THE DEVELOPMENT and EVALUATION of CRYPTOPHLEBIA LEUCOTRETA GRANULOVIRUS (Crlegv)
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THE DEVELOPMENT AND EVALUATION OF CRYPTOPHLEBIA LEUCOTRETA GRANULOVIRUS (CrleGV) AS A BIOLOGICAL CONTROL AGENT FOR THE MANAGEMENT OF FALSE CODLING MOTH, CRYPTOPHLEBIA LEUCOTRETA, ON CITRUS Submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY of RHODES UNIVERSITY by SEAN DOUGLAS MOORE March 2002 ii ABSTRACT A granulovirus isolated from Cryptophlebia leucotreta larvae was shown through restriction endonuclease analysis to be a novel strain (CrleGV-SA). No more than one isolate could be identified from a laboratory culture of C. leucotreta. However, a preliminary examination of restricted DNA profiles of isolates from different geographical regions indicated some minor differences. In surface dose bioassays on artificial diet, LC50 and LC90 values with neonate larvae 3 5 were estimated to be 4.095 x 10 OBs/ml and 1.185 x 10 OBs/ml respectively. LT50 and LT90 values with neonate larvae were estimated to be 4 days 22 h and 7 days 8 h, respectively. Detached fruit (navel orange) bioassays with neonate larvae indicated that virus concentrations that are likely to be effective in the field range from 1.08 x 107 to 3.819 x 1010 OBs/ml. In surface dose bioassays with fifth instar larvae LC50 and LC90 values were estimated to be 2.678 x 7 9 10 OBs/ml and 9.118 x 10 OBs/ml respectively. LT50 and LT90 values were estimated to be 7 days 17 h and 9 days 8 h, respectively. A new artificial diet for mass rearing the host was developed. Microbial contamination of diet was significantly reduced by adding nipagin and sorbic acid to the diet and by surface sterilising C. leucotreta eggs with Sporekill. Almost 20 % more eggs were produced from moths reared on the new diet compared to moths reared on the old diet. A further 9 % improvement in egg production and a reduction in the labour required to produce eggs, was made with the development of a new oviposition cage attached to the moth eclosion box. Virus was mass produced in fifth instar C. leucotreta larvae by surface inoculating diet with the LC90. When 300 individuals were placed onto inoculated diet, 56 % of them were recovered six to 11 days later as infected larvae. Mean larval equivalents was 1.158 x 1011 OBs/larva. When larvae and diet were harvested together, highest yields of virus were achieved at eight days after inoculation. Microbial contamination in semi-purified preparations of CrleGV ranged from 176211 to 433594 (OB:CFU ratio). Half-life of CrleGV in the field was estimated to be less than 1 day on the northern aspect of trees and between 3 - 6 days on the southern aspect. Original activity remaining (OAR) of the virus dropped below 50 % after 5 days on the northern aspect of trees and was still at 69 % on the southern aspect of trees after 3 weeks. In field trials, CrleGV reduced C. leucotreta infestation of navel oranges by up to 60 % for a period of 39 days. CrleGV in combination with augmentation of the C. leucotreta egg parasitoid, Trichogrammatoidea cryptophlebiae, reduced infestation by 70 %. The integration of CrleGV into an integrated pest management (IPM) system for the management of C. leucotreta on citrus is proposed. iii TABLE OF CONTENTS CHAPTER Page ABSTRACT ii TABLE OF CONTENTS iii LIST OF PLATES xi LIST OF FIGURES xiv LIST OF TABLES xviii LIST OF ABBREVIATIONS xxii ACKNOWLEDGMENTS xxvi 1 PROJECT BACKGROUND AND PROPOSAL 1 1.1 INTRODUCTION 1 1.2 THE HOST: CRYPTOPHLEBIA LEUCOTRETA 1 1.2.1 Taxonomy 1 1.2.2 Distribution 2 1.2.3 Biology and life history 2 1.2.4 Economic importance 5 1.2.4.1 Host range 5 1.2.4.2 Pest status on citrus 6 1.2.5 Pre-harvest control measures 8 1.2.5.1 Monitoring 8 1.2.5.2 Chemical control 9 1.2.5.3 Cultural control 11 1.2.5.4 Natural enemies 12 iv 1.2.5.5 Biological control 15 1.2.5.6 Sterile male release technique 15 1.2.5.7 Mating disruption 16 1.2.6 Post-harvest control 16 1.2.6.1 Cold sterilisation 16 1.2.6.2 Gamma radiation 17 1.3 THE PATHOGEN 17 1.3.1 Granuloviruses 17 1.3.1.1 Classification 17 1.3.1.2 Structure 19 1.3.1.3 Identification of baculoviruses 20 1.3.1.4 Host range 21 1.3.1.5 Gross pathology and symptomatology 22 1.3.1.6 Life cycle 23 1.3.1.7 Cytopathology and histopathology 25 1.3.1.8 Apoptosis regulation 26 1.3.1.9 Natural occurrence 27 1.3.1.10 Pathogenicity 28 1.3.2 Cryptophlebia leucotreta granulovirus (CrleGV) 28 1.4 MICROBIAL CONTROL 29 1.4.1 Microbial control of insect pests 29 1.4.2 Microbial control on citrus 30 1.4.2.1 Worldwide 30 1.4.2.2 South Africa 31 1.4.3 Microbial control with baculoviruses 33 1.4.3.1 An overview 33 1.4.3.2 Production of baculoviruses 39 1.4.3.3 Formulation of baculoviruses 40 1.4.3.4 Application of baculoviruses 41 1.4.4 Microbial control with Cryptophlebia leucotreta 42 granulovirus (CrleGV) v 1.5 PROJECT PROPOSAL 43 1.5.1 Justification 43 1.5.2 Objective 44 2 VIRUS IDENTIFICATION AND CHARACTERISATION 46 2.1 INTRODUCTION 46 2.2 MATERIALS AND METHODS 47 2.2.1 Virus identification 47 2.2.1.1 Symptomatology 48 2.2.1.2 Preliminary identification by light 48 microscopy 2.2.1.3 Identification of infected tissue sections by 48 electron microscopy 2.2.2 Characterisation of virus isolated from multiple 49 larvae 2.2.2.1 Virus purification 49 2.2.2.2 Purification using a sucrose gradient 50 2.2.2.3 Purification using a sucrose gradient 50 2.2.2.4 DNA extraction 51 2.2.2.5 Precipitation of DNA 51 2.2.2.6 Analysis of DNA concentration and purity 52 2.2.2.7 DNA digestion 52 2.2.2.8 Electrophoresis 53 2.2.2.9 Calculation of genome size 54 2.2.3 Characterisation of virus isolated from individual 54 larvae 2.2.3.1 Investigation for different isolates within 54 one laboratory culture 2.2.3.2 Investigation for different isolates from 55 different regions 2.2.3.3 Virus purification 55 vi 2.2.3.4 DNA extraction 56 2.2.3.5 Dialysis of DNA 56 2.2.3.6 DNA digestion 57 2.2.3.7 Electrophoresis 57 2.3 RESULTS AND DISCUSSION 58 2.3.1 Virus identification 58 2.3.1.1 Symptomatology 58 2.3.1.2 Preliminary identification by light 59 microscopy 2.3.1.3 Identification by electron microscopy 60 2.3.2 Characterisation of virus isolated from multiple 61 larvae 2.3.3 Characterisation of virus isolated from individual 72 larvae 2.3.3.1 Investigation for different isolates within 73 one laboratory culture 2.3.3.2 Investigation for different isolates from 73 different regions 2.4 CONCLUSION 75 3 BIOASSAYS 76 3.1 INTRODUCTION 76 3.2 MATERIALS AND METHODS 78 3.2.1 Surface dose bioassays with neonate larvae 78 3.2.1.1 General methodology 78 3.2.1.2 Dose-response bioassays 81 3.2.1.3 Time-response bioassays 83 3.2.2 Detached fruit bioassays 84 3.2.3 Surface dose bioassays with fifth instar larvae 87 3.2.3.1 General methodology 87 3.2.3.2 Dose-response bioassays 88 vii 3.2.3.3 Time-response bioassays 90 3.3 RESULTS AND DISCUSSION 91 3.3.1 Surface dose bioassays with neonate larvae 91 3.3.1.1 General methodology 91 3.3.1.2 Dose-response bioassays 93 3.3.1.3 Time-response bioassays 96 3.3.2 Detached fruit bioassays 101 3.3.3 Surface dose bioassays with fifth instar larvae 108 3.3.3.1 General methodology 108 3.3.3.2 Dose-response bioassays 109 3.3.3.3 Time-response bioassays 111 3.4 CONCLUSION 114 4 HOST REARING 116 4.1 INTRODUCTION 116 4.2 MATERIALS AND METHODS 118 4.2.1 Standard rearing protocol 118 4.2.2 Development of life stages 121 4.2.3 Oviposition 122 4.2.4 Surface sterilisation of eggs 124 4.2.5 Diet sterilisation 125 4.2.6 Diet improvement 126 4.2.7 Statistical analyses 129 4.3 RESULTS AND DISCUSSION 129 4.3.1 Development of life stages 129 4.3.2 Oviposition 133 4.3.3 Surface sterilisation 136 4.3.4 Diet sterilisation 140 4.3.5 Diet improvement 146 4.4 CONCLUSION 151 viii 5 VIRUS PRODUCTION 153 5.1 INTRODUCTION 153 5.2 MATERIALS AND METHODS 154 5.2.1 Virus replication 154 5.2.2 Virus harvesting 156 5.2.3 Semi-purification of virus from diet 156 5.2.4 Quantification of virus 157 5.2.5 Microbial contamination 159 5.3 RESULTS AND DISCUSSION 160 5.3.1 Virus replication 160 5.3.2 Virus harvesting 161 5.3.3 Semi-purification of virus from diet 163 5.3.4 Quantification of virus 163 5.3.5 Microbial contamination 167 5.4 CONCLUSION 169 6 FIELD TRIALS: PERSISTENCE AND EFFICACY 171 6.1 INTRODUCTION 171 6.2 MATERIALS AND METHODS 172 6.2.1 Persistence 172 6.2.2 Efficacy 174 6.3 RESULTS AND DISCUSSION 177 6.3.1 Persistence 177 6.3.2 Efficacy 185 6.4 CONCLUSION 193 7 INTEGRATION OF CrleGV-SA INTO A SYSTEM FOR THE 194 MANAGEMENT OF C.