UNIVERSITY OF GHANA COLLEGE OF BASIC AND APPLIED SCIENCES DIVERSITY AND SOME ASPECTS OF THE BIOECOLOGY OF APHIDS ON CABBAGE AND THEIR ASSOCIATION WITH A NOVEL NECROTIC DISEASE. BY FORCHIBE ETHELYN ECHEP 10513147 A THESIS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY IN ENTOMOLOGY. AFRICAN REGIONAL POSTGRADUATE PROGRAMME IN INSECT SCIENCE (ARPPIS) UNIVERSITY OF GHANA, LEGON GHANA Joint Interschool International Programme for the Training of Entomologists in West Africa. Collaborating Departments: Animal Biology and Conservation Science (School of Biological Sciences) and Crop Science (School of Agriculture), College of Basic and Applied Sciences, University of Ghana, Legon. DECEMBER, 2021 University of Ghana http://ugspace.ug.edu.gh I DECLARATION I hereby declare that this thesis is the original research work carried out by, Forchibe Ethelyn Echep for the award of Doctor of Philosophy (PhD) in Entomology at the African Regional Postgraduate Programme in Insect Science (ARPPIS) from the University of Ghana, Legon. All references to other peoples’ work have been duly acknowledged and the thesis has not been submitted in part or whole for the award of a degree elsewhere. University of Ghana http://ugspace.ug.edu.gh II ABSTRACT Agriculture serves as a significant source of livelihood for many small-scale farmers, who account for the greatest share of agricultural production. Of the numerous varieties of exotic vegetables grown in Ghana, cabbage (Brassica oleracea var. capitata) is widely grown to meet the high demand of the ever-growing urban market, due to the many health benefits associated with its consumption. However, the devastating impacts of pests such as aphids and diseases on cabbage have resulted in significant yield losses. Aphids achieve this fate by developing resistance to many insecticides by rendering them ineffective, heavy infestations, development of sooty mould and transmission of viral diseases. Management interventions have been largely dependent on calendar spray regimes, with little or no knowledge on pest species and bioecology. While these interventions are an integral component of an integrated pest management approach on cabbage, identifying pest species, understanding their bioecology is important in the development and success of these pest management interventions. The current study sought to understand the diversity and bioecology of aphids on cabbage, and their role in the aetiology of a novel necrotic disease in Ghana. Surveys were conducted from 2019 to 2020, in nine regions of Ghana across five agroecological zones, noted for the production of cabbage to determine aphid species associated with cabbage and their distribution. Their regional abundance and spatiotemporal variation across agroecological zones were also assessed. In each region, cabbage farms in three districts were selected for the survey. Sampling was done by randomly sampling 20 plants per farm and aphids were counted, and some collected into vials containing 70% alcohol for identification. Two aphid species were identified to occur on cabbage, i. e. Lipaphis erysimi pseudobrassicae and Myzus persicae, with the former being the more abundant in all the regions. The highest aphid distribution and abundance was recorded in the Southern part of the country. There was significant variation in the population of these aphids across the different agroecological zones, with the highest recorded in the Deciduous Forest and Coastal Savannah zones. Aphid abundance was influenced by agroecological zones, vegetative cover and climatic conditions. The distribution of L. e. pseudobrassicae and M. persicae, in Ghana could not be ascertained by manually sampling all cabbage growing regions, hence, the rationale for the use of species distribution models to understand their spatial patterns and the underlying contributing climatic factors to map their suitable habitats. Occurrence data collected during the survey was used to determine University of Ghana http://ugspace.ug.edu.gh III aphid spatial distribution. A tree-based supervised machine learning algorithm, Random Forest was used to infer the underlying contributing factors, and to map the habitat suitability of both aphid species. Temperature and isothermality were the most important weather variables that affected the distribution of L. e. pseudobrassicae and M. persicae. The Southern Ghana was most suitable for the occurrence of both aphid species. Species distribution models can inform researchers about particular ecological adaptations of aphids and other insect pests in targeted areas, which can optimize efficiency and cost in pest control programmes. Aphid dynamics on crops is often influenced by climatic factors and their association with natural enemies. Experimental fields were setup in Kpong (Coastal Savannah agroecological zone) and Aseseeso (Deciduous Forest agroecological zone), and populations of L. e. pseudobrassicae, M. persicae and their natural enemies were monitored and recorded for five growing seasons (2019-2021). The mean population and infestation of L. e. pseudobrassicae were largest during the minor rainy seasons of 2019 and 2020 in the Deciduous Forest agroecological zone, and during the dry season of 2019 and minor rainy season of 2020 in the Coastal Savannah agroecological zone. Largest mean population and infestation for M. persicae were recorded during the minor season of 2019 and the dry season of 2020 in the Deciduous Forest zone and during the minor seasons of 2019 and 2020 of the Coastal Savannah agroecological zone. Significant differences in average aphid count per leaf within and between seasons were recorded for both agroecological zones. The Deciduous Forest zone showed high natural enemy diversity. Lipaphis erysimi pseudobrassicae and M. persicae populations showed significant negative correlation with temperature in both agroecological zones, while significant positive correlation with precipitation and relative humidity were recorded with M. persicae in the Coastal Savannah zone. This study provides information on aphid incidence across different seasons and agroecological zones, peak aphid activity, in relation to climatic factors and natural enemies. To understand the growth and development of both aphid species on commonly grown cabbage varieties, the biological and population growth parameters were studied on three cabbage varieties (Oxylus, Fortune and Leader cross). The study was carried out in a screen house under ambient conditions at 30 ± 1ºC and 75 ± 5%RH from September to November, 2020. The preadult developmental period, survival rates, longevity, reproduction, and life table parameters were evaluated following the female age- specific life table. There were significant differences in the nymphal developmental time, longevity and fecundity on the different cabbage varieties for both aphid species. The highest University of Ghana http://ugspace.ug.edu.gh IV population growth parameters; net reproductive rate (R0), intrinsic rate of increase (r), and finite rate of increase (λ) were recorded on oxylus variety for both L. e. pseudobrassicae (R0 = 47.05 offspring, r = 0.314 d-1, λ = 1.368 d-1) and Myzus persicae (R0 = 23.75 offspring, r = 0.249 d-1, λ = 1.282 d-1). The lowest were recorded on leader cross variety (R0 = 6.46 offspring, r = 0.174 d-1, λ = 1.191 d-1) for L. e pseudobrassicae, and Fortune (R0 = 2.94 offspring, r = 0.088 d-1, λ = 1.092 d-1) for M. persicae. The results suggests that leader cross is a less suitable host for L. e pseudobrassicae, and fortune for M. persicae, and thus, should be considered as potential sources of host plant resistance in breeding programmes. Aphids were found associated with a necrotic disease on cabbage suspected to be of viral aetiology. To understand the role of L. e. pseudobrassicae, and M. persicae in the transmission and expression of the necrotic disease, diseased leaves were subjected to RT-PCR to identify the causal pathogen, after which transmission experiments were conducted following Koch’s postulate to confirm identified pathogen. A cabbage field was setup at Kpong to study aphid/disease interaction. Cucumber Mosaic Virus (CMV-GH) was identified to be associated with the observed disease on cabbage but could not be confirmed as the causal pathogen, following failed transmission experiments to demonstrate Koch’s postulates. A strong positive relationship between aphid occurrence and disease onset were recorded. These findings are consistent with a role for L. e. pseudobrassicae and M. persicae in the transmission of the novel necrotic disease of cabbage. Farmers’ perception of aphids as pests of cabbage, their associated damage and management in Southern Ghana was assessed. A total of 121 farmers were interviewed in person using semi-structured questionnaires. Farmers had a good knowledge on aphid dynamics; aphid occurrence and seasonal variation, their associated damage and management, and consequently viewed aphid as one of the most damaging pests of cabbage. Seven varieties of cabbage were cultivated by farmers which included oxylus, fortune, kk cross, super cross, santa, Copenhagen market and leader cross, but over 80% of them cultivated Oxylus variety, which was known as ‘farmers’ choice’. Farmers employed a wide range of control measure particularly pesticide use, sometimes rotating synthetic with biological pesticides, and other times, mixing a ‘cocktail’ of different pesticides. The findings confirm indigenous knowledge, which is relevant in pest management/breeding programmes, and in the development of new technologies and adaptive strategies to changing food systems. University of Ghana http://ugspace.ug.edu.gh V DEDICATION To my parents, Mr. Njoh Stephen Forchibe and Mrs. Forchibe Vivian Mawom, my lovely son, Ian-Jaiden Kojofoah Forchibe and my sister Forchibe Harriet Efong, Your support and Love throughout this journey have been insuperable. Thank you, I Love you forever. University of Ghana http://ugspace.ug.edu.gh VI ACKNOWLEDGEMENT Doing a PhD has never been easy, and comes with several frustrations and roadblocks, and infringement on social life. Though challenging and overwhelming at certain points, I received a great deal of support and encouragement through practical experiments to writing my dissertation. I am grateful to Abba Father for Grace, strength and immeasurable love showed me through every channel. First and foremost, I want to express my gratitude to my supervisors, Prof. Ken Okwae Fening, Prof. Kwame Afreh-Nuamah and Dr. Millicent Cobblah, for their invaluable advice, unwavering support, and patience throughout my PhD studies. Their vast knowledge and wealth of experience have inspired me throughout my studies and daily life. Additionally, your informative remarks encouraged me to improve my thoughts and raise the quality of my work. I’d also like to thank Prof. John P. Carr, Dr. Francis Wamonje, Dr. Alex Murphy and Mrs. Adrienne Pate of the University of Cambridge for teaching me the nitty gritty of virology and molecular biology, and for your amazing guidance and support. I am grateful to Prof. Fening and Prof. Carr for teaching me to celebrate all victories, whether big or small. This built my courage and motivation every step of the way. Thank you all for all the love and for making this journey worth finishing. I am grateful to the German Academic Exchange Program for believing in me and offering me a scholarship to pursue my PhD studies. Thank you for the needed assistance and unwavering support along the way when things were out of control, especially during the covid-19 pandemic. Thankful to the former ARPPIS coordinator, Dr. Billah, and staff for their contributions towards this milestone. University of Ghana http://ugspace.ug.edu.gh VII I am grateful for all the support from Mr. Shadrach and service personnel at the School of Agric Biotech unit, the amazing team at PPRSD, especially Mr. Davis and Mrs. Hannah, for taking me through the taxonomy know-how. Then there is Mr. Tegbe Richmond of SIREC Kpong, beyond words, thank you for all the assistance during every field experiment. I’d like to thank Mr. Isaac Newton of IFDC for your immense contribution in understanding species distribution modelling. I will also like to thank Mr. Esai Kpadonou for the lectures and enormous help during data analysis, and likewise Dr. Atalor Pascal. I want to also express my gratitude to my colleagues Mr. Adom, Mr. Osei, Mrs. Nkafu and Mr. Abunyewa, Ms. Lami for every word of encouragement. I am most grateful to Mr. Deric Tanka, Mr. Eric Nyudze, Mr. Aladu Ndome, Mr. Fabrice Gbenongsi and Ms. Grace Matina, for helping me through data collection. To every farmer in all the regions that gave me their farmland to conduct my experiments, I am indeed grateful. Special thanks to Mr. Fred, the most experienced vegetable farmer I know, at Aseseeso. You outdid yourself to make sure I had good data. I am grateful to Mr. Umar in the Northern regions, Mr. Sadiq and Mr. Adama in Ashanti, Mr. Ernest in the Central, Mr. Freeman in Volta and Mr. Mars in Brong Ahafo regions, for your amazing support during data collection. Finally, to my family and friends, I want to say a big thank you for your prayers, kind words and support. To my amazing son Ian-Jaiden Forchibe, thank you for understanding and for your encouragement, I owe you bigtime. I am grateful to my parents and siblings for your prayers, especially Harriet, thank you for crying with me and laughing with me and putting me just on the right gear every time. Thank you, Pastor T.D. Grace, Fonge Sandy, Floramae Ray and Deric Vershiyi, it’s been my greatest pleasure having you all on this journey. University of Ghana http://ugspace.ug.edu.gh VIII TABLE OF CONTENTS DECLARATION ....................................................................................................................... I ABSTRACT .............................................................................................................................. II DEDICATION ......................................................................................................................... V ACKNOWLEDGEMENT ...................................................................................................... VI TABLE OF CONTENTS ..................................................................................................... VIII LIST OF TABLES ............................................................................................................... XIV LIST OF FIGURES ............................................................................................................... XV LIST OF PLATES .............................................................................................................. XVII LIST OF APPENDICES .................................................................................................... XVIII LIST OF ABBREVIATIONS .............................................................................................. XIX CHAPTER 1 ............................................................................................................................. 1 1.0 GENERAL INTRODUCTION ....................................................................................... 1 1.1 Background .................................................................................................................. 1 1.2 Justification .................................................................................................................. 2 1.3 Objectives .................................................................................................................... 5 CHAPTER 2 ............................................................................................................................. 7 2.0 LITERATURE REVIEW ................................................................................................ 7 2.1 Origin and distribution of cabbage .............................................................................. 7 2.2 Nutritional attributes and economic importance of cabbage ....................................... 8 2.3 Constraints to the production of cabbage .................................................................... 9 2.4 Aphids as pests of crops .............................................................................................. 9 2.4.1 Major features of aphid identification................................................................. 11 2.5 Aphids that occur on cabbage .................................................................................... 12 2.5.1 The mustard aphid; Lipaphis erysimi pseudobrassicae (Davis, 1914) ............... 13 2.5.1.1 Taxonomy and distribution .......................................................................... 13 2.5.1.2 Life cycle of L. e pseudobrassicae ............................................................... 14 2.5.2 The cabbage aphid; Brevicoryne brassicae (Linnaeus, 1758) ............................ 14 2.5.2.1 Taxonomy and distribution .......................................................................... 14 2.5.2.2 Life cycle of B. brassicae ............................................................................. 15 2.5.3 The green peach aphid; Myzus persicae (Sulzer, 1776) ..................................... 15 2.5.3.1 Taxonomy and distribution .......................................................................... 15 University of Ghana http://ugspace.ug.edu.gh IX 2.5.3.2 Life cycle of M. persicae.............................................................................. 16 2.6 Variations in aphid populations on brassica due to biotic and abiotic factors .......... 16 2.7 Economic importance of aphids on cabbage ............................................................. 17 2.7.1 Aphids as vectors of plant viruses ...................................................................... 18 2.8 Management of aphids on cabbage ........................................................................... 19 2.8.1 Mechanical control.............................................................................................. 20 2.8.2 Monitoring and sampling .................................................................................... 21 2.8.3 Cultural control ................................................................................................... 22 2.8.4 Host plant resistance ........................................................................................... 22 2.8.5 Chemical control ................................................................................................. 23 2.8.6 Biological control................................................................................................ 25 2.8.6.1 Natural enemies of aphids on brassicas........................................................ 26 CHAPTER 3 ........................................................................................................................... 29 3.0 Diversity, distribution, and identification of aphids on cabbage in Ghana ................... 29 3.1 INTRODUCTION ..................................................................................................... 29 3.2 MATERIALS AND METHODS .............................................................................. 30 3.2.1 Study sites ........................................................................................................... 30 3.2.2 Sampling for aphids ............................................................................................ 33 3.2.3 Identification of aphids ....................................................................................... 34 3.2.3.1 Morphological identification ........................................................................ 34 3.2.3.2 Molecular identification ............................................................................... 35 3.2.4 Pairwise comparison ........................................................................................... 36 3.2.5 Mapping of aphid distribution ............................................................................ 37 3.2.6 Data analysis ....................................................................................................... 37 3.3 RESULTS .................................................................................................................. 37 3.3.1 Diversity of aphids on cabbage ........................................................................... 37 3.3.2 Distribution and abundance of aphids on cabbage in Ghana .............................. 43 3.3.3 Spatiotemporal variation of L. e. pseudobrassicae population across four agroecological zones .................................................................................................... 45 3.3.4 Spatiotemporal variation of M. persicae across four agroecological zones ....... 47 3.4 DISCUSSION ............................................................................................................ 50 CHAPTER 4 ........................................................................................................................... 54 University of Ghana http://ugspace.ug.edu.gh X 4.0 Spatial patterns and habitat suitability modelling of L.e. pseudobrassicae and M. persicae on cabbage in Ghana. ............................................................................................ 54 4.1 INTRODUCTION ..................................................................................................... 54 4.2 MATERIALS AND METHOD................................................................................. 56 4.2.1 Aphid occurrence data ........................................................................................ 56 4.2.2 Point Pattern Analysis ......................................................................................... 56 4.2.3 Environmental variables ..................................................................................... 58 4.2.4 Species distribution modelling ............................................................................ 60 4.2.5 Evaluation of model performance. ...................................................................... 61 4.3 RESULTS .................................................................................................................. 61 4.3.1 Aphid spatial patterns and point density distribution ......................................... 61 4.3.2 Contributing factors of aphid spatial distribution pattern ................................... 63 4.3.3 Aphid habitat suitability mapping ....................................................................... 65 4.4 DISCUSSION ............................................................................................................ 67 4.4.1 Spatial pattern of L.e pseudobrassicae and M. persicae and contributing climatic factors ........................................................................................................................... 67 4.4.2 Habitat Suitability of L.e. pseudobrassicae and M. persicae in Ghana .............. 68 CHAPTER 5 ........................................................................................................................... 70 5.0 Incidence and seasonal variation of aphids and associated natural enemies on cabbage ............................................................................................................................................. 70 5.1 INTRODUCTION ..................................................................................................... 70 5.2 MATERIALS AND METHODS .............................................................................. 71 5.2.1 Study sites ........................................................................................................... 71 5.2.2 Nursery establishment ......................................................................................... 71 5.2.3 Land preparation and experimental layout ......................................................... 72 5.2.4 Transplanting ...................................................................................................... 72 5.2.5 Study period ........................................................................................................ 73 5.2.6 Sampling for aphids and their natural enemies ................................................... 74 5.2.7 Meteorological data collection ............................................................................ 74 5.2.8 Insect identification. ............................................................................................ 75 5.2.9 Parasitism of aphids ............................................................................................ 75 5.2.10 Data analysis ..................................................................................................... 75 5.3 RESULTS .................................................................................................................. 76 5.3.1 Abundance of aphids........................................................................................... 76 University of Ghana http://ugspace.ug.edu.gh XI 5.3.2 Diversity and abundance of aphid natural enemies ............................................ 76 5.3.2.1 Abundance of aphid natural enemies ........................................................... 76 5.3.2.2 Shannon Weiner diversity indices and Shannon Evenness for natural enemies of aphids across five cropping seasons in two AEZs ........................................................... 79 5.3.3 Seasonal incidence and population fluctuation of aphids on cabbage ................ 80 5.3.3.1 Lipaphis erysimi pseudobrassicae (Davis) .................................................. 80 5.3.3.2 Myzus persicae (Sulzer) ............................................................................... 82 5.3.4 Seasonal incidence and population fluctuations of aphids’ natural enemies ...... 84 5.3.4.1 Paragus borbonicus ..................................................................................... 84 5.3.4.2 Cheilomenes spp. .......................................................................................... 86 5.3.4.3 Chrysoperla carnea ...................................................................................... 88 5.3.4.4 Aphidoletes aphidimyza................................................................................ 90 5.3.4.5 Aphelinus varipes ......................................................................................... 92 5.3.4.6 Paederus sp. ................................................................................................. 92 5.3.4.7 Spiders .......................................................................................................... 92 5.3.5 Factors affecting aphid abundance on cabbage .................................................. 95 5.3.5.1 Weather parameters ...................................................................................... 95 5.3.5.2 Natural enemies ............................................................................................ 98 5.3.5.3 Parasitism by the aphid parasitoid; Aphelinus varipes ............................... 101 5.4 DISCUSSION .......................................................................................................... 102 CHAPTER 6 ......................................................................................................................... 107 6.0 Comparative bionomics of Lipaphis erysimi pseudobrassicae (Davis) and Myzus persicae (Sulzer) on three varieties of cabbage (Brassica oleracea) in Ghana ................ 107 6.1 INTRODUCTION ................................................................................................... 107 6.2 MATERIALS AND METHODS ............................................................................ 109 6.2.1 Characteristics of cabbage varieties .................................................................. 109 6.2.2 Planting of host plants ....................................................................................... 109 6.2.3 Rearing of L. e. pseudobrassicae and M. persicae ........................................... 110 6.2.4 Bionomics study................................................................................................ 110 6.2.5 Lifetable calculations ........................................................................................ 111 6.2.6 Statistical analysis ............................................................................................. 112 6.2.7 Survival analysis ............................................................................................... 113 6.3 RESULTS ................................................................................................................ 113 University of Ghana http://ugspace.ug.edu.gh XII 6.3.1 Bionomics of Lipaphis erysimi pseudobrassicae and Myzus persicae ............. 113 6.3.2 Lifetable parameters of L. e. pseudobrassicae and M. persicae ....................... 116 6.3.2.1 Survival rate ............................................................................................... 116 6.3.2.2 Life expectancy .......................................................................................... 118 6.3.2.3 Fecundity .................................................................................................... 119 6.3.3 Population growth parameters .......................................................................... 120 6.4 DISCUSSION .......................................................................................................... 121 CHAPTER 7 ......................................................................................................................... 124 7.0 Confirmation and characterization of disease-causing pathogen associated with aphid infestation on cabbage ....................................................................................................... 124 7.1 INTRODUCTION ................................................................................................... 124 7.2 MATERIALS AND METHODS ............................................................................ 125 7.2.1 Collection of diseased-cabbage leaves .............................................................. 125 7.2.2 Bulking up virus strains in Nicotiana benthamiana and cabbage..................... 126 7.2.3 CMV Agristrip test ........................................................................................... 127 7.2.4 Pathogen Identification ..................................................................................... 127 7.2.4.1 RNA extraction .......................................................................................... 127 7.2.4.2 Genomic DNA removal ............................................................................. 128 7.2.4.3 Total RNA purification .............................................................................. 128 7.2.4.4 Analysis for RNA concentration and purity ............................................... 128 7.2.4.5 Reverse transcription Reactions ................................................................. 129 7.2.4.6 PCR Reactions............................................................................................ 129 7.2.4.7 Gel electrophoresis of DNA ....................................................................... 130 7.2.4.8 Sequencing ................................................................................................. 132 7.2.5 Phylogenetic analysis and pairwise comparison ............................................... 132 7.2.6 Aphid/disease interaction on cabbage ............................................................... 132 7.2.6.1 Sampling for disease incidence, severity and associated aphids ................ 132 7.2.7 Data analysis ..................................................................................................... 133 7.3 RESULTS ................................................................................................................ 133 7.3.1 Identification of cucumber mosaic virus (CMV) on cabbage ........................... 133 7.3.2 Characterization and phylogenetic analysis of the cucumber mosaic virus genome ....................................................................................................................... 134 7.3.3 Transmission experiments ................................................................................ 139 University of Ghana http://ugspace.ug.edu.gh XIII 7.3.4 Relationship between aphid incidence and disease onset ................................. 139 7.3.5 Relationship between aphid abundance, viral disease incidence and severity . 140 7.4 DISCUSSION .......................................................................................................... 140 CHAPTER 8 ......................................................................................................................... 144 8.0 Farmers’ perception of aphids as pests of cabbage and their associated damage ....... 144 8.1 INTRODUCTION ................................................................................................... 144 8.2 MATERIALS AND METHODS ............................................................................ 145 8.2.1 Administration of questionnaires ...................................................................... 145 8.2.1.1 Interviews ................................................................................................... 145 8.2.1.2 Knowledge and perception of aphids and their damage to cabbage .......... 146 8.2.1.3 Management practices ................................................................................ 146 8.2.2 Data analysis ..................................................................................................... 147 8.3 RESULTS ................................................................................................................ 147 8.3.1 Demographics of respondents ........................................................................... 147 8.3.2 Knowledge on cabbage production ................................................................... 149 8.3.3 Knowledge of aphids and their damage on cabbage ......................................... 150 8.3.4 Management practices ...................................................................................... 151 8.4 DISCUSSION .......................................................................................................... 153 CHAPTER 9 ......................................................................................................................... 157 9.0 GENERAL DISCUSSION .......................................................................................... 157 9.1 INTRODUCTION ................................................................................................... 157 9.2 Diversity and distribution of aphids on cabbage in Ghana ...................................... 158 9.3 Aphid spatial pattern and habitat suitability ............................................................ 160 9.4 Incidence and seasonal variation of L.e. pseudobrassicae and M. persicae ........... 162 9.5 Bionomics of L.e. pseudobrassicae and M. persicae on three cabbage varieties.... 165 9.6 Identification and characterization of the disease-causing pathogen on cabbage ... 167 9.7 Famer perception of aphids as pests of cabbage, and associated damages ............. 170 CHAPTER 10 ....................................................................................................................... 174 10.0 CONCLUSION AND RECOMMENDATIONS ...................................................... 174 10.1 Conclusion ............................................................................................................. 174 10.2 Recommendations ................................................................................................. 177 APPENDIX ........................................................................................................................... 179 REFERENCES ..................................................................................................................... 192 University of Ghana http://ugspace.ug.edu.gh XIV LIST OF TABLES Table 3.1: Regions/Districts from which study sites were selected in Ghana ........................ 32 Table 3.2: Major morphological distinguishing features of aphids on cabbage. .................... 34 Table 3.3: Occurrence of aphids and their mean (±SE) population per leaf in each agroecological zone. ............................................................................................................... 49 Table 4.1: Bioclimatic variables used in the model ................................................................ 59 Table 5.1: Sampling periods for incidence and seasonal variation of aphids and their natural enemies. .................................................................................................................................. 73 Table 5.2: Abundance of aphid natural enemies in the CS and DF AEZs across five cropping seasons .................................................................................................................................... 78 Table 5.3 Shannon Weiner diversity indices and Evenness for aphid natural enemies .......... 79 Table 5.4: Summary of generalised linear model showing relationship between weather parameters and aphid densities on cabbage ............................................................................ 97 Table 5.5: Summary of generalised linear model showing relationship between natural enemies and aphid densities on cabbage ............................................................................... 100 Table 5.6: Seasonal and cumulative percentage parasitism of aphids by the parasitoid Aphelinus varipes in Coastal Savannah and Deciduous Forest agroecological zones .......... 101 Table 6.1: Mean of the developmental duration (days) and reproduction period of L. e. pseudobrassicae reared on three cabbage varieties .............................................................. 115 Table 6.2: Mean of the developmental duration (days) and reproduction period of M. persicae reared on three cabbage varieties ........................................................................... 116 Table 6.3 Population growth parameters of Lipaphis erysimi pseudobrassicae and Myzus persicae on three cabbage varieties ...................................................................................... 120 Table 7.1: Primers used to test for the presence of virus in cabbage leaf samples ............... 131 Table 8.1: Demographic parameters of respondents............................................................. 147 Table 8.2: List of pesticides used against aphids on cabbage in Southern Ghana. ............... 152 University of Ghana http://ugspace.ug.edu.gh XV LIST OF FIGURES Figure 2-1: Generalised life cycle of aphids (Source: Flint, 2000)......................................... 11 Figure 3-1: Study sites sampled for aphids during the study .................................................. 31 Figure 3-2: Colony of aphids on cabbage leaves A) L. e. pseudobrassicae, B) M. persicae. 38 Figure 3-3: Aphids identified on cabbage in Ghana. A) L. e. pseudobrassicae (Davis 1814), B) M. persicae (Sulzer). .......................................................................................................... 39 Figure 3-4: Distinguishing features of L. e. pseudobrassicae (A) and M. persicae (B) as seen under the Leica EZ digital microscope (X35 Mag.). .............................................................. 40 Figure 3-5: Gel electrophoresis showing 700bp fragment of 5’ region of the mitochondria cytochrome oxidase I – (CO-I) gene for identification of aphid species. ............................... 41 Figure 3-6: Pairwise comparison of CO1 gene sequences for Lipaphis erysimi pseudobrassicae and Myzus persicae. .................................................................................... 42 Figure 3-7: Distribution and abundance of Lipaphis erysimi pseudobrassicae and Myzus persicae. .................................................................................................................................. 44 Figure 3-8: Mean number of Lipaphis erysimi pseudobrassicae across four agroecological zones of Ghana, from April to October 2019 (A) and 2020 (B). ............................................ 46 Figure 3-9: Mean number of Myzus persicae across four agroecological zones of Ghana, from April to October 2019 (A) and 2020 (B). ....................................................................... 48 Figure 4-1: Point density distribution of L. e. pseudobrassicae (A) and M. persicae (B) ...... 62 Figure 4-2: Random Forest variable importance plots for (A) Lipaphis e. pseudobrassicae and (B) M. persicae modelling ............................................................................................... 64 Figure 4-3: Random Forest current habitat suitability map for Lipaphis erysimi pseudobrassicae (A) and Myzus persicae (B). ....................................................................... 66 Figure 5-1: Seasonal incidence and population fluctuation of Lipaphis erysimi pseudobrassicae (LEP) (±SE) in the Deciduous Forest (A) and Coastal Savannah (B) agroecological zones across five cropping seasons (2019-2021). .......................................... 81 Figure 5-2: Seasonal incidence and population fluctuations of M. persicae (±SE) in the Deciduous Forest (A) and Coastal Savannah (B) agroecological zones across five cropping seasons (2019-2021). .............................................................................................................. 83 University of Ghana http://ugspace.ug.edu.gh XVI Figure 5-3: Seasonal incidence and population fluctuations of the hoverfly Paragus borbonicus (±SE) in the Deciduous Forest (A) and Coastal Savannah (B) agroecological zones across five cropping seasons (2019-2021). ................................................................... 85 Figure 5-4: Seasonal incidence and population fluctuations of Cheilomenes spp (±SE) in the Deciduous Forest (A) and Coastal Savannah (B) agroecological zones across five cropping seasons (2019-2021). .............................................................................................................. 87 Figure 5-5: Seasonal incidence and population fluctuations of Chrysoperla carnea (±SE) in the Deciduous Forest (A) and Coastal Savannah (B) agroecological zones across five cropping seasons (2019-2021). ............................................................................................... 89 Figure 5-6: Seasonal incidence and variation in the population of Aphidoletes aphidimyza (±SE) in the Deciduous Forest (A) and Coastal Savannah (B) agroecological zones across five cropping seasons (2019-2021). ........................................................................................ 91 Figure 5-7: Seasonal incidence and population fluctuation of spiders (±SE) in the Deciduous Forest (A) and Coastal Savannah (B) zones across five cropping seasons (2019-2021)........ 94 Figure 5-8 Climatic factors and aphid densities on cabbage crops in the Coastal Savannah and Deciduous Forest agroecological zones in Ghana. Values shown are the average number of individuals recorded on 20 plants ....................................................................................... 96 Figure 5-9 Natural enemies and aphid densities on cabbage crops in the Coastal Savannah and Deciduous Forest agroecological zones in Ghana. Values shown are the average number of individuals recorded on 20 plants ....................................................................................... 99 Figure 6-1: Kaplan–Meier nymphal survival curve for a) Lipaphis erysimi pseudobrassicae and b) Myzus persicae on three cabbage varieties. ............................................................... 117 Figure 6-2: Kaplan–Meier adult survival curve for a) Lipaphis erysimi pseudobrassicae and b) Myzus persicae on three cabbage varieties. ...................................................................... 118 Figure 6-3: Life expectancy (ex) of Lipaphis erysimi pseudobrassicae and b) Myzus persicae on three cabbage varieties. .................................................................................................... 119 Figure 7-1: Phylogenetic analysis of CMV using selected RNA1 sequences from GenBank and CMV Isolate GH (K RNA1 SJ3R). ................................................................................ 136 Figure 7-2: Phylogenetic analysis of CMV using selected RNA2 sequences from GenBank and CMV Isolate GH (K RNA2 SJ5F). ................................................................................ 137 Figure 7-3: Phylogenetic analysis of CMV using selected RNA3 sequences from GenBank and CMV Isolate GH (K3 3CMVr3 3F) ............................................................................... 138 Figure 8-1: Percentage cultivation of different cabbage varieties. ....................................... 149 University of Ghana http://ugspace.ug.edu.gh file:///C:/Users/User/Desktop/Thesis%20final/Forchibe%20final_post%20viva%20cor.docx%23_Toc120046517 file:///C:/Users/User/Desktop/Thesis%20final/Forchibe%20final_post%20viva%20cor.docx%23_Toc120046517 XVII LIST OF PLATES Plate 5.1: Nursery in screen house (Kpong). .......................................................................... 72 Plate 6.1: A) Clip cage used for clipping aphids B) Cabbage plants with aphids under clip cages in screen house. ........................................................................................................... 111 Plate 7.1: Plants showing symptoms of disease/ collection of disease plant samples .......... 126 Plate 7.2: Inoculation of (i) N. benthamiana and (ii) cabbage plants at the Plant Science Department, University of Cambridge .................................................................................. 127 Plate 7.3: Gel electrophoresis showing 400bp fragment of the CMV coat protein gene ...... 134 Plate 7.4: Negative immunodiagnostic strips for CMV antibodies ...................................... 139 University of Ghana http://ugspace.ug.edu.gh XVIII LIST OF APPENDICES Appendix 1: Surveyed Districts with geo-referenced positions ........................................... 179 Appendix 2: Blast results for aphid identification ................................................................ 180 Appendix 3: Bioclimatic variables used in modelling .......................................................... 182 Appendix 4: Average Nearest Neighbour visualised results for L.e. pseudobrassicae points (A) and M. persicae points (B) ............................................................................................. 183 Appendix 5: Primers used to sequence the Cucumber mosaic virus genome....................... 184 Appendix 6: CMV Isolates used in phylogenetic analysis ................................................... 185 Appendix 7: Correlation coefficients for disease severity and aphid abundance ................. 186 Appendix 8: Questionnaire on farmers’ perception of aphids as pests of cabbage .............. 187 University of Ghana http://ugspace.ug.edu.gh XIX LIST OF ABBREVIATIONS FAO - Food and Agriculture Organization of the United Nations USDA – United States Department of Agriculture MoFA – Ministry of Agriculture AEZ - Agroecological zone PCR – Polymerase chain reaction CMV – Cucumber mosaic virus TuMV – Turnip mosaic virus BWYV – Beet western yellows virus CaMV – Cauliflower mosaic virus RT-PCR - Reverse transcription-polymerase chain reaction RNA – Ribonucliec acid gDNA – genomic deoxyribonucleic acid cDNA – complementary deoxyribonucleic acid dNTP - deoxy-nucleoside triphosphates SDM - Species distribution modelling RF – Random Forest ANN – Average nearest neighbour University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER 1 1.0 GENERAL INTRODUCTION 1.1 Background Cabbage, Brassica oleracea var. capitata L. is a crop belonging to the family Brassicaceae, and crops of this family are of great economic importance worldwide (Chiang et al., 1993; Bhatia et al., 2011). Its cultivation provides livelihood for both rural and urban dwellers (Abbey and Manso, 2004; Owusu-Boateng and Amuzu, 2013; Amoabeng et al., 2017). It is an exotic vegetable known native to Southern Europe and the Mediterranean regions (FAO, 2000). It is an important source of food and nutrients globally and has been listed as one of the most important green vegetables worldwide (FAO, 2020). It is grown throughout the world in more than 90 countries including the tropics (Sawant et al., 2010; FAO Stats, 2011). This vegetable has valuable nutrients such as vitamins, dietary fiber, calcium, fatty acids; and it is being used across different cultures around the world for medicinal purposes (Ahuja et al., 2013). For example, the antioxidant property of cabbage is known to fight cancer (Liu, 2004; Tajalli et al., 2020). Several varieties of cabbage are grown around the world and are known to be well adapted to a wide range of climatic conditions (Shinohara, 1965). In Ghana, the major varieties grown include Oxylus, Fortune, KK cross, Copenhagen market, Super cross, Santa F1, and Vantar F1 hybrid of which oxylus and fortune are often intensively grown to meet the ever-growing demand (Adenka et al., 2020). It is typically eaten raw (in salads, sandwiches, and hamburgers) or cooked, in sauces (Baidoo et al., 2012). Cabbage is noted as one of the major staple vegetables grown in Ghana (GhanaVeg, 2014). University of Ghana http://ugspace.ug.edu.gh 2 Cabbage is susceptible to attack by insect pests and diseases during its cultivation, which causes a significant reduction in yield and quality, thereby affecting its market value (Zehnder et al., 1997; Fening et al., 2013). Adenka et al. (2021) reported up to 100% yield loss of cabbage, attributed to insect pests and diseases. Of the different insect pests that attack cabbage, aphids are considered as major pests of economic importance and great concern because in addition to the physical damage they cause, they are also vectors of plant viruses such as Cauliflower mosaic virus (CaMV), Cucumber mosaic virus (CMV), and Turnip mosaic virus (TuMV), which cause huge losses in Brassica crop production worldwide (Rohilla and Kumar, 1991; Munthali et al., 2004), thus drawing attention as a major pest of economic importance. 1.2 Justification Aphids are ‘small, pear-shaped, delicate insects with soft, fragile bodies’, belonging to the superfamily Aphidoidea (Blackman and Eastop, 2000). They may be winged or wingless, with their nymphs resembling the adults, and extensive colour variation even within a single colony, a phenomenon known as colour polymorphism. (Blackman and Eastop, 2000; VanEmden and Harrington, 2007). Colour polymorphism often affects the fitness of aphids and in some cases, affects their susceptibility to an array of natural enemies (Caillaud and Losey, 2010). Aphids cause severe damage to crops and their ability to inflict direct and indirect damage on crops categorises them as one of the most detrimental insect pests on cultivated plants (Sorenson, 2009). Direct plant damage includes stunting, distortion, yellowing, and wilting, usually resulting from sucking sap from infested plants (Hughes, 1963; Aslam et al., 2011). Indirect damage includes dark sooty mold formed from fungus growth on honeydew excretion, the transmission of plant viruses that results in diseases, and plant deformations because of toxic saliva secretions (Blackman and Eastop, University of Ghana http://ugspace.ug.edu.gh 3 2000). Apart from preventing adequate photosynthesis, sooty mold and honeydew also reduce the aesthetic value and marketability of crops (Fening et al., 2013; Forchibe et al., 2017). The pest status of aphids is enhanced by their capability to reproduce fast, and adapt to new environments (Myburgh, 1993). The major aphids of economic importance that occur on cabbage worldwide are the cabbage aphid, Brevicoryne brassicae (Linnaeus), (Hemiptera: Aphididae), the Indian mustard or false cabbage aphid; Lipaphis erysimi pseudobrassicae (Davis), and the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae), (Liu and Sparks, 2001; Mpumi et al., 2020). Brevicoryne brassicae and L. e. pseudobrassicae are specialist aphids feeding solely on brassica crops, whereas M. persicae is a generalist, feeding on several crop families (Blande et al., 2004; Van Emden, 2007; Fening et al., 2016). In Ghana, L. e. pseudobrassicae and M. persicae have been reported to be the major aphids of economic importance on cabbage (Fening et al., 2016; Forchibe et al., 2017; Adenka et al., 2021). Lipaphis e. pseudobrassicae was first recorded in Ghana in 2016, as one of the major aphids on cabbage, causing devastating losses often leading to little or no marketable cabbage heads (Fening et al., 2016; Forchibe et al., 2017). It is mostly found in association with M. persicae, an extremely polyphagous aphid feeding on a wide variety of plant species in over 40 different families (Eastop and Hille, 1976; Blackman and Eastop 2000). Its attack often leads to severe damages in associated plants and has been implicated in transmitting over 40 viruses to different crops (Blackman and Eastop, 2000). The cabbage aphid; B. brassicae (Linnaeus), has also been cited by many authors as occurring on cabbage in Ghana (Fening et al., 2014; Amoabeng et al., 2013; Ngosong et al., 2020), although it is known as a temperate and subtropical species. This therefore raises concerns about its occurrence and distribution in Ghana as they are sometimes mistaken for the false cabbage aphid, L. e. pseudobrassicae due to their similarity in appearance University of Ghana http://ugspace.ug.edu.gh 4 (Blackman and Eastop, 2000). Additionally, there are no detailed studies on aphids in Ghana to firmly attest that this species was present in the country. Thus, proper identification of aphid species on brassica crops in Ghana was essential for their subsequent control and implementation of quarantine measures. Traditionally, morphological characteristics have been used to identify aphids (Van Emden and Harrington, 2007). However, due to their small sizes, the presence of immature stages, the presence of cryptic species, the similarity between species, and loss of key morphological characteristics after collection (e.g., damaged specimens), identification has become almost impossible (Foottit et al., 2008; Miller and Foottit, 2009; Lee et al., 2011). Additionally, ‘the presence of cryptic species in a genus that constitutes significant agricultural pests represents a serious gap in the management of aphid pest species’ (Valenzuela et al., 2009). The complexity of the life cycle of aphids which involves pathogenesis and high polymorphisms, causes different morphs to exist within one species, thus complicating species identification (Blackman and Eastop, 2000; Miller and Foottit, 2009). These further buttresses the need to clarify the species status of aphids found on cabbage in Ghana using molecular tools to confirm what has been already identified using morphological methods. As vectors of plant viruses, aphid occurrence on crops is a major cause for concern. Studies has shown that aphids generally account for the transmission of up to 50 % of insect-vectored viruses worldwide (Brunt et al., 1996; Nault, 1997; Hogenhout et al., 2008). Their piercing-sucking mouthparts coupled with their high reproductive rates make them agents of permeating disease epidemics and spreading of plant viruses (Stafford et al., 2012). Viral transmission could be persistent, semi-persistent, or non-persistent depending on the viral pathogen (Brunt et al., 1996). Cucumber mosaic virus (CMV) for example, is transmitted by M. persicae non-persistently, and University of Ghana http://ugspace.ug.edu.gh 5 is known as one of the most prevalent plant viral pathogens, infecting over one thousand (1,000) plant species (Ng and Perry, 2004; Akhtar et al., 2010). Recently, aphids were found to be associated with a novel necrotic disease on cabbage in Ghana, characterized by mosaic, necrosis, stunting and subsequently death of plants (Fening et al., 2016; Forchibe, 2016, Adenka et al., 2021). The disease is suspected to be of viral aetiology, and has been reported to cause complete yield loss once reported on cabbage fields (Adenka et al., 2021). Thus, it is important to identity the disease causal pathogen as well as understand the role of aphids in the transmission of the novel necrotic disease, as it is unsure if aphids are the authentic vectors or if the presence of the disease on cabbage predisposes them to attack by aphids. There was therefore the need to study the diversity of aphids on cabbage in Ghana, important aspects of their bioecology and understand their association with the novel necrotic disease, as a prerequisite to developing quarantine measures and sustainable management interventions. 1.3 Objectives The main objective of this study was to determine the diversity of aphids present on cabbage in Ghana and understand some important aspects of their bioecology in association with a novel necrotic disease on cabbage. The specific objectives of the study were to: I. ascertain the species of aphids present on cabbage, their distribution, and density in five agroecological zones of Ghana. II. determine the spatial density patterns and model the habitat suitability of identified aphids. III. determine the incidence and seasonal variation of aphids and their associated natural enemies on cabbage. University of Ghana http://ugspace.ug.edu.gh 6 IV. study the bionomics of the two dominant aphids on three cabbage varieties commonly grown in Ghana. V. identify and characterise the disease-causing pathogen associated with aphids’ infestation on cabbage VI. assess farmers’ perception of aphids as pests of cabbage in Southern Ghana. University of Ghana http://ugspace.ug.edu.gh 7 CHAPTER 2 2.0 LITERATURE REVIEW 2.1 Origin and distribution of cabbage Cabbage, Brassica oleracea var. capitata L. (Family: Brassicaceae), is an important leafy vegetable derived from the wild type, Brassica oleracea L. (also known as colewort) (Nieuwhof, 1969). Other varieties of the brassica family include cauliflower, broccoli, kale, and brussels sprout. Cabbage is indigenous to Southern Europe and the Mediterranean regions, but is now cultivated throughout the world (FAO, 2000; Bhatia et al., 2011). It is considered as one of the oldest vegetables and was mentioned in early literature to be in use as early as 2000 or 2500BC by ancient Romans and Greeks (Chiang et al., 1993; Economic Research Service (ESR), 2002). Different types and varieties of cabbage show considerable variation with respect to the size, shape, color, and texture of the head (Singh et al., 2006). Earlier classifications by Nieuwhof (1969), categorized cultivated cabbage into red cabbage, savoy cabbage, and white cabbage. It is unclear when the cultivation of cabbage commenced in Ghana (Cobblah et al., 2012) but is believed to have been introduced with the incursion of the British on the Gold Coast in the 1940s (Sinnadurai, 1992). Since then, there has been a growing popularity of cabbage in Ghana especially in urban areas, and several varieties have been developed to suit consumers’ tastes and resist environmental stress. Due to its high nutritional content, cabbage has now become a major part of peoples’ diet in the country. Timbilla and Nyarko (2004) recorded over nine varieties of cabbage cultivated throughout the country (Capitata, Copenhagen market, Oxylus, KK cross, Holland, Tenzier, KY cross, Gloria, and Master globe) but in recent times, newer varieties such as Leader University of Ghana http://ugspace.ug.edu.gh 8 cross, Santa, Fortune, Tropica cross, Super cross, Tropica king, Super comet, and Milor (Agriseed Ltd, Ghana) have been developed to suit the changing climatic conditions. 2.2 Nutritional attributes and economic importance of cabbage The top ten major producers of cabbage include China, India, Russia, Japan, South Korea, Ukraine, Indonesia, Poland, Romanian, and the USA, with China being the largest; with about 50% of the world’s production (FAO stats, 2011; World atlas, 2017). Cabbage is considered an important crop with a huge demand from the culinary industry across the globe. It is rich in nutrients such as carbohydrates, vitamins, alkaloids, tannins, minerals, and glutamine (Oguwike et al., 2014; Parveen et al., 2016). Its richness in phytochemical components makes it have anti-inflammatory, antioxidant, and anticarcinogenic effects (Tajalli et al., 2020). In West Africa, cabbage is mostly grown by small-scale farmers for the local and the surplus for the international markets (Kouamé et al., 2006; Probst et al., 2012; Owusu-Boateng and Amuzu, 2013). It is mostly cultivated in peri-urban areas and supplied into major cities. In Ghana, Timbilla and Nyarko (2004) reported that cabbage is grown in all regions of the country except the Upper East and Western regions with up to 49% of farmers growing all year round. Other studies have indicated an increase in the cultivation of cabbage across the country, attributed to an increase in demand and the population of foreigners in some urban cities (Osei et al., 2013). The cultivation of cabbage in Ghana provides livelihood for a large number of small-scale farmers, thus, contributing to food security and income generation leading to poverty reduction (Timbilla and Nyarko, 2004; Mochiah et al., 2011). University of Ghana http://ugspace.ug.edu.gh 9 2.3 Constraints to the production of cabbage Cabbage, though important with enormous benefits for consumption, faces severe biotic and abiotic constraints during production. Of the biotic constraints, are insect pests whose attack causes a significant reduction in yield and quality of cabbage, often leading to frequent pesticide applications (Zehnder et al., 1997; Mpumi et al., 2020). Pesticides have been reported to have adverse effects on the environment, health and non-target organisms, thus, purported as an issue of great concern (Obeng-Ofori, 2007).Major pests that attack cabbage include; Diamondback moth (Plutella xylostella), Aphids (Brevicoryne brassicae, Lipaphis pseudobrassicae, Myzus persicae), cabbage webworm (Hellula undalis), Flea beetles (Phyllotreta sp.), Cabbage looper (Trichoplusia ni), cabbage white butterfly (Pieris rapae), Whitefly (Bemisia tabaci) (Shelton et al., 1988; Amoabeng et al., 2013; Fening et al., 2014). Of this complex of arthropod pests attacking cabbage are aphids whose ability to vector plant viruses makes them one of the most economically important groups of pests in horticulture or agriculture in many parts of the world (Dixon, 1985; Guerrieri and Digilio, 2008). Abiotic constraints identified with cabbage production includes; availability of land, irregular water supply, weed competition, lack of credit facilities, high cost of inputs, and marketing of crops (Bangnikon and Ofori, 1996; Timbilla and Nyarko, 2004; Osei et al., 2013). 2.4 Aphids as pests of crops Aphids are among the major insect pest groups, globally causing significant economic damage to many food and commodity crops. Those of agricultural importance are known to be mostly from the subfamily Aphidinae, with more than 4000 species identified and over 250 species noted as University of Ghana http://ugspace.ug.edu.gh 10 crop pests (Blackman and Eastop, 2007). Aphids damage crops in several ways: firstly, as phloem- feeders, they suck plant sap which leads to stunting and sometimes death of plants in heavy infestations. Secondly, aphids excrete ‘honeydew’ which is suitable for the growth of a black sooty mold which reduces the cosmetic appearance of crops and photosynthetic activity (Hughes, 1963). Thirdly, they also inject saliva into plants during feeding which could be phytotoxic (Dedryver et al., 2010). Lastly, aphids are major vectors of plant viruses, transmitting over half of insect- vectored viruses (Nault, 1997). Their enhanced ability to reproduce rapidly, for example; one mature aphid can give birth to 2 to 5 live young per day which matures between 5 to 7 days, further raises their pest status (Myburgh, 1993). Aphids have complex and varied life cycles. Major taxa exhibit two major life cycles which include holocyclic (periods of very low temperatures and reduced photoperiods; where males are produced) and anholocyclic (during warm periods, by parthenogenesis) (Fig 2.1) (Kessing and Mau 1991; Blackman and Eastop, 2000; Agarwala, 2007). Holocyly is characterized by the presence of an egg stage which is usually deposited before winter. It then hatches into a fundatrix (a wingless female aphid) which subsequently produces daughter clones (Flint, 2000). These females then produce both males and females which mate, and the females lay eggs that enter diapause to survive the winter. In anholocycly, female aphids reproduce parthenogenetically, giving birth to live clones of themselves that are already carrying a developing aphid embryo (Flint, 2000). Nevertheless, cosmopolitan species follow both holocyclic and anholocyclic life cycles, concurrently in different geographical locations (Bhatia et al., 2011). University of Ghana http://ugspace.ug.edu.gh 11 Figure 2-1: Generalised life cycle of aphids (Source: Flint, 2000). 2.4.1 Major features of aphid identification Aphids can be primarily recognized by several shared morphological characteristics such as siphunculi, five or six-segmented antennae, two-segmented tarsi, and cauda often used for thrusting off honeydew from the anus (Blackman and Eastop, 1984; Van Emden and Harrington, 2007). However, these features may vary depending on environmental and ecological factors (Saguez et al., 2013). The waxy secretion of some aphids is important for identification (Blackman and Eastop, 1984). However, inadequate distinguishing morphological traits or high levels of polymorphism exhibited by aphids makes it difficult for them to be morphologically differentiated (Blackman and Eastop, 1984). For this reason, further suggestions were made by Blackman and Eastop (2000) that non-physically confusing features be used for morphological delimitation among aphids. Furthermore, recent advances in molecular methods and tools for species identification would be the best option for ascertaining the identity of a particular species. University of Ghana http://ugspace.ug.edu.gh 12 2.5 Aphids that occur on cabbage Thirteen species of aphids have been recorded as feeding on Brassica oleraceae worldwide, Blackman and Eastop (2000), which include: Aphis craccivora, fabae, gossypii, nasturtii, spiraecola; Aulacorthum prasinum, solani; Brachycaudus helichrysi; Brevicoryne brassicae; Dyaphis montemartinii ghanii; Hyalopterus pruni; Lipaphis pseudobrassicae (erysimi); Macrosiphum euphorbiae; Myzus antirrhinii, ascalonicus, ornatus, persicae; Pemphigus populitransversus; Protaphis middletonii; Rhopalosiphoninus latysiphon; Sitobion miscanthi and Smynthurodes betae. Of these, the green peach aphid, Myzus persicae (Sulzer), the Indian mustard aphid; Lipaphis erysimi pseudobrassicae (Davis), and cabbage aphid, Brevicoryne brassicae (Linnaeus) are of economic importance in cabbage cultivation. Nevertheless, as with other pest species, their distribution, abundance and economic status vary globally depending on climatic factors and food availability (Bebber et al., 2014). The polyphagous aphid, M. persicae has successfully colonized hundreds of plant species belonging to over 40 families (Flanders et al., 1992; Blackman and Eastop 2000; CABI, 2015). Lipaphis e. pseudobrassicae and B. brassicae are known to feed specifically on cruciferous crops, and attack cabbage from the nursery till harvest, while M. persicae attacks cabbage before heading begins (Elwakil and Mossler, 2013). In the tropics, L. e. pseudobrassicae is noted as the most damaging aphid on brassica and is often referred to as the false cabbage aphid due to its close resemblance to B. brassicae (Ronquist and Ahman, 1990) which is also noted in the temperate and subtropical regions as the most damaging aphid on brassica (Blackman and Eastop, 2007). University of Ghana http://ugspace.ug.edu.gh 13 2.5.1 The mustard aphid; Lipaphis erysimi pseudobrassicae (Davis, 1914) 2.5.1.1 Taxonomy and distribution Lipaphis erysimi pseudobrassicae (Hemiptera: Aphididae) (=Lipapahis erysimi or Lipaphis pseudobrassicae) is a major brassica pest, native to Asia where it has an extensive distribution (Bakhetia and Sohi, 1980), but has over the years been introduced into other countries around the world. This insect was first confused for B. brassicae in North America until Davis (1914) identified it as a different species (Essig, 1948). The Lipaphis group constitutes mainly of two morphologically similar but geographically distinct subspecies; the European form (Lipaphis erysimi erysimi) and the Asian form (Lipaphis erysimi pseudobrassicae) (Müller, 1986; Ronquist and Ahman 1990). The European forms only occur on wild crucifers, are holocyclic, and not usually associated with brassica crops (Müller 1986). Between the 1970s-2000s, both sub-species were referred to as Lipaphis erysimi due to difficulty in distinguishing them (Müller, 1986; Liu et al., 1997; Blackman and Eastop, 2000). Nonetheless, some studies still refer to Lipaphis erysimi as the widely distributed pest of brassica (Srivastava and Gularia, 2003; Rana, 2005; James et al., 2010, Saethre et al., 2011; Adhab and Schoelz 2015; Patel et al., 2017a). L e. pseudobrassicae is distributed in both the tropical and temperate regions of the world feeding mainly on brassica crops. The apterae are yellowish, grey, or olive green with a waxy layer which becomes denser in humid conditions. They have a body length of 1.5-2.3 mm and occur in large colonies under cabbage leaves, leaving the leaves wrinkled. ‘Winged females have dusky green abdomens with dark lateral stripes’ (Blackman and Eastop, 1984). The antennae are dark, and pale at the base (Blackman and Eastop, 1984). The major distinguishing features from other aphids are University of Ghana http://ugspace.ug.edu.gh 14 the non-convergence of the frontal tubercles; the pale cornicles which are longer than the cauda; the tongue-shaped cauda and a thin layer of white waxy secretion on the colonies (Blackman and Eastop, 1984; Adhab and Schoelz, 2015). 2.5.1.2 Life cycle of L. e pseudobrassicae Lipaphis erysimi pseudobrassicae undergoes anholocyclic reproduction in most regions of the world (Blackman and Eastop, 2000) and males are very rare. However, holocycly has been recorded on cabbage in Western Honshu, Japan (Kawada and Murai, 1979). Without much evidence of the occurrence of oviparous females, only ovoviviparous forms have been reported to occur on crops throughout the year even in both warm and cold climates. The mustard aphid is highly prolific and can have up to 11–25 and 35 generations annually in temperate and tropical conditions respectively (Capinera, 2008). 2.5.2 The cabbage aphid; Brevicoryne brassicae (Linnaeus, 1758) 2.5.2.1 Taxonomy and distribution Brevicorryne brassicae (Hemiptera: Aphididae) has a body length of 1.6 to 2.8 mm, covered with a greyish waxy covering, with thick and very short siphunculi and broad triangular cauda (Blackman and Eastop 1984; Carter and Sorensen 2013). The short cornicles and thick greyish waxy secretion are the major distinguishing features from the other aphids (Blackman and Eastop 1984; Opfer and McGrath, 2013). A concise account of the biology and pest status of the cabbage aphid has been provided by Gabrys et al. (2008) in the encyclopedia of entomology. It is known University of Ghana http://ugspace.ug.edu.gh 15 to be native to Europe and is distributed throughout the temperate and warm temperate regions of the world (Blackman and Eastop, 2000). 2.5.2.2 Life cycle of B. brassicae Reproduction in B. brassicae is anholocyclic during warm periods, (Kessing and Mau 1991) and holocyclic during lower temperatures; during which there is the production of males (Blackman and Eastop, 1984). After mating occurs, the females lay eggs that overwinter, and nymphal development takes 7-10 days and differs from the adults in having less developed cauda and siphunculi. Several generations occur within the life of one aphid, with over 15 generations during one cropping season (Hafez, 1961; Hines and Hutchison 2013). 2.5.3 The green peach aphid; Myzus persicae (Sulzer, 1776) 2.5.3.1 Taxonomy and distribution Myzus persicae (Hemiptera: Aphididae) is an extremely polyphagous and highly polymorphic aphid, with colours varying from pale greenish-yellow to pink, red, or almost black and has an average body length of 1.2-2.3 mm (Blackman and Eastop, 1984; Van Emden and Harrington, 2007). The distinguishing features from other brassica aphid species include prominent and inward-pointing antennal tubercles, and the base of the cornicles are swollen, and they are longer than the cauda (Blackman and Eastop, 1984). Winged forms have a shiny black fleck on the rear part of the abdomen. It is presumed to originate from Asia, where its primary host, Prunus persica (L.) Batsch, is native, and is now distributed worldwide on diverse plant species (Jansson, 2003; Van Emden and Harrington, 2007). University of Ghana http://ugspace.ug.edu.gh 16 2.5.3.2 Life cycle of M. persicae Myzus persicae is heteroecious holocyclic (host alternating, with sexual reproduction during part of its life cycle) between Prunus (usually peach) and summer host plants, but anholocyclic on secondary (summer) hosts in many parts of the world with favorable environmental conditions (Broadbent, 1949; Blackman, 1974; Blackman and Eastop 2000). It undergoes anholocycly in the tropics and sub-tropics on a wide variety of plants, including many vegetable crops such as cabbage and its Brassica relatives, potato and other crops of the family Solanaceae and celery, mustard, pepper, pumpkin, okra, corn, sunflower, and other flower crops (Blackman, 1974; Margaritopoulos et al., 2002). An aphid starts producing about 12days after birth and there can be 20 generations in a year during warmer climates (Blackman, 1972). 2.6 Variations in aphid populations on brassica due to biotic and abiotic factors Colonies of aphids on cabbage consist mainly of apterous females and nymphs during the major growing seasons and several alate forms in unfavorable conditions. Their abundance on crops fluctuates based on host plant quality, inter-specific and intra-specific abundance, host plant availability and density, and environmental factors (Van Emden and Bashford, 1971; Dixon, 1977; Agarwala and Datta, 1999) which can be termed biotic and abiotic factors respectively. Therefore, different seasons affect physiological processes, population build-up, distribution, and colonization of new habitats. They usually exhibit an exponential growth rate when favourable environmental factors are fairly constant (Honěk, 1987). However, as overcrowding becomes evident their rate of increase decreases due to phenological alterations of their host plants as well as other climatic factors or they may migrate (Moran, 1992). Amongst the abiotic factors that affect University of Ghana http://ugspace.ug.edu.gh 17 insect abundance and distribution, temperature and relative humidity stand out to be the most constraining factors (Savopoulou-Soultani et al., 2012). Studies have shown the influence of abiotic factors on aphid population on cruciferous crops (Fidelis et al., 2018; Soh et al., 2018; Shonga and Getu, 2020). Biotic factors that affect aphid populations include plant phenology, natural enemies such as predators, parasitoids, and naturally occurring entomopathogens. Associated natural enemies of aphids are discussed in Section 2.8.6 of this review. 2.7 Economic importance of aphids on cabbage The economic importance of aphid species is dependent on the type and extent of damage caused, and on the economic importance of the hosts (Dedryver et al., 2010). Losses due to aphid infestation on brassica crops have been estimated at approximately 35 – 91 % under different environmental conditions (Singh and Sachan, 1994). The intensity of this damage is often influenced by the plant growth stage, the duration of infestation, plant vigour, and environmental conditions. Munthali et al. (2004) recorded rapid population build-up on cabbage in Botswana during warm weather, accompanied by severe damages to the crops. Rana (2005) reported 10-90% damage caused by Lipaphis erysimi depending on the severity of infestation and plant stage while in India, Singh and Sharma (2002) reported damage ranging from 9-96% in different agroecological zones. Losses due to B. brassicae and L. erysimi were estimated at 70-80% on oilseed brassica in Pakistan (Razaq et al., 2011). Other brassicas like Brassica napus, Brassica juncea, and Brassica carinata had severe losses from aphid infestation of up to 75.06%, 77.25%, and 81.86% respectively (Razaq et al., 2011). Brevicoryne brassicae has been reported to cause total crop loss in leafy brassica crops in Ethiopia (Munthali and Tshegofatso, 2014). In Senegal and the Democratic Republic of Congo, major aphids occurring on cabbage are often M. persica, University of Ghana http://ugspace.ug.edu.gh 18 B. brassicae, and L. erysimi which causes rolling of young leaves, chlorosis, deformation, and thickening of leaves in severe outbreaks (Yarou et al., 2017). Yarou et al. (2017) further reported that the most important damage by these pests on cabbage is the transmission of viruses and sooty mold formation following honeydew production. Mersha et al. (2014) reported that aphids reduce the yield of cabbage and its aesthetic and nutritional content. In Ghana, the two major aphid species reported to be causing huge losses (20-100%) and damage in major cabbage-producing regions are M. persicae, and L. pseudobrassicae (Forchibe et al., 2017). Adenka et al. (2021), reported 100% loss to aphids in six different cabbage varieties in Ghana. Some reports have concluded that aphid infestation often reduces plant growth and in cabbage, it usually leads to the death of plants (Munthali and Tshegefatso, 2014; Embaby and Lotfy, 2015). 2.7.1 Aphids as vectors of plant viruses Transmission of viruses by insects is one of the major problems associated with the intensification of vegetable and fruit production often leading to huge losses due to quality, with several unmarketable products (Moreno et al., 2004; Dedryver et al., 2010). Tomlinson (1987) reported that the major economically important viruses transmitted by aphids in field-grown vegetables, includes the Cucumber mosaic virus (CMV), Lettuce mosaic virus (LMV), Papaya ringspot virus (PRSV), Turnip mosaic virus (TuMV), and Potato virus Y (PVY). The efficiency of virus spread by aphids is linked to their capacity to make intracellular probes on leaves without killing the cell (Wamonje et al., 2020). When favorable content is found, aphids probe deeper into sieve elements, and in the process, acquire virus particles (Tjallingii and Esch, 1993). Probing on the epidermal cells of leaves often suffices for the acquisition of non-persistently transmitted viruses while phloem-feeding is usually correlated with the acquisition of persistently transmitted viruses (Brault University of Ghana http://ugspace.ug.edu.gh 19 et al., 2010). Virus transmission by aphids involves acquisition, retention, inoculation, and association with the vectors (Brault et al., 2010). The major viruses infecting cabbage includes Cauliflower mosaic virus (CaMV), Turnip mosaic virus (TuMV), Beet western yellows virus (BWYV), and sometimes Cucumber mosaic virus (CMV), all of which are vectored and transmitted by aphids (Moreno et al., 2004; Sevik and Deligoz, 2017). CMV (Moreno et al., 2004) and TuMV (Nguyen et al., 2013) are noted as the most widespread and damaging pathogens of brassicas worldwide, followed by the CaMV (Spence et al., 2007). The former is transmitted by M. persica, L. erysimi and B. brassicae non-persistently (Day and Venables, 1961), and BWYV is persistently transmitted by M. persicae (Kyriakou et al., 1983). Brevicoryne brassicae is known to vector and transmit over 23 viral pathogens in a non-persistent mode (Eastop, 1977). Myzus persicae transmits over 100 plant viruses in many economically important crops (CABI Plantwise, 2020), and is considered among the most successful aphid vectors of plant viruses due to its polyphagous nature (Ng and Perry, 2004). Lipaphis e. pseudobrassicae is reported to vector and transmit at least 16 plant viruses (Blackman and Eastop, 1984). Ghosh et al. (2017) reviewed aphids as vectors of plant viruses. 2.8 Management of aphids on cabbage Aphid management on cabbage had earlier been geared towards the use of insecticides because of their accessibility, availability, and rapid action (Dewar et al., 2017). In Ghana, insecticides remain the main control option for aphids on many crops, however, there has been a push for more environmentally friendly practices as a segment of an Integrated Pest Management (IPM) strategy (Ntow et al., 2006; Horna et al., 2008; Forchibe et al., 2017). IPM aims to augment beneficial University of Ghana http://ugspace.ug.edu.gh 20 insect populations, avert pest outbreaks, pest resurgence, and disease spread, while maintaining the natural environment (Mangan and Mangan, 1998; Afreh-Nuamah, 2003; Naranjo et al., 2015). IPM combines different pest management strategies with judicious pesticide use, usually as the last resort, to achieve production goals. Multiple IPM approaches have been suggested for the management of aphids on brassica crops (Farooq and Tasawar, 2008; James et al., 2010; Liu et al., 2014; Reddy, 2017). 2.8.1 Mechanical control Mechanical control measures are usually recommended to be considered first when pest populations have not exceeded threshold limits. They often include a wide range of practices such as the use of traps, hand crushing, erecting barriers, removing infested plants or leaves, and conducting activities that disrupt pests feeding and multiplication (Pesticide Research Institute). For example, Griffin and Williamson (2012) suggested that fields be furrowed immediately after harvest to prevent aphids spreading to other crops. Clearing alternative host plants like mustard and other cruciferous weeds around fields also reduces the probability of infestation (Natwick, 2009). Other reports have shown that trap crops also prevent aphid infestation of the main crop. Crops like dill, nasturtiums, and timothy grass planted close to the main crop have been reported to deter aphids (Philbrick and Gregg, 2012). Aphids can also be washed off plants using a high- pressure water hose with jet-set dial nozzles. The Landscape IPM showed that using jet or shower setting nozzles easily dislodges insects from plants. Although mechanical control measures as important, they are only practical in small gardens and cannot be adopted in large-scale cultivation. University of Ghana http://ugspace.ug.edu.gh 21 2.8.2 Monitoring and sampling Monitoring of aphids is a useful tool, aimed at providing information for timely and efficient implementation of management strategies. Several methods are used in aphid monitoring depending on the purpose. As vectors of plant viruses, there is almost zero tolerance for aphids on brassica crops, hence the need for monitoring. Harrington et al. (2007) provided a review of techniques in aphid monitoring. In their review, sampling methods were grouped into direct which includes: in situ plant counts, destructive plant counts, vacuum sampling, and indirect which includes the use of coloured water pan traps, clear or plant-coloured water pan traps, sticky traps, filter nets, and suction traps. Direct sampling methods are often used in cabbage production because they provide a measure of absolute abundance per plant, which is usually requisite for population density estimates. Monitoring provides threshold data for the implementation of pest management interventions. In USA and UK, treatment thresholds have been established for B. brassicae (Ellis et al., 1999; Collier and Finch, 2007) and L. pseudobrassicae in brassica crops in Asia (Singh and Malik, 1998). Pest management guidelines for the USA suggested that cabbage crops should be treated as soon as 1-2% of plants were infested with B. brassicae since it has a low action threshold (Collier and Finch, 2007). However, infestations of up to 100 aphids/plant for M. persicae could be tolerated (Eastman et al., 2005). In Kenya, Pests Management Decision Guide suggests the institution of control measures when 1 to 2% of plants are infested with either of the three major aphids (Murage et al., 2014). Despite the availability of this information in some countries, and it is incumbent on the growers to utilize them, nevertheless, information on how widely adopted and used these thresholds are have not been determined (Collier and Finch, 2007). However, such action thresholds are very useful for the implementation of IPM approaches. University of Ghana http://ugspace.ug.edu.gh 22 2.8.3 Cultural control Cultural control of aphids involves the reduction of aphid damage through manipulations of the physical and biological environment of the crop before and during growth (Wratten et al., 2007). Some methods are aimed at improving plant vigour, crop yield, etc, which may not directly have impacts on the aphids (Perring et al., 2018). Farm sanitation for example is very effective for the improvement of plant growth and at the same time kills over overwintering aphid eggs in temperate climates and prevents farm re-infestation (Hines and Hutchison, 2013). Sowing dates have been shown to have a significant effect on aphid populations while the crops sown late in the season suffer aphid infestation (Sidiqui et al., 2009; Dharavath et al., 2016). Intercropping is a cultural approach employed by several growers to manage aphids in the production of brassica crops. This approach is such that non-host plants offer resources to natural enemies, thus augmenting their populations, and interfere with the host finding ability of specialist herbivores such as aphids (Root, 1973). Subsequently, several authors have reported on the effects of intercropping in controlling pests of cabbage (Ponti et al., 2007; Mochiah et al., 2011; Baidoo et al., 2012). Ben-Issa et al. (2017) reviewed companion plants used in aphid pest management in the categories of trap plants, alteration of host plant selection, and natural enemy attractants. 2.8.4 Host plant resistance Plant resistance to insects is often a result of heritable characters that result in less damage compared to plants without these characters. Stout (2014) in his book, explores and discusses host plant resistance (HPR) approaches and integration into IPM strategies, outlining the major steps involved in development and implementation. In a review by Van Emden (2007), 12 mechanisms University of Ghana http://ugspace.ug.edu.gh 23 of resistance to aphids were discussed and classified under the three major classes: antixenosis, antibiosis, and tolerance. Breeding for plant resistance against insects is not common as is the case of breeding disease-resistant cultivars partly due to the availability of a wide range of rapid control measures. Sharma and Ortiz (2002) attributed this scarcity to the difficulty involved in screening insect-resistant plant varieties, the high cost of rearing programmes, and long years of technology development which may not yield the expected results. Bhatia et al. (2011) suggested that breeding for genetic resistance against aphids is difficult, due to the absence of resistant sources within the crossable germplasms and insufficient knowledge in the associated genetics. Several studies have been carried out with cabbage lines to screen for resistant varieties to B. brassicae, L. pseudobrassicae and M. persicae (Ahmed et al., 2018; Taghizadeh, 2019; Hong et al., 2019; Ali et al., 2021). HPR can be used an important strategy for the integrated management of aphids on cabbage. 2.8.5 Chemical control The use of chemicals in the management of crop pests dates back to the times of the organochlorines, organophosphates, carbamates, and recently pyrethroids and neonicotinoids (Ware and Whitacre, 2004; Davies et al., 2007). With frequent food safety concerns with the use of synthetic insecticides, alternative options have been recommended for pest control (Fening et al., 2013, 2014; Forchibe et al., 2017). Tolerance to aphids as a pest is minimal, owing to their rapid reproduction and resistance to insecticides (Ahmad and Akhtar, 2013; Bass et al., 2014). In this light, several studies have been done to develop new compounds for their management (Tang et al., 2019; Singh and Joshi, 2020). Dewar and Denholm (2007) reviewed the chemical control of aphids stating the progressive shift from more persistent pesticides to less persistent and University of Ghana http://ugspace.ug.edu.gh 24 environmentally friendlier options. With the increasing demand for cabbage, most farmers have relied on the use of insecticides as it gives fast results (Ntow et al., 2006; Owusu-Boateng & Amuzu, 2013; Machekano et al., 2019). However, with the increasing concern on the negative impacts of these chemicals on the environment and non-target organisms, there is a gradual shift to safer options. Development of insecticides moved from broad-spectrum persistent materials to short-lived, effective, sometimes expensive, and specific materials which are environmentally friendly (Perring et al., 2018). Several authors have evaluated the efficacy of different insecticides against aphids on brassicaceous crops; effects of imidacloprid (Neonicotinoid) on aphids on cabbage (Djomaha et al., 2016), effects of flupyradifurone (Butenolide) on the green peach aphid, M. persicae (Tang et al., 2019), effects of acetamiprid (Neonicotinoid) on L. pseudobrassicae (Maia et al., 2020), effects of cyantraniliprole (Diamide) and imidacloprid on B. brassicae, M. persicae (Ahumada and Chorbadjian, 2019), among others. With the growing demand for organic vegetables, less toxic options for pest management have been suggested to meet up with the growing market. Much research work has been done on the effects of botanicals on the population of aphids on cabbage: Baidoo and Adam (2012) recorded larger yield in cabbage when neem seeds and Lanthana camara leaf extracts were used to control pest, Fening et al. (2014) observed insecticidal effects of garlic and hot pepper extracts against insect pests on cabbage with minimal effects on natural enemies, Djomaha et al. (2016) and Forchibe et al. (2017) recorded positive control of aphids on cabbage using neem seed extract, Pissinati and Ventura (2015) suggested neem oil and kaolin oil as alternatives for the control of cabbage aphids on kale. Pereira et al. (2019) reported insecticidal properties in Agave americana against aphids on cabbage. He further recommended its extract as a good alternative to synthetic insecticide against aphids on cabbage. A study by Jabran et al. University of Ghana http://ugspace.ug.edu.gh 25 (2016), reported control of the cabbage aphid by 50% through allelopathic water extracts of sorghum, sunflower, brassica and mulberry. Great success has been recorded for many botanicals against insect pests and some; especially neem pesticides are now commercially produced and exported for use in vegetable production. The safety and efficacy of these neem-based pesticides have been tested and recommended for use by several researchers (Obeng-Ofori, 2008; Lokanadhan et al., 2012). Insect growth regulators (IGRs) are also a group of chemical compounds that alter growth and development in insects; as juvenile hormones, as precocenes, and as chitin synthesis inhibitors (Ishaaya, 2001). Juvenile hormones (JH) include ecdysone (the moulting hormone), JH mimic, JH analog (JHA), and are known by their broader synonyms, juvenoids and juvegens. They disrupt immature development and emergence as adults. Precocenes interfere with the normal function of glands that produce juvenile hormones. Chitin synthesis inhibitors, (conventional benzoylureas, buprofezin and cyromazine), affect the ability of insects to produce new exoskeletons when molting (Horowitz and Ishaaya, 2004). Neem products have been reported to act as IGRs and are very useful in controlling insect pests (Lokanadhan et al., 2012). Though IGRs seem to be very effective in their mode of action, they are non-specific and often affect beneficial arthropods. Cabral et al. (2008) reported that buprofezin (a chitin synthesis inhibitor) affected the survival, fecundity, fertility and egg hatching of the major aphid predator Coccinella undecimpunctata. 2.8.6 Biological control Biological control is the use of natural enemies; predators, parasitoids and pathogens to control insect pests. The natural enemy populations of aphids are often numerous enough to keep their University of Ghana http://ugspace.ug.edu.gh 26 population below economic damage thresholds (Rabasse and van Steenis, 1999; Mandal and Patnaik, 2008). Sometimes, wasp parasites are the most effective; in other cases, predators, such as the hoverflies, lady beetles, or entomopathogens such as fungi cause the most mortality (Liu et al., 2014; Fidelis et al., 2018). Although there is a rich diversity of natural enemies of aphids, biological control is often not efficient (Dixon, 2000; Sampaio et al., 2017). Perring et al. (2018) explained that this unsatisfactory result gotten from biological control can be attributed to the incompetence of aphid natural enemies as agents of biological control. Pal and Singh (2012) rather suggested that, under natural conditions, ‘before parasitoid populations are large enough to be effective, the aphid population has usually exceeded the Economic Injury Level (EIL).’ Other factors include hyperparasitism of main aphid parasitoids, the complexity of implementation of a biological control programme as it takes into consideration all factors that affect the pest unlike using insecticides. Thus, implementation involves high levels of skilled personnel. 2.8.6.1 Natural enemies of aphids on brassicas The natural enemies of aphids in brassicas range from entomopathogenic fungi such as those of the genus Lecanicillium, (Vu et al., 2007) and Entomophthorales (Scorsetti et al., 2007) to Braconid and Aphelinid parasitoids (Geiger et al., 2005; Sampaio et al., 2017) and predators, of the families Coccinellidae, Syrphidae and Chrysopidae (Debaraj & Singh, 1998; Nieto et al., 2006). Additionally, some Aranae are good predators of cabbage pests (Amoabeng et al., 2013; Grez et al., 2014; Forchibe et al., 2017). They hunt or use webbing to catch their prey, and experiments have demonstrated their ability to control aphids (Madsen et al., 2004; Dippenaar- Schoeman et al., 2013). Liu et al. (2014) discussed the diversity of natural enemies associated with University of Ghana http://ugspace.ug.edu.gh 27 brassica vegetables in China, detailing the bioecology and some ecological aspects of major natural enemies in the region. Sæthre et al. (2011) observed the following predators to be associated with L. erysimi and M. persicae on cabbage and turnip in Benin; Cheilomenes propinqua, Hippodamia varigata, Ischiodon aegyptius, and Cheilomenes sulphurea. Fidelis et al. (2018) reported the following natural enemies associated with B. brassicae on cabbage; Allograpta exotica, Pseudodorus clavatus, Ocyptamus gastrostactus (Syrphidae larvae) Coccinella septempunctata, Cycloneda sanguinea, Eriopis connexa and Harmonia axyridis (Coccinellidae). He concluded that predation is the major mortality factor of B. brassicae in cabbage crops. Of the huge diversity of predators associated with aphids on cabbage reporte