- Title
- Baculovirus synergism: investigating mixed alphabaculovirus and betabaculovirus infections in the false codling moth, thaumatotibia leucotreta, for improved pest control
- Creator
- Jukes, Michael David
- Subject
- Baculoviruses
- Subject
- Cryptophlebia leucotreta -- Biological control
- Subject
- Citrus -- Diseases and pests -- South Africa
- Subject
- Pests -- Integrated control
- Subject
- Nucleopolyhedroviruses
- Subject
- Natural pesticides
- Subject
- Cryptophlebia leucotreta granulovirus (CrleGV)
- Date Issued
- 2018
- Date
- 2018
- Type
- text
- Type
- Thesis
- Type
- Doctoral
- Type
- PhD
- Identifier
- http://hdl.handle.net/10962/61797
- Identifier
- vital:28061
- Description
- Baculovirus based biopesticides are an effective and environmentally friendly approach for the control of agriculturally important insect pests. The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is indigenous to southern Africa and is a major pest of citrus crops. This moth poses a serious risk to export of fruit to foreign markets and the control of this pest is therefore imperative. The Cryptophlebia leucotreta granulovirus (CrleGV) has been commercially formulated into the products Cryptogran™ and Cryptex®. These products have been used successfully for over a decade as part of a rigorous integrated pest management (IPM) programme to control T. leucotreta in South Africa. There is however, a continuous need to improve this programme while also addressing new challenges as they arise. An example of a rising concern is the possibility of resistance developing towards CrleGV. This was seen in Europe with field populations of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), which developed resistance to the Mexican isolate of the Cydia pomonella granulovirus (CpGV-M). To prevent such a scenario occurring in South Africa, there is a need to improve existing methods of control. For example, additional baculovirus variants can be isolated and characterised for determining virulence, which can then be developed as new biopesticides. Additionally, the potential for synergistic effects between different baculoviruses infecting the same host can be explored for improved virulence. A novel nucleopolyhedrovirus was recently identified in T. leucotreta larval homogenates which were also infected with CrleGV. This provided unique opportunities for continued research and development. In this study, a method using C. pomonella larvae, which can be infected by the NPV but not by CrleGV, was developed to separate the NPV from GV-NPV mixtures in an in vivo system. Examination of NPV OBs by transmission electron microscopy showed purified occlusion bodies with a single nucleopolyhedrovirus morphology (SNPV). Genetic characterisation identified the novel NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which was recently isolated from the litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae). To begin examining the potential for synergism between the two viruses, a multiplex PCR assay was developed to accurately detect CrleGV and/or CrpeNPV in mixed infections. This assay was applied to various samples to screen for the presence of CrpeNPV and CrleGV. Additionally, a validation experiment was performed using different combinations of CrpeNPV and/or CrleGV to evaluate the effectiveness of the mPCR assay. The results obtained indicated a high degree of specificity with the correct amplicons generated for each test sample. The biological activity of CrpeNPV and CrleGV were evaluated using surface dose bioassays, both individually and in various combinations, against T. leucotreta neonate larvae in a laboratory setting. A synergistic effect was recorded in the combination treatments, showing improved virulence when compared against each virus in isolation. The LC90 for CrpeNPV and CrleGV when applied alone against T. leucotreta was calculated to be 2.75*106 and 3.00*106 OBs.ml"1 respectively. These values decreased to 1.07*106 and 7.18*105 OBs.ml"1 when combinations of CrleGV and CrpeNPV were applied at ratios of 3:1 and 1:3 respectively. These results indicate a potential for developing improved biopesticides for the control of T. leucotreta in the field. To better understand the interactions between CrleGV and CrpeNPV, experiments involving the serial passage of these viruses through T. leucotreta larvae were performed. This was done using each virus in isolation as well as both viruses in different combinations. Genomic DNA was extracted from recovered occlusion bodies after each passage and examined by multiplex and quantitative PCR. This analysis enabled the detection of each virus present throughout this assay, as well as recording shifts in the ratio of CrleGV and CrpeNPV at each passage. CrleGV rapidly became the dominant virus in all treatments, indicating a potentially antagonistic interaction during serial passage. Additionally, CrpeNPV and CrleGV were detected in treatments which were not originally inoculated with one or either virus, indicating potential covert infections in T. leucotreta. Occlusion bodies recovered from the final passage were used to inoculate C. pomonella larvae to isolate CrpeNPV from CrleGV. Genomic DNA was extracted from these CrpeNPV OBs and examined by restriction endonuclease assays and next generation sequencing. This enabled the identification of potential recombination events which may have occurred during the dual GV and NPV infections throughout the passage assay. No recombination events were identified in the CrpeNPV genome sequences assembled from virus collected at the end of the passage assay. Lastly, the efficacy of CrpeNPV and CrleGV, both alone and in various combinations, was evaluated in the field. In two separate trials conducted on citrus, unfavorable field conditions resulted in no significant reduction in fruit infestation for both the virus and chemical treatments. While not statistically significant, virus treatments were recorded to have the lowest levels of fruit infestation with a measured reduction of up to 64 %. This study is the first to report a synergistic effect between CrleGV and CrpeNPV in T. leucotreta. The discovery of beneficial interactions creates an opportunity for the development of novel biopesticides for improved control of this pest in South Africa.
- Format
- 181 pages
- Format
- Publisher
- Rhodes University
- Publisher
- Faculty of Science, Biochemistry and Microbiology
- Language
- English
- Rights
- Jukes, Michael David
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Jukes, Michael (Dr)
RU Department of Biochemistry and Microbiology (2015-)
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