i Deployment of the cowpea aphid resistance gene for cowpea improvement in Ghana By Francis Kusi Master of Philosophy in Entomology, Insect Science Programme University of Ghana, Legon This Thesis is Submitted to the University of Ghana, Legon in Partial Fulfilment of the Requirements for the Award of Doctor of Philosophy Crop Science (Entomology) Degree University of Ghana Legon July 2014 University of Ghana http://ugspace.ug.edu.gh ii Declaration of Originality I certify that the substance of this thesis has not been submitted for any degree and it is not being submitted for any other degree. I certify that to the best of my knowledge any help received in preparing this thesis and all sources used have been acknowledged in this thesis. ……………………………………… Francis. Kusi (Student) …………………………… …………………………………… Dr. S.K. Asante Dr. F. K. Padi (Supervisor) (Supervisor) ……………………………………….. Professor D. Obeng-Ofori (Supervisor) University of Ghana http://ugspace.ug.edu.gh iii Dedication This work is dedicated to my wife, Lydia and our children Prince, Emmanuel, Francis and Bright for their love and support during my studies. University of Ghana http://ugspace.ug.edu.gh iv Acknowledgements I thank the Lord Almighty for his care, protection and guidance throughout my studies. My sincere and grateful acknowledgement goes to Professor D. Obeng-Ofori, Dr. S.K. Asante and Dr. F.K Padi, my supervisors, whose guidance, invaluable suggestions and constructive criticisms have made this work possible. The Kirkhouse Trust sponsored the study and provided molecular laboratory at Savanna Agricultural Research Institute (SARI) for this work. The reliable supply of laboratory equipment and consumables by Kirkhouse Trust facilitated the timely implementation of the study timelines. I am grateful to Professor Mike Timko and Dr. Robert Koebner (Consultants of Kirkhouse Trust) for their immense contribution to the study. The cooperation and support I received from Bernard Armooh and Eric Brenya, the staff on the Kirkhouse Mobile Laboratory Van and all the staff of the molecular laboratory at Cocoa Research Institute, Ghana, is highly appreciated. The committed and hardworking staff of the molecular laboratory and the field staff of Entomology section at SARI especially Abor Awudu, Agyare Yaw Richard, Frederick Awuku Justice and Frederick Agemga provided technical support. The Head of Department, lecturers and the staff of Crop Science Department of School of Agriculture, College of Agriculture and Consumer Sciences, University of Ghana, provided useful suggestions during seminar presentations of the work. I am grateful to the Savanna Agricultural Research Institute of the Council for Scientific and Industrial Research (SARI-CSIR) for granting me study leave to pursue this programme of study. Finally, I thank my wife, Lydia and our children, Prince, Emmanuel, Francis and Bright for their love and support during my studies. University of Ghana http://ugspace.ug.edu.gh v TABLE OF CONTENTS Page Title page------------------------------------------------------------------------------------------------i Declaration of originality-----------------------------------------------------------------------------ii Dedication---------------------------------------------------------------------------------------------iii Acknowledgements-----------------------------------------------------------------------------------iv Table of Contents--------------------------------------------------------------------------------------v List of Tables-------------------------------------------------------------------------------------------x List of Figures-----------------------------------------------------------------------------------------xi Abbreviations-----------------------------------------------------------------------------------------xii Abstract-----------------------------------------------------------------------------------------------xiv CHAPTER ONE---------------------------------------------------------------------------------------1 1.0. Introduction---------------------------------------------------------------------------------------1 1.1 Justification----------------------------------------------------------------------------------------4 1.2. Objectives-----------------------------------------------------------------------------------------5 CHAPTER TWO--------------------------------------------------------------------------------------6 2.0. Literature Review--------------------------------------------------------------------------------6 2.1. Origin and Cultivation of Cowpea-------------------------------------------------------------6 2.2. Production Level---------------------------------------------------------------------------------6 2.3. Uses and Nutritional Value of Cowpea -------------------------------------------------------8 2.4. Insect Pests of Cowpea--------------------------------------------------------------------------8 2.4.1. Aphid-------------------------------------------------------------------------------------------12 2.4.1.1. The Cowpea Aphid, Aphis craccivara Koch-------------------------------------------13 2.4.1.2. Host Plants of the Cowpea Aphid--------------------------------------------------------14 2.4.1.3. Damage and Economic Importance------------------------------------------------------14 University of Ghana http://ugspace.ug.edu.gh vi 2.4.1.4. Reproduction of A. craccivora -----------------------------------------------------------15 2.4.1.5. Growth, Development, Fecundity and Longevity of Cowpea Aphid---------------16 2.4.1.6.. Management of A. craccivora -----------------------------------------------------------17 2.4.2. Host-Plant Resistance------------------------------------------------------------------------19 2.4.2.1. Advantages and Disadvantages of Host-Plant Resistance----------------------------20 2.4.2.2. Classification of Host-Plant Resistance-------------------------------------------------21 2.4.2.3. Mechanisms of Resistance----------------------------------------------------------------22 2.4.2.4. Resistance of Cowpea to A. craccivora-------------------------------------------------24 2.4.2.5. Antibiosis------------------------------------------------------------------------------------24 2.5. Application of Molecular Markers in Crop Improvement---------------------------------25 2.5.1. Success in the deployment of aphid resistance loci in soybean improvement-------27 2.5.2. Genetic Markers------------------------------------------------------------------------------29 2.5.2.1. What are Genetic Markers? --------------------------------------------------------------29 2.5.2.1.2. Marker-Assisted Selection (MAS) ----------------------------------------------------31 2.5.2.1.3. Advantages of MAS---------------------------------------------------------------------31 2.5.2.1.4. Cost/Benefit Analysis of MAS-------------------------------------------------------- 32 2.5.2.1.5. Marker-Assisted Backcrossing---------------------------------------------------------32 CHAPTER THREE----------------------------------------------------------------------------------35 3.0. Introduction--------------------------------------------------------------------------------------35 3.1. Materials and Methods ------------------------------------------------------------------------36 3.2. Selection of Resistant and Susceptible Progenies -----------------------------------------36 3.3. Identification of DNA Marker(s) Linked to the Cowpea Aphid Resistance Gene -------------------------------------------------------------------------------------37 3.3.1. FTA Protocol ---------------------------------------------------------------------------------38 3.3.2. PCR Amplification --------------------------------------------------------------------------39 University of Ghana http://ugspace.ug.edu.gh vii 3.3.3. Casting the Gel -------------------------------------------------------------------------------40 3.4. Testing the Reliability of the Marker, CP 171F/172R, in F2 Segregating Population---------------------------------------------------------------------------------------------41 3.5. Genetic Analysis -------------------------------------------------------------------------------42 3.6. Introgression of Aphid Resistance Locus into Ghanaian Cowpea Cultivars ---------------------------------------------------------------------------------------------42 3.6.1. Polymorphism Test--------------------------------------------------------------------------42 3.6.2. Marker Assisted Backcrossing-------------------------------------------------------------43 3.6.2.1. Development and Advance of Backcross Progenies ---------------------------------43 3.7. Determining the Stability of the Aphid Resistance Locus across the Major Cowpea Belts in Ghana---------------------------------------------------------------------44 CHAPTER FOUR-----------------------------------------------------------------------------------46 4.0. Stability of the cowpea aphid resistant genotype across the major cowpea growing zones in Ghana-------------------------------------------------------------------46 4.1. Introduction--------------------------------------------------------------------------------------46 4.2. Materials and Methods-------------------------------------------------------------------------48 4.3. Data Collection and Analysis-----------------------------------------------------------------51 4.4. Results--------------------------------------------------------------------------------------------52 4.4.1. Seedling Mortality----------------------------------------------------------------------------52 4.4.2. Seedling Vigour-------------------------------------------------------------------------------52 4.5. Discussion---------------------------------------------------------------------------------------54 CHAPTER FIVE-------------------------------------------------------------------------------------56 5.0. Genetic Mapping and Inheritance of the Aphid Resistance Locus in Cowpea---------56 5.1. Introduction--------------------------------------------------------------------------------------56 5.2. Materials and Methods ------------------------------------------------------------------------58 University of Ghana http://ugspace.ug.edu.gh viii 5.2.1. Plant Materials Used in the Study----------------------------------------------------------58 5.2.2. Identification of Markers Linked to the Aphid Resistance Locus---------------------58 5.3. Data Analyses-----------------------------------------------------------------------------------59 5.4. Results--------------------------------------------------------------------------------------------59 5.4.1. Inheritance of Aphid Resistance in Line SARC 1-57-2---------------------------------59 5.4.2. Identification of Markers Linked to the Aphid Resistance Locus---------------------60 5.5. Discussion---------------------------------------------------------------------------------------62 CHAPTER SIX--------------------------------------------------------------------------------------64 6.0. Introgression of Aphid Resistance Locus into Ghanaian Cowpea Cultivars-----------64 6.1. Introduction-------------------------------------------------------------------------------------64 6.2. Materials and Methods------------------------------------------------------------------------65 6.2.1. Polymorphism Test--------------------------------------------------------------------------65 6.2.2. Marker Assisted Backcrossing-------------------------------------------------------------65 6.3. Results-------------------------------------------------------------------------------------------66 6.3.1. Test for Polymorphism----------------------------------------------------------------------66 6.3.2. Determination of Plants from a Successful Cross (F1 lines) ---------------------------66 6.3.3. Genotyping to Select Heterozygotes from the Backcross Populations---------------67 6.3.4. Genotyping of BC4F2 to Select Homozygous Lines ------------------------------------67 6.4. Discussions--------------------------------------------------------------------------------------68 CHAPTER SEVEN---------------------------------------------------------------------------------71 7.0. Yield loss assessment of ten Cowpea varieties---------------------------------------------71 7.1. Introduction-------------------------------------------------------------------------------------71 7.2. Materials and Methods------------------------------------------------------------------------72 7.3. Data Analysis-----------------------------------------------------------------------------------74 7.4. Results-------------------------------------------------------------------------------------------74 University of Ghana http://ugspace.ug.edu.gh ix 7.4.1. Grain Yield-----------------------------------------------------------------------------------74 7.4.2. Biomass Production-------------------------------------------------------------------------76 7.4.3. Days to Flowering---------------------------------------------------------------------------77 7.4.4. Maturity Period------------------------------------------------------------------------------79 7.5. Discussion--------------------------------------------------------------------------------------80 CHAPTER EIGHT---------------------------------------------------------------------------------83 8.0. General Discussion----------------------------------------------------------------------------83 8.1. Conclusion--------------------------------------------------------------------------------------85 8.2. Recommendations-----------------------------------------------------------------------------85 REFERENCES--------------------------------------------------------------------------------------87 APPENDICES--------------------------------------------------------------------------------------121 University of Ghana http://ugspace.ug.edu.gh x List of Tables Table Page 2.1. Major insect pest species found on cowpea worldwide----------------------------------10 2.2. Cowpea growth stages and pest incidence-------------------------------------------------12 2.3. Percentage of recurrent parent genome after backcrossing-------------------------------33 3.1. SSR primers used and their sequences------------------------------------------------------37 3.2. Preparation of 100 ml 5% acrylamide gel--------------------------------------------------40 3.3. Locations in the six regions where cowpea aphids were sampled-----------------------45 4.1. Characteristics of study areas in the six regions-------------------------------------------49 4.2. Description of the five genotypes of cowpea by parentage or source-------------------50 4.3. Mean seedling mortality and plant vigour score following aphid infestation on five cowpea genotypes at 18 locations in Ghana-----------------------------------------------------53 7.1. Description of the 10 cultivars of cowpea by parentage or source----------------------74 7.2. Grain yield and percentage grain yield loss (kg) ha-1 of 10 cowpea cultivars evaluated under aphid infestation and no infestation------------------------------------------------------76 7.3. The dry biomass yield and percentage dry biomass yield loss (kg) ha-1 of the 10 cultivars evaluated under aphid infestation and uninfested conditions---------------------77 7.4. The number of days to 50% flowering under infested and uninfested condition of the ten cultivars-----------------------------------------------------------------------------------------76 7.5. The number of days to maturity under infested and un-infested conditions-----------80 University of Ghana http://ugspace.ug.edu.gh xi List of Figures Figure Page 4.1. The Kirkhouse Trust Mobile Screen House-------------------------------------------------51 4.2. Responses of the susceptible lines and resistant lines to aphid attack------------------53 5.1. Silver stained PAGE showing the DNA of resistant and susceptible parents---------61 5.2. A silver stained PAGE showing the DNA patterns of F2 plants------------------------61 6.1. Tests for polymorphism for marker CP 171F/172R on four elite cultivars-----------66 6.2. The F1 plants from the crosses between SARC1-57-2 and Zaayura genotype---------67 6.3. Successive backcross populations genotyped to selected heterozygote individual---67 6.4. BC4F2 genotyped with the marker CP 171F/172R----------------------------------------68 University of Ghana http://ugspace.ug.edu.gh xii Abbreviations ANOVA Analysis of Variance APS Ammonium persulphate BC Backcrossing bp Base pair CABMV Cowpea aphid-borne mosaic virus CGIAR Consultative Group on International Agricultural Research CRIG Cocoa Research Institute of Ghana CSIR Council for Scientific and Industrial Research DNA Deoxyribonucleic acid DRC Democratic Republic of Congo EtBr Ethidium Bromide FAO Food and Agriculture Organization of the United Nations FTA Fast technology for analysis GPS Geographic position system h-PAGE horizontal-Polyacrylamide gel electrophoresis IITA International Institute of Tropical Agriculture MAS Marker-assisted selection NARS National agricultural research system PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction PLABSIM PLAnt Breeding SIMulation QTL Quantitative trait loci SARI Savannah Agricultural Research Institute SAT Semi Arid Tropics University of Ghana http://ugspace.ug.edu.gh xiii SSA Sub Sahara African SSR Single sequence repeat TBE Tris/Borate/EDTA TEMED Tetramethylethylenediamine UV Ultraviolet University of Ghana http://ugspace.ug.edu.gh xiv Abstract Resistance to the cowpea aphid is important component of integrated pest management of cowpea cropping systems most especially at the vegetative stage. The objective of this study was to demonstrate the effectiveness of the aphid resistance locus identified in advanced breeding line SARC 1-57-2 in reducing damage from the cowpea aphid in Ghana. Using an F2 population developed from Apagbaala x SARC 1-57-2, the resistance locus was tagged with the SSR marker CP 171F/172R with a recombination fraction of 5.91%. Based on the CP 171F/172R, recurrent marker assisted backcrossing was carried out to introduce the resistance locus into the susceptible cultivar, Zaayura. This led to the development of several BC4F3 lines that are isogenic except for the region of the resistance locus. In field tests under no insecticide protection, the BC4F3 lines carrying the dominant marker allele suffered 3% loss of biomass and 4% loss of grain yield compared with plots protected with recommended insecticides. The BC4F3 lines carrying the recessive marker allele recorded 12% loss of biomass and 33% reduction in grain yield compared with the sprayed plots. The resistance locus did not influence the number of days to flowering or maturity and no pleiotropic effects were observed in terms of plant morphology or seed characteristics. In all segregating populations analysed, the locus segregated as a single Mendelian gene. Stability of the resistance locus was conducted at 18 locations covering six important cowpea growing Regions in Ghana. The range of damage by the pest on resistant and susceptible progenies were consistent across locations, and did not support the hypothesis of existence biotypes of the insect (based on differences in feeding damage on different varieties) in Ghana. This stability in performance places a premium on the resistance locus in improving cowpea cultivars developed for different agro-climatic regions of the country for resistance to the pest. The study has demonstrated the effectiveness of an insect resistance locus in significantly reducing insect damage under typical cowpea production conditions in Ghana. University of Ghana http://ugspace.ug.edu.gh xv University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0. Introduction Cowpea, Vigna unguiculata (L.) Walp is an important source of protein for human nutrition in many parts of the semi-arid tropics (SAT) (Rachie, 1985; Githiri et al., 1996; Bashir et al., 2002). It is eaten in the form of dry seeds, green pods, green seeds, and tender green leaves (Githiri et al., 1996). Cowpea is also an important source of protein for animal nutrition; it is used for pasture, hay, silage, or green manure (Singh, 1990). Nigeria, Brazil, Niger and Burkina Faso are among the major producers and account for over 70 % of the world crop (FAO, 2008). Nigeria is the largest producer and consumer of cowpea, with about 5 million ha and over 2 million mt production annually, followed by Niger (650,000 mt) and Brazil (490,000 mt) (Timko et al., 2008: FAO, 2008; Asare, 2012). However, yields at farmers level are low (Jackai and Dacoust, 1986; Motimore et al., 1997: Asare, 2012). The major cause of the low yields are insect pests, diseases, drought and low soil fertility, of which insect pests constitute the major constraint (Nampala et al., 1999; Asare, 2012). Cowpea suffers serious insect pest infestation from the time of planting through harvesting and during storage (Obeng-Ofori, 2007). The crop therefore suffers severe attack of pre- harvest and post-harvest infestation which if not controlled could lead to total crop failure. The major field pests of cowpea are aphids (Aphis craccivora Koch), flower bud thrips (Megalurothrips sjostedti Trybom), the legume pod borer (Maruca vitrata Fab), pod- sucking bugs including Clavigralla tomentosicollis Stål, Anoplycnemis curvipes Fab., Mirperus jaculus Thunbeng and Nezera viridula Linnaeus (Singh and Jackai, 1985; Jackai and Adalla, 1997; Obeng-Ofori, 2007: Egho, 2011). The cowpea aphid, A. craccivora, is an important pest of cowpea in Africa (Singh and Jackai, 1985; Kusi et al., 2010a; Souleymane University of Ghana http://ugspace.ug.edu.gh 2 et al., 2013). The pest primarily infests the seedlings of cowpea and causes direct damage to the crop by sucking plant sap, resulting in stunted plants and distorted leaves (Bohlen, 1978; Jackai and Daoust, 1986; Ofuya, 1997a). Aphids are usually found in clusters around stems, young leaves and on young shoots. The infested leaves are often cupped or distorted and become more or less yellow (Singh and Jackai, 1985). In heavy infestation the plant dies, especially under water stress (Ofuya, 1995). High numbers of cowpea aphids can produce a significant amount of honeydew and sooty mould which reduce the photosynthetic ability of the leaves (Baute, 2004). Indirectly, cowpea aphid transmits aphid- borne cowpea mosaic viruses (Singh and Jackai, 1985; Thottappilly and Rossel, 1985; Shoyinka et al., 1997). Estimated yield losses of 20% to 40% in cowpea due to A. craccivora infestation in Asia and up to 35% in Africa have been reported (Singh and Allen 1980; Kusi et al., 2010b). In eastern region of the Democratic Republic of Congo, Aphis craccivora (Hemiptera: Aphididae) is a major pest of cowpea and groundnut (Munyuli et al., 2007) where about 35-65% of yield losses are associated with this pest species (Munyuli et al., 2008; Munyuli, 2009). The cowpea aphid can be controlled by various methods including the use of insecticides, cultural practices and biological control (Singh and Jackai, 1985). However, growing of aphid resistant cultivars offers one of the simplest and most convenient methods of pest control for the resource-poor farmers (Dent, 1991; Orawu et al., 2013). Host plant resistance as indicated by Painter (1951) is a relationship between the plant feeding insects and their host-plants. It is the property that enables a plant to avoid, tolerate or recover from injury by insect populations that will cause greater damage to other plants of the same species under similar environmental conditions (Kogan, 1975; Tingey, 1986). Kumar (1984) and Dent (1991) defined host plant resistance as the inherent ability of crop University of Ghana http://ugspace.ug.edu.gh 3 plants to restrict, retard or overcome pest infestation and thereby improve yield and/or quality of the harvested product. Host plant resistance has proved to be a successful tool against insect pests that attack many crops (van Emden, 1991; Thomas and Waage, 1996; Felkl et al., 2005; Orawu et al., 2013). Plant genotypes, either due to environmental stress or genetic makeup, possess physiological and biochemical differences which alter the nutritional value (primary metabolites) and may also cause changes in the levels of secondary metabolites that could affect the behaviour of phytophagous insects (Eckey- Kaltenbach et al., 1994; Karban and Baldwin, 1997: Siemens et al., 2002; Städler, 2002; Theis and Lerdau, 2003). Three mechanisms of plant resistance originally defined by Painter (1951) are non- preference, antibiosis and tolerance. The non-reference has since been replaced by antixenosis (Kogan and Omar, 1978). Antixenosis is the inability of a plant to serve as host to an insect herbivore. The basis of this resistance mechanism can be morphological (e.g. leaf hairs, surface waxes, tissue thickness) or chemical (e.g. repellents or antifeedants) (Kogan and Omar, 1978). These plants would have reduced initial infestation and/or higher emigration rate of the insect than susceptible plants. Antibiosis is the mechanism that describes the negative effects of a resistant plant on the biology of an insect which has colonized the plant (e.g. adverse effect on development, reproductive and survival) (Painter, 1951; Kogan and Omar, 1978). Both chemical and morphological plant defences can induce antibiosis effects (Painter, 1951; Kogan and Omar, 1978). The consequences of antibiosis resistance may vary from mild effect that influences fecundity, development time and body size to acute direct effect resulting in death (Painter, 1951; Kogan and Omar, 1978). University of Ghana http://ugspace.ug.edu.gh 4 Plant tolerance is the degree to which a plant can support an insect population that under similar conditions would severely damage a susceptible plant (Painter, 1951; Kogan and Omar, 1978). When two cultivars are equally infested the less tolerant one will produce low yield. Cowpea aphids are easily controlled by the use of aphid resistant varieties (Singh, 1977; Obeng-Ofori, 2007). Several aphid-resistant cowpea lines have been identified at the IITA and have been tested against aphid populations from several locations in Africa and Asia (Chari et al., 1976; Dhanorkar and Daware, 1980; Karel and Malinga, 1980; MacFoy and Dabrowski, 1984; Manawadu, 1985; Ofuya, 1988a; 1993). Antibiosis has been shown as the main mechanism responsible for aphid resistance in cowpea (Singh, 1977; Ansari, 1984; Ofuya, 1988b,) and is controlled by a single dominant gene (Singh and Ntare, 1985; Bata et al., 1987; Ombakho et al., 1987; Singh, et al., 1987; Pathak, 1988). Additionally, a large number of aphid-resistant lines have been developed, and have been evaluated in international yield trials (MacFoy and Dabrowski, 1984; Manawadu, 1985; Ofuya, 1988a; 1993). 1.1. Justification Farmers have over-relied on chemical insecticides over the years to control cowpea aphid which has resulted in misuse of chemicals, high cost of production, poisoning of human beings, the environment and development of resistance to most of the insecticides leading to resurgence of the aphids (Dent, 1991; Singh and Jackai, 1985). More recently, Kusi et al. (2010a) identified new sources of cowpea genotypes (SARC1- 57-2) resistant to A. craccivora. Segregation ratio in F2 population generated between a resistant line and Apagbaala (a susceptible parent) suggested that a single dominant gene University of Ghana http://ugspace.ug.edu.gh 5 controlled resistance to the aphid in the breeding line. This presents a valuable source of resistance for developing cowpea cultivars with resistance to the cowpea aphid in the field. Ongoing efforts at mapping the cowpea genome presents an opportunity to tag the resistance locus with co-dominant PCR based markers to facilitate marker-based selection of aphid resistant progenies in large segregating populations. 1.2. Objectives The main objective of the study was to demonstrate the effectiveness of an aphid resistance locus in the cowpea line SARC 1-57-2 at controlling aphid in Ghana and specifically: 1. Identify DNA marker(s) tightly linked to locus controlling resistance to the cowpea aphid in a resistant breeding line, SARC 1-57-2. 2. Deploy the DNA marker(s) to improve at least one cowpea cultivar through marker- assisted backcrossing. 3. Assess the stability of the cowpea aphid resistant line in the major cowpea growing regions in Ghana. 4. Determine yield loss due to aphid infestation in near isogenic lines developed from the resistant line SARC 1-57-2 and Zaayura University of Ghana http://ugspace.ug.edu.gh 6 CHAPTER TWO 2.0. Literature Review 2.1. Origin and Cultivation of Cowpea Cowpea (Vigna unguiculata (L.) Walp), is said to have originated in Africa, where it has become an integral part of traditional cropping systems, particularly in the semi-arid West African savannah (Steele, 1972). The history of cowpea dates to ancient West African cereal farming, 5 to 6 thousand years ago, where it was closely associated with the cultivation of sorghum and pearl millet (Davis et al., 2003). Cowpea is an annual legume and is also commonly referred to as southern pea, blackeye pea, crowder pea, lubia, niebe, coupe or frijole. Worldwide cowpea production has increased dramatically in the last 25 years (Davis et al., 2003). It is widely grown in Africa, Latin America, Southeast Asia and in the Southern United States (Fery, 1985; Mishra et al., 1985; Singh and Ntare, 1985). 2.2. Production Level Cowpea, a native crop of West Africa, is one of the most important food legume crops now grown in the semi-arid tropics covering Asia, Africa, southern Europe and Central and South America (Akibode and Maredia, 2011). Total cowpea area harvested has risen by 38% between 1994-06 and 2006-08 (Akibode and Maredia, 2011; FAO, 2011). World cowpea production has increased 88% and yields have increased by 35% in the same time period. This increase in area, production and yield has been made possible by a similar trend in Sub Sahara African (SSA), which dominates the world scene (Akibode and Maredia, 2011; FAO, 2011). Despite the dramatic increase in production in SSA, cowpea yields remain one of the lowest among all food legume crops, averaging at 450 kg/ha in 2006-08 (Akibode and Maredia, 2011; FAO, 2011). University of Ghana http://ugspace.ug.edu.gh 7 The top five cowpea growing countries in African are all in West Africa. Nigeria and Niger have maintained the top first and second position over the past 14 years, together covering more than 80% of total cowpea area in the world (FAO, 2011). Other important cowpea growing countries include Burkina Faso (6%), Mali (2%) and Senegal (2%). These five West African countries share more than 90% of the world cowpea area harvested in 2006- 08 (Akibode and Maredia, 2011; FAO, 2011). While area cultivated has stayed stable in Burkina Faso, Mali and Senegal over the last 14 years, it has fluctuated significantly in Niger and Nigeria with drops and increments at the scale of more than 1 million ha (FAO, 2011). The average yields in Nigeria have steadily increased since mid-1990s and have reached around 700 kg/ha in recent years (Akibode and Maredia, 2011: FAO, 2011). Compared to Nigeria, all the other top cowpea growing countries in West Africa have significantly lower yields (almost by 200-300 kg/ha). Except, for Nigeria and Niger, these countries have either experienced a decline in average yields or yields have remained stagnant over the past 14 years (Akibode and Maredia, 2011; FAO, 2011). Cowpea is one of the most widely grown grain legumes in Ghana but, commercial production is restricted to some parts of the Volta, Northern, Upper East, Upper West and Brong-Ahafo regions (Tweneboah, 2000). In Ghana the estimated researcher-managed on-farm yields of 1.8 t ha- 1 are more than double the average farm level yields (SARI, 1999). Reasons for the low yields in most countries include use of low yielding traditional varieties, poor soil fertility, unfavorable weather, and insect pest and disease attack (Diehl and Sipkins, 1985; Montimore et al., 1997; Blade et al., 1997; Asare, 2012). University of Ghana http://ugspace.ug.edu.gh 8 2.3. Uses and Nutritional Value of Cowpea Cowpea is one of the five most important legumes in the tropics and provides the source of protein for most people in the region. According to Jackai and Singh (1983), 100 g raw mature seeds typically contain 11.4 g moisture, 338 kcal (1415kj) of energy, 22.5 g protein, 1.4 g fat, 61.0 g total carbohydrate, 5.4 g fiber, 3.7 g ash, 104 mg Ca, 416 g P, 0.08 mg thiamine, 0.09 mg riboflavin, 4.0 mg niacin, and 2 mg ascorbic acid. Cowpea has many uses, in fresh form, the young leaves, immature pods and peas are used as vegetables, while several snacks and main meal dishes are prepared from the grain (Jackai and Singh, 1983). All parts of the plant that are used for food are nutritious, providing protein, vitamins (notably vitamin B) and minerals. The cowpea haulm is also an important source of livestock feed, and therefore of great value to farmers. 2.4. Insect Pests of Cowpea Cowpea suffers serious insect pest infestation from the time of planting through harvesting and during storage (Obeng-Ofori, 2007). Several pre-harvest and post-harvest insect pests are associated with the crop (Tables 2.1 and 2.2), which if not controlled could lead to total crop failure. In general, cowpea suffer more damage as a monocrop than as a mixed crop which is the traditional method of production (Singh and Jackai, 1985). Studies have shown that in unprotected monocrops, yield losses due to the major field pests may range from 20- 100% (Youdeowei, 1989). The pest problem is more serious in Africa than in Asia or Latin America (Singh and Jackai, 1985). In Ghana, Agyen-Sampong (1978) reported that, there were more than 150 species of insects recorded to be associated with cowpea in both field and storage, but only few were of major economic importance. The pest complex of cowpea in Ghana includes leafhoppers, University of Ghana http://ugspace.ug.edu.gh 9 Empoasca spp, aphids, Aphis craccivora Koch, flower bud thrips, Megalurothrips sjostedti (Trybom), pod borers, Maruca vitrata (Fab), pod-sucking bugs Clavigralla tomentosicollis Stål, Nezera viridula Linnaeus, Leptoglossus spp and bruchids Callobruchus spp. In Africa the major field pests of cowpea are aphid (A. craccivora), legume flower thrips (M. sjostedti), legume pod borer (M. vitrata) and pod-sucking bugs (C. tomentosicollis, Leptoglossus spp and N. viridula) (Singh and Jackai, 1985; Jackai and Adalla, 1997; Obeng- Ofori, 2007). The principal storage pest of cowpea grain in Sub-Saharan Africa is the cowpea beetle Callosobruchus maculatus Walp (Taylor, 1981). In low resource farms, C. maculatus infestation starts in the field and continues in storage. Another bruchid pest of cowpea is C. chinensis L. (Taylor, 1981). Other storage pests of cowpea include Acanthoselides obtectus (Say), A. clandestinus (Mots), C. analis (F), C. rhodesianus Pic and Zabrotis subfasciatus (Boheman), all in the family Bruchidae (Obeng-Ofori, 2007). University of Ghana http://ugspace.ug.edu.gh 10 Table 2.1. Major Insect Pest Species Found on Cowpea Worldwide Pest Species (Order: Family) Geographical Distribution Plant Part Attacked Importance Callosobruchus sp. (Coleoptera: Bruchidae) Cosmopolitan Seed (Storage) Key Chalcodermus sp. (Coleoptera : Curculionidae USA South America Pods Key Ophiomyia phaseoli (Trybom) (Diptera: Agromizidae) Asia, Africa Leaves, Stem Key, Sporadic Clavigralla tomentosicollis Stål (Hemiptera : Coreidae) Africa Asia South America Pods Pods Pods Key Minor Minor Crinocerus sanctus (Fab) (Hemiptera: Coreidae) South America Pods Key Leptoglossus sp. (Hemiptera : Coreidae) USA Pods Sporadic Lygus hysperus (Hemiptera : Miridae) USA Pods, Leaves Key Nezera viridula Linnaeus (Hemiptera : Pentatomidae) USA Africa Asia South America Pods Pods Pods Pods Key Sporadic Sporadic Sporadic Aphis craccivora Koch (Homoptera: Aphididae) Cosmopolitan Foliage, flowers, pod Key Empoasca biguitula (Shiraka) Asia Leaves Unknown University of Ghana http://ugspace.ug.edu.gh 11 Table2.1. Continued Empoasca dolichi Paoli (Homoptera: Cicadelidae) Africa Leaves Key Empoasca kraemri Ross and Moore (Homoptera:Cicadelidae) South America Leaves Key Amsacta moorei (Butler) (Lepidoptera: Arctiidae) Africa (Senegal) Leaves Sporadic Elasmopalpus lignosellus (Zeller) (Lepidoptera: Pyralidae South America Stem Key Etiella zinckenella (Treitschke) (Lepidoptera : Pyralidae) Asia Pods, Flowers Sporadic Maruca vitrata (Fab) (Lepidoptera : Pyralidae) Cosmopolitan (Rare in America) Stems, Flowers, Pods Key Megalurothrips sjostedti (Trybom) (Thysanoptera: Thripidae) Africa, Asia, Americas Floral structures Floral structures Floral structures Key Not important Unknown Thrips palmi (Thysanoptera: Thripidae) Asia Floral structures Sporadic Thrips tabasi Lindeman (Thysanoptera: Thripidae) Asia South America Floral structures Sporadic Source: Jackai and Adalla, (1997) (re-arranged according to the order of the pests in alphabetical order) University of Ghana http://ugspace.ug.edu.gh 12 Table 2.2. Cowpea growth stages and pest incidence Growth stages Days after planting 20 30 40 50 60 70 Insect pests Foliage ▬▬▬▬ Aphis, Leafhoppers, Foliage Beetle Flower Budding ▬▬▬ Flower Thrips Flowering ▬▬▬ Flower Thrips, pod borers Podding ▬▬▬ Pod sucking bugs, pod borers Late Podding ▬▬▬ Pest population decline due to crop senescence Spraying by growth stage • • • • Source: Jackai and Adalla (1997). 2.4.1 Aphids Aphids are small soft-bodied insects of the order Hemiptera and sub-order Homoptera that feed on the fluid in the plant phloem (Summers et al., 2006). Aphids are plant sucking bugs which occur throughout the world (Jackai and Adalla, 1997). The greatest number of species is in the temperate regions, where few higher plants are free from aphid attack (Dixon, 1977). They differ from other plant sucking bugs of the Aphidoidea in that the females of at least a few generations are parthenogenetic and viviparous (Dixon, 1977). Although many species are small and inconspicuous, they frequently become abundant. As many as 2000 University of Ghana http://ugspace.ug.edu.gh 13 million aphids per acre (0.4 ha) may live on the above-ground parts of plants, and the roots may support a further 260 million (Dixon, 1977). Aphids, like many other insects, are capable of migrating great distances (up to 1300 km) by means of wind (Dixon, 1977). Aphids exhibit polymorphism. Asexual aphids of some species can either possess wings (in which case they are termed alatae), or lack wings (these morphs are called apterae). Typically, there are several structurally different morphs in a species, including both sexual and asexual forms (Dixon, 1977). Aphids exhibit complex life cycles. It is estimated that approximately 10% of the species alternate between a primary (usually woody) host plant and a secondary (herbaceous) host plant (Blackman and Eastop, 1994). Non-host alternating species are usually monophagous but may feed on a range of related host plants (Blackman and Eastop, 1994). Aphids display high reproductive peculiarities in their reproductive biology (Braendle et al., 2006). First, female aphid reproduces parthenogenetically, obviating the need for males. Secondly, during their parthenogenic generations, the embryos initiate development immediately after the budding of the oocyte from the germarium and are born as fully developed first-instar nymphs (i.e. they are viviparous). Finally, the oldest embryos also contain embryos, so that adult parthenogenetic aphids carry not only their daughters but also some of their granddaughters within them (Braendle et al., 2006). 2.4.1.1 The Cowpea Aphid, Aphis craccivora Koch. The cowpea aphid is cosmopolitan in distribution, occurs in the temperate, subtropical and tropical regions of the world (Jackai and Adalla, 1997). The cowpea aphid is a soft-bodied, pear-shaped insect, has antennae which are shorter than the body length (about two third as long as the body) and a pair of cornicles (tailpipe-like appendages) (Summers et al., 2006). University of Ghana http://ugspace.ug.edu.gh 14 They may be winged (alate) or wingless (apterous) but the wingless forms are most common (Summers et al., 2006). It has a shiny black body with white appendages and blackish tips and ranges from 1.5 to 2.5 mm long. In general, the cowpea aphid is a relatively small aphid, less than 2 mm long. Though smaller than adults, nymphs resemble the apterous forms in shape. Cowpea aphid nymphs are pale green to grey with powdery coating (Summers et al., 2006). 2.4.1.2. Host Plants of A. craccivora The host plants of the cowpea aphid include alfalfa, apple, carrot, cotton, cowpea, kidney bean, lettuce, lima bean, pinto bean, peanut, pepperweed, pigweed, red clover, wheat, white sweet clover and yellow sweet clover (A’Brook, 1964; Hamid et al., 1977; Kumar, 1984; Ofuya, 1989). Studies also indicate that a common pattern in the tropics is for A. craccivora to spend the dry season on wild hosts and weedy species such as Medicago spp., Melilotus spp., Trifolium spp., Euphorbia spp., Boerhaavia spp as well as volunteer species of other legume crops (A’Brook, 1964; Hamid et al., 1977; Kumar, 1984; Ofuya, 1989). 2.4.1.3. Damage and Economic Importance Cowpea aphid is an important pest of cowpea in Africa (Singh and Jackai, 1985). The pest primarily infests the seedlings of cowpea and causes direct damage on the crop by sucking plant sap, resulting in stunted plants and distorted leaves. The aphids are usually found in clusters around stems, young leaves and on young shoots. The infested leaves are often cupped or distorted and become more or less yellow (Singh and Jackai, 1985). In heavy infestation the plant dies, especially under water stress conditions. High numbers of cowpea aphids can produce a significant amount of honeydew, excreted by aphids which lead to the development of sooty mold, a dark coloured fungus. This covers the surface of the leaves, University of Ghana http://ugspace.ug.edu.gh 15 affecting photosynthesis and results in yield loss (Baute, 2004). Ultimately these feeding effects of aphid result in reduced vegetative biomass and reduced grain yield. Estimated yield losses of 20% to 40% in cowpea due to A. craccivora infestation in Asia and up to 35% in Africa have been reported (Singh and Allen, 1980; Kusi et al., 2010b). In eastern region of the Democratic Republic of Congo, Aphis craccivora (Hemiptera: Aphididae) is a major pest of cowpea and groundnut (Munyuli et al., 2007) where about 35-65% of yield losses are associated with this pest species (Munyuli et al., 2008; Munyuli, 2009). Aphis craccivora causes indirect damage by transmitting aphid-borne cowpea mosaic viruses. The cowpea aphid-borne mosaic virus (CABMV) is a cosmopolitan, economically significant seed-borne virus of cowpea (Thottappilly and Rossel, 1985; Shoyinka et al., 1997). It can cause a yield loss of 13-87% under field conditions depending upon crop susceptibility, virus strain and the environmental conditions (Thottappilly and Rossel, 1985; Shoyinka et al., 1997). CABMV has spread world-wide through the exchange of virus- infected germplasm materials. The virus-infected seed provides the initial inoculum and aphids are responsible for the secondary spread of the disease under field conditions (Obeng-Ofori, 2007). 2.4.1.4. Reproduction of A. craccivora The reproductive system of the parthenogenetic aphid consists of ovarioles (the number of which is determined prior to birth) that contain the developing embryos (Lees, 1959; Osteo and Helms, 1971). Each ovariole usually contains several embryos at different stages of development (Dixon, 1985). There are many factors, both intrinsic and extrinsic, that can affect an aphid’s reproductive capacity. The reproductive capacity is correlated positively with adult weight (Murdie, 1969; Dixon, 1970; 1971; Dixon and Wretten, 1971; Taylor, University of Ghana http://ugspace.ug.edu.gh 16 1975; Kempton et al., 1980; Wellings et al., 1980). It may either increase or decline with nutritional quality (Wellings et al., 1980), and may be correlated positively (Dixon and Oharma, 1980; Leather and Welling, 1981) or negatively (Dixon, 1977) with ovariole number. Fecundity has been used to measure aphid’s responses to environmental conditions. showed that Uroleucon jacae, L. that fed on high quality host plants had more embryo that were larger and more sclerotised than when fed on low-quality plants (host plant grown in a mixture of sand, gravel and compost in equal volumes (low quality treatment or in compost only (high quality treatment. Stadler (1995) also found that aphids feeding on low-quality plants selectively controlled the development of only a few old embryos. Ward and Dixon (1982) and Leather et al. (1983) showed that if adult aphid (Megoura viviae and Aphis fabae Scoop.) were starved, they would resorb their smallest embryo and concentrate their effort on producing a few large embryos that were more likely to survive than the smaller embryos. 2.4.1.5. Growth, Development, Fecundity and Longevity of A. craccivora The growth, development, fecundity and longevity of A. craccivora vary with weather conditions, soil fertility, soil moisture and host plants. The adult may live up to 15 days or less. Daily progeny production can be as high as 20, and female fecundity may reach 100 or more (Suranyi et al., 1998; Mackean, 2006). Developmental time from the first instar to adulthood takes an average of 3 to 5 days (Ofuya, 1997b; Mackean, 2006). Feeding and reproduction increase with warm weather. At temperatures of about 11.5°C, nymphs develop into adults in about 22 days. At warmer temperatures of about 28.5°C, development takes only 5 days. Under conditions of abundant food and favourable climate, parthenogenetic apterous adult females are successively produced (Ofuya, 1997b; Dagg, University of Ghana http://ugspace.ug.edu.gh 17 2002; Mackean, 2006). Most nymphs mature into wingless females, but periodically, winged females (alatae) develop and migrate to new host plants (Dixon, 1977). The alatae immigrants reproduce parthenogenically and thereby colonize new plants (Dagg, 2002). Many generations are produced each year. The insect passes through four nymphal instars before reaching adulthood. 2.4.1.6. Management of A. craccivora Aphis craccivora can be controlled by various methods including use of insecticides, cultural practices, biological control and host plant-resistance (Singh and Jackai, 1985: Erbaugh et al., 1995; Jackai and Adalla, 1997; Omongo et al., 1997; Stoddard et al., 2010). The majority of African farmers still rely on indigenous pest control approaches to manage pest problems, although many government extension programs encourage the use of pesticides. However, current pest management research activities carried out by national and international agricultural research programmes in Africa focus on biological control and breeding of resistance host plants (Abate et al., 2000). Aphids are generally susceptible to most insecticides (Hill, 1983). Insecticides that have been reported to be effective against A. craccivora include: carbofuran granules which when applied to the soil gives good control of A. craccivora infestation at the cowpea seedling stage (Jackai and Dacoust, 1986). Foliar application of phosphamidon, dimethoate, thiometon and pirimicarb are effective against the pest (Jackai and Dacoust, 1986). Lambda cyhalothrin, a synthetic pyrethroid, at the rate of 20 g active ingredient ha-1 has been one of the most common insecticides used in Ghana for the control of cowpea aphid (Kusi et al., 2010b). Generally, the use of natural enemies for the control of the insect pests of cowpea has not been given adequate attention (Singh et al., 1990). The potential of biological control is University of Ghana http://ugspace.ug.edu.gh 18 much higher in tropical than in temperate countries due to the high arthropod diversity and year-round activity of natural enemies (Gullan and Cranston, 1994). In nature, A. craccivora is attacked by many parasitoids, predators and pathogens (Booker, 1963; Bohlen, 1978; Jang and Yun, 1983; Singh and Jackai, 1985; Ofuya, 1991). Coccinellid adults and larvae, and syrphid larvae are the most numerous predators. Parasitoids encountered include species in the genera Aphidius, Trioxys and Psyllaephagus. Entomophagous fungi are the main pathogens (Ofuya, 1990; 1995). Ofuya and Akingbohungbe (1988) have shown that Cheilomenes lunata (Fabricius) and Cheilomenes vicina (Mulsant) could be good candidates for the biological control of the aphid in Nigeria. Trioxys indicus (Shubba Rao and Sharma), a hymenopterous endoparasitoid of aphids, has also been reported to show promise as an effective control agent for A. craccivora in India. In the green house, Neozygites fresenii (Nowakowski) Remaudiere and Kellet, an entomophagous fungus, has been observed to kill effectively A. craccivora in all instars especially high densities of aphids under high humidity (Singh and Jackai, 1985). Two common aphid parasites, Lysiphlebus spp. and Diaraetiella spp. have been identified from both the high and low desert areas of California (Summers et al., 2006). Although parasitism as high as 95% has been documented, fields with high aphid infestation can cause significant injury to the plants. From observations made in Malawi, Farrell (1976a, 1976b) concluded that the natural enemies of A. craccivora reduce population densities only after the aphid population starts to decline towards the end of the season, as a result of the deterioration of the host plants. Ofuya (1997a) recommended the conservation approach of judicious use of insecticides to avoid significant natural enemy mortality. This method may include use of less toxic chemicals to the natural enemies, reduction of frequency of University of Ghana http://ugspace.ug.edu.gh 19 applications and reduction of dosage levels. Pirimicarb has been reported as a selective aphicide (Jackai and Dacoust, 1986). 2.4.2. Host-Plant Resistance Host-plant resistance has been variously defined by different authors. Snelling (1941), defined resistance as “including those mechanisms which enable a plant to avoid, tolerate or recover from attacks of insects under conditions that will cause great injury to other plants of the same species”. Painter (1951), defined plant resistance to insect as the amount of heritable qualities possessed by the plant, which influences the ultimate degree of damage done by the insect. Beck (1965), defined plant resistance as “collective heritable characteristics by which a plant species, race, clone or individual may reduce the probability of successful utilization of the plant as a host by insect species, race, biotype or individual. Kumar (1984), also defined resistance as the inherent ability of crop plant to restrict, retard or overcome pest infestation and thereby improve the yield and/or the quality of the harvestable crop product. Crop plants may also avoid damage from a pest species through the mechanism of escape where the sensitive phases of development do not coincide with the optimum conditions for the pest’s development (Cuartera et al., 1999). When a pest cannot establish a compatible relationship under any condition with a certain plant genotype, then the genotype is said to be immune or absolutely resistant to the pest. Resistance shown by non-host plants is termed non-host resistance, basic resistance, or basic incompatibility. Non-host resistant plants can exhibit resistance to their specific pests. If a plant expresses some resistance to all isolates or races of a pest, it has non-race-specific resistance. If it expresses resistance to only one isolate or pest race it has race-specific resistance. University of Ghana http://ugspace.ug.edu.gh 20 From the point of view of the farmer, horticulturalist and others, the use of resistant cultivars represents one of the simplest and the most convenient methods of insect pest control provided that the cultivar does not require expensive input of fertilizer in order to guarantee high yields. 2.4.2.1. Advantages and Disadvantages of Host-Plant Resistance Some of the many advantages of pest control by use of resistant cultivars over control by application of pesticides include (i) the technique is easy to apply because the grower only has to buy seeds of resistant cultivars; (ii) it is relatively inexpensive. In the majority of cases, seed of resistant cultivars is not more expensive than seed of non-resistant cultivars; (iii) completely resistant cultivars need no chemicals for pest control and even partially resistant cultivars need much less to control pests; (iv) resistant cultivars can be incorporated into integrated pest management programmes and when combined with biological control they give a cumulative effect; (v) adverse environmental effects are minimal or nil, pollution is much reduced; and (vi) resistant cultivars, except transgenic cultivars, are acceptable to the public. Some of the disadvantages of resistant cultivars are: (i) it takes a long time to develop a resistant cultivar; (ii) resistant cultivars may control only one pest, while pesticides are often effective against several pests; (iii) resistance must be introduced in each new cultivar; and (iv) the pest may adapt to the resistance and this limits the durability of resistant cultivars. The disadvantages of resistant cultivars are, however, much less than their advantages. Public concerns about the effects of pesticides have compelled governments to make laws to reduce the use of pesticides. The best way to avoid or reduce the use of pesticides in crop University of Ghana http://ugspace.ug.edu.gh 21 production is to introduce integrated pest management techniques that include the use of resistant cultivars. Consequently the prospects for the future development of many more resistant cultivars appear promising. 2.4.2.2. Classification of Host-Plant Resistance In terms of infestation levels and degree of damage, resistance can be classified as Immunity: a variety that cannot be infested or injured at all by specific insect species under any known condition, anything less than immunity is resistance. High resistance: varieties which suffer little damage by a specific insect under a given set of conditions. Low level of resistance: varieties of species which are damaged less by a pest than the average damage for the crop. Susceptible: varieties which show average or more than average damage by an insect pest. Highly susceptible: varieties which are readily infested and suffer considerably more damage than the average by an insect pest under consideration (Painter, 1951; Obeng- Ofori, 2007). Johnson and Law (1975) proposed the term durable resistance to describe long-lasting resistance. Durability does not imply that resistance is effective against all variants of a pest, but that the resistance has merely given effective control for many years in environmental conditions favourable to the pest (Russell, 1978). Where susceptible cultivars are grown, the pest population comprises a set of races in dynamic equilibrium, but one or two of the races will tend to predominate. If a resistant cultivar is introduced, the predominant races either will not propagate, or their propagation rate will be substantially less than normal. In both cases, if one or some races can propagate effectively on the resistant cultivar, their proportions in the pest population will increase because they no longer have competition from the other races. A new outbreak of the pest will occur because the resistance will have been effectively “broken” (Cuartera et al., 1999). University of Ghana http://ugspace.ug.edu.gh 22 It is difficult to determine whether a pest population is composed of a mixture of races, some present in very small proportions, or whether the pest produces virulent mutants that disappear from the pest population unless there is a compatible resistant host plant in which they can propagate (Cuartera et al., 1999). In theory, when the introduced resistance is complete, the predominant races will disappear and more virulent races will spread. The spread will be faster than when the introduced resistance is only partial because the virulent and dominant races will compete (Cuartera et al., 1999). 2.4.2.3. Mechanisms of Resistance Three mechanisms of plant resistance originally defined by Painter (1951) are non- preference (interference with insect behaviour), antibiosis (interference with insect biology) and tolerance. The non-preference has since been replaced by antixenosis (Kogan and Omar, 1978). Antixenosis is the inability of a plant to serve as host to an insect herbivore. The basis of this resistance mechanism can be morphological (e.g. leaf hairs, surface waxes, tissue thickness) or chemical (e.g. repellents or antifeedants). These plants would have reduced initial infestation and/or higher emigration rate of the insect than susceptible plants. Some plant morphological characteristics that can interfere with or modify the behaviour of the insect are colour, shape, type of cuticle wax and the hairiness of plant stalks and leaves (Kogan and Omar, 1978). Antibiosis is the mechanism that describes the negative effects of a resistant plant on the biology of an insect which has colonized the plant (e.g. adverse effect on development, reproduction and survival). Both chemical and morphological plant defences can induce antibiosis effects. The consequences of antibiosis resistance may vary from mild effect that University of Ghana http://ugspace.ug.edu.gh 23 influences fecundity, development time and body size through to acute direct effect resulting in death (Kogan and Omar, 1978). Plant tolerance is the degree to which a plant can support an insect population that under similar conditions would severely damage a susceptible plant. When two cultivars are equally infested, the less tolerant one produces low yield. A tolerant plant may be colonized by a pest to the same extent as susceptible plants, but there is no reduction in yield both in quantity and quality. The usual patterns of insect approach, landing, probing, feeding and egg-laying on a susceptible plant can be disturbed by resistance and induce non-preference or non- acceptance. These disturbances modify the behaviour of the insect and so protect a plant in the initial phase of an attack. Many examples of plant substances with repellent, deterrent or anti-feedant properties are known (Cuartera, et al., 1999). Several groups of toxic, secondary plant compounds like alkaloids, flavonoids and terpenoids may adversely affect the growth, development, generation-time and fertility of the insects. The gene-for-gene interaction produces absolute resistance, or absolute susceptibility, of the host plant against a race of the pest. This race-specific response is termed vertical resistance and is very effective, but only against certain biotypes of a particular pest species. If the resistance is effective against all genotypes of the pest species without differential interaction, the resistance would be race-non-specific or horizontal resistance (Cuartera et al., 1999). University of Ghana http://ugspace.ug.edu.gh 24 2.4.2.4. Resistance of Cowpea to A. craccivora Cowpea aphids are easily controlled by the use of aphid resistant varieties (Obeng-Ofori, 2007). Several aphid-resistant cowpea lines have been identified at the IITA and had been tested against aphid populations from several locations in Africa and Asia (Chari et al., 1976; Dhanorkar and Daware, 1980; Karel and Malinga, 1980; MacFoy and Dabrowski, 1984; Manawadu, 1985; Ofuya, 1988a; 1993). Antibiosis has been shown as the main mechanism responsible for aphid resistance in cowpea (Singh, 1977; Ansari, 1984; Ofuya, 1988b) and is controlled by a single dominant gene (Singh and Ntare, 1985; Bata et al., 1987; Ombakho et al., 1987; Singh et al., 1987; Pathak, 1988). A large number of aphid-resistant lines have been developed and evaluated in international yield trials. These lines, which need no insecticide protection against aphids include, IT8S- 728-5, IT83S-728-13, IT83S-742-2, IT84E-1-108 (Obeng-Ofori, 2007). Others include TVu 36, TVu300, TVu 310, TVu408, TVu410, TVu2996, TVu3000, IT 84S-2246, IT87S- 1459, IT 84S-2049 and IT 93K-503-1 (Bata, et al., 1987; Ofuya, 1997b). Other aphid resistant genotypes include: IT90K-59, IT90K-76, IT97K-499-35 and IT00K-1251 (Singh, 2004). The resistance in genotype IT84S-2246 is the source of resistance in genotypes IT90K-59, IT90K-76, IT97K-499-35 and IT00K-1251 (Singh, 2004). 2.4.2.5. Antibiosis The antibiosis category of plant resistance occurs when the negative effects of a resistant plant affect the biology of an arthropod attempting to use that plant as a host. The antibiotic effects of a resistant plant range from mild to lethal, and may result from both chemical and morphological plant defensive factors (Smith et al., 2004). The effects on an arthropod feeding on a plant with antibiosis mechanism of resistance may be death of the neonate University of Ghana http://ugspace.ug.edu.gh 25 (larva or nymph), reduced food consumption resulting in a lower weight, increased development time, low food reserves, death in pre-pupal or pupal stages, reduced weight of pupae and/or reduced fecundity (Wiseman, 1999). According to Schultz (2002), antibiosis mechanism of resistance is offered by certain endogenously produced compounds like phenolics, jasmonic acid, oxilipins, terpenoids, etc. These compounds are essential for resistance to pests and diseases in plants. The author identified that the resistance of certain varieties of sugarcane to woolly aphid was due to the presence of large quantities of phenolic acid and terpenoids in these varieties. Dahms (1972), illustrated the antibiotic effects of resistant plant on differential rate of aphid development. Nymphs matured in 5 days (susceptible variety), 10 days (intermediate antibiosis) and 20 days (high antibiosis). Mortality of immature arthropods was one of the most important factors limiting the increase of arthropod populations, which was also illustrated by Dahms (1972). 2.5. Application of Molecular Markers in Crop Improvement Many agriculturally important traits such as yield, quality and some forms of disease resistance are controlled by many genes and are known as quantitative traits (also ‘polygenic,’ ‘multi-factorial’ or ‘complex’ traits). The regions within genomes that contain genes associated with a particular quantitative trait are known as quantitative trait loci (QTLs). The identification of QTLs based only on conventional phenotypic evaluation is not possible. A major breakthrough in the characterization of quantitative traits that created opportunities to select for QTLs was initiated by the development of DNA (or molecular) markers in the 1980s (Mc Couch and Doerge, 1995; Mohan et al., 1997; Paterson, 1996a,b). University of Ghana http://ugspace.ug.edu.gh 26 One of the main uses of DNA markers in agricultural research has been in the construction of linkage maps for diverse crop species. Linkage maps have been utilised for identifying chromosomal regions that contain genes controlling simple traits (controlled by a single gene) and quantitative traits using QTL analysis (reviewed by Mohan et al., 1997). The process of constructing linkage maps and conducting QTL analysis to identify genomic regions associated with traits is known as QTL mapping (also ‘genetic,’ ‘gene’ or ‘genome’ mapping) (Mc Couch and Doerge, 1995; Mohan et al., 1997; Paterson, 1996a,b). DNA markers that are tightly linked to agronomically important genes (called gene ‘tagging’) may be used as molecular tools for marker-assisted selection (MAS) in plant breeding (Ribaut and Hoisington, 1998). MAS involves using the presence/absence of a marker as a substitute for, or to assist in phenotypic selection, in a way which may make it more efficient, effective, reliable and cost-effective compared to the more conventional plant breeding methodology. The use of DNA markers in plant (and animal) breeding has opened a new realm in agriculture called ‘molecular breeding’ (Rafalski and Tingey, 1993). DNA markers are widely accepted as potentially valuable tools for crop improvement in rice (Mackill et al., 1999; McCouch and Doerge, 1995), wheat (Eagles et al., 2001; Van Sanford et al., 2001; Koebner and Summers, 2003), maize (Stuber et al., 1999; Tuberosa et al., 2003), barley (Thomas, 2003; Williams, 2003), tuber crops (Fregene et al., 2001; Gebhardt and Valkonen, 2001; Barone, 2004), pulses (Weeden et al., 1994; Svetleva et al., 2003; Kelly et al., 2003), oilseeds (Snowdon and Friedt, 2004), horticultural crops (Mehlenbacher, 1995; Baird et al., 1996, 1997) and pasture species (Jahufer et al., 2002). Some studies suggest that DNA markers will play a vital role in enhancing global food production by improving the efficiency of conventional plant breeding programs (Kasha, University of Ghana http://ugspace.ug.edu.gh 27 1999; Ortiz, 1998). Although there has been some concern that the outcomes of DNA marker technology as proposed by initial studies may not be as effective as first thought, many plant breeding institutions have adopted the capacity for marker development and/or MAS (Lee, 1995; Kelly and Miklas, 1998; Eagles et al., 2001). A thorough understanding of the basic concepts and methodology of DNA marker development and MAS, including some of the terminology used by molecular biologists, will enable plant breeders and researchers working in other relevant disciplines to work together towards a common goal – increasing the efficiency of global food production. 2.5.1. Success in the deployment of aphid resistance loci in soybean improvement Soybean is the second highest cash crop following corn in the United States. Farmers annually produced on average nearly 2.8 billion bushels, valued at more than $15 billion, on 72.4 million acres during the 2000–2002 period (Kim et al., 2008). Most soybeans produced in the United States are used by domestic consumers and the livestock sector, with any remainder exported to foreign consumers. Exports from the 2003 crop were 887 million bushels out of a total crop of 2,454 million bushels, or 36 percent of production (World Agricultural Outlook Board, 2008). However, this valuable crop for U.S. farmers has come under attack by invasive species-the soybean aphid from the North and soybean rust from the South (Livingston et al., 2004, Lee et al., 2006). Soybean aphid is an economically damaging pest in most parts of the North Central United States (McCarville et al., 2013). They are capable of reaching densities of over 1,000 per plant in the field and can reduce soybean yields by 14–40%. Soybean aphids cause damage, including plant stunting, reduced pod and seed counts, and puckering and yellowing of plant leaves. Additionally, soybean aphids are capable of transmitting viruses, including alfalfa mosaic, soybean mosaic, and bean yellow mosaic (Grau et al., 2002). University of Ghana http://ugspace.ug.edu.gh 28 Since its discovery in North America in 2000, economically damaging populations of soybean aphids have developed in parts of Iowa in the past twelve years. It is however not economical to treat soybean aphids with insecticides due to the high cost of insecticides and the high number of soybean aphids that can be found on a single plant (McCarville et al., 2013). Reports suggest that before soybean growers faced severe economic losses from this invasive insect, greater efforts were made to develop new high-yielding seed varieties that are resistant to the soybean aphid. However, without the successful development of soybean aphid resistant varieties through conventional breeding, soybean growers suffered greater economic losses from soybean aphid infestations. There was therefore the need to breed for resistance to soybean aphid through marker assisted selection. These varieties incorporate one or more genes conferring resistance to the soybean aphid. For instance, in 2004, scientists from USDA’s Agricultural Research Service (ARS) and the University of Illinois collaborated on the discovery of genes (Rag1, Rag2, Rag3 and Rag4) which confer resistance to soybean aphids (Suszkiw, 2005, Wang et al., 2005). These genes suppress aphid growth and reproduction causing their populations to develop much slower, often preventing them from reaching economically damaging levels. Soybean aphid-resistant varieties slow the rate at which soybean aphids populations increase. The resistant plants will not be aphid free, but they will have fewer aphids than susceptible plants. This development has set the stage for seed companies to breed for high-yielding cultivars that are resistant to the soybean aphid (Hill et al., 2006). University of Ghana http://ugspace.ug.edu.gh 29 2.5.2. Genetic Markers 2.5.2.1. What are Genetic Markers? Genetic markers represent genetic differences between individual organisms or species. Generally, they do not represent the target genes themselves but act as ‘signs’ or ‘flags’. Genetic markers that are located in close proximity to genes (i.e. tightly linked) may be referred to as gene ‘tags’. Such markers themselves do not affect the phenotype of the trait of interest because they are located only near or ‘linked’ to genes controlling the trait. All genetic markers occupy specific genomic positions within chromosomes (like genes) called ‘loci’ (singular ‘locus’) (Winter and Kahl, 1995; Jones et al., 1997). There are three major types of genetic markers: (1) morphological (also ‘classical’ or ‘visible’) markers which themselves are phenotypic traits or characters; (2) biochemical markers, which include allelic variants of enzymes called isozymes; and (3) DNA (or molecular) markers, which reveal sites of variation in DNA (Winter and Kahl, 1995; Jones et al., 1997). Morphological markers are usually visually characterized phenotypic characters such as flower colour, seed shape, growth habits or pigmentation. Isozyme markers are differences in enzymes that are detected by electrophoresis and specific staining. The major disadvantages of morphological and biochemical markers are that they may be limited in number and are influenced by environmental factors or the developmental stage of the plant (Winter and Kahl, 1995). However, despite these limitations, morphological and biochemical markers have been extremely useful to plant breeders (Weeden et al., 1994; Eagles et al., 2001). DNA markers are the most widely used type of marker predominantly due to their abundance. They arise from different classes of DNA mutations such as substitution University of Ghana http://ugspace.ug.edu.gh 30 mutations (point mutations), rearrangements (insertions or deletions) or errors in replication of tandemly repeated DNA (Paterson, 1996a). These markers are selectively neutral because they are usually located in non-coding regions of DNA. Unlike morphological and biochemical markers, DNA markers are practically unlimited in number and are not affected by environmental factors and/or the developmental stage of the plant (Winter and Kahl, 1995). Apart from the use of DNA markers in the construction of linkage maps, they have numerous applications in plant breeding such as assessing the level of genetic diversity within germplasm and cultivar identity (Weising et al., 1995; Winter and Kahl, 1995; Baird et al., 1997; Henry, 1997; Jahufer et al., 2003). DNA markers may be broadly divided into three classes based on the method of their detection: (1) hybridization-based; (2) polymerase chain reaction (PCR)-based and (3) DNA sequence-based (Winter and Kahl, 1995; Jones et al., 1997; Gupta et al., 1999; Joshi et al., 1999). Essentially, DNA markers may reveal genetic differences that can be visualised by using a technique called gel electrophoresis and staining with chemicals (ethidium bromide or silver) or detection with radioactive or colourimetric probes. DNA markers are particularly useful if they reveal differences between individuals of the same or different species. These markers are called polymorphic markers, whereas markers that do not discriminate between genotypes are called monomorphic markers. Polymorphic markers may also be described as codominant or dominant. This description is based on whether markers can discriminate between homozygotes and heterozygotes. Codominant markers indicate differences in size whereas dominant markers are either present or absent. Strictly speaking, the different forms of a DNA marker (e.g. different sized bands on gels) are called marker ‘alleles’. Codominant markers may have many different alleles whereas a dominant marker has only two alleles. University of Ghana http://ugspace.ug.edu.gh 31 2.5.2.1.2. Marker-Assisted Selection (MAS) Selecting plants in a segregating progeny that contain appropriate combinations of genes is a critical component of plant breeding (Weeden et al., 1994; Ribaut and Betran, 1999). Moreover, plant breeders typically work with hundreds or even thousands of populations, which often contain large numbers (Ribaut and Betran, 1999; Witcombe and Virk, 2001). ‘Marker-assisted selection’ (also ‘marker-assisted breeding’ or ‘marker-aided selection’) may greatly increase the efficiency and effectiveness in plant breeding compared to conventional breeding methods. Once markers that are tightly linked to genes or QTLs of interest have been identified prior to field evaluation of large numbers of plants, breeders may use specific DNA marker alleles as a diagnostic tool to identify plants carrying the genes or QTLs (Michelmore, 1995; Young, 1996; Ribaut et al., 1997). 2.5.2.1.3. The Advantages of MAS Some of the important advantages of MAS include the following: •Time saving from the substitution of complex field trials (that need to be conducted at particular times of year or at specific locations, or are technically complicated) with molecular tests • Elimination of unreliable phenotypic evaluation associated with field trials due to environmental effects • Selection of genotypes at seedling stage • Gene ‘pyramiding’ or combining multiple genes simultaneously • Avoiding the transfer of undesirable or deleterious genes (‘linkage drag; this is of particular relevance when the introgression of genes from wild species is involved) • Selecting for traits with low heritability University of Ghana http://ugspace.ug.edu.gh 32 • Testing for specific traits where phenotypic evaluation is not feasible (e.g. quarantine restrictions may prevent exotic pathogens to be used for screening) 2.5.2.1.4. Cost/Benefit Analysis of MAS The cost of using ‘tools’ in breeding programs is a major consideration. The cost of using MAS compared to conventional plant breeding varies considerably between studies. Dreher et al. (2003) indicated that the cost effectiveness needs to be considered on a case by case basis. Factors that influence the cost of utilizing markers include: inheritance of the trait, method of phenotypic evaluation, field/glasshouse and labour costs, and the cost of resources. In some cases, phenotypic screening is cheaper compared to marker-assisted selection (Bohn et al., 2001; Dreher et al., 2003). However, in other cases, phenotypic screening may require time-consuming and expensive assays, and the use of markers will then be preferable. Some studies involving markers for disease resistance have shown that once markers have been developed for MAS, it is cheaper than conventional methods (Yu et al., 2000). In other situations, phenotypic evaluation may be time-consuming and/or difficult and therefore using markers may be cheaper and preferable (Young, 1999; Yu et al., 2000; Dreher et al., 2003). An important consideration for MAS, often not reported, is that while markers may be cheaper to use, there is a large initial cost in their development. An estimate for the cost to develop a single SSR marker was AUD$ 100,040 (Langridge et al., 2001). 2.5.2.1.5. Marker-Assisted Backcrossing Using conventional breeding methods, it typically takes 6–8 backcrosses to fully recover the recurrent parent genome. The theoretical proportion of the recurrent parent genome after n generations of backcrossing is given by: (2n+1−1)/2n+1 (where n = number of University of Ghana http://ugspace.ug.edu.gh 33 backcrosses; assuming an infinite population size). The percentages of recurrent parent recovery after each backcross generation are presented in Table 2.3. The percentages shown in Table 2.3 are only achieved with large populations; the percentages are usually lower in smaller population sizes that are typically used in actual plant breeding programmes Table 2.3. Percentage of recurrent parent genome after backcrossing Generation Recurrent parent genome (%) BC1 75.0 BC2 87.5 BC3 93.8 BC4 96.9 BC5 98.4 BC6 99.2 Although the average percentage of the recurrent parent genome is 75% for the entire BC1 population, some individuals possess more of the recurrent parent genome than others. Therefore, if tightly-linked markers flanking QTLs and evenly spaced markers from other chromosomes (i.e. unlinked to QTLs) of the recurrent parent are used for selection, the introgression of QTLs and recovery of the recurrent parent may be accelerated. This process is called marker-assisted backcrossing. The use of additional markers to accelerate cultivar development is sometimes referred to as ‘full MAS’ or ‘complete line conversion’ (Ribaut et al., 2002; Morris et al., 2003). Simulation studies using PLABSIM (a computer program that simulates recombination during meiosis) indicate that efficiency of recurrent parent recovery using markers is far University of Ghana http://ugspace.ug.edu.gh 34 greater compared to conventional backcrossing (Frisch et al., 1999; Frisch and Melchinger, 2000). Therefore, considerable time savings can be made by using markers compared to conventional backcrossing. Although the initial cost of marker-assisted backcrossing would be more expensive compared to conventional breeding in the short term, the time savings could lead to economic benefits. This is an important consideration for plant breeders because the accelerated release of an improved variety may translate into more rapid profits by the release of new cultivars in the medium to long-term (Morris et al., 2003). University of Ghana http://ugspace.ug.edu.gh 35 CHAPTER THREE 3.0. Introduction Following the release of two early maturing varieties Apagbaala (ITX P48-2) and Marfo- Tuya (Sul 518-2) by the Savanna Agricultural Research Institute (Padi et al., 2004), several advanced breeding lines were generated from crosses between these adapted parents and exotic lines. These very popular varieties have been found to be susceptible to aphid infestation in the field. To further improve on the yield and agronomic characteristics of these cultivars, each genotype was crossed with an exotic line as Apagbaala × UCR 01-11- 52 and UCR 01-15-127-2 × Marfo-Tuya. Ten advanced breeding lines (F6) from the Apagbaala × UCR 01-11-52 population and six from the UCR 01-15-127-2 × Marfo-Tuya population were selected as lines with the highest yield potential in northern Ghana. Information on the reaction of these lines to field pests was, however, lacking. A study was therefore carried out to evaluate the reaction of the 16 advanced breeding lines and their adapted parents to infestation by the cowpea aphid. A local variety in northern Ghana, SARC-L02, and three varieties developed by the International Institute of Tropical Agriculture (IITA) were used as controls. At least the IT 97 K-499-35 variety from the IITA was known to have been developed with emphasis on resistance to the cowpea aphid (Singh 2004). Progress has been achieved in identifying a source of resistance to the cowpea aphid, Aphis craccivora in an advanced breeding cowpea line and in identifying a marker linked to the resistance locus. The resistance gene imparts antibiosis to the cowpea plant such that the aphid’s fecundity is significantly reduced when fed in a no-choice experiment on resistant plants (Kusi et al., 2010a). With the progress so far made, it is now possible to use this gene to improve upon the field resistance of existing cowpea cultivars in Ghana. University of Ghana http://ugspace.ug.edu.gh 36 3.1. Materials and Methods 3.2. Selection of Resistant and Susceptible Progenies The resistant genotype, SARC 1-57-2, was crossed to the susceptible genotype, Apagbaala. The susceptible parent served as the mother parent whilst the resistant genotypes served as the male in the cross. The F1, F2 and F2-3 seeds from the cross were developed in the screen house of the Council for Scientific and Industrial Research-Savannah Agricultural Research Institute (CSIR-SARI), Nyankpala. The seeds of parental F1 and F2 populations were sown in plastic buckets containing sandy loam soil from the experimental field of SARI and kept in the insectary. Three to four days after emergence, each seedling was infested with five, four-day old nymphs using camel hair brush (Bata et al., 1987; Githiri et al., 1996; Kusi et al., 2010a). On the eighth day after infestation, the genotypes were classified into various levels of resistance based on percentage of dead or heavily damaged seedlings (Bata et al., 1987; Githiri et al., 1996; Kusi et al., 2010a). In order to get seeds from the non-segregating susceptible progenies, seedlings that had shown symptoms of susceptibility to the aphid attack were rescued by applying lambda cyhalothrin (Lambda Super®), a synthetic pyrethroid, at the rate of 20 g active ingredient ha-1 6 and 7 days after infestation to control the aphids. The rescued susceptible seedlings were maintained to produce non-segregating susceptible seeds. The resistant seedlings that survived the progeny test of the F2 population were maintained to generate F3 seeds which were also screened using the same method described above. The screening of the F3 seedlings which were generated from the resistant seedlings from the F2 screening were further tested for their reaction to the aphid using procedures as outline above. Seedlings killed by factors other than aphid infestation such as disease infection were removed before University of Ghana http://ugspace.ug.edu.gh 37 the seedlings were classified into resistant and susceptible groups. Remnant seeds of the resistant F3 segregants were kept for further analysis. 3.3. Identification of DNA Marker(s) Linked to the Cowpea Aphid Resistance Gene A total of 50 DNA markers were screened and these are presented in Table 3.1. Table 3.1. SSR primers used and their sequences NAME SEQUENCE ANNEALING TEMPERATURE NAME SEQUENCE ANNEALING TEMPERATURE MS3F GTTGGCTTCTGTTGTGG CAT 56 Y26F CTAAATTATAATATTCGT CGGTC 52 MS3R GTTACACCAATGCCAA AAAC Y26R GGTTAAGGAAAAGAGGG TAGG MS26F TGCGGTTGAGATTTTGA CGT 56 Y31F CTATTGGAATCTTGCCGT TG 56 MS26R CGTGAAGTTGAATGTG AAT Y31R CTTTACCTTTATGCAAAC CAATTC SM29F TTGATTAGTTGGCTCTT AGGGGC 56 Y45F CGATTATCCTGGCTAAC GATG 56 MS29R GAGGACTTAATTAGAA CAAACTTTG Y45R GGATCTGAGATAGTGTG AC MS31F GTGACTACAATGGCGG AACT 56 CP115F GGGAGTGCTCCGGAAAG T 56 MS31R GGAGGTACCGAAAAGA AAG CP116R TTCCCTATGAACTGGGA GATCTAT Kad1F TAGACCAGATGACATT GTAATTC 52 CP117F GTGGAAGGAATGGGTCC AG 56 Kad1R GTCGTAACTGGGCACA ATAG CP118R AGGAAATTTGCATTCCCT TGT Kad26F CCCAAAATCCTGACCC ACTA 56 CP163F CACTTTCTCCTAAGCACT TTTGC 56 Kad26R TTTTCACGATGGAACAG TGC CP164R AAGTGAAGCATCATGTT AGCC Kod7F CTAGAACGTTCCATCTT AATATTAC 55 CP171F GTAGGGAGTTGGCCACG ATA 56 Kod7R TAACTATTTAAAGATGA TTTC CP172R CAACCGATGAAAAAGTG GACA KOD17F CTTATCGTGATAACATG TATTT 55 CP181F GGGTGCTTTGCTCACATC TT 56 KOD17 R CAACAAGTATAAAATG AGTGTGAG CP182R TCCATGTGTTTATGACGC AAA Kod21F GACTTTTACATTTATTA CATG 52 CP197F TGAATGGAGCAACTTCT TGGA 56 Kod21R TAATCACCATACCCACT CTAC CP198R GTTGCACTGGTTGCCCTA T MS43F CATTTTTTAATCCATTT TTATC 52 CP201F GGTTTCCTAGTTGGGAA GGAA 56 MS43R AAGTTTTTAGGGGCTAT GGC CP202R ATTATGCCATGGAGGGT TCA MS50F TTTAAAATGGTCCCTCC CGT 56 CP215F CAGAAGCGGTGAAAATT GAAC 56 MS50R CCTAAACGAATTCTACC TGG CP216R GCATGTTGCTTTGACAAT GG MS52F CCAGCAGTATATACAT AAGA 50 CP239F CACCCCCGTACACACAC AC 56 MS52R GCAAGCCAAGACAAAA TAGTG CP240R CACTTAAATTTTCACCAG GCATT MS53F GAAAGTATATGTTGTTA ACTCT 50 CP333F CAAAGGGTCATCAGGAT TGG 56 MS53R AAGAGTGACAAGAAAG ATTT CP334R TTTAAGCAGCCAAGCAG TTGT MS98F GATAAAGAGGAAAATA GACA 50 CP359F TGAAAACAACGATATGC AGAAG 56 MS98R AAAATGTGGCAGATAA GGAA CP360R TCAGTCTTAGAATTGATG GGGCTTCG University of Ghana http://ugspace.ug.edu.gh 38 Table 3.1 Continued MS111F TAATAAAGCAAAGATG GTCG 50 CP391F TGCTATGCTTATGCCTGT G 56 MS111R AATAACATAATAACGC GTGC CP392R GATGCCTGTTACTTGCCT TCT MS113F GTTAAAGTTTTCTTCAT CAT 50 CP393F GCGGATGAAATTACGAT AAAACA 56 MS113R ATCTTGATCCAGAAAAT GTTT CP394R GTGCAGAACAATGCAAA GGA MS120F TTTCTAGGCAGTGAAG ATAATCA 50 CP395F GTTGTGAGCTTCCCCAG ATG 56 MS120R AAACAAAATACCAACT ACCA CP396R AATTTTGAACCCACCAC CAG MS121F GGTTGTTCCGAAAAACT TATACG 50 CP397F TCATGGGTTAAATTTGCT TCAA 56 MS121R GATAGAAGTTTTAACAT TACTC CP398R AAACCATGTGGTTGTTG CAC MS128F CGTAATTTGTAATGTGT AGG 50 CP403F TGCAATATGGACCAGAA GAAA 56 MS128R AACCCTAAACAAACTTT TGGTAG CP404R ATGCCCCAACAACAACA TTT MS138F AACACATGGATAACAG AAAT 50 CP431F CCTCAACACCTTTTGGAA GGA 56 MS138R GATCTCGTCCACAAAC AACA CP432R CAAATGCACCTCCTGTG CTA MS143F ATGTTTCAGATCGGTTT AGA 52 CP433F CAACTTCACAGCCCTCA A 56 MS143R GAGCTGAAAAAATCGG TGTC CP434R TTGAAGGTATGGCCTTTT GTTT MS144F GTGAGTTTAATGACATT TAC 50 CP435F TGCTCATCGTGCTTTGTC TT 56 MS144R GACTGCTATGTCATAAT ATT CP436R CACTTCAGACTTAGAGC GAAGAA Y16F CACATTAACTCAAGTCC ACACC 52 CP443F GCTCGGATATGGTCCTG AAA 56 Y16R CCAGTGAAATCATGTC AAAT CP444R TCAGTGTCAGCACCATC CC Y1F GATATAGAATAGCATA TTTAACATATTAG 52 CP573F CAGAATCCTTGTGAACC TG 56 Y1R GTTGAAAGTTTGATAGT AAAGTGG CP574R TTTCGCAATATGCCCTTT TC Y21F GAGAACTTCACGCACA ATAG 56 CP605F AAAGAGATACACATGCC TAACA 56 Y21R CGCGGTAGCATGATTG AATTTTG CP606R GACCAACAGCGACTTTG AGC 3.3.1. FTA Protocol The DNA from the samples used for the present study was extracted using fast technology for analysis (FTA) of nucleic acids card supplied by Kirkhouse Trust following the methodology described below: The Leaf was cleaned with ethanol and then was placed over the marked circle (underside of the leaf facing down) on top of the FTA matrix card. The leaf was overlaid with parafilm and a small porcelain pestle was used to apply moderate pounding for 15 seconds over each sample circle area to burst the cell walls of the plant tissue. To visually verify that the plant material has transferred sufficiently into the FTA card matrix, the back of the FTA card University of Ghana http://ugspace.ug.edu.gh 39 matrix was checked to see if the plant tissue was being drawn through the matrix. When plant tissue transfer was complete, the FTA card was air dried for a minimum of one hour at room temperature. The FTA matrix card was placed on FTA sample mat and using 2.0 mm harries micro punch tool, a disc was removed from the centre of the dried sample area into1.5 ml microfuge tube. Care was taken when transferring the dry FTA discs into the microfuge tube because the static charge that could develop on some plastic laboratory ware could repel the discs. Alcohol was used to wipe the punch tip between samples. The disc in each tube was washed with 70 % ethanol for 5 minutes; the washing with ethanol was repeated until the disc turned white. About 200 µl of FTA purification reagent was added to each tube, capped, inverted twice and incubated for 4-5 minutes at room temperature. After the incubation the FTA reagent was pipetted up and down twice, to ensure that the disc remained in the tube. A pipette was used to remove and discard as much of the reagent as possible. This was repeated for two FTA reagent washes. The discs were allowed to completely air dry for a minimum of one hour at room temperature. Whenever the PCR amplification could not be conducted within three hours of discs drying, the discs were stored at 4 °C or -12 °C. This was due to the fact that the DNA purification process removed the protective chemistry of the FTA technology. 3.3.2. PCR Amplification A PCR amplification mix containing Top DNA Polymerase, dNTPs, reaction buffer, tracking dye, and patented stabilizer and was obtained from Bioneer. About 20 µl of the complete PCR amplification mix was added directly to the PCR tube containing the dried disc, assuming the DNA volume used was zero. Each of the 50 DNA markers was used to run the set of DNA samples extracted from the resistant parent (SARC1-57-2), susceptible parent (Apagbaala) and the resistant and susceptible progenies. PCR amplification was University of Ghana http://ugspace.ug.edu.gh 40 carried out in ABI 2720 thermal cycler (Applied Biosystems). PCR conditions consisted of denaturing at 94°C for 3 minutes, annealing at temperatures (Table 3.1.) for each primer for 30 seconds and extension at 72°C for 30 seconds. This cycle was repeated 35 times and final extension at 72°C for 10 minutes. The PCR products were further run on horizontal polyacrylamide gel electrophoresis (h-PAGE) (81-2325 by Galileo Biosciences, dimension of tank: 32 cm W x 37.5 cm L x 10.5 cm H; dimension of plate: 24.5 cm W x 27.5 cm L) to separate and resolve the bands with the protocol indicated below. The 5% acrylamide gel was prepared as shown in Table 3.2. Table 3.2. Preparation of 100 ml 5% acrylamide gel Reagent Volume 40% acrylamide solution 12.5 ml 5X TBE Buffer 20 ml 10% Ammonium persulphate (APS) 0.7 ml Distilled Water 66.68 ml TEMED 0.12 ml Total Volume 100 ml 3.3.3 Casting the Gel Gel was cast in a tray (27.5 cm 24.5 cm) with barriers to retain acrylamide and a 50 well- forming combs were inserted to create wells. A lid was used to cover the tank with the gel to prevent oxygen inhibition of polymerisation. The 100 ml volume required to fill the tank was determined by weighing the tank before and after filling with water. The monomer and University of Ghana http://ugspace.ug.edu.gh 41 catalyst mix were prepared as shown in the Table 3.1. The mix was poured into the tank and distributed across the whole surface, removing bubbles. The comb was inserted, the lid was laid on the tank and lowered carefully and pressed gently against the comb. This was allowed to polymerise. The whole assembly was transferred into electrophoresis tank and the comb was removed when the assembly was submerged in buffer (3-5 mm above the lid). The PCR products were loaded into the wells. During loading care was taken to avoid the ‘skirt.’ of polyacrylamide that might fall into the well. The lid was left in place during loading and electrophoresis. The gel was run at to at least half way to the end of the glass and a spatula was used to prise off the lid after running the gel. The gel was stained with a solution of ethidium bromide, 0.5 µg/ml for 30 minutes using the same volume used to make the gel. The gel was photographed under Ultraviolet light. Polymorphic primer pairs were noted for further analysis. 3.4. Testing the Reliability of the Marker, CP 171F/172R, in F2 Segregating Population Upon identification of the marker CP 171F/172R as being polymorphic out of the 50 markers, the marker was tested for its effectiveness in selecting aphid resistant lines out of a large segregating F2 population. The resistant parent (SARC1- 57-2) was crossed to the susceptible parent (Apagbaala) to generate F1 population. The F1 population was advanced to F2 population by selfing. A total of 169 F2 plants were screened for reaction to aphid infestation. The 169 seeds were planted individually in plastic pots in the screen house and each plant was labeled. Leaf samples were taken from each plant after emergence for DNA extraction using FTA paper. Each plant was subsequently infested with five, four-day old aphids. After the infestation, the aphids were monitored within 24 hours to ensure that the five aphids settled on each plant. University of Ghana http://ugspace.ug.edu.gh 42 The resistant and the susceptible parents were also planted with the F2 population, and after every 10 individuals of F2 lines, the resistant and the susceptible parents were planted in the experimental set up. The infested plants were observed until about 90% of the plants of the susceptible parent were killed by the aphids. The experiment was terminated and the numbers of dead and live seedlings of the F2 lines were recorded. The ratio of dead seedlings (susceptible) to the live seedlings (resistant) was determined. Chi square test was carried out to determine the goodness of fit. 3.5. Genetic Analysis The FTA cards with the leaf samples were sent to the Kirkhouse Trust Mobile Molecular Laboratory stationed at the Cocoa Research Institute of Ghana (CRIG) for genetic analysis. The experimental procedure described above was used to wash the FTA cards, run the PCR using the marker CP 171F/172R and the PCR product was run on h-PAGE as described above to separate and resolve the bands. The bands were photographed and classified in relation to the band pattern of the resistant and the susceptible parents. 3.6. Introgression of Aphid Resistance Locus into Ghanaian Cowpea Cultivars 3.6.1. Polymorphism Test Polymorphism test was conducted to determine whether the marker CP 171F/172R could separate the resistant parent and four cowpea cultivars recommended for Northern Ghana. The cultivars were Padituya (SARC3-122-2), Zaayura (SARC4-75), Songotra (IT97K-499- 35) and Bawutawuta (IT95K-193-2). DNA samples were taken from the leaves of the four (4) cultivars together with the resistant and susceptible parents. The samples were taken two weeks after planting using FTA card. The standard procedure detailed in Sub-sections 3.2.1- 3.2.4 was used to wash the FTA cards and the PCR was run using the marker CP 171F/172R. University of Ghana http://ugspace.ug.edu.gh 43 The PCR products were run on a non-denaturing h-PAGE II. The photographed bands of the parents, and the four cultivars were analysed to determine which of the cultivars is polymorphic with SARC 1-57-2 for the marker CP 171F/172R, to enable it to be improved for aphid resistance in a marker assisted backcrossing. 3.6.2. Marker Assisted Backcrossing The following procedures were followed to introgress the cowpea aphid resistance locus into the cowpea cultivar Zaayura. 3.6.2.1. Development and Advance of Backcross Progenies From a cross between SARC1-57-2 and Zaayura, the F1 was backcrossed to Zaayura (the recurrent parent) to generate 20 BC1 lines. All individuals were genotyped to select heterozygous plants. These plants were screened in the screen house to confirm their reaction to the aphid. Three resistant BC1 plants were used for backcrossing to the recurrent parent to generate BC2 individuals. This cycle of crossing, identification of heterozygous lines using SSR marker, CP 171F/172R, and screen house confirmation of resistance was followed till BC4 plants were obtained. A heterozygous BC4F1 plant was selfed to generate 100 BC4F2 lines. These were genotyped with the CP 171F/172R to select plants that were homozygous dominant for the region of the resistance locus. Twenty-five BC4F2 plants carrying homozygous resistant alleles based on marker CP 171F/172R were screened further with the aphid to confirm resistance. Five plants showing resistance to the aphid were selected for multiplication. This was bulked for larger scale field testing. University of Ghana http://ugspace.ug.edu.gh 44 3.7. Determining the Stability of the Aphid Resistance Locus Across the Major Cowpea Belts in Ghana The cowpea aphids were sampled from cowpea plants in farmers’ fields in six cowpea growing regions in Ghana. These were Upper East, Upper West, Northern, Brong Ahafo, Central and Volta Regions (Table 3.2). Based on the seedling screening technique developed during the course of this research, the resistant lines SARC1-57-2 and SARC1-91-1, and IITA line IT 97K-499-35 together with known susceptible varieties (Apagbaala and Zaayura) were tested in each region with aphids in the region. Each of the cowpea lines were replicated 9 times in each experimental setup. The reaction of the test genotypes informed the possible existence of biotypes within A. craccivora in Ghana. University of Ghana http://ugspace.ug.edu.gh 45 Table 3.3. Locations in the six regions where cowpea aphids were sampled Region Areas within the region GPS Coordinates Upper East Bawku Navrongo Sakote 11°03′N 0°14′W 10°53′5″N 1°5′25″W 10° 44′ N 0°36′247″W Upper West Nandom Tumu Wa 10°51′00″N 2°45′00″W 10°53′N 1°59′W 10°04′N 02°30′W Northern Nyankpala Walewale Yendi 9° 24' 0"N 0° 59' 0"W 10° 21' 0"N 0° 48' 0"W 9°25′57″N 0°0′15″W Volta Kpeve Nkwanta Sogakope 6°41′1″N 0°20′1″E 8°16′N 0°31′E 6°00′N 0°36′E Central Asowanzi Mankesim UCC 5°53′0″N 1°13′0″W 5°16′N 1°01′W 5°06′N 1°15′W Brong Ahafo Kintampo Nkoranza Wenchi 8°3′8″N 1°44′5″W 07°34′00″N 01°42′00″W 07°34′38″N 01°55′45″W A 5-point scale of score of seedling vigour was used where: 1 = dead seedling due to aphid damage, 2 = seedling with weak stem and leaves with symptoms of aphid damage, 3 = seedling showing symptoms of aphid damage, 4 = seedling with aphids without symptoms of damage and 5 = seedling with no aphid (as in control pots) (Kusi, 2008). University of Ghana http://ugspace.ug.edu.gh 46 CHAPTER FOUR 4.0. Stability of the cowpea aphid resistant genotype across the major cowpea growing zones in Ghana 4.1. Introduction Cowpea, Vigna unguiculata (L.) Walpers is cultivated mostly in tropical Africa and the edible seeds constitute a major source of protein in the human diet (Diouf and Hilu, 2005; Kamara et al., 2010). Cowpea is the most important in the genus Vigna in terms of planting area. Production area of cowpea is about 14 million hectares worldwide and annual global production of cowpea is approximately 3.3 million tons (CGIAR, 2011). West and Central Africa is the leading cowpea producing region in the world, producing about 64%. Nigeria is the largest producer and consumer, and accounts for 61% of production in Africa and 58% worldwide (Quin 1997; IITA, 2009). A major biological constraint to the production of cowpea in Africa is severe infestation and damage by various insect pests in the field and during storage (Kamara et al., 2007). The cowpea aphid, A. craccivora Koch is considered to be an important field pest of cowpea in Africa, Asia and Latin America (Kusi et al., 2010a; Benchasri et al., 2012; Kamphuis et al., 2012). It is the most important worldwide pest of cowpea causing significant yield losses when either young seedlings or the pods of adult plants are attacked (Annan et al., 2000). Singh and Allen (1980) estimated yield losses of 20% to 40% in cowpea due to A. craccivora infestation in Asia and up to 35% in Africa. Cowpea aphid can be controlled by various methods including use of insecticides, cultural practices and biological control (Erbaugh et al., 1995; Jackai and Adalla, 1997; Omongo et al., 1997; Stoddard et al., 2010). Public concerns about the effects of pesticides have stimulated the search for more environmentally safe methods such as the use of host plant resistance to control the pest. University of Ghana http://ugspace.ug.edu.gh 47 Host plant resistance is easy to apply, relatively inexpensive, needs no chemicals for pest control, and can be incorporated into integrated pest management programmes. When combined with biological control it gives a cumulative effect. The use of resistant cultivar minimises or eliminates adverse environmental effects caused by pesticides and is generally acceptable to the public. Antibiosis has been shown as the main mechanism responsible for aphid resistance in cowpea (Ansari, 1984; Ofuya, 1988b; Singh, 1977; Laamari et al., 2008) and is controlled by a single dominant gene (Bata et al., 1987; Ombakho et al., 1987; Singh, et al., 1987; Pathak, 1988; Nualsri et al., 2012). The dominance nature of aphid resistance genes in cowpea means that resistant progeny can easily be identified in segregating populations, thus making selection in a breeding process easy (Githiri et al., 1996). However, the major problem with single gene inheritance is that insects can develop biotypes very fast which could overcome the resistant cultivars (Githiri et al., 1996). This problem can easily be encountered with aphids which have a very short life cycle and reproduce parthenogenetically (Githiri et al., 1996). Three biotypes of A. craccivora have been reported from West Africa where cowpea is widely grown (IITA, 1981). Biotypes A and B occur in Nigeria and Biotype K in Upper Volta (Burkina Faso) (IITA, 1981). Kusi et al. (2010a) found a cowpea line, IT97K-499-35 known to be resistant in Nigeria (Singh, 2004) to be susceptible to aphids in Ghana. They attributed this to the possible existence of a biotype of A. craccivora in Ghana or in the Guinea Savannah zone of Ghana that is more virulent than the biotype that existed in Nigeria where IT97K-499-35 was developed and evaluated earlier. Other researchers have reported the presence of more aggressive aphid biotypes in Burkina Faso and other West African countries than in Nigeria University of Ghana http://ugspace.ug.edu.gh 48 (Martyn, 1991; van Emden, 1991; Ofuya, 1997a). The California types of cowpea aphids that are not controlled by the ‘IITA’ type of aphid resistance have also been reported (Messina et al., 1985). In related studies of Chari et al. (1976), Ombakho et al. (1987) and Martyn (1991), it has been reported that at least three distinct biotypes of the cowpea aphid may occur in Africa and Asia, and another biotype occurring in the United States and they all require different resistance genes to control them. SARC 1-57-2 and SARC 1-91-1 were identified as resistant to A. craccivora among 22 cowpea genotypes evaluated with seedling screening method (Kusi et al., 2010a). SARC 1- 57-2 significantly recorded the least percentage of seedlings killed by aphids among the 22 genotypes eight days after aphid infestation. SARC 1-57-2 was again evaluated among nine other genotypes to assess the rate of growth and reproduction of aphids on each of these genotypes. Significantly, SARC 1-57-2 recorded the least number of aphids per seedling at 3, 6 and 9 days after infestation (Kusi et al., 2010a). The differential resistance response exhibited by cowpea lines developed with resistance to aphid in different growing eco-regions raised the concern for the existence of biotypes of the insect that may require different resistance genes. The objective of this study was therefore is to test for the stability of the aphid resistance gene in SARC 1-57-2 in all the major cowpea growing zones in Ghana. The reaction of the test genotypes would inform the possible existence of biotypes within A. craccivora in Ghana. 4.2. Materials and Methods The experiments were carried out between July 2011 and January 2012. The experiments were conducted in six Regions in Ghana namely, Upper East, Upper West, Northern, Brong Ahafo, Central and Volta (Table 4.1). University of Ghana http://ugspace.ug.edu.gh 49 Table 4.1. Characteristics of study areas in the six regions Region Areas within the region GPS Coordinates Agro- ecological Zone Rainfall (mm/yr) Length of growing Season (Days) Dominant land use Main food crops Upper East Bawku Navrongo Sakote 11°03′N 0°14′W 10°53′5″N 1°5′25″W 10° 44′ N 0°36′247″W Sudan Savannah 1000 150-160 Annual food crops Sorghum , millet, cowpea , maize Upper West Nandom Tumu Wa 10°51′00″N 2°45′00″W 10°53′N 1°59′W 10°04′N 02°30′W Guinea Savannah 1100 180-200 Annual food crops, cash crops and livestock maize, yam, Groundn ut sorghum, millet, cowpea Northern Nyankpala Walewale Yendi 9° 24' 0"N 0° 59' 0"W 10° 21' 0"N 0° 48' 0"W 9°25′57″N 0°0′15″W Guinea Savannah 1100 180-200 Annual food crops, cash crops and livestock Ground nut maize, yam, Sorghum , millet, cowpea Volta Kpeve Nkwanta Sogakope 6°41′1″N 0°20′1″E 8°16′N 0°31′E 6°00′N 0°36′E Coastal Savannah/ Transitional 800-1300 Major season: 100-160 Minor season: 50- 90 Annual food crops, cash crops livestock Maize, roots, cowpea Central Asowanzi Mankesim UCC 5°53′0″N 1°13′0″W 5°16′N 1°01′W 5°06′N 1°15′W Coastal Savannah 800 Major season: 100-110 Minor season: 50 Annual food and cash crops Maize, roots and cowpea Brong Ahafo Kintampo Nkoranza Wenchi 8°3′8″N 1°44′5″W 07°34′00″N 01°42′00″W 07°34′38″N 01°55′45″W Transitional 1300 Major season: 150-160 Minor season: 90 Annual food and cash Crops Maize, root and tuber, cowpea. Ground nut University of Ghana http://ugspace.ug.edu.gh 50 The cowpea genotypes used for the study were SARC1-57-2, SARC1-91-1, Apagbaala, IT97K-499-35 and Zaayura (Table 4.2). Table 4.2. Description of the five genotypes of cowpea by parentage or source Genotype Description by pedigree or source Apagbala (control) Prima/TVu 4552/California Black eye No.5/7977. Cultivar, released in 2002 in Ghana. Largely of exotic background SARC 1-57-2 Apagbaala/ UCR 01-11-52. Breeding line of SARI SARC 1-91-1 Apagbaala/ UCR 01-11-52 Breeding line of SARI IT97K-499-35 Breeding line from the IITA, Ibadan Nigeria. Cultivar, released in 2008 in Ghana as Songotra. Zaayura Marfo-Tuya/ UCR 01-15-127-2. Cultivar, released in 2008 in Ghana. Each Region was zoned into 3 areas to provide 18 test locations. Aphid sampling and screening were carried out as described in (3.6). Screening was done in a fabricated mobile screen house (Fig. 4.1) which could easily be dismantled and conveyed from one location to the other. Three mobile screen houses were constructed for the study so that the experiments in each of the three locations in a Region were conducted concurrently. The seedling screening method (Bata et al., 1987; Githiri et al., 1996; Kusi et al., 2010a) was used to evaluate the cowpea genotypes. The aphids from the farmers’ field were further sub-sampled with soft painter’s brush to remove all predators that were sampled with the aphids from the field. The predator free sub-samples were used to infest the cowpea seedlings at 3-4 days after emergence. University of Ghana http://ugspace.ug.edu.gh 51 The experimental design used was the completely randomised design with nine replications. The seedlings were raised in plastic containers measuring 20 cm deep and 20 cm wide. Each replication consisted of ten seedlings of the cowpea variety. Mobile Screen House Fig. 4.1. The Kirkhouse Trust Mobile Screen house 4.3. Data Collection and Analysis Data collected included the percentage of seedlings killed and seedling vigour 10 days after infesting the seedlings with aphids. Seedling vigour was rated on a five-point scale (Kusi, 2008) where: 1 = dead seedling due to aphid damage, 2 = seedling with weak stem and leaves with symptoms of aphid damage, 3 = seedling showing symptoms of aphid damage, 4 = seedling with aphids without symptoms of damage and 5 = seedling with no aphid. University of Ghana http://ugspace.ug.edu.gh 52 The data were subjected to analysis of variance (ANOVA) using Genstat statistical program (9th edition). Fisher’s LSD was used to separate the means after ANOVA showed significant differences. 4.4. Results There were no significant interactions between the cowpea varieties and the zones within the regions where the studies were conducted in terms of seedling mortality and plant vigour score. The zone main effect also did not show significant difference across the regions for seedling mortality and plant vigour score. Data were therefore presented for the cowpea variety main effects for seedling mortality and plant vigour. 4.4.1. Seedling Mortality Significant differences were observed among the cowpea varieties for the percentage seedling mortality at each of the 18 locations (Table 4.3). Significantly (P < 0.001), SARC 1-57-2 recorded the least number of seedlings killed by the aphids across the regions and locations. On the other hand, Apagbaala, IT97K-499-35 and Zaayura recorded significantly higher number of seedlings killed by the aphids. At all the 18 locations, the three genotypes maintained their susceptibility to aphids. Apagbaala and IT97K-499-35 recorded as high as 96% seedlings death with Zaayura recording 85% mortality 10 days after infestation. 4.4.2. Seedling Vigour Similar to the results on the mortality of seedlings, the vigour of the varieties 10 days following aphid infestation was highest in SARC 1-57-2 and least in Apagbaala and IT97K- 499-35 (Table 4.3). SARC1-57-2 and SARC 1-91-1 maintained average vigour score of 3.8 and 3.4, respectively across the locations. The less vigorous genotypes were Apagbaala and IT97K-499-35 and Zaayura with average score of 1.16, 1.16 and 1.89, respectively. The University of Ghana http://ugspace.ug.edu.gh 53 general responses of the resistant and the susceptible lines due to the aphid infestation as manifested in the leaves colour and plant growth is represented in Fig. 4.2. Table 4.3. Mean seedling mortality and plant vigour score following aphid infestation on five cowpea genotypes at 18 locations in Ghana Variety % Seedling killed Seedling vigour score Apagbaala 96.17 1.167 SARC1-57-2 3.70 3.802 SARC1-91-1 7.99 3.444 IT97K-499-35 96.54 1.167 Zaayura 85.56 1.870 Mean 58.0 2.3 s.e.d. 0.413 0.043 CV% 0.7 4.6 Fig. 4.2. Response of the susceptible lines (A and C), resistant lines (B) and D comparing leaf colour of resistant and susceptible lines due to aphid attack in the regions University of Ghana http://ugspace.ug.edu.gh 54 4.5. Discussion Insect species containing biotypes have been described by their ability to damage crops with host plant resistance genes (Puterka et al., 1992; Gallun, 1977; Porter et al., 1997). Insect biotypes are intra-specifically classified based on biological rather than morphological characteristics, and they are generally morphologically indistinguishable (Shufran et al., 2007). The cowpea aphid infests seedlings of cowpea and causes direct damage on the crop by sucking plant sap, resulting in stunted plants and distorted leaves. The infested leaves are often cupped or distorted and become more or less yellow. In heavy infestation the plant dies, especially under water stress. Indirectly, cowpea aphid transmits aphid-borne cowpea mosaic viruses. The performance of the genotypes in response to aphid infestation across the regions and locations was stable. The resistant genotypes (SARC 1-57-2 and SARC 1-91-1) and the susceptible genotypes (Apagbaala, Songotra and Zaayura) maintained their status across the regions and the locations within the regions. Between 10 to 15 days after aphid infestation, the growth and development of the aphids on the resistant genotypes were very slow. The seedlings of the resistant genotypes grew vigorously under aphids attack, they also maintained greenness in their leaves and eventually survived aphid attack. On the other hand, the susceptible genotypes showed heavy aphid population build-up, stunted growth and eventually resulted in the death of the seedlings. The number of aphids under artificial infestation is usually higher than that observed under natural field infestation so the mortality of some resistant seedlings is not expected to occur under natural field conditions. The results did not show that biotypes of A. craccivora exist within the cowpea belt of Ghana distinguishable by differences in damage to cowpea genotypes. Even if there exist University of Ghana http://ugspace.ug.edu.gh 55 differences in the A. craccivora population in Ghana, they may be different in genetic composition but not in biological attribute that could make them overcome the resistant genotypes (Saxena and Barrion, 1987). The current study has therefore confirmed that the aphid resistance gene in SARC1-57-2 is stable against A. craccivora in all the major cowpea growing belts in Ghana. SARC1-57-2 is thus a very important breeding material for cowpea breeders in Ghana aiming at breeding for cowpea aphid resistant varieties in any part of the country. Similarly, SARC1-57-2 may be important to the international cowpea breeding centres such as IITA and University of California, Riverside. The cowpea aphid resistant genotype could feature prominently in their efforts to develop elite cowpea lines for evaluation and adoption in the National Agricultural Research Stations (NARS). Besides being resistant to A. craccivora, SARC1-57-2 is early maturing, has white seed coat colour (the most preferred seed coat colour in Ghana) and medium seed size. These additional attributes of SARC1- 57-2 make it most suitable and easy to use breeding material for cowpea breeders compared to varieties with non-white coloured seed coats. Introgression of the aphid resistance gene into most of the commercially important cowpea varieties in Ghana has therefore been identified as a follow up studies to the current study using marker-assisted backcrossing methodology. The marker-assisted backcrossing methodology is proposed to be used in order to get the improved varieties that can be released to the farmers as quickly as possible. University of Ghana http://ugspace.ug.edu.gh 56 CHAPTER FIVE 5.0. Genetic Mapping and Inheritance of the Aphid Resistance Locus in Cowpea 5.1. Introduction In cowpea cultivation, attack by insect pests represents an important constraint to obtaining economic yields (Blade et al., 1997; Montimore et al., 1997). In the savanna regions of West Africa where the bulk of the crop is produced, A. craccivora is the most important insect pest during the vegetative phase of the crop (Singh et al., 1990; Obeng-Ofori, 2007). The pest primarily infests the seedlings of cowpea and causes direct damage on the crop by sucking plant sap, resulting in stunted plants and distorted leaves. Aphis craccivora also causes indirect damage by transmitting aphid-borne cowpea mosaic viruses (Singh and Jackai, 1985). Research at the International Institute of Tropical Agriculture (IITA) identified resistant sources in cowpea against A. craccivora, with antibiosis as the main basis for resistance (Ansari, 1984; Singh, 1977). Following this, a number of breeding lines with resistance to the pest were developed at IITA and distributed to cowpea breeding stations worldwide (Bata et al., 1987; Ofuya, 1997a; Singh, 2004). Field tests in many locations including Ghana had shown that the IITA type of resistance was not effective against local biotypes of the aphid in many locations (Messina et al., 1985; Kusi et al., 2010a). Resistance tests in Ghana with IT 97K-499-35, bred with the IITA source of resistance, for example, had been shown to be highly susceptible to A. craccivora (Kusi et al., 2010a). On-going research at the CSIR-Savanna Agricultural Research Institute (CSIR-SARI) to identify resistance sources in cowpea to the cowpea aphid has uncovered a number of advanced breeding lines with high levels of resistance to the pest (Kusi et al., 2010a). University of Ghana http://ugspace.ug.edu.gh 57 In these tests, lines with the IT 84S-2246 source of resistance that was identified at IITA were not more resistant than the susceptible Ghanaian cultivar, Apagbaala. The advanced breeding line SARC 1-57-2 was able to grow and yield successfully after manually infesting plants with A. craccivora. On-going efforts at sequencing the cowpea genome (Timko, 2009), have provided the opportunity to obtain a large number of co-dominant PCR based markers for genome analysis. This also presents an opportunity to tag loci underlying key traits of agronomic importance with markers to facilitate marker-assisted breeding of the crop. Marker-based selection enhances the efficiency of breeding for simple inherited traits such as aphid resistance. Phenotypic screening for aphid resistance for instance is laborious, expensive and dependent on favourable environmental conditions. Availability of tightly linked markers will therefore facilitate early generation selection, reducing the effective size of breeding populations and enhancing the overall efficiency of cultivar development. Knowledge of the inheritance of insect resistance is useful in the design of appropriate breeding procedures to develop resistant cultivars and for the identification of biotypes that may already exist or develop over time (Smith, 1989). This study sought to determine the mode of inheritance of aphid resistance in the line SARC 1-57-2 and to identify markers linked to the aphid resistance locus in cowpea to facilitate the use of this source in breeding cowpea for resistance to A. craccivora. University of Ghana http://ugspace.ug.edu.gh 58 5.2. Materials and Methods 5.2.1. Plant Materials Used in the Study The plant materials used were progenies of the cross between the Ghanaian cowpea cultivar, Apagbaala (Padi et al., 2004) and an advanced breeding line SARC 1-57-2. SARC 1-57-2 is an inbred line (F8) selected from the cross between Apagbaala and a line with exotic pedigree, UCR 01-11-52 (Padi and Ehlers, 2008). SARC 1-57-2 was observed to be resistant to A. craccivora under both screen house and field conditions among a large number of test lines (Kusi et al., 2010a). One hundred and sixty-nine F2 lines of the cross between Apagbaala and SARC 1-57-2 were tested for their reaction to aphid infestation in a screen house facility following standard protocols (Bata et al., 1987; Kusi et al., 2010a) as described in (3.3). As soon as individual lines could be unambiguously classified into resistant or susceptible classes, they were sprayed with lambda cyhalothrin (Lambda Super®) to control the aphids. Recovered plants were maintained to generate F3 seeds for progeny testing. DNA was obtained from each of the field-grown lines and stored in a refrigerator until needed. Each F2-derived F3 family (F2:3) was tested further for their reactions to the aphid using 20 seedlings per family. 5.2.2. Identification of Markers Linked to the Aphid Resistance Locus Fifty simple sequence repeat (SSR) primer pairs randomly selected across the cowpea genome were provided by Professor Mike Timko of the University of Virginia. The primers were tested for their ability to generate reproducible banding patterns in the parents of the mapping population. The sub-set of primers that produced clear reproducible bands were tested on two groups of five resistant and five susceptible individuals based on classification of F2 plants infested with the aphids. Primer pairs that showed polymorphism between the University of Ghana http://ugspace.ug.edu.gh 59 two sets of lines following denaturing polyacrylamide gel electrophoresis were tested further on all the 169 individuals. 5.3. Data Analyses Chi-square tests were performed to test the goodness of fit of observed segregations among F2 plants and F2:3 families to that of a single dominant gene. Similarly, the segregation pattern of SSR markers was tested for their fit to that of a single locus. Segregation among F2:3 families was analyzed after classifying each family as homozygous resistant (all plants showing same vigour as non-infested controls), homozygous susceptible (all plants dead by 10 days after infestation), and heterozygous (both resistant and susceptible plants were identified). 5.4. Results 5.4.1. Inheritance of Aphid Resistance in Line SARC 1-57-2 Only the parental phenotypes (Apagbaala, susceptible; SARC 1-57-2, resistant) were observed in the F2 population of Apagbaala × SARC 1-57-2. On resistant plants, aphid colonies increased in numbers slowly on inoculated trifoliates within the first 10 days that it was easy to count the total numbers per plant. On susceptible individuals, infested plants were overcrowded with the insect and death of seedlings began after the 10th day of inoculation. The observed segregation ratio was 123 resistant plants to 46 susceptible plants which fits a 3:1 ratio (χ2 = 0.44; P = 0.505). After spraying the plants with insecticides to kill the aphids, only 128 plants could establish successfully in the field and produce the minimum of 20 seeds. This represented 108 resistant plants (88% recovery of plants) and 20 susceptible plants (43% recovery of plants). The F2:3 families therefore did not accurately represent the F2 population. The segregation ratio was 35 homozygous resistant families, 73 University of Ghana http://ugspace.ug.edu.gh 60 heterozygous families and 20 homozygous susceptible families. Because of the different recovery rates of F2 plants in different resistance classes, only the ratio of resistant to segregating families was tested. The ratio of 35:73 resistant: segregating or heterozygous F2:3 families significantly fit the 1:2 resistant/segregating (heterozygote) ratio expected for a monogenic dominant gene (χ2 = 0.042; P = 0.838) 5.4.2. Identification of Markers Linked to the Aphid Resistance Locus Of the 50 SSR markers tested, only 31 amplified the cowpea lines in the two sets of five resistant and five susceptible classes, and produced reproducible banding patterns on denaturing PAGE. However, only four primer pairs (CP171F, CP172R; MS50F, MS50R; Y31F, Y31R and CP573F, CP573R) showed polymorphism between the two classes of resistant and susceptible lines. Out of these four, only CP 171F/172R (left sequence: 5’-TAGGGAGTTGGCCACGATA-3’; right sequence: 5’- CAACCGATGTAAAAAGTGGACA-3’) displayed a segregation pattern consistent with the phenotypic scores obtained following aphid infestation of the 128 lines (Fig 5.1). The expected band size of 176 bp based on information in the cowpea genomics database (http://cowpeagenomics.med.virginia.edu/CGKB/) was observed following PAGE. To determine the degree of linkage between CP 171F/172R and the aphid resistance locus, the F2 individuals were analyzed (Fig 5.2). CP 171F/172R was co-dominant and segregated in the expected 1:2:1 fashion following Chi square analysis (χ2 = 0.25; P = 0.856). Based on the SSR data, there were 10 misclassified plants that were possible single crossover events between marker CP 171F/172R and the resistance locus. University of Ghana http://ugspace.ug.edu.gh 61 Fig 5.1. A silver stained PAGE showing the DNA banding patterns of resistant and susceptible parents and their progenies amplified by SSR marker CP 171F/172R. The faster migrating band is that of the susceptible parent (Apagbaala). Fig. 5.2. A silver stained PAGE showing the DNA banding patterns of F2 plants derived from Apagbaala × SARC 1-57-2 amplified by SSR marker CP 171F/172R. The faster migrating band is that of Apagbaala. 176 bp University of Ghana http://ugspace.ug.edu.gh 62 5.5. Discussion In peasant crop farming, as is cowpea production in West Africa, the use of insect resistant cultivars offers one of the simplest and most convenient methods of pest control (Dent, 1991). The use of resistant varieties appears to be the best option for the small scale farmers of the Semi-Arid Tropics (SAT) owing to its low cost, compatibility with other control methods, and to the low incomes realised by the farmers (Dent, 1991). The discovery of resistance in cowpea to the aphid in a local × exotic cross (Apagbaala × UCR 01-11-52, Kusi et al, 2010a) provided impetus to initiate breeding for resistance to the insect to improve existing cultivars. Previously reported sources of resistance were found ineffective in Ghana (Singh, 2004) and because Apagbaala is highly susceptible, the source of the resistance will be UCR 01-11-52. Due to the simple inheritance of the gene found in SARC 1-57-2 and the ease of distinguishing resistant from susceptible plants in aphid resistance bioassays, cowpea breeders will be able to rapidly convert existing cowpea cultivars into aphid resistant cultivars using efficient backcross breeding procedures. In spite of the ease of distinguishing resistant and susceptible plants in phenotypic screen, conducting the entire resistance bioassay in large populations is tedious due to the need to maintain aphids on live plants, and use of nymphs of the same age. Moreover, relative humidity and temperature do influence the growth and survival of the aphids under screen-house conditions which may lead to inefficiencies in selection. Discovery of co-dominant SSR markers linked to the aphid resistance locus will facilitate marker-assisted selection (MAS) which is simpler than phenotypic screening, saves time, resources and effort (Akhtar et al., 2010). Moreover, homozygous and heterozygous resistant plants cannot be distinguished by the phenotypic screening, requiring further progeny testing to select desirable plants. Considering that the University of Ghana http://ugspace.ug.edu.gh 63 marker is some distance away from the gene locus, the most practical use of CP 171F/172R may be its application in reducing the size of plants in a population prior to phenotypic screening. To my knowledge, this is the first account of linkage between a co-dominant SSR marker and aphid resistance locus with strong effect on A. craccivora infestation in cowpea. The map location of CP 171F/172R is the tip of linkage group 2 (Mike Timko, personal communication). With knowledge of its map location, markers within the vicinity will need to be tested to uncover others more closely linked to the resistance locus. The identification of this single resistance locus and a position on the cowpea genetic map will facilitate the deployment of resistance as a component of integrated management of A. craccivora in West Africa. University of Ghana http://ugspace.ug.edu.gh 64 CHAPTER SIX 6.0. Introgression of Aphid Resistance Locus into Ghanaian Cowpea Cultivars 6.1. Introduction For the introgression of qualitative traits such as pathotype-specific disease resistance, which are typically controlled by single dominant genes, backcross breeding (BC) has been used for a long time (Allard, 1960; Fabio et al., 2004). It allows the transfer of one or few genes from a donor genotype (typically with poor agronomic traits) into an elite recipient genotype, the recurrent parent (Welz and Geiger, 1999). Classical backcross breeding can be termed as phenotypic background selection (Welz and Geiger, 1999). In each BC generation, carriers of the target gene would be directly identified by a phenotype-based assay and the portion of unwanted donor genes would be halved. For the transfer of a single dominant gene, six BC generations would normally be conducted to recover 99% of the recurrent parent genome, a time-consuming procedure (Welz and Geiger, 1999). The use of genetic and genomic analysis to help identify DNA regions tightly linked to agronomic traits in crops can improve the efficiency of breeding strategies for crop improvement. The use of molecular markers for indirect selection of improved crops speeds up the selection process by alleviating time-consuming approaches of direct screening under greenhouse and field conditions (Dita et al., 2006). Molecular markers are particularly useful when targeting characters controlled by several genes (Dita et al., 2006). The potential to map different Quantitative Trait Loci (QTL) contributing to a trait of agronomic importance and to identify linked molecular markers opens up the possibility to transfer simultaneously several QTLs and to pyramid QTLs for several important traits in one improved cultivar (Dita et al., 2006). However, Yu et al. (2004) outlined the following factors to be considered as prerequisites on studies to identify and validate potential University of Ghana http://ugspace.ug.edu.gh 65 markers: (a) level of polymorphism existing between parental lines, (b) unclear expression of some markers inherent to the marker class used, (c) false-positive markers, (d) discrepancy between the presence of the marker and target gene, which requires testing the gene with conventional screening and (e) presence of multiple genes scattered over several linkage groups. The current study sought to deploy the co-dominant SSR marker, CP 171F/172R, linked to the aphid resistance locus to facilitate marker-assisted backcrossing to improve an elite cowpea variety, Zaayura for resistance to the aphid. 6.2. Materials and Methods 6.2.1. Polymorphism Test Tests for polymorphism for marker CP 171F/172R were conducted with the methodology in (3.5.1.) on cowpea cultivars that had previously been shown to be susceptible to the cowpea aphid. The cultivars were developed and released for general cultivation in 2004 by the CSIR-Savanna Agricultural Research Institute (CSIR-SARI). The varieties were Padi Tuya, Zaayura, Songotra, and Bawutawuta. 6.2.2. Marker Assisted Backcrossing Among the four cultivars tested, only Zaayura was polymorphic at the CP 171F/172R locus and was therefore selected as the recurrent parent for introgression of the aphid resistance locus. The methodologies described in (3.5.2.1) were used to introgress the cowpea aphid resistance locus into Zaayura. University of Ghana http://ugspace.ug.edu.gh 66 6.3. Results 6.3.1. The Test for Polymorphism The results of the polymorphism test between SARC 1-57-2 and the four cultivars, Padi Tuya, Zaayura, Songotra and Bawutawuta are presented in Fig. 6.1. Among the four cultivars that were tested, the marker CP 171F/172R showed polymorphism between Zaayura and the resistant cultivar, SARC 1-57-2. Fig. 6.1. DNA banding patterns for marker CP 171F/172R on four cowpea cultivars and susceptible and resistant checks. 6.3.2. Determination of Plants from a Successful Cross (F1 Plants) The results from F1 plants between SARC1-57-2 and Zaayura which were genotyped with CP 171F/172R prior to the backcrossing to the recurrent parent, Zaayura to determine which of the plants were from a successful cross is presented in Fig. 6.2. Four samples of the susceptible parent (Zaayura) and three samples of the resistant parents (SARC1-57-2) were included as checks. With the exception of the susceptible and resistant checks, all the other samples were heterozygous for the CP 171F/172R locus (F1 plants). Apagbaala Bawuta Padi Tuya Zaayura Songotra SARC1-57-2 University of Ghana http://ugspace.ug.edu.gh 67 Fig. 6.2. DNA banding patterns of the F1 plants from the crosses between SARC1-57- 2 and Zaayura genotyped with CP 171F/172R. 6.3.3. Genotyping to Select Heterozygotes from the Backcross Populations The results from individual plants from successive backcross populations (BC1 to BC4) genotyped for their banding pattern at the CP 171F/172R locus is presented in Fig. 6.3. Based on Chi-square tests for goodness of fit, the segregation ratios fit the expected 1:1 ratio for heterozygous and homozygous susceptible individuals (χ2 = 0.138; P = 0.710). Fig.6.3. DNA banding patterns of successive backcross populations genotyped to select heterozygote individuals at the CP 171F/172R locus 6.3.4. Genotyping of BC4F2 to Select Homozygous lines A BC4F1 individual was selfed to generate BC4F2 lines. BC4F2 were genotyped with the marker CP 171F/172R to select individual plants that are homozygous dominant for the marker locus (Fig. 6.4). Out of sixty BC4F2 plants screened, the segregation at the marker locus was 13:31:16 (homozygous resistant: heterozygous: homozygous susceptible) which fits the expected ratio of 1:2:1 for a single dominant gene using χ2 tests (χ2 = 0.37; P = 0.83). Successful F1 Progenies Susceptible Parent Resistant parent Heterozygote indidvidals Zaayura SARC1-57-2 176 bp 176 bp University of Ghana http://ugspace.ug.edu.gh 68 Fig. 6.4. DNA banding patterns of BC4F2 genotyped with the marker CP 171F/172R 6.4. Discussion The tests for polymorphism between the donor of the resistace locus (SARC 1-57-2) and the cultivars determined that only the cultivar Zaayura was suitable as the recurrent parent for improvement. The size of the populations created and screened for resistance in the screenhouse studies in the various generations were reduced significantly in size by selecting only for segregants heterozygous for the marker locus. This reduced the total amount of time spent in selecting individuals that will serve as parents for a subsequent generation, and the overall effeciency of the backcross method of transfering the resistance locus. Segregation distortion, though common in mapping populations (Lorieux et al., 1995) was not observed in the current study. At each generation, the Chi square tests showed a good fit to the expected Mendelian ratio for a single locus. At the BC4F1 where over 90% of the background of the recurrent parent is recovered and the aphid resistance gene is more or less fixed, the BC4F1 was selfed to generate BC4F2 individuals. The BC4F2 population when genotyped afford the opportunity to select individuals having the aphid resistance gene in a homozygous resistance state. These individuals were tagged and subjected to further phenotypic selection based on the feature Heterozygote Homozygote Susceptible Homozygote Resistant 176 bp University of Ghana http://ugspace.ug.edu.gh 69 of the recurrent parent (vegetative, podding and seed) and the seeds of the improved individuals were subsequently multiplied. In a field evaluation of the improved Zaayura it was realised that the mproved Zaayura has recovered all the physical features of the original Zaayura. This is an indication that the BC4 was adequate to retain the background of the recurrent parent. The multiplied seeds of the improved Zaayura have been presented for assessment and approval to be released as a variety. Inspite of the limitation of the SSR marker CP 171F/172R, it has successfully been used in combination with intermittent aphid screening in a coordinated backcrossing programe to improve the field resistance of Zaayura. This was achieved within two years which could not have been possible under conventional approach. Conventional plant breeding is primarily based on phenotypic selection of superior individuals among segregating progenies resulting from hybridization. It is often time consuming as breeding a new variety in some crops takes between eight and twelve years and even then, the release of improved variety is not guaranteed (Ibitoye and Akin-Idowu, 2010). Hence, breeders are extremely interested in new technologies that could make this procedure more efficient. Molecular marker-assisted selection offers such a possibility by adopting a wide range of novel approaches to improving the selection strategies in crop breeding (Ibitoye and Akin-Idowu, 2010). Thus, molecular markers bring a systematic basis to traditional breeding, enhancing its precision and expediting the process (Kumar, 1999; Collard et al., 2005). Unwanted self-pollination in field or during crossing programs is one of the major sources of impurity of hybrid seeds that interferes with trait improvement via conventional breeding programs or variety improvement via backcross scheme (Asadollah and Mehdi, 2010). Conventional characterization of hybrid seeds based on specific morphological and University of Ghana http://ugspace.ug.edu.gh 70 agronomic data is time-consuming, restricted to a few characteristics, and is influenced by environment. In contrast, DNA-based markers are highly heritable, available in high numbers, and exhibit enough polymorphism; hence they can be used to trace that alleles came from a given parent or to discriminate closely related genotypes of a plant (Yashitola et al., (2002); Wang et al., (2005); Asadollah and Mehdi ( 2010)). This achievement underscores the importance of marker-assisted selection (MAS) in plant breeding in the national agricultural research system (NARS). The current efforts in capacity building in both human and infrastructure in most of the NARS in Ghana and Africa as a whole should therefore be given the necessary support by the state as a bold step towards achieving food security. University of Ghana http://ugspace.ug.edu.gh 71 CHAPTER SEVEN 7.0. Yield Loss Assessment of Ten Cowpea Varieties 7.1. Introduction Aphis craccivora Koch, a phloem feeding insect, is a major insect pest of cowpea with a worldwide distribution (Obeng-Ofori, 2007). They are economically important insect pests causing serious damage to several crop plants (Obeng-Ofori, 2007, Kusi et al., 2010a). They feed mainly by sucking plant sap from the succulent parts of the plant including leaves, stem, terminal shoots, petioles, flowers and pods (Asiwe et al., 2005). Damage is caused by both nymphs and adults sucking plant sap from seedling to pod bearing stage (Kusi, 2008). The harmful effects of cowpea aphid can be assessed either directly or indirectly. The direct damage on the crop due to the sucking of plant sap causes stunted growth and distorted leaves (Fatokun, 2002; Kusi, 2008; Souleymane et al., 2013). In a field study, Kusi et al. (2010b) observed delayed flowering and maturity in medium to late cowpea cultivars and attributed it to stunted growth as a result of the sucking of plant sap by A. craccivora. Aphid feeding interferes with the amount of photosynthates translocated for leaf production, flower initiation and podding (Annan et al., 1995; Giordanengo et al., 2010). Ofuya (1993) indicated that, heavy feeding of aphids can kill young plants, distort leaves, delay flower initiation and reduce pod set in plants which survive aphid attack. Leaf distortion as a result of aphid infestation reduces the photosynthetic area of the leaves with consequent reduction in the photosynthetic rate of the plant resulting in stunted growth and low yield (Annan et al., 1995; Giordanengo et al., 2010). Aphids have also been found to severely damage cowpea indirectly through the transmission of cowpea aphid-borne mosaic virus (Laamari et al., 2008). Cowpea aphid-borne mosaic University of Ghana http://ugspace.ug.edu.gh 72 virus is considered an important limitation in cowpea production. It is an economically important virus causing yield losses exceeding 87% under field conditions (Bashir and Hampton, 1996). The combined effects of cowpea aphid-borne mosaic virus and stunted growth has been reported to be the most damaging effect of cowpea aphid (Laamari et al., 2008). Blaney et al. (1990) indicated that the feeding action of aphids lower the yield, quality and marketability of cowpea by transmitting plant viruses. The intensity of damage caused by insect pests varies greatly with the intensity of infestation, duration of occurrence and stage of growth of the plant (Dent 1991; Kusi et al., 2010b). Dent (1991) indicated that the combination of these three factors in relation to the crop affect crop yield. Yield loss due to aphids is assessed by comparing the yield of cowpea from protected and unprotected fields. The yield from the protected field represents the attainable yield while the yield from the unprotected plot represents the actual yield with the difference between the two yields accounting for the yield reduction due to aphid infestation. The current study therefore assessed yield loss of 10 cowpea cultivars due to aphid infestation. 7.2. Materials and Methods The assessment of the yield loss of the 10 genotypes was carried out at the Manga (Upper East Region) Station of CSIR-Savanna Agricultural Research Institute. The genotypes (Table 7.1) include advanced breeding lines from the IITA (SARC1-57-2, IT99K-573-3-2- 1, SARC1-91-1 and IT99K-573-1-1) and cultivars (Apagbaala, Padituya, Songotra (IT97K499-35) and Zaayura) and two advanced breeding lines developed during the course of this study. The advanced backcross progeny were selected based on SSR marker scores at the aphid resistance locus either as resistant or susceptible. The treatments were replicated University of Ghana http://ugspace.ug.edu.gh 73 six times in a randomized complete block design. Each plot consisted of 2 rows of 4 m long. Plots were separated from each other at a distance of 1 m. Three seeds were sown per stand at 60 cm between rows and 20 cm within rows and were thinned to one plant per stand at 10 days after planting. There were two trials, one trial serving as the control was sprayed on three occasions (seedling, flowering and podding phases) against insect pests with lambda cyhalothrin (Lambda Super®), a synthetic pyrethroid, at the rate of 20 g active ingredient ha-1. The other trial was sprayed on two occasions (flowering and podding phases). The cowpea seedlings of the second trial were infested with five, four-day old aphids per seedling (Annan et al., 1995; Bosque-Perez and Schotzko, 2000; Kusi et al., 2010b; Benchasri et al., 2012) two weeks after planting. During the period of infestation, the seedlings were confined under insect proof net in order to limit the damage of the seedlings to only aphids and to prevent effects of predators and parasitoids on the aphids. The aphids were allowed to form colonies and fed on the seedlings until symptoms of damage were observed on the susceptible seedlings. When the susceptible seedlings became stunted with distorted and yellowing leaves at sixteen days after infestation (30 days after planting), the aphids were killed by spraying with lambda cyhalothrin (Lambda Super ®). At plant maturity (65 days after planting), the pods were harvested, dried, threshed and grain weight recorded using an electronic balance. Other agronomic data recorded include: days to 50% flowering, days to maturity, weight of pods, and weight of vegetative biomass at maturity. Percentage grain yield reduction due to aphid infestation was calculated as: 100plot uninfestedin Yield plot infestedin Yield -plot uninfestedin Yield X University of Ghana http://ugspace.ug.edu.gh 74 Table 7.1. Description of the 10 cultivars of cowpea by parentage or source Variety Description APAGBALA Prima/TVu 4552/California Blackeye No.5/7977. Cultivar, released in 2002 in Ghana. Largely of exotic background. IT99K-573-1-1 Breeding line from the IITA, Ibadan Nigeria PADITUYA Apagbaala/ UCR 01-11-52 released in 2008 in Ghana SARC1-91-1 Apagbaala/ UCR 01-11-52 Breeding line of SARI BC4F3 (Zaayura //(Zaayura x SARC1-57-2) susceptible Marfo-Tuya/ UCR 01-15-127-22/SARC1-57-2 ZAAYURA Marfo-Tuya/ UCR 01-15-127-2 released in 2008 in Ghana IT99K-573-3-2-1 Breeding line from the IITA, Ibadan Nigeria IT97K499-35 (Songotra) Breeding line from the IITA, Ibadan Nigeria. Cultivar, released in 2004 in Ghana as Songotra. BC4F3 (Zaayura //(Zaayura x SARC1-57-2) resistant Marfo-Tuya/ UCR 01-15-127-2/SARC1-57-2 SARC1-57-2 Apagbaala/ UCR 01-11-52 Breeding line of SARI 7.3. Data Analysis GenStat statistical program (9th edition) was used to analyze the data. Fisher’s LSD was used to separate the means after ANOVA showed significant differences. 7.4. Results 7.4.1. Grain Yield Grain yield and percentage yield loss of 10 cowpea cultivars evaluated under aphid infestation and no infestation are presented in Table 7.2. The grain yield recorded under uninfested plots ranged between 775 and 1086 kg ha-1. Padituya recorded significantly University of Ghana http://ugspace.ug.edu.gh 75 (P<0.05) higher grain yield than six of the cultivars, however, there was no significant differences among Padituya, improved Zaayura, Apagbaala, SARC 1-57-2 and SARC 1-91- 1. Apart from Padituya, there was no significant difference among the rest of the cowpea cultivars evaluated in the study under uninfested conditions. On the other hand, under infested conditions, Padituya recorded the highest grain yield whereas the susceptible progeny recorded the lowest grain yield. However, there were no significant differences among Padituya, Improved Zaayura, SARC 1-57-2, SARC 1-91-1 and IT97K-499-35. On the other hand, there were also no significant differences among susceptible progeny, Zaayura, IT99K-573-1-1, IT99K-573-3-2-1, Apagbaala, IT97K-499-35 and SARC1-91-1 Generally, all the cultivars yielded higher grains under uninfested conditions than under aphid infested conditions. The percentage yield loss ranged between 4.9% and 32.8%. The three cultivars that recorded the lowest yield loss due to aphid infestation were improved Zaayura, SARC 1-57-2 and SARC 1-91-1 which recorded 3.8%, 4.9% and 9.8%, respectively. On the other hand, the cultivars that recorded the highest yield loss were the susceptible progeny, IT99K-573-1-1 and Apagbaala with yield loss of 32.8%, 32.1% and 30.3%, respectively. The other cultivars that also suffered substantial yield loss due to aphid attack were Zaayura (26.6%) and IT99K573-3-2-1 (27.0%). Two cultivars, IT97K-499-35 (17.1%) and Padituya (16.1%) suffered moderate yield losses. University of Ghana http://ugspace.ug.edu.gh 76 Table 7.2. Grain yield and percentage grain yield loss (kg) ha-1 of 10 cowpea cultivars evaluated under aphid infestation and no infestation Variety Uninfested Infested Grain yield (Kg)/ha Grain yield (Kg)/ha % yield loss (Kg)/ha APAGBALA 897 625 30.3 IT99K-573-1-1 806 547 32.1 PADITUYA 1086 911 16.1 SARC1-91-1 850 767 9.8 BC4F3 (Zaayura //(Zaayura x SARC1-57-2) susceptible 806 542 32.8 ZAAYURA 775 569 26.6 IT99K-573-3-2-1 803 586 27.0 IT97K499-35 (Songotra) 814 675 17.1 BC4F3 (Zaayura //(Zaayura x SARC1-57-2) resistant 936 900 3.8 SARC1-57-2 850 808 4.9 Mean s.e.d CV% 862.3 120.9 24.3 693.0 126.0 31.7 7.4.2. Biomass Production The dry biomass per hectare and percentage vegetative biomass loss of the 10 cultivars evaluated under aphid infested and uninfested conditions are presented in Table 7.3. The improved Zaayura recorded the highest dry biomass yield; however, there were no significant differences (P>0.05) among the improved Zaayura and Padituya, susceptible progeny and Zaayura. The lowest dry biomass yield was recorded for Apagbaala which was not significantly different (P>0.05) from IT99K-573-3-2-1, IT99K-573-1-1, IT97K-499-35, SARC1-57-2 and SARC1-91-1. Under the infested conditions, the improved Zaayura had the highest dry biomass yield, although it was not significantly different (P>0.05) from Padituya and Zayura. The cultivar IT99K-573-3-2-1 recorded the lowest dry biomass yield; however, it was not significantly University of Ghana http://ugspace.ug.edu.gh 77 different from IT99K-573-1-1, IT97K-499-35, SARC1-57-2 and SARC1-91-1, Apagbaala and SARC1-57-2. The percentage dry biomass yield loss ranged between 3.3% and 14.3% (Table 7.3). The improved Zaayura recorded the least dry biomass yield loss while Zaayura suffered the highest yield loss. The cultivars that suffered dry biomass yield loss above 10% were Padituya (10.5%), the susceptible progeny (12.3%), Zaayura (14.1%) and IT99K-573-3-2- 1 (14.3%). Four cultivars that suffered less than 10% dry biomass yield loss were SARC 1- 57-2 (6.1%), IT99K-573-1-1 (8.9%), SARC 1-91-1 (9.4%) and IT97K-499-35 (9.5%). Table 7.3. The dry biomass yield and percentage dry biomass yield loss (kg) ha-1 of the 10 cultivars evaluated under aphid infested and uninfested conditions Variety Uninfested Infested Biomass (Kg)/ha Biomass (Kg)/ha % yield loss APAGBALA 3236 2942 9.1 IT99K-573-1-1 3528 3214 8.9 PADITUYA 4947 4428 10.5 SARC1-91-1 3439 3117 9.4 BC4F3 (Zaayura //(Zaayura x SARC1-57-2) susceptible 4528 3972 12.3 ZAAYURA 4900 4211 14.1 IT99K-573-3-2-1 3278 2808 14.3 IT97K499-35 (Songotra) 3719 3367 9.5 BC4F3 (Zaayura //(Zaayura x SARC1-57-2) resistant 5028 4864 3.3 SARC1-57-2 3667 3444 6.1 Mean s.e.d CV% 4027 310.8 13.4 3637 324.2 15.4 7.4.3. Days to Flowering The number of days to 50% flowering under both infested and uninfested conditions of the 10 cultivars is presented in Table 7.4. Significantly (P<0.05) SARC 1-57-2 and IT97K-499- University of Ghana http://ugspace.ug.edu.gh 78 35 were early to flower although there was no significant difference between IT97K-499- 35 and IT99K-573-3-2-1. On the other extreme, Zaayura, the susceptible progeny and Padituya significantly (P<0.05) flowered late among the cultivars. However, there were no significant differences between Padituya and the improved Zaayura. The rest of the cultivars fell between the two extremes which flowered after 40 to 42 days. Under uninfected conditions, SARC 1-57-2 and IT99K-573-3-2-1 significantly flowered earlier among the 10 cultivars; however, there were no significant differences between IT99K-573-3-2-1 and IT99K-573-1-1, SARC 1-91-1, IT97K-499-35 and Apagbaala. The cultivars that significantly (P<0.05) flowered late among the 10 genotypes were Zaayura, the susceptible progeny and Padituya. Table 7.4. The number of days to 50% flowering under infested and uninfested conditions of the 10 cultivars Variety Uninfested Infested Days to 50% flowering Days to 50% flowering APAGBALA 38 42 IT99K-573-1-1 38 40 PADITUYA 42 47 SARC1-91-1 37 40 BC4F3 (Zaayura //(Zaayura x SARC1-57-2) susceptible 42 48 ZAAYURA 42 48 IT99K-573-3-2-1 37 40 IT97K499-35 (Songotra) 38 39 BC4F3 (Zaayura //(Zaayura x SARC1-57-2) resistant 41 46 SARC1-57-2 36 38 Mean s.e.d CV% 39 0.4 1.5 43 0.6 2.5 University of Ghana http://ugspace.ug.edu.gh 79 7.4.4. Maturity period The number of days to maturity under both infested and uninfested conditions of the 10 cultivars is presented in Table 7.5. Under the infested conditions, SARC 1-57-2, IT97K- 499-35 and IT99K-573-1-1 significantly (P<0.05) matured earlier among the cultivars. However, IT97K-499-35 and IT99K-573-1-1 were not significantly different from IT99K- 573-3-2-1. Similarly, IT99K-573-1-1, IT99K-573-3-2-1 and SARC 1-91-1 were not significantly different in terms of days to maturity. On the other hand, Zaayura significantly (P<0.05) recorded the longest days to maturity (70 days). The susceptible progeny and the improved Zaayura came next to Zaayura which also recorded significantly longer days to maturity than the remaining cultivars. Padituya and Apagbaala, although were significantly different, fell between the extremes with 64 and 68 days to maturity, respectively. Under the uninfested conditions, SARC 1-57-2, IT99K-573-3-2-1 and IT99K-573-1-1 significantly (P<0.05) matured earlier among the cultivars. Significant differences were not observed among IT99K-573-3-2-1, IT99K-573-1-1, IT97K-499-35, SARC 1-91-1 and Apagbaala. Padituya, the susceptible progeny, the improved Zaayura and Zaayura recorded the highest number of days to maturity ranging between 65 and 66. University of Ghana http://ugspace.ug.edu.gh 80 Table 7.5. The number of days to maturity under infested and uninfested conditions of the 10 cultivars Variety Uninfested Infested Days to maturity Days to maturity APAGBALA 61 64 IT99K-573-1-1 60 63 PADITUYA 65 68 SARC1-91-1 61 63 BC4F3 (Zaayura //(Zaayura x SARC1-57- 2) susceptible 65 69 ZAAYURA 65 70 IT99K-573-3-2-1 60 63 IT97K499-35 (Songotra) 61 63 BC4F3 (Zaayura //(Zaayura x SARC1-57- 2) resistant 65 69 SARC1-57-2 59 62 Mean s.e.d CV% 62 0.6 1.6 65 0.3 0.9 7.5. Discussion Resistance to the cowpea aphid among advanced breeding lines selected from recurrent backcross selection using Zaayura as the recurrent parent has significantly been improved with the resistance from SARC 1-57-2. This was achieved within a period of two years using marker-assisted backcrossing methodology, which could otherwise be achieved in not less than five years using the conventional selection methods. The improved resistance of the selected lines was manifested in the reduced percentage of yield loss under no insecticide protection during the vegetative phase, The extrapolation of this observation to field conditions will be improved stability of yield under typical farmers' production conditions. The resource poor farmers can hardly afford the high cost of insecticides and in some cases do not have access to the insecticides even University of Ghana http://ugspace.ug.edu.gh 81 when they have the money to buy. With the improvement of the field resistance of Zaayura, the farmers will not need to spray against aphids at the vegetative stage. Since aphid infestation is a major production constraint of cowpea at the vegetative stage (Singh and Jackai, 1985; Jackai and Adalla, 1997; Fatokun, 2002; Obeng-Ofori, 2007; Souleymane et al., 2013) the resource poor farmers could reduce up to 30% of the cost of insecticides and labour to spray, limiting insecticide protection to only the flowering and podding phases. The farmers will not only save cost on insecticide and labour, exposure to the harmful chemicals, contamination of water bodies, the environment, resurgence of insects and other hazards associated with the use of chemical insecticides will also be reduced. In particular, the profitability of dry season production of cowpea on residual soil moisture or under irrigation is expected to increase considerably using the aphid resistant lines developed. In the major cowpea production belt in Northern Ghana for example, farmers move to the banks of the White Volta River between October and December after the rainy season farming activities to make use of the residual moisture in the soil to grow cowpea. Moreover, the bulk of the cowpea in the Upper East region is produced using the residual moisture as a result of the flooding due to the spillage from the Bagre dam in Burkina Faso. Under these conditions, lack of rainfall that is critical in the control of aphids worsens the pest problem and necessitates additional insecticide sprays to produce the crop. The high dry biomass yield recorded against the improved Zaayura even under infested conditions is of great significance to the crop/livestock intensification production system in Northern Ghana. The northern savannah zone is a predominantly crop and livestock production area. However, feeding of the livestock during the long dry season of up to seven months in the Sudan and Guinea Savannah zones is a major constraint to livestock farming. University of Ghana http://ugspace.ug.edu.gh 82 The nature of the livestock feeding during the dry season is both of inadequacy and imbalance of the feed. The animals are kept on free range to feed on limited crop residues of cereals, most of which are also destroyed by bush fire, with little or no source of leguminous crop residues. The combination of good grain and biomass yield by the improved Zaayura even under aphid infestation indicates that the farmers will not only increase their income from cropping the improved Zaayura, but will also have cowpea haulm to improve the cereal crop residue to feed their animals during the long dry season. Improved feeding of livestock has been identified to increase growth, fecundity and productivity of livestock (Akbar et al., 2000; FAO, 2012). This will eventually lead to increased income and improved livelihood to the resource poor farmers as a result of improved crop/livestock intensification system. The original Zaayura has been widely adopted by the farmers as a result of its bigger seed size, white seed coat, high yielding and early maturity. The marker-assisted backcrossing methodology used to develop the improved Zaayura has retained all these features in the improved cultivar. With the exception of its ability to withstand pressure of cowpea aphid infestation and ability to combine good grain yield and biomass, the farmers may not be able to identify any other distinguishing features between the original Zaayura and the improved Zaayura. Therefore, very simple dissemination strategies such as on-farm demonstrations and field days could be enough to get the farmers adopt the improved Zaayura. It is also recommended that the field resistance of the improved Zaayura to Striga gesnerioides should be improved to further increase its yield stability. University of Ghana http://ugspace.ug.edu.gh 83 CHAPTER EIGHT 8.0. General Discussion Host-plant resistance is the relative proportions of heritable characteristics of a plant that influence the degree of damage produced by a pest (Painter, 1951; Cuartero et al., 1999; Dent, 2000; Meyer, 2003). It has proved to be a successful tool against insects in many crops (Felkl et al., 2005). Resistant crop varieties provide an inherent control that involves no environmental problems, and they are generally compatible with other insect-control methods (Kfir et al., 2002; Kusi, 2008). The cultivation of resistant crop plants is not subject to the vagaries of weather as are chemical-control measures, and in certain circumstances it is the only effective means of control. Resistant varieties control even a low pest density, whereas insecticide use is justifiable only when the density reaches the economic injury level (Kfir et al., 2002; Kusi, 2008). Identification of SARC 1-57-2 has therefore become an important breakthrough as an effective, affordable and sustainable management of cowpea aphid. The use of host plant resistance can be described as effective means of controlling pests because it addresses problems of chemical insecticide control such as sub dosal application of insecticides. This usually results in ineffective control of the insect pest which in most cases leads to the insects developing resistance to the insecticide and eventually leading to resurgence of insects. The use of host plant resistance is a sustainable means of pest control due to its compatibility with other pest control measures. It does not lead to resurgence of insect pests and it is environmentally friendly. Identification of host plant resistance and its deployment in plant breeding or in insect management programme are two different things. In most cases, the protectionist identifies materials with various levels of resistance to major pests of crops and that ends it all. In most cases the genotypes found to be resistant to a major pest of a crop tend to trade off in University of Ghana http://ugspace.ug.edu.gh 84 terms of yield, growth habits and seed quality which limits their direct use in production. There is therefore the need for a strong collaboration between the protectionist and the plant breeder to deploy the resistant gene to improve the field resistance of elite genotypes that meet all qualities desired by the farmers. The current study identified the aphid resistant cowpea genotype (SARC 1-57-2) and introgressed the aphid resistance gene into one of the cowpea cultivars released by CSIR-SARI (Zaayura) using marker-assisted backcrossing methodology. Marker-assisted selection (MAS) is time saving from the substitution of complex field trials (that need to be conducted at particular times of the year or at specific locations, or are technically complicated) with molecular tests. This leads to elimination of unreliable phenotypic evaluation associated with field trials due to environmental effects. Selection of genotypes is possible with MAS at seedling stage as well as gene ‘pyramiding’ or combining multiple genes simultaneously. Marker assisted-selection also limits transfer of undesirable or deleterious genes. Other advantages for using MAS include: selecting for traits with low heritability, testing for specific traits where phenotypic evaluation is not feasible (e.g. quarantine restrictions may prevent exotic pathogens to be used for screening). The current study embarked on a search for a DNA marker that is tightly linked with the aphid resistance gene. The search led to the identification of CP 171F/172R which showed polymorphism between the aphid resistant genotype (SARC 1-57-2) and a susceptible genotype (Apagbaala). Zaayura was subsequently selected following a polymorphism study between SARC 1-57-2 and four elite varieties released by CSIR-SARI. The marker CP 171F/172R showed polymorphism between Zaayura and SARC 1-57-2. Marker-assisted backcrossing methodology was therefore used to improve the field resistance of Zaayura to University of Ghana http://ugspace.ug.edu.gh 85 cowpea aphid. The advanced breeding lines selected from the recurrent backcrossing of Zaayura are resistant to the cowpea aphid. In a replicated study, the new Zaayura maintained all the phenotyped features of the original Zaayura. These include high grain yield, early maturity, large seed size, white seed coat and brown eye colour. In addition, it combined both high grain yield and biomass yield even under no spray conditions. The improved Zaayura has been presented for assessment and release by the National Variety Release Committee. 8.1. Conclusion To my knowledge, the current success in improving the field resistance of Zaayura to cowpea aphid using marker-assisted backcrossing is the first report of marker-assisted selection in cowpea leading to direct deployment in the field. This was achieved in the space of two years which would have taken a minimum of five years using the conventional breeding approach. Incorporation of marker-assisted breeding efforts into the breeding programmes is advocated on the strength of this study. Further investments in laboratory infrastructure are advocated to help improve the effectiveness of the breeding process, particularly for simply inherited traits. 8.2. Recommendations Large-scale deployment of varieties with a single locus conditioning resistance may lead to breakdown of resistance, due to directional selection for virulent races that may be present at low frequencies within the pest population. A search for cowpea varieties that possess resistance conditioned by other loci is therefore recommended to reduce the chances of breakdown of the resistance. In this regard, it is also recommended that follow up studies University of Ghana http://ugspace.ug.edu.gh 86 should include the determination of the sources of resistance in SARC1-57-2 and SARC1- 91-1 for possible pyramiding of genes if they are found to be different. With the report of the existence of different biotypes of cowpea aphids in West Africa, it is recommended that future studies should aim at assembling the various sources of cowpea aphid resistant genotypes across West Africa. The similarity or otherwise of the genes controlling the resistance in the different genotypes should be determined for pyramiding of genes if they are found to be different genes. It is also recommended that further search for DNA marker(s) closer than CP171F/172R to the aphid resistance gene in SARC 1-57-2 should be carried out to enhance efficiency in its deployment in marker-assisted selection. The biochemistry that underline the cowpea aphid resistance in SARC 1-57-2 is also very important to be investigated in follow up studies. This could help make clearer the mechanism of resistance and could lead to development of biochemical assays to rapidly identify other resistant sources in the cowpea germplasm. The importance of resistant cultivar as a principal component of integrated pest management cannot be over emphasized, owing to its compatibility with other crop and pests management practices. It is therefore recommended that a search for sources of natural resistance in germplasm of field crops should be given further attention, and be more vigorously pursued in integrated pest management programmes. University of Ghana http://ugspace.ug.edu.gh 87 References Abate, T. van Huis, A. and Ampofo, J.K.O. (2000). Pest Management Strategies in traditional agriculture: An African perspective. Annual Review of Entomology 45, 631-659. A'Brook, J. (1964). The effect of planting date and spacing on the incidence of groundnut rosette disease and of the vector, Aphis craccivora Koch, at Mokwa, Northern Nigeria. Annals of Applied Biology 54, 199 -208. Agyen-Sampong, M. (1978). Pests of cowpea and their control in Ghana. In Singh. S. R., Van Emden, H.F. and Taylor, T.A., (eds.), Pest of grain legumes: Ecology and control, London/New York, Academic press, pp. 85-92. Akbar, M.A., Islam, M.S., Bhuiya, M.S.U. and Hossain, M.A. (2000). Integration of fodder legumes into rice-based cropping systems in Bangladesh: Production of Lathyrus sativus and its use as a supplement to straw-based rations of dairy cows. Proceeding of the 9th AAAP/ASAP Congress held in Sydney, Australia, 2–7 July (2000), pp. 1–4. Akhtar, S., Bhat, M.A., Shafiq, A. Wani, Bhat, K. A., Chalkoo, S., Mir, M.R. and Shabir, A.W. (2010). Marker assisted selection in rice. Journal of Phytology 2(10), 66-8. Akibode, S. and Maredia, M. (2011). Global and Regional Trends in Production, Trade and Consumption of Food Legume. Report Submitted to SPIA, 87 pp. Allard, R. W. (1960). Principles of plant breeding. Wiley and Sons, Incorporated, John, 486 pp. University of Ghana http://ugspace.ug.edu.gh 88 Annan, I. B., Schaefers, G. A. and Tingey, W. M. (1995). Impact of density of Aphis craccivora Koch. (Aphididae) on growth and yield of susceptible and resistant cowpea cultivars. Annals of Applied Biology 128 (2), 185-193. Annan, I.B., Tingey, W.M., Schaefers, G.A., Tjallingii, W.F., Backus, E.A. and Saxena, K.N. (2000). Stylet penetration activities by Aphis craccivora (Homoptera: Aphididae) on plants and excised plant parts of resistant and susceptible cultivars of cowpea (Leguminosae). Annual Review of Entomology Society of America 93, 133-140. Ansari, A.K. (1984). Biology of Aphis craccivora Koch and varietal resistance of cowpeas, PhD Thesis, University of Reading, UK, 273 pp. Yashitola, J., Thirumurgan, T., Sundaram, R. M., Naseerullah, M. K., Ramesha, M. S., Sarma, N. P., and Sonti, R. V., (2002). Crop Science. 42, 1369-1373. Asare, K. B. (2012). Inheritance of resistance to flower bud thrips (Megalurothrips sjostedti) in cowpea. Master of Science Thesis, Kwame Nkrumah University of Science and Technology, 80 pp. Asiwe, J.A.N., Nokoe, S., Jackai, L.E.N. and Ewete, F.K. (2005). Does varying cowpea spacing provide better protection against cowpea pests? Crop Protection 24 (5): 461–471. Baird, W.V., Ballard, R.E., Rajapakse, S. and Abbott, A.G. (1996). Progress in Prunus mapping and application of molecular markers to germplasm improvement. Horticultural Science, 31, 1099–1106. University of Ghana http://ugspace.ug.edu.gh 89 Baird, W.V., Abbott, A., Ballard, R., Sosinski, B. and Rajapakse, S. (1997). DNA Diagnostics in Horticulture, Current Topics in Plant Molecular Biology Technology Transfer of Plant Biotechnology. CRC Press, Boca Raton, pp 111–130. Barone, A. (2004). Molecular marker-assisted selection for potato breeding. American Journal of Potato Research, 81, 111–117. Bashir, M., Ahamad, Z. and Ghafoor, A. (2002). Cowpea aphid-borne mosaic potyvirus: a review. International Journal of Pest Management, 48, 155–168. Bashir, M. and Hampton, R.O. (1996). Sources of genetic resistance in cowpea (Vigna unguiculata (L.) Walp.) to cowpea aphid-borne mosaic potyvirus. European Journal of Plant Pathology, 102, 411–419. Bata, H. D., Singh, B. B., Singh, R. S. and Ladeinde, T. A. O. (1987). Inheritance of resistance to aphid in cowpea. Crop Science, 27, 892 – 894. Baute, T. (2004). Soybean aphid. Available online at: Http//:www.gov.on.ca/omefra/english/crops/facts/soyaphid.htm; verified 21 June 2005. Beck, S.D. (1965). Resistance of plants to insects. Annual Review of Entomology, 10, 207- 232. University of Ghana http://ugspace.ug.edu.gh 90 Benchasri, S., Bairaman, C. and Nualsri, C. (2012). Evaluation of yardlong bean and cowpea for resistance to Aphis craccivora Koch in Southern Part of Thailand. Journal of Animal and Plant Sciences, 22 (4), 1024-1029. Blackman, R.L. and Eastop, V.F. (1994). Aphids on the World’s Trees: An Identification and Information Guide. CAB International, Wallingford, 466 pp. Blade, S.F., Shetty, S.V.R., Terao, T. and Singh, B.B. (1997). Recent development in cowpea cropping research. In Singh, B. B., Mohan Raj, D. R., Dashiell, K. E. and Jackei, L. E. N. (Eds.), Advances in Cowpea Research. International Institute of Tropical Agriculture, Ibadan, Nigeria, pp. 114-128. Blaney, W.M., Simmonds, M.S.J., Ley, S.V., Anderson, J.C. and Toogood, P.L. (1990). Antifeedant effects of azadirachtin and structurally related compounds on lepidopterous larvae. Entomologia Experimentalis Applicata, 55, 149-160. Bohlen, E. (1978). Crop pests in Tanzania and their control. Berlin-Hambrug: Verlag Paul Parey, 142 pp. Bohn, M., Groh, S., Khairullah, M.M., Hoisington, D.A., Utz, H.F. and Melchinger, A.E. (2001). Re-evaluation of the prospects of marker-assisted selection for improving insect resistance against Diatraea spp. In: Tropical maize by cross validation and interdependent validation. Theoretical and Applied Genetics, 103, 1059–1067. University of Ghana http://ugspace.ug.edu.gh 91 Booker, R.H. (1963). The effect of sowing date and spacing on rosette disease of groundnut in northern Nigeria, with observations on the vector, Aphis craccivora. Annals of Applied Biology, 52, 125-31. Bosque-Perez, N. A. and Schotzko, D. J. (2000). Wheat genotype, early plant growth stage and infestation density effects on Russian wheat aphid (Homoptera: Aphididae) population increase and plant damage. Journal of Entomological Science, 35, 22-38. Braendle, C., Davis, G.K., Brisson, J.A. and Stern, D.L. (2006). Wing dimorphism in aphids. Heredity, 97 (3), 192-201. CGIAR. (2011). Consultative Group on International Agricultural Research (CGIAR). Cowpea (Vigna unguiculata). [Online] Available: http://www.cgiar.org/impact/research /cowpea.html (Accessed on 23 April, 2011). Chari, M.S., Patel, P.N. and Raj, S. (1976). Evaluation of cowpea lines for resistance to aphid, Aphis craccivora, Koch. Gujarat Agricultural University Research Journal, 1, 130- 132. Collard, B. C. Y., Jahufer, M. Z. Z., Brouwer, J. B. and Pang, E. C. K. (2005). An introduction to markers, quantitative trait loci (QTL) mapping and marker-assisted selection for crop improvement: The Basic Concepts. Euphytica, 142: 169-196. Cuartera, J., Laterrot, H. and van Lenteren, J.C. (1999). Host-Plant Resistance to Pathogens and Arthropod Pests. Integrated Pest and Disease Management in Greenhouse Crops. Albajes, R. (ed.), Academic Publishers. Printed in the Netherlands, pp. 124-138. University of Ghana http://ugspace.ug.edu.gh 92 Dagg, J. (2002). Strategies of Sexual Reproduction in Aphids. Dissertation, ontionstudium der Biologie, Universität Göttingen, Germany, 63 pp. Dahms, R. G. (1972). The role of host plant resistance in integrated insect control. In: The control of sorghum shoot Fly. M.G. Jotwani and W. R. Young, Eds. Delhi: Oxford and IBH, pp 152-167. Davis, D.W., Oelke, E.A., Oplinger, E.S., Doll, J.D., Hanson, C.V. and Putnam, D.H. (2003). Alternative field crop manual. Center for Alternative Plant and Animal Products, Minnesota Extension Service, University of Minnesota, http://www.hort.purdue.edu/newcrop/afcm/cowpea.html, last updated: Wed May 28 19:38:04 UTC+0100 2003 Dent, D. (1991). Insect pest management. CAB international, Wallingford, UK, 604 pp. Dent, D. (2000). Insect Pest Management. 2nd edition. CABI Publishing 410 pp. Dhanorkar, B.K. and Daware, D.G. (1980). Differences in number of aphids found on lines of cowpea in a replicated trial. Tropical Grain Legume Bulletin, 19, 3 - 4. Diehl, L. and Sipkins, L. (1985). The Development of Mixed Cropping Technologies in Northern Ghana. In: Ohm, H. W. and Nagy, J. G. (Eds.), Appropriate technologies for farmers in semi-arid West Africa. International Programme in Agriculture. Purdue University. West Lafayette, Indiana, USA. pp 260-268. University of Ghana http://ugspace.ug.edu.gh 93 Diouf, D. and Hilu, K.W. (2005). Microsatellites and RAPD markers to study genetic relationship among cowpea breeding lines and local varieties in Senegal. Genetic Resources and Crop Evolution, 52, 1057-1067. Dita, M.A., Rispail, N., Prats, E., Rubiales, D. and Singh, K.B. (2006). Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes. Euphytica, 147, 1- 24. Dixon, A.F.G. (1970). Quality and availability of food for a sycamore aphid population. Animal populations in relation to their food resources Watson, A. (Ed.), Blackwell, Oxford, pp. 271-285. Dixon, A.F.G. (1971). The life-cycle and host preference of the bird charry-oat aphid, Rhopalosiphum padi L. and their bearings on the theories of host alteration in aphids. Annals of Applied Biology, 68, 135 – 147. Dixon, A.F.G and Wretten, S.D. (1971). Laboratory studies on aggregation, size and fecundity of the black bean aphid, Aphis fabae Scop. Bulletin of Entomological Research 61, 91-111. Dixon, A.F.G. (1977). Aphid Ecology: Life-cycles, polymorphisms, and population regulation. Annual Review of Ecology and Systematics, 8, 329-353. Dixon, A.F.G. and Oharma, T.I. (1980). Number of ovarioles and the fecundity in the black bean aphid, Aphis fabae. Entomologia Experimentalis et applicata, 28, 1- 14. University of Ghana http://ugspace.ug.edu.gh 94 Dixon, A.F.G. (1985). Aphid Ecology. 1st edn. Blackie, Glasgow, 157 pp. Dreher, K., Khairallah, M., Ribaut J., Morris, M. (2003). Money matters (I): costs of field and laboratory procedures associated with conventional and marker-assisted maize breeding at CIMMYT. Molecular Breeding, 11, 221–234. Eagles, H., Bariana, H., Ogbonnaya, F., Rebetzke, G., Hollamby, G., Henry, R., Henschke, P., Carter, M. (2001). Implementation of markers in Australian wheat breeding. Australian Journal of Agricultural Research, 52, 1349–1356. Eckey-Kaltenbach, H., Ernst, D., Heller, W. and Sandermann, H. Jr, (1994). Biochemical plant responses to ozone: IV. Cross-induction of defensive pathways in parsley (Petroselinum crispum L.) plants. Plant Physiology, 104, 67-74. Egho, E. O. (2011). Management of major field insect pests and yield of cowpea (Vigna unguiculata (L) Walp) under calendar and monitored application of synthetic chemicals in Asaba, southern Nigeria. American Journal of Scientific and Industrial Research, 2(4), 592- 602. Erbaugh, J.M., Willson, H. and Kyamanywa, S. (1995). Participatory Appraisal: Iganga and Kumi districts, Uganda. Working paper 95-96 of the IPM/CRSP, Virginia Polytechnic Institute and State University, Blacksburg, Virginia. Pp. 28-30. University of Ghana http://ugspace.ug.edu.gh 95 Fabio, F.G., Ragagnin, V.A., Moreira, M. A. and Barros E.G. de. (2004). Use of molecular markers to accelerate the breeding of common bean lines resistant to rust and anthracnose. Euphytica. 138 (3), 213–218. F.A.O. (2008). Food and Agriculture Organization of the United Nations, Rome. FAOSTAT Database. F.A.O. (2011). Food and Agriculture Organization of the United Nations, Rome. FAOSTAT Database. http://faostat.fao.org/default.aspx, accessed December 2010 to March 2011. F.A.O. (2012). Balanced feeding for improving livestock productivity – Increase in milk production and nutrient use efficiency and decrease in methane emission, by M.R. Garg. FAO Animal Production and Health Paper, 173, 46 pp. Farrell, J.A.K. (1996a). Effect of groundnut sowing date and plant spacing on rosette virus by Aphis craccivora Koch (Hemiptere, Aphididae) in Malawi, Bulletin of Entomological Research, 66, 159-171. Farrell, J.A.K. (1996b). Effect of intersowing with beans on the spread of groundnut rosette virus by Aphis craccivora (Hemiptere, Aphididae) in Malawi. Bulletin of Entomological Research, 66, 331-333. Fatokun, C.A. (2002). Breeding cowpea for resistance to insect pests: attempted crosses between cowpea and Vigna vexillata. Challenges and Opportunities for Enhancing University of Ghana http://ugspace.ug.edu.gh 96 Sustainable Cowpea Production. Proceedings of the World Cowpea Conference III. 4-8th September 2000. C. A. Fatokun, S. A., Tarawali, B. B., Singh, P. M., Kormawa, and M. Tamo. (Eds.). International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Pp. 52-61. Felkl, G., Jensen, E.B., Kristiansen, K., Andersen, S.B. (2005). Tolerance and antibiosis resistance to cabbage root fly in vegetable Brassica species. Experimental Entomology, 116, 65-71. Fery, R.L. (1985). Improved cowpea cultivars for the horticultural industry for the USA. In: Cowpea research, production and utilization, Singh S.R. and Rachie K.O (eds.), John Wiley and Sons, Chichester, UK, Pp. 129-136. Fregene, M., Okogbenin, E., Mba, C., Angel, F., Suarez, M.C., Janneth, G., Chavarriaga, P., Roca, W., Bonierbale, M. and Tohme, J. (2001). Genome mapping in cassava improvement: Challenges, achievements and opportunities. Euphytica, 120,159– 165. Frisch, M., Bohn, M., Melchinger, A.E. (1999). Comparison of selection strategies for marker-assisted backcrossing of a gene. Crop Science, 39, 1295–1301. Frisch, M. and Melchinger, A.E. (2000). The length of the intact donor chromosome segment around a target gene in marker-assisted backcrossing. Genetics, 157, 1343-1356. University of Ghana http://ugspace.ug.edu.gh 97 Gallun, R.L. (1977). Genetic basis of Hessian fly epidemics. Annals of the New York Academy of Science, 287, 223-229. Gebhardt, C. and Valkonen, J.P.T. (2001). Organization of genes controlling disease resistance in the potato genome. Annual Review of Phytopathology, l39, 79–102. Giordanengo, P., Brunissen, L., Rusterucci, C., Vincent, C., van Bel, A., Dinant, S., Girousse, C., Faucher, M., Bonnemain, J.L. (2010). Compatible plant-aphid interactions: How aphids manipulate plant responses. Comptes Rendus Biologies, 333, 516-523. Githiri, S.M., Ampong-Nyarko, K., Osir, E.O. and Kimani, P.M. (1996). Genetics of resistance to Aphis craccivora in cowpea. Kluwer Academic Publishers. Euphytica, 89. 371- 376. Grau, C., B. Jensen, S. Myers, and J. Wedberg. (2002). “Soybean Aphid.” Fact Sheet, Team Grains Publication No. 1:1, University of Wisconsin, Madison, WI Gullan, P.J. and Cranston, P.S. (1994). The Insect: An Outline of Entomology. London: Chapman and Hall, 491 pp. Gupta, P.K., Varshney, R.K., Sharma, P.C. and Ramesh, B. (1999). Molecular markers and their application in wheat breeding: a review. Plant Breeding. 118, 369-390. Hamid, S., Shah, M.A. and Anwar, A.M. (1977). Some ecological and behavioural studies on Aphis craccivora Koch (Hem.: Aphididae). CIBC Technical Bulletin. 18, 99 -111. University of Ghana http://ugspace.ug.edu.gh 98 Henry, R. (1997). Molecular markers in plant improvement In: Practical Applications of Plant Molecular Biology, Chapman and Hall, London. pp. 99–132. Hill, D.S. (1983). Agricultural Insect Pests of the Tropics and their control. Cambridge: Cambridge University Press, 746 pp. Hill, C.B., Y. Li, and G.L. Hartman. (2006). “A Single Dominant Gene for Resistance to the Soybean Aphid in the Soybean Cultivar Dowling.” Crop Science 46: 1601–1605. Ibitoye, D. O. and Akin-Idowu, P. E. (2010). Marker-assisted-selection (MAS): A fast track to increase genetic gain in horticultural crop breeding. African Journal of Biotechnology, 9, (52), pp. 8889-8895. IITA. (1981). Annual report for 1980. Ibadan, Nigeria, pp 59 - 60. IITA. (2009). Annual report for 2008/2009. Ibadan, Nigeria, 46 pp. Jackai, L.E.N. and Adalla, C.B. (1997). The management practices in cowpea: a review. In: Advances in Cowpea Research, B.B. Singh, D.R. Mohan Raj, K.E. Dashiell and L.E.N. Jackai (eds.), Co-publication of International Institute of Tropical Agriculture (IITA) and Japan International Research Center for Agricultural Sciences (JIRCAS), IITA Ibadan, Nigeria, pp. 240-258. University of Ghana http://ugspace.ug.edu.gh 99 Jackai, L.E.N. and Singh, J.R. (1983). Insect pests of cowpea in Africa: Their life cycle, economic importance and potential control. In: cowpea production and utilization. John Wiley and Sons, New York, pp. 217 - 231. Jackai, L.E.N. and Dacoust, R.A. (1986). Insect pests of cowpeas. Annual Review of Entomology. 31, 95-119. Jahufer, M., Barret, B., Griffiths A. and Woodfield, D. (2003). DNA fingerprinting and genetic relationships among white clover cultivars. In: J. Morton (Ed.), Proceedings of the New Zealand Grassland Association, 65, 163–169. Jang, Y.D. and Yun, Y.N. (1983). A study on the biology of primary parasites of the cowpea aphid, Aphis craccivora (Aphididae, Homoptera) and its hyperparasites. Korea Journal of Plant Protection 22, 237 - 243. Johnson, R. and Law, C.N. (1975). Genetic control of durable resistance to yellow rust Puccinia striiformis) in the wheat cultivar Hybrid de Bersce. Annals of Applied Biology, 81, 385-392. Jones, N., Ougham, H. and Thomas, H. (1997). Markers and mapping: We are all geneticists now. New Phytologist, l137, 165–177. Joshi, S., Ranjekar, P. and Gupta, V. (1999). Molecular markers in plant genome analysis. Current Science, 77, 230–240. University of Ghana http://ugspace.ug.edu.gh 100 Kamara, A.Y., Chikoye, D., Omoigui, L. O. and Dugje, I. Y. (2007). Influence of insecticide spraying regimes and cultivar on insect pests and yield of cowpea in the dry savannas of north-eastern Nigeria. Journal of Food, Agriculture and Environment. 5 (1), 154-158. Kamara, A.Y., Ekeleme, F., Omoigui, L.O., Abduolaye, T., Amaza, P., Chikoye, D. and Dugje, Y. (2010). Integrating planting date with insecticide spraying regime to manage insect pests of cowpea in north-eastern Nigeria. International Journal of Pest Management, 56 (3) 243-253. Kamphuis, L.G., Gao, L. and Singh, K.B. (2012). Identification and characterization of resistance to cowpea aphid (Aphis craccivora Koch) in Medicago truncatula. BMC Plant Biology, 4, 12, pp. 101. Karban, R. and Baldwin, I. T. (1997). Induced Responses to Herbivory. The University of Chicago Press, Chicago, 330 pp. Karel, A.K. and Malinga, Y. (1980). Leafhopper and aphid resistance in cowpea varieties. Tropical Grain Legume Bulletin, 20: 10 – 11. Kasha, K.J. (1999). Biotechnology and world food supply. Genome, 42, 642–645. Kelly, J.D. and Miklas, P.N. (1998). The role of RAPD markers in breeding for disease resistance in common bean. Molecular Breeding, 4, 1–11. University of Ghana http://ugspace.ug.edu.gh 101 Kelly, J.D., Gepts, P., Miklas, P.N. and Coyne, D.P. (2003). Tagging and mapping of genes and QTL and molecular marker-assisted selection for traits of economic importance in bean and cowpea. Field Crops Research, 82, 135–154. Kempton, R.A., Lowe, H.J.B. and Bintcliffe, E.L.B. (1980). The relationship among fecundity and adult weight in Myzus persiae. Journal of Animal Ecology, 49, 917 – 926. Kfir, R., Overholt, W.A., Khan, Z.R. and Polaszek, A. (2002). Biology and management of economically important Lepidopteran cereal stem borers in Africa. Annual Review of Entomology, 47, 701-731. Kim, C.S., Schaible, G., Garrett, L., Lubowski, R. and Lee, D. (2008). Economic Impacts of the U.S. Soybean Aphid Infestation: A Multi-Regional Competitive Dynamic Analysis. Agricultural and Resource Economics Review 37(2):227–242 Koebner, R.M.D. and Summers, R.W. (2003). 21st century wheat breeding: plot selection or plate detection? Trends of Biotechnology, 21, 59–63. Kogan, M. (1975). Plant resistance in pest management. In: Introduction to insect pest management. R. L. Metealf and W. H. Luckman (Eds.). Wiley and sons. New York. USA, 103-46. Kogan, M. and Omar, E.E. (1978). Antixenosis – a new term proposed to replace painter‘s ‘Nonpreference’ modality of resistance. ESA Bulletin, 24. University of Ghana http://ugspace.ug.edu.gh 102 Kumar, L. S. (1999). DNA markers in plant improvement: An overview. Biotechnology Advances, 17: 143-182. Kumar, R. (1984). Insect pest control with special reference to African Agriculture. Edward Arnold, London, 298 pp. Kusi, F. (2008). Screening of cowpea genotypes for resistance to cowpea aphids, Aphis craccivora Koch. M.Phil thesis, University of Ghana Legon, 119 pp. Kusi, F., Obeng-Ofori, D., Asante, S. K. and Padi, F. K. (2010a). New sources of resistance in cowpea to the cowpea aphid (Aphis craccivora Koch) (Homoptera: Aphididae). Journal of Ghana Science Association, 12 (2), 95-104. Kusi, F., Obeng-Ofori, D., Asante, S. K. and Padi, F. K. (2010b). The compensatory and susceptive responses of cowpea genotypes to infestation by Aphis craccivora Koch. Journal of Science and Technology, 30 (3), 27-34. Laamari, M., Khelfa, L. and Acier, A.C. (2008). Resistance source to cowpea aphid (Aphis craccivora Koch) in broad bean (Vicia faba L.) Algerian landrace collection. Africa Journal of Biotechnology, 7(14), 2486-2490. Langridge, P., Lagudah, T., Apples, R., Sharp, P. and Chalmers, K. (2001). Trends in genetics and genome analysis in wheat: A review. Australian Journal of Agricultural Research, 52, 1043-1077. University of Ghana http://ugspace.ug.edu.gh 103 Leather, S.R. and Wellings, P.W. (1981). Ovariole number and fecundity in aphids. Entomologia Experimentalis et Applicata, 30, 128 - 133. Leather, S.R., Ward, S.A. and Dixon, A.F.G. (1983). The effect of nutrition stress on some life history parameters of the black bean aphid, Aphis fabae, Scop. Oecologia, 57, 156 – 157. Lees, A.D. (1959). The role of photoperiod and temperature in the determination of the parthenogenetic and sexual forms in the aphid Magoura viciae Buckton,1. The influence of these factors on the apterous virginoparae and their progeny. Journal of Insect Physiology, 3, 93 – 117. Lee, D., Kim, C.S. and Schaible, G. (2006). “Estimating the Cost of Invasive Species on U.S. Agriculture: The U.S. Soybean Market.” Selected paper, American Agricultural Economics Association annual meetings, Long Beach, CA (July 23–26). Lee, M. (1995). DNA Markers and Plant Breeding Programs, Advances in Agronomy, 55: 265–344. Livingston, M., Johansson, R., Daberkow, S., Roberts, M., Ash, M. and Breneman, V. (2004). “Economic and Policy Implications of Wind-Borne Entry of Asian Soybean Rust in the United States.” Report No. OCS-04D-02, Economic Research Service, U.S. Department of Agriculture, Washington, D.C. University of Ghana http://ugspace.ug.edu.gh 104 Lorieux, M., Goffinet, B., Perrier, X., Gonzalez, de Leon, D. and Lanaud, C. (1995). Maximum-likelihood models for mapping genetic markers showing segregation distortion in backcross populations. Theoretical and Applied Genetics, 90, 73–80. MacFoy, C.C. and Dabrowski, Z.T. (1984). Preliminary studies of cowpea resistance to Aphis craccivora Koch (Homoptera: Aphididae). Journal of Applied Entomology, 97, 202- 209. Mackean, D.G (2006). Biology teaching resources. http://www.biology-resources.com/insect-01.html, Retrieved on: 23rd August, 2008. Mackill, D.J., Nguyen, H.T. and Zhan, J. (1999). Use of molecular markers in plant improvement programs for rain fed lowland rice, Field Crops Research, 64: 177-185. Manawadu, D. (1985). Varietal susceptibility of cowpea to Aphis craccivora. Tropical Grain Legume Bulletin, 30, 15 - 20. Martyn, K.P. (1991). Genetic performance and behavioral variation among clones of A. craccivora Koch, with specific reference to resistance in Vigna unguiculata (L) Walp. (cowpea). Ph.D Thesis, University of London. The years work english studies 72 (1), 593- 616. McCarville, M., Hodgson, E. and O’Neal, M. (2013). Soybean aphid-resistant soybean varieties in Iowa. Extension and Outreach. pp. 1-4. University of Ghana http://ugspace.ug.edu.gh 105 McCornack, B.P., Ragsdale, D.W. and Venette, R.C. (2004). “Demography of Soybean Aphid (Homoptera: Aphididae) at Summer Temperatures.” Journal of Economic Entomology 97(3): 854–861. McCouch, S.R. and Doerge, R.W. (1995). QTL mapping in rice. Trends of Genetics, 11, 482-487. Mehlenbacher, S.A. (1995). Classical and molecular approaches to breeding fruit and nut crops for disease resistance. Horticultural Science, 30, 466–477. Messina, F.J., Renwick, J.A.A. and Barmore, J.L. (1985). Resistance to Aphis craccivora (Homoptera: Aphididae) in selected varieties of cowpea. Journal of Entomological Science, 20, 263 - 9. Meyer, J.R. (2003). Pest Control Tactics: North Carolina State ENT 425 Chapter 19. http://www.cals.ncsu.edu/course/ent425/text19/cultural.html Michelmore, R. (1995). Molecular approaches to manipulation of disease resistance genes. Annual Review of Phytopathology, 33(1), 393-427. Mishra, S.N., Verma, J.S. and Jayasekara, S.J.B.A. (1985). Breeding cowpea to suit Asian cropping systems and consumer tastes. In: Cowpea research, production and utilization, S.R. Singh and K.O. Rachie (eds.), John Wiley and Sons, Chichester, UK, pp. 117 – 123. University of Ghana http://ugspace.ug.edu.gh 106 Mohan, M., Nair, S., Bhagwat, A., Krishna, T.G., Yano, M., Bhatia, C.R. and Sasaki, T. (1997). Genome mapping, molecular markers and marker-assisted selection in crop plants. Molecular Breeding, 3, 87–103. Mortimore, M. J., Singh, B. B., Harris, F and Blade, S. F. (1997). Cowpea in Traditional Cropping Systems. In: Singh, B. B., Mohan-Raj, D. R; Dashiell, K. E., Jackai, L. E. N (eds.), Advances in Cowpea Research, Co publication of International Institute of Tropical Agriculture (IITA) and Japan International Research Center for Agricultural Sciences (JIRCAS) IITA, Ibadan, Nigeria, pp. 99 - 113. Morris, M., Dreher, K., Ribaut, J.M. and Khairallah, M. (2003). Money matters (II): costs of maize inbred line conversion schemes at CIMMYT using conventional and marker assisted selection. Molecular Breeding, 11, 235-247. Munyuli, .M.B., Luther, G.C. and Kyamanywa, S. (2007). Effects of cowpea cropping systems and insecticides on arthropod predators in Uganda and Democratic Republic of the Congo. Crop Protection, 26, 114-126. Munyuli, T.M.B., Luther, G.C. and Kyamanywa, S. (2008). Effects of groundnut genotypes, cropping systems and insecticides on the abundance of native arthropod predators from Uganda and Democratic Republic of the Congo. Bulletin of Insectology, 67, 11-19. Munyuli, T. (2009). Effects of Native Insect Predators on Population Densities of Aphis craccivora and Yields of Vigna unguiculata and Arachis hypogeae Grown under Various University of Ghana http://ugspace.ug.edu.gh 107 Cropping Systems, in Kivu Province, Eastern Democratic Republic of Congo. Tunisian Journal of Plant Protection, 208 (4), 197-210. Murdie, G. (1969). Some causes of size variation in pea aphid, Acyrthosiphum pisum Harris. Transactions of the Royal Entomological Society of London, 121, 423 – 442. Nampala, P., Ogenga-Latigo, M. W., Kyamanywa, S., Adipala, E., Karunji, J., Oyobo, N., Obuo, J. E. and Jackai L.E.N. (1999). Integrated management of major field pests of cowpea in eastern Uganda. Africa Crop Science Journal, 7, 479-486. Obeng-Ofori, D. (2007). Pests of grain legumes. In: Major pests of food and selected fruit and industrial crops in West Africa, D. Obeng-Ofori (ed.) The City publishers Ltd, Accra, pp. 81-112. Ofuya, T.I. (1988a). Resistance of some cowpea varieties to the cowpea aphid, Aphis craccivora Koch (Homoptera: Aphididae) under field and screenhouse conditions in Nigeria. Tropical Pest Management, 34, 445 - 447. Ofuya, T.I. (1988b). Antibiosis in some cowpea varieties resistant to the cowpea aphid, Aphis craccivora Koch (Homoptera: Aphididae). Integrated Pest Control, 30, 68 - 69. Ofuya, T.I. (1989). Studies on infestation, occurrence, growth and survival of Aphis craccivora Koch on cowpea and other alternative hosts in Nigeria. Nigerian Journal of Basic Application of Science, 3, 19 - 25. University of Ghana http://ugspace.ug.edu.gh 108 Ofuya, T.I. (1990). Observation on the biology of Cheilomenes vicina (Mulsant) (Coleoptera: Coccinellidae), a predator for the control of the cowpea aphid, Aphis craccivora Koch (Homoptera: Aphididae) in Nigeria. Nigerian Journal of Science, 24, 171 - 173. Ofuya, T.I. (1991). Aspects of the ecology of predation in two coccinellid species on the cowpea aphid in Nigeria. In L. Polgar, R.J. Chambers, A.F.G. Dixon and I. Hodek (eds.). Behaviour and Impact of Aphidophaga. The Hague: SPB Academic Publishing bv, pp. 213 - 220. Ofuya, T.I. (1993). Evaluation of selected cowpea varieties for resistance to Aphis craccivora Koch (Homoptera: Aphididae) at the seedling and podding phase. Ann. Appl. Boil. 123(1), 19-23. Ofuya, T.I. (1995). Studies on the capability of Cheilomenes lunata (Fabricius) (Coleoptera: Coccinellidae) to prey on the cowpea aphid, Aphis craccivora Koch (Homoptera: Aphididae) in Nigeria. Agricultural Ecosystem Environment, 52, 35 - 38. Ofuya, T. I. (1997a). Control of cowpea aphid, Aphis craccivora Koch (Homoptra: Aphididae), in cowpea, Vigna unguiculata (L.) Walp. Integrated Pest Management Reviews, 2, 199 – 207. Ofuya, T.I. (1997b). Effect of some plant extracts on two coccinellid predators of the cowpea aphid, Aphis craccivora (Hom.: Aphididae). Entomophaga, 42, 279 - 284. University of Ghana http://ugspace.ug.edu.gh 109 Ofuya, T.I. and Akingbohungbe, A.E. (1988). Functional and numerical responses of Cheilomenes lunata (Fabricius) (Coleoptera: Coccinellidae) feeding on the cowpea aphid, Aphis craccivora Koch (Homoptera: Aphididae). Insect Science and its Application, 9, 543 -546. Ombakho, G. A., Tyagi, A. P. and Pathak, R. S. (1987). Inheritance of resistance to the cowpea aphid in cowpea. Theoretical and Applied Genetics, 74 (6), 817 - 819. Omongo, C.A., Ogenga-Latigo, M.W., Kyamanywa, S. and Adipala, E. (1997). Effects of seasons and cropping systmes on occurrence of cowpea pests in Uganda. African Crop Science Conference Proceedings, 3, 1111-1116. Orawu, M., Melis, R., Laing, M. and Derera, J. (2013), Genetic inheritance of resistance to cowpea aphid-borne mosaic virus in cowpea. Euphytica, 189, 191–201. Ortiz, R. (1998). Critical role of plant biotechnology for the genetic improvement of food crops: perspectives for the next millennium. Electron Journal of Biotechnology, 1(3), 1-8. Osteo, C.T. and Helms, T.I. (1971). Embryonic and post-paturienic reproductive systems development in Schizoaphis graminum (Hemiptera: Aphididae), Annals of the Entomological Society of America, 64, 603 – 609. Padi, F.K., Denwar, N.N., Kaleem, F.Z., Salifu, A.B., Clottey, V.A., Kombiok, J., Haruna, M., Hall, A.E. and. Marfo, K.O. (2004). Registration of ‘Apagbaala’ cowpea. Crop Science, 44, 1486-1487. University of Ghana http://ugspace.ug.edu.gh 110 Padi, F.K. and Ehlers, J.D. (2008). Effectiveness of early generation selection in cowpea for grain yield and agronomic characteristics in semiarid West Africa. Crop Science 48, 533 – 540. Painter, R.H. (1951). Insect Resistance in Crop Plants, Macmillan, New York, 520pp. Paterson, A.H. (1996a). Making genetic maps. In: A.H. Paterson (Ed.). Genome mapping in plants. Landes Company, San Diego, California: Academic Press; Austin, Texas. Pp. 23- 39. Paterson, A.H. (1996b). Mapping genes responsible for differences in phenotype. In: A.H. Paterson (Ed.). Genome mapping in plants. Landes Company, San Diego, California: Academic Press; Austin, Texas. Pp. 41-54. Pathak, R.S. (1988). Genetics of resistance to aphid in cowpea. Crop Science, 28, 474 - 476. Porter, R.D., Burd, J.D., Shufran, K.A., Webster, J.A. and Teetes, G.L. (1997). Greenbug biotypes: selected by resistant cultivars or preadapted opportunists? Journal of Economic Entomology, 90, 1055-1065. Puterka, G.J., Burd, J.D. and Burton, R.L. (1992). Biotypic variation in a worldwide collection of Russian wheat aphid (Homoptera: Aphididae). Journal of Economic Entomology, 85, 1497-1506. University of Ghana http://ugspace.ug.edu.gh 111 Quin, F.M. (1997). Introduction. In: B.B. Singh, D.R. Mohan Raj, K.E. Dashiel and L.E.N. Jackai (Eds.). Advances in cowpea research. Co-publication of International Institute of Tropical Agriculture (IITA) and Japan International Research Centre for Agricultural Sciences (JIRCAS), Ibadan, Nigeria. Rachie, K.O. (1985). Introduction, In: Cowpea research production and utilization. Singh S. R. and Rachie, K.O. (Eds.). Wiley and Sons. London. Rafalski, J. and Tingey, S. (1993). Genetic diagnostics in Plant breeding: RAPDs, microsatellites and machines. Trends of Genetics, 79, 275-280. Recovery of the recurrent parent genome (2008): http://mcclintock.generationcp.org/index.php?option=com_content&task=view&id=109& Itemid=115. Date sited: 20th November 2013. Ribaut, J.M., Hu, X., Hoisington, D. and Gonza´ lez-de-Leon, D. (1997). Use of STS and SSRs as rapid and reliable preselection tools in a marker-assisted backcross selection scheme. Plant Molecular Biology, 15, 154–162. Ribaut, J.M. and Hoisington, D. (1998). Marker assisted selection: new tools and strategies. Trends Plant Science, 3, 236-239. Ribaut, J.M. and Betran, J. (1999). Single large-scale marker-assisted selection (SLS– MAS) Molecular Breeding, 5,531–541. University of Ghana http://ugspace.ug.edu.gh 112 Ribaut, J.M., Jiang C. and Hoisington, D. (2002). Simulation experiments on efficiencies of gene introgression by backcrossing. Crop Science, 42,557–565. Russell, G.E. (1978). Plant breeding for pest and disease resistance. Butterworth, London, pp 485. SARI. (1999). Annual Report of 1998. SARI, Tamale, Ghana 276 pp. Saxena, R.C. and Barrion, A.A. (1987). Biotypes of insect pests of agricultural crops. Insect Science and its Applications, 8, 453-458. Schultz, T. C.(2002). How plants fight dirty. Nature,416, 267 pp. Shoyinka, S.A., Bozarth, R.F., Rees, J. and Okusanya, B.O. (1997). Field occurrence and identification of southern bean mosaic virus (cowpea strain) in Nigeria. Turrialba, 29 (2), 111 – 116. Shufran, K. A., Mornhinweg, D. W., Baker, C. A. and Porter, D. R. (2007). Variation to cause host injury between Russian wheat aphid (Homoptera: Aphididae) clones virulent to Dn4 wheat. Journal of Economic Entomology, 100 (5), 1685 -1691. Siemens, D.H., Garner, S.H., Mitchell-Olds T. and Callaway, R.M. (2002). Cost of defense in the context of plant competition: Brassica rapa may grow and defend. Ecology, 83, 505-517. University of Ghana http://ugspace.ug.edu.gh 113 Singh, B.B. (2004). Achievements so far in improving cowpea productivity through conventional breeding. A conference proceeding at an international workshop for strategies for application of molecular technologies to the breeding of cowpea in Africa for increased productivity. Jointly convened by AATF, NGICA and Kirkhouse Trust. 15-17 November, 2004, Cresta Royal hotel – Accra, 6 pp. Singh, S.R. (1977). Cowpea cultivars resistant to insect pests in world germplasm collection. Tropical Grain Legume Bulletin, 9, 3 - 7. Singh, S.R. (1990). Insect pests of tropical food legumes. John Wiley and Sons Ltd, New York, S.R. Singh (ed.), pp. 43 - 89. Singh, S.R. and Allen, D.J. (1980). Pests, diseases, resistance and protection in cowpea. In: Advances in Legume Science. Summerfield, R.J. and Bunting, H.H. (Eds.), pp. 419-433. Royal Botanical Garden, Kew, Ministry of Agriculture, Fisheries and Food, London. Singh, S.R. and Jackai, L.E.N. (1985). Insect pests of cowpea in Africa; Their life cycle, economic importance, and potential for control. In: Cowpea Research, Production, and Utilization. Singh, S.R. and Rachie, K.O. (Eds.), pp. 217 – 231. Singh, S.R. and Ntare, B.R. (1985). Development of improved cowpea varieties in Africa. In S.R. Singh and K.O. Rachie (Eds.). Cowpea Research, Production and Utilization. Chichester: John Wiley & Sons, pp. 106 – 115. University of Ghana http://ugspace.ug.edu.gh 114 Singh, B.B., Thottappilly, G. and Rossel, H.W. (1987). Breeding for multiple virus resistance in cowpea. Agronomy Abstract. American Society of Agronomy, Madison, Wisconsin, USA, Crop Science 25, 736 – 739. Singh, S.R., Jackai, L.E.N., Dos Santos, J.H.R. and Adalia, C.B. (1990). Insect pests of cowpea. In: S.R. Singh (ed.). Insect Pests of Tropical Food Legumes. Chichester: John Wiley & Sons, pp. 43-89. Smith, C.M. (1989). Plant resistance to insects: A fundamental approach. John Wiley & Sons, New York, 286 pp. Smith, C.M., Belay, T., Stauffer, C., Stary, P., Kubeckova, I. and Starkey, S. (2004). Identification of Russian wheat aphid (Homoptera: Aphididae) populations virulent to the Dn4 resistance gene. Journal of Economic Entomology, 97 (3), 1112-1117. Snelling, R.O. (1941). Resistance of plants to insect attack. Botanical Research, 7, 543 – 586. Snowdon, R. and Friedt, W. (2004). Molecular markers in Brassica oilseeds breeding. Current status and future possibilities, Plant Breeding, 123, 1–8. Souleymane, A., Aken’Ova, M.E., Fatokun, C. A. and Alabi O. Y. (2013). Screening for resistance to cowpea aphid (Aphis craccivora Koch) in wild and cultivated cowpea (Vigna unguiculata Walp.) accessions. International Journal of Science, Environment and Technology, 2 (4), 611 – 621. University of Ghana http://ugspace.ug.edu.gh 115 Stadler, B. (1995). Adaptive allocation of resources and life-history trade-offs in aphid relative to plant quality. Oecologia, 102, 246 – 254. Städler, E. (2002). Plant chemical cues important for egg deposition by herbivorous insects. In: Hilker, M., and Meiners, T. (eds). Chemoecology of insect eggs and egg deposition. Blackwell Verlag, Berlin, pp. 171-204. Steele, W.M. (1972). Cowpea in Nigeria. PhD thesis, University of Reading, UK, 242 pp. Stoddard, F. L., Nicholas, A. H., Rubiales, D., Thomas, J. and Villegas-Ferna´ndez, A.M. (2010). Integrated pest management in faba bean. Field Crops Research, 115, 308– 318. Stuber, C. W., Polacco, M. and Senior, M.L. (1999). Synergy of empirical breeding, marker-assisted selection and genomics to increase crop yield potential. Crop Science, 39, 1571-1583. Summers, C.G., Godfrey, L.D., Rethwisch, M., Goodell, P.B. and Long, R.F. (2006). UC IPM Pest Management Guidelines. UC ANR Publication 3430, 72, (2), 4 pp. Suranyi, R., Ragsdale, D. and Radcliffe, T. (1998). Aphid Alert. Published by Department of Entomology and Plant Pathology, College of Agricultural, Food and Environmental Sciences. University of Minnesota. http://ipmworld.umn.edu/aphidalert/alert3.htm, Last updated: 2003 University of Ghana http://ugspace.ug.edu.gh 116 Suszkiw, J. (2005). “Resistance Gene to Fortify Soybean Against Exotic Pest.” Agricultural Research, 1105. Available at http://www.ars.usda.gov/is/AR/archive/nov05/soy[-] Svetleva, D., Velcheva, M. and Bhowmik, G. (2003). Biotechnology as a useful tool in common bean (Phaseolus vulgaris L) improvement. Euphytica, 131, (2), 189-200. Taylor, T.A. (1981). Distribution, ecology and importance of bruchid attacking grain legumes and pulses in Africa. In: The bruchids attacking legumes (pulses). Labeyrie, V. and Junk, W. (Eds.). The Hague, Netherlands, pp. 199 – 203. Taylor, L.R. (1975). Longevity, fecundity and size; control of reproductive potential in the polymorphic migrant, Aphis fabae Scop. Journal of Animal Ecology, 44, 135 – 165. Theis, N. and M. Lerdau. (2003). The evolution of function in plant secondary metabolites. International Journal of Plant Science, 164 (3), 93-102. Thomas, M.B. and Waage, J.K. (1996). Integration of Biological Control and Host Plant Resistance Breeding: A Scientific and Literature Review (Technical Centre for Agricultural and Rural Cooperation of the European Union, Wageningen, The Netherlands). Thomas, W. (2003). Prospects for molecular breeding of barley. Annals of Applied Biology, 142, 1–12. University of Ghana http://ugspace.ug.edu.gh 117 Thottappilly, G. and Rossel, H.W. (1985). World-wide occurrence and distribution of virus diseases. In: Cowpea research, production, and utilization. S.R. Singh and K.O. Rachie (Eds.). John Wiley and Sons, Chichester, UK, pp. 155–171. Timko, M.P. and Singh, B.B. (2008). Cowpea, a multifunctional legume. In: Genomics of tropical crop plants. Moore, P.H. and Ming, R. (eds.), Springer, New York, pp. 227-257. Timko, M. P. (2009). Cowpea genomics initiative. Department of Biology at university of Virginia. http://cowpeagenomics.med.virginia.edu/, sited on 1st March 2013. Tingey, W.M. (1986). Plant Insect Interactions. In: J.A. Miller and T.A. Miller (Eds.). New York, USA, Springer, pp. 251-284. Tuberosa, R., Salvi, S., Sanguineti, M.C., Maccaferri, M., Giuliani, S. and Landi, P. (2003). Searching for quantitative trait loci controlling root traits in maize: A critical appraisal, Plant and Soil, 255, 35–54. Tweneboah, C.K. (2000). Legumes. In: Morden agriculture in the tropics with special reference to Ghana. Co-Wood publishers, pp. 182 - 237. van Emden, H.F. (1991). The role of host plant resistance in insect pest mis-management. Bulletin of Entomological Research, 81, 123 (6), 1497 - 1506. van Sanford, D., Anderson, J., Campbell, K., Costa, J., Cregan, P., Griffey, C., Hayes, P. and Ward, R. (2001). Discovery and deployment of molecular markers linked to University of Ghana http://ugspace.ug.edu.gh 118 Fusarium head blight resistance: an integrated system for wheat and barley. Crop Science, 41: 638–644. Wang, Y., Xue Y. and Li, J. (2005). Trends in Plant Science. 10, 610-614. Wang, Y., Hobbs, H.A., Hill, C.B., Domier, L.L., Hartman, G.L. and Nelson. R.L. (2005). “Evaluation of Ancestral Lines of U.S. Soybean Cultivars for Resistance to Four Soybean Viruses.” Crop Science, 45: 639–644. Ward, S.A. and Dixon, A.F.G. (1982). Selective resorption of aphid embryos and habitat changes relative to life-span. Journal of Animal Ecology, 51 (3), 59 - 864. Weeden, N., Timmerman, G. and Lu, J. (1994). Identifying and mapping genes of economic significance. Euphytica, 73, 191–198. Weising, K., Nybom, H., Wolff, K. and Meyer, W. (1995). Applications of DNA fingerprinting in plants and fungi. CRC Press, Boca Raton, 336 pp. Wellings, P.W., Leather, S.R. and Dixon, A.F.G. (1980). Seasonal variation in reproductive potential: a programmed feature of aphid life cycles. Journal of Animal Ecology, 49, 975 – 985. Welz, H.G. and Geiger, H.H. (1999). Principles of marker-assisted selection. In: Haussmann B.I.G., Geiger H.H., Hess D.E., Hash C.T. and Bramel-Cox P. (eds.). Application of molecular markers in plant breeding. Training manual for a seminar held at University of Ghana http://ugspace.ug.edu.gh 119 IITA, Ibadan, Nigeria, from 16-17 August 1999. International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India. Williams, K.J. (2003). The molecular genetics of disease resistance in barley. Australian Journal of Agricultural Research, 54, 1065–1079. Winter, P. and Kahl, G. (1995). Molecular marker technologies for plant improvement. World Journal of Microbiology and Biotechnology, 11, 438–448. Wiseman, B. R. (1999). Commulative effects of antibiosis on five parameters of fall armyworm. Florida Entomology, 82. 277-283. Witcombe, J.R. and Virk, D.S. (2001). Number of crosses and population size for participatory and classical plant breeding. Euphytica, 122: 451-462. Yashitola, J., Thirumurgan, T., Sundaram, R. M., Naseerullah, M. K., Ramesha, M. S., Sarma, N. P., and Sonti, R. V., (2002). Crop Science. 42, 1369-1373. Youdeowei, A. (1989). Major arthropod pests of food and industrial crops in Africa and their economic importance. In: J. S. Yaninek and H. R. Herren (eds.) Biological Control: A sustainable solution to the crop pests’ problem in Africa, Ibadan, IITA, pp. 31 - 50. Young, N. (1996). QTL mapping and quantitative disease resistance in plants. Annual Review of phytopathology, 34(1), 479-501. University of Ghana http://ugspace.ug.edu.gh 120 Young, N.D.A. (1999). Cautiously optimistic vision for marker-assisted breeding. Molecular Breeding, 5, 505–510. Yu, K. F., Park, S. J. and Poysa V. (2000). Marker-assisted selection of common beans for resistance to common bacterial blight: efficacy and economics. Plant Breeding, 119, 411–415. Yu, K.F., Park, S.J., Zhang, B.L., Haffner, M. and Poysa, V. (2004). An SSR marker in the nitrate reductase gene of common bean is tightly linked to a major gene conferring resistance to common bacterial blight. Euphytica, 138, 89–95. University of Ghana http://ugspace.ug.edu.gh 121 Appendices The stability of the resistant genotype across cowpea growing zones in Ghana Analysis of variance Variate: Vigour score Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum 8 8.1432 1.0179 6.80 Rep.*Units* stratum Trt 4 1023.8642 255.9660 1710.87 <.001 Zone 17 2.3765 0.1398 0.93 0.533 Trt.Zone 68 11.9136 0.1752 1.17 0.172 Residual 712 106.5235 0.1496 Total 809 1152.8210 Variate: Percentage of seedlings killed Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum 8 111.91 13.99 1.01 Rep.*Units* stratum Trt 4 1482422.84 370605.71 26791.14 <.001 Zone 17 167.19 9.83 0.71 0.793 Trt.Zone 68 1063.83 15.64 1.13 0.228 Residual 712 9849.20 13.83 Total 809 1493614.97 University of Ghana http://ugspace.ug.edu.gh 122 Yield loss assessment of ten cowpea varieties Aphid infested treatment Variate: Grain yield (kg/ha) Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum 5 131911. 26382. 0.55 Rep.*Units* stratum Cultivar 9 1109644. 123294. 2.55 0.018 Residual 45 2174691. 48326. Total 59 3416246. Variate: Biomass weight (kg/ha) Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum 5 235276. 47055. 0.15 Rep.*Units* stratum Cultivar 9 25815793. 2868421. 9.10 <.001 Residual 45 14186831. 315263. Total 59 40237900. Variate: Days to 50% flowering Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum 5 3.933 0.787 0.68 Rep.*Units* stratum Cultivar 9 842.067 93.563 81.39 <.001 Residual 45 51.733 1.150 Total 59 897.733 University of Ghana http://ugspace.ug.edu.gh 123 Variate: Days to maturity Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum 5 4.2833 0.8567 2.71 Rep.*Units* stratum Cultivar 9 544.0833 60.4537 191.35 <.001 Residual 45 14.2167 0.3159 Total 59 562.5833 No aphid infestation Treatment Variate: Grain yield (kg/ha) Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum 5 185702. 37140. 0.85 Rep.*Units* stratum Cultivar 9 462052. 51339. 1.17 0.336 Residual 45 1973262. 43850. Total 59 2621016. Variate: Biomass weight (kg/ha) Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum 5 560130. 112026. 0.39 Rep.*Units* stratum Cultivar 9 29205604. 3245067. 11.20 <.001 Residual 45 13036752. 289706. Total 59 42802486. University of Ghana http://ugspace.ug.edu.gh 124 Variate: Days to 50% flowering Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum 5 3.9500 0.7900 2.19 Rep.*Units* stratum Cultivar 9 355.4833 39.4981 109.60 <.001 Residual 45 16.2167 0.3604 Total 59 375.6500 Variate: Days to maturity Source of variation d.f. s.s. m.s. v.r. F pr. Rep stratum 5 19.883 3.977 3.87 Rep.*Units* stratum Cultivar 9 332.017 36.891 35.87 <.001 Residual 45 46.283 1.029 Total 59 398.183 University of Ghana http://ugspace.ug.edu.gh