anisopliae strains respectively. Nankinga (1994) in studies on the potential o f indigenous fungal pathogens for the biological control o f the banana weevil in Uganda, and Traore (1995) in Benin reported mortalities up to 100% for C. sordidus adults exposed to the infective units o f B. bassiana and M. anisopliae. The two authors reported that the rate of mortality of C. sordidus depended on isolate, conidial concentration in formulation, mode of formulation and delivery system. These studies showed the potential of B. bassiana and M. anisopliae in the microbial control o f C. sordidus. 2.10. The entom opathogens Beauveria bassiana and M etarh izium anisopliae 2.10.1. Taxonom y Most fungal pathogens with good potential for development into mycopesticides belong to either the class Hyphomycetes or order Entomophthorales (Class Zygomycetes) (Moore and Prior, 1993). There are more than 700 species of entomogenous fungi in approximately 100 genera but only six species are currently registered for use in pest control (Goettel et al., 1990; Robert et al., 1990). Ainsworth (1973) divided fungi into two divisions Myxomycota for plasmodial forms and Eumycota for non-plasmodial forms that are frequently mycelial. Entomopathogenic fungi are found in the division Eumycota and in the following subdivisions Mastigomycotina, Zygomycotina, Ascomycotina, Basidiomycotina and Deuteromycotina. Most entomopathogenic fungi are found in Zygomycotina, class Zygomycetes, order Entomophthorales; in Ascomycotina, Class Pyrenomycetes, order Sphaeriales; in Class Laboulbeniomycetes, order Laboulbeniales and in the University of Ghana http://ugspace.ug.edu.gh 20 Deuteromycotina, Class Hyphomycetes, order Moniliales (Tanada and Kaya, 1993). Selected examples o f entomopathogenic fungi genera are given in Table 2. Table 2. Outline classification o f some entomopathogenic fungi. Subdivision Class Order Selected genera Mastigomycotina Chytridiomycetes Blastocladiales Coelomomyces Zygomycotina Zygomycetes Entomophthorales Entomophaga, Entomophthora, Erynia, Neozygites Ascomycotina Pyrenomycetes Sphaeriales Cordyceps Basidiomycotina Phragm obasidiomycetes Septobasidiales Septobasidium Deuteromycotina Hyphomycetes Moniliales Beauveria, Hirsutella, Metarhizium, Nomuraea, Paecilomyces, Sorosporella, Verticillium, Fusarium Source Tanada and Kaya (1993) The genera Beauveria and Metarhizium are in the class Hyphomycetes. The genus Beauveria has four species of which B. bassiana and B. brongniartii are the most commonly studied (McCoy et al., 1988). The genus Metarhizium has two species, M. anisopliae and M. flavoviride W. Gams & J. Rozsypal. The species M. anisopliae has two varieties, M. anisopliae var. major (Johnston) Tulloch and M. anisopliae var. anisopliae Tulloch (Tulloch, 1976). The genus Metarhizium is currently under revision. 2.10.2. Infection processes by Beauveria and M etarhizium Entomopathogenic fungi such as species o f Beauveria and Metarhizium , unlike bacteria and viruses that pass through the gut wall from contaminated food, infect their host by contact. They reach the haemocoel by penetrating the cuticle or possibly through the mouth parts and other external openings of an insect (Ferron, 1981; McCoy et al., 1988; Tanada and Kaya, 1993). Three phases have been recognised in the development of insect mycosis (Samson et al., 1988, Tanada and Kaya, 1993); (i) University of Ghana http://ugspace.ug.edu.gh 21 adhesion and germination of the spores to the host cuticle, (ii) penetration o f the insect integument by a germ tube and (iii) development of the fungus inside the insect body, generally resulting in death of the infected host. The adhesion of the spores onto the insect’s cuticle may be a passive mechanism and involve mucilagenous material and spore surface structures (Samson et al., 1988). Spore germination on the cuticle surface is affected by micro-climatic factors, especially temperature and humidity (Tanada and Kaya, 1993). For example, Ferron (1981) reported that the optimal growth temperature is 23-25°C for Beauveria and 27- 28°C for Metarhizium. Penetration o f hyphae through the cuticle occurs after the spores have met favourable conditions for their germination. The mode o f penetration mainly depends on the property of the cuticle, it thickness, sclerotization, and the presence o f antifungal and nutritional substances (Chamley, 1984). The hyphal penetration process involves both mechanical and enzymatic factors (Ferron, 1981; Samson et al., 1988). The biochemical basis for pathogenesis by mycosis was discussed by Mclnnis in 1975 (Ferron, 1981) and Samson et a/.(1988). Several enzymes, including lipase, protease, amylase and chitinase, are reported from B. bassiana that enable the hydrolysis o f the protein-chitin complex o f the integument (Samsinakova et al., 1971; Smith et a l , 1981; St Leger et al., 1986). The chitnase activity occurs mainly at the time o f fungal growth and conidia formation (Coudron et al., 1984). Different toxins can be produced by B. bassiana. The toxin beauvericin. a depsipeptide (Hamill et al., 1969) and other toxins in B. bassiana have an antibacterial effect which serves to reduce bacterial competition during the saprophytic phase of growth on the host cadaver (Ferron. 1978). The mycosis produced by the entomopathogenic fungi imperfecti (Moniliales) B. bassiana, B. brongniartii (= B. tenella) and M. anisopliae var. anisopliae and var. major is known as white muscardine or green muscardine depending on the colour of the spores. University of Ghana http://ugspace.ug.edu.gh 22 2.10.3. D evelopm ent o f m uscardine disease Once the fungus has penetrated the integument, the muscardine caused by Beauveria spp. or Metarhizium spp. develops in the haemocoel in the presence o f cellular defensive reactions of the host (Seryczynska and Bajan, 1975). Plasmotocytes surround the mycelium as pseudo-tissue or granuloma as described by Vey et al. (1975) in invertebrate cell culture (Ferron, 1981). Beauveria and Metarhizium produce toxins which erode the granuloma and allow blastospores to invade the haemocoel. Hyphal bodies proliferate only just before death o f the host (Ferron, 1981). The role of entomogenous toxins is particularly important (Roberts, 1966; Evlakhova 1974). Several toxic cyclodepsipeptides, such as destruxins A, B, C and D and desmethyl- destruxin have been isolated from M. anisopliae cultures (Suzuki et al., 1970, 1971). Also beauvericin has been isolated from B. bassiana (Hamill et al., 1969) and beauvellide from B. brongniartii (Frappier et al., 1975 cited by Ferron, 1981). The toxin production in different strains o f B. bassiana is positively correlated with their virulence (Sikura and Bezenko, 1972, cited by Ferron, 1981). Within the same species of fungus, different strains can have very different spectra o f activity (Ferron et al., 1972). Another factor that can affect muscardine infection is the quantity o f spores in contact with the insects. There is a positive correlation between the number o f infective spores and mortality by mycosis (Ferron, 1978; Nankinga, 1994; Traore, 1995). For small, short-lived and soil insects, more inoculum (spores) o f B. bassiana or M. anisopliae must be used, for example 1016 to 1017 conidia/ha (Muller-Kogler and Stein. 1970, cited by Ferron, 1981). With fewer spores, muscardine disease develops slowly and affects only the older larvae or adults and disturbance in fecundity and diapause of the surviving adults can occur (Faizy, 1978 cited by Ferron, 1981). In the laboratory the disease normally develops after contamination of insects either directly by spore suspensions (10'1 to 108 spores/ml) or by mixing soil with 105 to 10* spores/g or cm3 for soil insects (Ferron, 1981). University of Ghana http://ugspace.ug.edu.gh 23 2.10.4. Saprophytic developm ent o f m uscardine The parasitic phase o f fungal development ends with death o f host. The mycelium then grows saprophytically through all the insect tissues in competition with the intestinal bacterial flora (Ferron, 1981). White muscardine produces oosprein, a red antibiotic pigment which colours the cadaver and curbs bacteria (Ferron, 1981). Mclnnes et al. (1974) also identified two yellow pigments in white muscardine, bassianin and tenellin. 2.10.5. M ass production o f species o f Beauveria and M etarhizium Metarhizium spp. and Beauveria spp. are naturally-occurring in C. sordidus affected areas and survive as saprophytes in the absence o f the hosts (Charles, 1941). This makes them suitable candidates for microbial insecticide development (Brady, 1981; Roberts, 1989; Moore and Prior, 1993). Cheap and effective mass production technologies have already been developed in some parts o f the world for control o f insect pests. There are, however, many more production systems in operation than are fully described in the literature. Often a successful production system is kept secret to promote commercial exploitation (Jenkins, 1995). The ability to mass produce a pathogen becomes crucial in augmentative or inundative microbial control, where the pathogen is used as a biological insecticide (Jenkins, 1995). There are two basic methods for in-vitro production o f entomopathogenic fungi: liquid fermentation and production on solid substrates. In general, a large surface area is needed for sporulation of fungi. Therefore grains such as rice which have a high surface area to volume ratio, are often used (Marques et al., 1981; M endonga, 1992) and have been shown to be superior to other substrates such as sweet potato, tapioca, papaya or coconut (Ibrahim and Low, 1993). The production technique for B. bassiana (Samsinakova et al, 1971) and B. hrongniartii (Blachere et al., 1973 cited by Ferron, 1981) in submerged culture was developed a long time ago but was abandoned because of the difficulties o f storing the University of Ghana http://ugspace.ug.edu.gh 24 infective units (spores) (Ferron, 1981). Liquid media for mass production o f B. brongniartii blatospores in a fermentor (Blachere et al., 1973, cited by Ferron, 1981) and the composition of aqueous fermentation media for the mass production o f B. bassiana conidia (Goral, 1971, cited by Ferron, 1981) was established. A two stage technique for mass production of B. bassiana conidiospores was used in the USSR (Zakharchenko et al., 1963 cited by Ferron, 1981). The first stage involved the production of mycelium in a fermentor. The second phase was a surface-culture on nutrient medium in trays for sporulation. A pilot-factory in Krasnodar has produced annually 22 tons o f Boverin (B. bassiana conidia plus an inert carrier, standardised at 6 x 109 conidia/g) (Ferron, 1981). A similar technique is used for the mass production of M. anisopliae (Goral and Lappa, 1973). In Brazil, the same technique was used but the trays were replaced with autoclaved polypropylene bags containing rice grains as a nutritive substrate (Guagliumi et al., 1974). M. anisopliae is being produced on rice grains using a liquid-solid phase technique in IITA/Abomey-Calavi near Cotonou in Republic o f Benin (Jenkins, 1995). Other cereals grains such as maize (Nankinga, 1994) have been suggested for the mass production o f several entomopathogenic fungi imperfecti (Villacorta, 1976 cited by Ferron, 1981) but have not been used on a large-scale. 2.10.6. Form ulation One of the major limitations to the development o f fungi for insect control is the lack of readily available formulation technology (Goettel and Roberts, 1992). It is through formulation that improved shelf life, persistence, efficacy, and field targeting can be achieved (Goettel and Roberts, 1992). The entomopathogenic fungi are living organisms and need to be formulated prior to application. The main aims of formulation are to provide an economical and easily useable form o f the active ingredient with long shelf life, and if possible to enhance the effectiveness o f the active ingredient (Auld, 1992). An active ingredient may be applied in the dry state as dust or granules or as a liquid or in the presence o f liquid (Auld, 1992). Formulation University of Ghana http://ugspace.ug.edu.gh 25 o f fungi may incorporate additives such as wetter, stickers, humectants, UV protectants, and thixotropic agents (Goettel and Roberts, 1992). Since spores o f B. bassiana and M. anisopliae can be mass-produced and dried, they can be applied as dry material or formulated in wettable powder or mixed with an inert carrier and applied as bait. B. bassiana was successfully formulated for grasshopper control in wheat bran baits and oil (Goettel and Roberts, 1992). The formulation must ultimately be chosen on the basis o f mode o f infection, target host habitat, crop and application method. 2.10.7. Delivery system s Fungal propagules are microscopic and most can be applied as conventional chemical insecticides. The application technique depends on the type o f formulation, the insect host habitat, the insect host behaviour and host-pathogen relationship (Tanada and Kaya, 1993). Application technology involves the mechanisms of proper placement of a desirable concentration o f active agent on the target site to obtain maximum effective control of the target insect (Tanada and Kaya, 1993). Microbial pesticides such as Bacillus thuringiensis, baculoviruses, protozoans, and fungi (e.g. B. bassiana and M. anisopliae) must be ingested or be in contact with their hosts to be effective. The short residual activity of these agents also requires that the pest consumes or comes into contact with the pathogen soon after application and this is accomplished by thorough coverage at the site o f insect feeding. Microbial pesticides are applied with equipment and technology developed for chemical pesticides such as hand-held spinning disk sprayers (i.e. Ultta low volume (ULV) sprayers) and rotary atomisers mounted on vehicles or aircaft (Tanada, 1967, Bateman, 1997). Moreover, simple techniques are also used. For example, B. bassiana production on rice is commercialised in Brazil. The fungus on rice grains is applied to banana stumps against adult banana weevils (Gert Roland Fischer’s Company COINBIOL-GRF, Guide lor the use of B. bassiana to control banana weevil in University of Ghana http://ugspace.ug.edu.gh 26 Brazil). Cubans also report the delivery of B. bassiana formulated in solid or in liquid form with the conventional sprayer o f chemical insecticides (C.S. Gold, personal communication, 1998). 2.11. Practical and econom ic feasibility o f b iocontrol using entom opathogenic fungi Tanada and Kaya (1993) pointed out that although chemical control o f pests has been efficacious, the drawbacks such as pesticide resistance; resurgence o f the target organism or emergence of secondary pests to primary pest status because o f the destruction o f parasitoids and predators; impact on non-target organisms, including humans; environmental pollution through the accumulation o f pesticides in soil, water, and air; and residues on agricultural products and animals, have necessitated the development of more selective control methods compatible with the environment. Insect pathogens overcome many problems o f chemical pesticides but are not extensively used despite their many positive features (Falcon, 1985). The production o f entomopathogens in the group o f Deuteromycetes such as Beauveria spp. and Metarhizium spp. can be undertaken using cheap media (i.e. cassava products, oil palm kernel cake) available in the developing countries. The conidia of such fungi are formulated in water or oil and applied with a conventional equipment o f chemical application such as ULV sprayers (Bateman, 1997). The conidia can also be produced on solid substrates and applied with the substrates which can maintain the growth o f the fungi at the application site. It was reported by Ferron (1981) that many field experiments reveal the potential uses of muscardine fungi (i.e. Beauveria spp. and Metarhizium spp.). The efficacy o f muscardine fungi is comparable to that o f chemical insecticides, with additional advantage o f noticeable long-term insect pest limitation (Ferron, 1981). Once a muscardine fungus is applied on the target pest, it can multiply and persist on the pest that it kills. The infected insect can also carry the disease from one point to another in a field, thus spreading the disease among the healthy population. For example, B. brongniartii caused an epizootic to Melolontha melolontha L. (Coleoptera: Scarabaeidae) one year after its University of Ghana http://ugspace.ug.edu.gh 27 application and 4 years later the muscardine appeared again in M melolontha populations and caused a noticeable pest reduction (Keller, 1992). In Africa, the development of microbial pesticides is still at an early stage. It is only in recent years that IITA/CABI has developed a microbial pesticide “Green M uscle” in which the active ingredients are conidia o f M. anisopliae, for the control o f grasshoppers and locusts (Lomer et al. 1997; Neethling and Dent, 1998). Even though this product was a success, the production is not economical, partly because of problems o f contamination during the production processes (Cherry et al., 1999). In contrast, little or no contaminant appears during the production o f B. bassiana (author’s observation). For this reason it may be possible to produce B. bassiana at the farmers level with little assistance. This makes B. bassiana a promising candidate for insect pests control at the level o f resource-limited farmers. Therefore the main goal in the present study is to determine the potential and practicability o f the use of B. bassiana in managing C. sordidus on plantain in West Africa. University of Ghana http://ugspace.ug.edu.gh 28 CHAPTER 3 BIOLOGY AND SPATIAL DISTRIBUTION OF C. sordidus ON PLANTAIN IN GHANA 3.1. INTRODUCTION The banana weevil, Cosmopolites sordidus (Germar) is an important insect pest on plantain and banana (Afreh-Nuamah, 1993a; Gold et al, 1993). The female bores a small hole in the corm at the ground level and after preparing an incubation chamber deposits a single egg (Froggatt, 1925; Cuille, 1950; Kranz et al., 1977). As soon as the egg hatch the larva starts feeding by tunnelling preferably on the corm tissue. The destruction o f the cortical tissue o f the corm generally affects the plant nutrition and physiology (Treverrow et al., 1991). Therefore the plant becomes very weak especially during the dry season and may topple (Sikora et al., 1989; Swennen, 1990). Infestation o f young plants causes stunting o f growth, disruption o f fruiting or death. This usually occurs when infected suckers are planted or clean suckers are grown in heavily infested fields (Harris, 1947; Cuille, 1950). In Uganda for example 60% o f the suckers planted in heavily infested field may die due to weevil attack (P. Speijer, personal communication, 1997). In West and Central Africa a range of damage levels and yield losses due to C. sordidus have been reported. For example, in Cote d 'Ivoire, yield reductions o f 30 to 60% were found to be common (Vilardebo, 1973). Lescot (1988) reported yield reductions o f 20 to 90% in Cameroon. In Ghana. Afreh-Nuamah (1993a) reported that one month after planting, percentage weevil infestation ranged from 0 to 82.5% depending on the origin o f planting material (i.e. nursery material or ratoon material), history of land (cropped land or forest land) and cultivars. Also, Udzu (1997) reported yield reduction o f 33.3% due to weevil infestation only and 86.1% yield reduction when the effect o f nematodes and weevil infestation was combined Prior to any control measure against C. sordidus, its biology and behaviour should be well understood. Froggatt (1925), Cuille (1950) and Kranz et al (1977) reported that University of Ghana http://ugspace.ug.edu.gh 29 the duration of the different stages of C. sordidus vary widely according to season and locality. In Ghana, recognition of C. sordidus as a major insect pest on plantain has occurred recently and research and information on C. sordidus such as its biology and the spatial distribution pattern of different stages on plantain is limited (Afreh-Nuamah, 1993b). It is based on this background that the present study was initiated. The focus was to determine the duration and the spatial distribution of the different stages o f C. sordidus on plantain as a first step towards the evaluation o f the efficacy o f B. bassiana in controlling the weevil. 3.2. MATERIALS AND METHODS 3.2.1. Duration o f different developm ental stages o f C. sordidus The study was conducted at the Agricultural Research Station (ARS), Kade o f the University o f Ghana located about 120 km north west o f Accra (6°09’N, 0°55’W) (Gary, 1987). The climate at ARS, Kade is characterised by two wet and two dry seasons. The major wet season extends from March to mid-July followed by a minor dry season between mid-July and early September. The second wet season extends from mid-September to the end o f November while the main dry season is from November to February. The average annual rainfall is approximately 1650 mm (Obeng, 1959). The vegetation of the station is representative of the moist semi- deciduous tropical rain forest o f Ghana (Taylor, 1960). For the determination of the duration o f different stages o f C. sordidus, three different batches o f adult weevils (16 females and 4 males; 9 females and 3 males; 16 females and 4 males) were collected from the same plantain field at ARS, Kade, on different days. The weevils were collected by means o f pseudostem traps. The traps were 15 cm long stems, split in half (lengthways) and placed at the bases o f the plantain, split side facing downwards (Ogenga-Latigo and Bakyalire, 1993). The batches o f adult weevils collected were each used to infest ten pieces of corm (12 x 6 x 2 cm each) in University of Ghana http://ugspace.ug.edu.gh 30 separate plastic buckets in the laboratory where temperatures ranged between 22- 29°C. Eggs laid on corm pieces by the first batch o f weevils were collected every fourth day after infestation, over a 12-day period. For the second batch o f weevils, eggs were collected, once on the fourth day and twice a day after infestation. Lastly eggs from the third batch were collected one day after infestation. Eggs were collected from the pieces o f corm by scratching the outer tissue layer with a knife. The corm pieces were replaced with new ones after egg collection. Eggs collected 4 days after infestation could not be used to determine the developmental duration o f the egg stage since the date on which eggs were laid was unknown. These eggs could only be used to give the developmental duration o f larval and pupal stages. Eggs collected one day after corm infestation could however be used to determine the developmental duration o f the egg, larval and pupal stages. Each egg collected from the pieces o f corm was transferred onto a new separate piece o f corm ( 6 x 5 x 2 cm) through a window made with a knife. The new pieces o f corm were labelled (date of collection, date o f transfer and egg number). These pieces o f corm were put into plastic bowls with lids. Water or moist tissue paper was put around the pieces o f corm to keep them from drying (Plate 1.). Daily observations were made on each egg and the date of hatching recorded. Two weeks after hatching of the eggs, the piece of conn was split, the larva removed and then transferred into a new piece o f conn through a small hole made with a knife. The size o f the holes made depended on the stage o f larva and were as follow: 0.5 x 1 x 0.5 cm for two week-old larvae and 2 x 1 x 0.5 cm for three week-old to mature larvae. The hole was covered with a slender piece o f split corm held in place with cello-tape. At weekly intervals the cello-tape was removed and the piece o f corm was split and the larva transfened to a new piece o f conn by the same technique described above. Larvae close to the pupal stage ceased to feed and could not bore holes in the pieces o f corm and were thus exposed. This facilitated daily observations on mature larvae until pupal and adult stages were obtained. Dates at which stages ended were recorded. Knowing the beginning o f the stage and its end, it was easy to calculate the developmental period of each stage. Data collected from the different batches o f adult University of Ghana http://ugspace.ug.edu.gh 31 100 mm i______ i Plate 1. Plastic bowls containing pieces o f corm on which C. sordidus eggs were incubated. Note: (a) Moist tissue papers were placed between the pieces of corm to prevent them from drying. (b) During egg incubation period the plastic bowls were covered. University of Ghana http://ugspace.ug.edu.gh 32 weevils used were combined to establish the average duration o f the developmental period of egg, larval and pupal stages and also the developmental period from egg to adult stage. 3.2.2. Spatial distribution o f the different stages o f C. sordidus on plantain A trial was conducted at ARS, Kade, on cropped land that had been fallowed for two years and mainly colonised by Chromolaena odorata (L.) King & Rob. The land was cleared by slashing the weeds with a cutlass to the ground level. Three experimental plots o f 15 x 15 m each were demarcated and pegged. Planting holes were dug in the experimental plots at 3 m spacing. Planting holes were 20 cm deep. The experimental plots were weeded monthly by hand using a cutlass. A widely grown plantain cultivar in Ghana, Apantu-pa (false horn) (Schill et al., 1997) was used as planting material. Sword suckers bought from farmers were freed from different stages of C. sordidus by paring (removal o f surface tissue o f the rhizome) and removing the old leaf sheath. The leaves on suckers were pruned. Adult weevils o f C. sordidus required for the experiment were collected from farmers’ fields at Akanteng, about 45 km South East o f ARS, Kade. Adult weevils were collected by hand from rotten plantain leaf sheaths and stumps and by means of pseudostem traps (Section 3.2.1). After collection, the adult weevils were kept in the laboratory at ARS, Kade in plastic buckets with lids, containing pieces o f plantain corm at room temperature (25-28°C). The collected weevils were kept under laboratory conditions for about 2 weeks prior to use. The three experimental plots were each planted with 25 plantain suckers. After 4 months, 20 adult banana weevils (4 males and 16 females) were released and confined at the base of each plant with grass mulch. To monitor the distribution of a particular stage o f C. sordidus within the plantain plant, one of the experimental plots was selected at random and all the 25 plants were University of Ghana http://ugspace.ug.edu.gh 33 uprooted. The sampling time for each stage was chosen on the basis o f results obtained from the experiments on the developmental period o f the different stages o f C. sordidus (Table 4.). The levels at which the plants were sampled for the stages o f C. sordidus were as shown in Figure 1. i) Pseudostem (within 5 cm from the collar (ps)) ii) The rooting zone of the rhizome (ca) iii) The remaining part o f the rhizome (cb). Egg distribution: Five days after infestation, the number o f eggs deposited by the released females o f C. sordidus was counted at each level selected. Larval distribution: 34 days after infestation, data were collected on: number o f larvae number o f pupae number o f eggs The number o f pupae and eggs o f C. sordidus collected during the larval distribution study was few, therefore was not analysed nor reported. Pupal distribution: 42 days after plant infestation, data were taken on: number o f pupae number o f larvae number o f eggs number o f adults The number o f eggs, larvae and adults o f C. sordidus collected was few during the pupal distribution study, therefore was not analysed nor reported. University of Ghana http://ugspace.ug.edu.gh 34 Figure 1. Levels at which uprooted plantains were sampled for weevil stages. Note: ps = pseudostem sampled within 5 cm from the plant collar, ca = rooting zone of the rhizome sampled, cb = remaining part of the rhizome sampled. University of Ghana http://ugspace.ug.edu.gh 35 Data were analysed using SAS version 6.12 for Windows®. Counts o f egg and larvae o f C. sordidus at the different sampling levels o f the plant were subjected to Generalized Linear Models (GENMOD procedure, log linear models) analysis for comparison (SAS, 1997). The pupae of C. sordidus were found only at the corm level. Thus the counts of pupa were not analysed. 3.3. RESULTS 3.3.1. D uration o f different developm ental stages o f C. sordidus The number o f eggs collected from adults o f C. sordidus and percentage o f viable eggs are shown in Table 3. Egg viability varied from 40 to 69% with a mean o f 55 ± The mean duration o f egg incubation period, larval and pupal developmental period and the developmental period from egg to adult o f C. sordidus was 6.3 ± 0.2, 28 ± 0.6, 7.1 ± 0.3 and 40.4 ± 0.7 days respectively (Table 3.). The duration for which most o f the egg, larval and pupal stages ended are shown in Figure 2., 3 and 4 respectively. 3.3.2. Spatial d istribution o f different stages o f C. sordidus on plantain Figures 5., 6 and 7 show the percentage o f eggs, larvae and pupae respectively at pseudostem and corm levels. Eggs and larvae o f C. sordidus were found more on the underground part (corm) than on the pseudostem part of the plantain. Pupae o f C. sordidus were not found at all at the pseudostem level. The numbers o f eggs at the different sampling levels of the plant were not significantly different (P > 0.05) (Appendix 1.). The number of larvae of C. sordidus at the pseudostem level was significantly lower (P < 0.05) than that at the corm level ca (Table 5. and Appendix 2.). The pupae, as mentioned above, were all found at the conn level. University of Ghana http://ugspace.ug.edu.gh 36 Table 3. Percentage viability of eggs laid by adults o f Cosmopolites sordidus on corm tissue under laboratory conditions. Insect batch Days after infestation (days) No. o f female No. o f eggs adults collected No o f eggs hatched Percentage egg viability (%) 1 4 16 20 12 60 4 28 16 57 4 9 5 56 2 4 9 41 20 49 1 19 10 53 1 5 2 40 3 1 16 49 34 69 Mean ± SE 55 ± 9% SE = Standard Error Table 4. Mean duration of different developmental stages o f C. sordidus under laboratory conditions. Stage Duration (days) No. observed Range Mean . ± SE Egg 46 5-9 6.3 0.2 Larval 52 21-40 28.0 0.6 Pupal 41 3-12 7.1 0.3 Egg to adult 26 33-51 40.4 0.7 SE = Standard Error Table 5. Parameter estimates from Generalized Linear Models (GENMOD) analysis o f the number of larvae at three levels on growing plantain (Figure 1.). Parameter df Estimate SE chi-square P > Chi Level ca Level cb Level ps 1 1.6582 0.5455 1 1.1787 0.5718 0 0.0000 0.0000 9.2391 4.2494 0.0024 0.0393 University of Ghana http://ugspace.ug.edu.gh 37 ■O 30 a 25 Days after incubation _____ I Figure 2. Duration o f egg of C. sordidus incubation on corm pieces in the laboratory. Note: The arrow shows the period at which a great number o f the eggs hatched- Days after egg hatching Figure 3. Duration of larval stage o f C. sordidus in corm pieces in the laboratory. Note: The arrow shows the period at which most of the larvae pupated- 16 a 14 1 12 8> 10 I 8- © 6 j \ CJ t 4 I 2 o — \ ^ l 2 3 4 5 6 7 8 9 10 11 12 13 days after larval stage Figure 4. Duration of pupal stage of C. sordidus in corm pieces in the laboratory. Note: The straight line shows the range of time (days) where most o f the pupal stages lasted. University of Ghana http://ugspace.ug.edu.gh 38 -O 100 80 60 40 20 . 1 1 ps ca cb Sampling levels Figure 5. Percentage o f eggs o f C. sordidus at three sampling levels (Figure 1.) on plantain. tj 100 i 80 60 -2 40 20 - IJ ps ca cb Sampling levels Figure 6. Percentage o f larvae o f C. sordidus at three sampling levels (Figure 1.) on plantain. i 10 80 60 40 - 20 ps ca cb Sampling levels Figure 7. Percentage o f pupae o f C. sordidus at three sampling levels (Figure 1.) on plantain. University of Ghana http://ugspace.ug.edu.gh 39 3.4. DISCUSSION The duration o f developmental periods for egg and pupa were within the ranges reported by Cuille (1950), Woodruff (1969), Bakyalire and Ogenga-Latigo (1992), Hill (1983), Afreh-Nuamah (1993b) and Traore (1995). However, the range o f larval stage duration differed from that observed by Afreh-Nuamah (1993b) who conducted his experiment where the present experiment was conducted. The materials and methods used, environmental conditions (i.e. temperature) (Traore, 1995), seasonal variation (Cuille, 1950) and even the biotype o f C. sordidus may account for such differences. The results from the present work can be used in laboratory and field trials to guide data collection on the different stages since the experiment was conducted within the temperature range prevailing in the field. Similarly, the feeding material used to follow the developmental period o f the larva was the same as in nature but one variation might be that cut conns are more palatable as they start rotten to larvae than growing ones. The spatial distribution of eggs observed in this study agree with that reported by Froggatt (1925). The adults o f C. sordidus deposited their eggs both on the pseudostem and on the corm of plantain but more eggs were laid on the rhizome. The results obtained for the present study contrast with that of Abera et al. (1997) who found more eggs on the pseudostem than on the corm. One o f the reasons for such dissimilarity may be the plant nature (i.e. morphology) since the trials were earned out on two different Musa spp. (banana and plantain) and another reason may be differences in soil type. The spatial distribution of the larvae of C. sordidus (Figure 6.) followed that reported by Knowles (1918), Moznette (1920), Frogatt (1925), Cuille (1950) and Abera et al (1997). Cuille (1950) observed that on the growing banana, tunnels o f larvae o f C. sordidus were downward in the rhizome and the grub rarely occurred in the University of Ghana http://ugspace.ug.edu.gh 40 pseudostem because the high moisture content o f the pseudostem is not favourable. This observation is supported by that o f Abera et al (1997) who found that even though more eggs were laid in the pseudostem, more than 80% o f the banana weevil larvae were located within the corm. Cuille (1950), on the other hand reported that larvae were often found in fallen dehydrated pseudoste