Baculovirus synergism: investigating mixed alphabaculovirus and betabaculovirus infections in the false codling moth, thaumatotibia leucotreta, for improved pest control
- Authors: Jukes, Michael David
- Date: 2018
- Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61797 , 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.
- Full Text:
- Date Issued: 2018
- Authors: Jukes, Michael David
- Date: 2018
- Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61797 , 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.
- Full Text:
- Date Issued: 2018
The implementation of a push-pull programme for the control of Eldana saccharina (Lepidoptera: Pyralidae) in sugarcane in the coastal regions of Kwazulu-Natal, South Africa
- Authors: Mulcahy, Megan Marie
- Date: 2018
- Subjects: Pyralidae -- South Africa -- KwaZulu-Natal , Pests -- Integrated control , Sugarcane -- Diseases and pests -- South Africa -- KwaZulu-Natal , Stem borers -- Effect of habitat modification on -- South Africa -- KwaZulu-Natal , Insect-plant relationships -- South Africa -- KwaZulu-Natal
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63290 , vital:28390
- Description: Eldana saccharina, an indigenous lepidopteran stemborer, is considered the most damaging pest of sugarcane in South Africa. Researchers have advocated the use of an area-wide integrated pest management (AW-IPM) programme as a means of improving the control of this pest. A push-pull strategy was developed as a component of this AW-IPM approach. The push-pull strategy in sugarcane is a habitat management method of pest control that uses plants that are both repellent (Melinis Minutiflora) and attractive (Cyperus dives, Cyperus papyrus and Bt-maize) to E. saccharina. Previous research into push-pull has shown that this strategy is an effective tool for the control of E. saccharina. Push-pull has been implemented successfully in the Midlands North sugarcane growing region of KwaZulu-Natal (KZN), South Africa. Despite the proven efficacy of push-pull, very little push-pull research has been conducted in the coastal sugarcane growing regions of KZN, and adoption of this technology has been poor in these regions. Therefore, the aim of this research was to facilitate the implementation of push-pull for the management of E. saccharina in sugarcane in the coastal regions of KZN. This was done by focussing on on-farm field trials and farmer participatory research. On-farm push-pull field trials were conducted on five model farms in the North and South Coast sugarcane growing regions of KZN. High levels of E. saccharina were recorded during this study. The push-pull treatment sites showed a significant reduction of E. saccharina damage on four of the five farms used in the study. Mean percentage of stalks damaged decreased by up to 50 % in the presence of the repellent grass species, M. minutiflora. The number of E. saccharina found per 100 stalks also decreased significantly at these farms. The farm which did not show a significant reduction in E. saccharina populations or damage had low numbers of this pest in the sugarcane throughout the experiment. This demonstrates that push-pull is more effective in areas that have high levels of E. saccharina. Stem borer surveys in wetlands on sugarcane farms revealed that high numbers of E. saccharina were found within the pull plants, C. papyrus and C. dives, in comparison to the push-pull sites. This verifies that the pull plants do work efficiently to attract E. saccharina away from sugarcane. Additionally, eight parasitoids emerged from E. saccharina larvae collected in wetland sedges. The beneficial roles that push-pull plants play in attracting and maintaining natural enemies in the agroecosystem are discussed, and these findings further demonstrate the important ecosystem, and pest management services that wetlands provide on sugarcane farms. The success of the push-pull trials in this study show that this technology can be an effective tool for controlling E. saccharina in the coastal sugarcane growing regions. The timing of the planting of push-pull plants was shown to play a role in the efficacy of this technology. The study also confirmed that push-pull should be used as a component of AW-IPM in conjunction with good crop management practices. Surveys were undertaken to determine large-scale sugarcane growers' (LSGs) knowledge and perceptions of E. saccharina and other pests. Research regarding the farmers' perceptions of push- pull was also conducted to better understand the drivers and barriers to adoption of push-pull, and other new technologies. The surveys found that large-scale farmers in the coastal regions suffer from high infestations of E. saccharina. As such there is scope for the introduction of new pest management practices such as push-pull in this area. Farmers also demonstrated a good basic knowledge of E. saccharina and IPM. However, LSGs had a poor understanding of push-pull and how it works, as well as the plants that make up the push-pull system that is being implemented against E. saccharina in South Africa. A dearth in practical knowledge regarding the implementation of push-pull was seen as a major barrier to the adoption of this strategy, as was financial instability, farmer attitudes and poor institutional support. Farmers recommended collaboration amongst stakeholders, improved education, proof of the efficacy of push-pull and incentives as tools to improve the implementation of this strategy in the coastal sugarcane growing regions of KZN. Farmers preferred direct contact with extension personnel and experiential learning opportunities when acquiring information about push- pull and other new pest management practices. If opportunities for push-pull education are increased through direct contact with extension personnel, and through on-farm demonstrations, and if inputs are provided in the form of push-pull plants, it is likely that push-pull will succeed amongst coastal LSGs, especially since farmers had an overall positive attitude towards the technology. Surveys amongst small-scale sugarcane growers (SSGs) showed that sugarcane is important in the lives of these farmers. The SSGs perceive pests to be a major constraint to their farming systems, and they identified E. saccharina as a major pest of sugarcane. The farmers also demonstrated good knowledge of sugarcane pests and vegetable pests. However, SSGs lacked knowledge regarding pest management practices and beneficial insects. Extension and advisory services should to continue concentrating on pest management practices to educate SSGs on the variety and application of pest control strategies. SSGs were found to employ complex, diverse and integrated agricultural systems that are well-suited to the implementation of IPM technologies such as push-pull. Since insect pests act were found to be a major constraint to SSG sugarcane production, push-pull was deemed a feasible pest management strategy for coastal farmers and its implementation by SSGs should be further explored. SSGs in this study were also concerned about vegetable pests, therefore if push-pull can be adapted to help protect additional crops, adoption of this technology by small-scale growers will improve.
- Full Text:
- Date Issued: 2018
- Authors: Mulcahy, Megan Marie
- Date: 2018
- Subjects: Pyralidae -- South Africa -- KwaZulu-Natal , Pests -- Integrated control , Sugarcane -- Diseases and pests -- South Africa -- KwaZulu-Natal , Stem borers -- Effect of habitat modification on -- South Africa -- KwaZulu-Natal , Insect-plant relationships -- South Africa -- KwaZulu-Natal
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/63290 , vital:28390
- Description: Eldana saccharina, an indigenous lepidopteran stemborer, is considered the most damaging pest of sugarcane in South Africa. Researchers have advocated the use of an area-wide integrated pest management (AW-IPM) programme as a means of improving the control of this pest. A push-pull strategy was developed as a component of this AW-IPM approach. The push-pull strategy in sugarcane is a habitat management method of pest control that uses plants that are both repellent (Melinis Minutiflora) and attractive (Cyperus dives, Cyperus papyrus and Bt-maize) to E. saccharina. Previous research into push-pull has shown that this strategy is an effective tool for the control of E. saccharina. Push-pull has been implemented successfully in the Midlands North sugarcane growing region of KwaZulu-Natal (KZN), South Africa. Despite the proven efficacy of push-pull, very little push-pull research has been conducted in the coastal sugarcane growing regions of KZN, and adoption of this technology has been poor in these regions. Therefore, the aim of this research was to facilitate the implementation of push-pull for the management of E. saccharina in sugarcane in the coastal regions of KZN. This was done by focussing on on-farm field trials and farmer participatory research. On-farm push-pull field trials were conducted on five model farms in the North and South Coast sugarcane growing regions of KZN. High levels of E. saccharina were recorded during this study. The push-pull treatment sites showed a significant reduction of E. saccharina damage on four of the five farms used in the study. Mean percentage of stalks damaged decreased by up to 50 % in the presence of the repellent grass species, M. minutiflora. The number of E. saccharina found per 100 stalks also decreased significantly at these farms. The farm which did not show a significant reduction in E. saccharina populations or damage had low numbers of this pest in the sugarcane throughout the experiment. This demonstrates that push-pull is more effective in areas that have high levels of E. saccharina. Stem borer surveys in wetlands on sugarcane farms revealed that high numbers of E. saccharina were found within the pull plants, C. papyrus and C. dives, in comparison to the push-pull sites. This verifies that the pull plants do work efficiently to attract E. saccharina away from sugarcane. Additionally, eight parasitoids emerged from E. saccharina larvae collected in wetland sedges. The beneficial roles that push-pull plants play in attracting and maintaining natural enemies in the agroecosystem are discussed, and these findings further demonstrate the important ecosystem, and pest management services that wetlands provide on sugarcane farms. The success of the push-pull trials in this study show that this technology can be an effective tool for controlling E. saccharina in the coastal sugarcane growing regions. The timing of the planting of push-pull plants was shown to play a role in the efficacy of this technology. The study also confirmed that push-pull should be used as a component of AW-IPM in conjunction with good crop management practices. Surveys were undertaken to determine large-scale sugarcane growers' (LSGs) knowledge and perceptions of E. saccharina and other pests. Research regarding the farmers' perceptions of push- pull was also conducted to better understand the drivers and barriers to adoption of push-pull, and other new technologies. The surveys found that large-scale farmers in the coastal regions suffer from high infestations of E. saccharina. As such there is scope for the introduction of new pest management practices such as push-pull in this area. Farmers also demonstrated a good basic knowledge of E. saccharina and IPM. However, LSGs had a poor understanding of push-pull and how it works, as well as the plants that make up the push-pull system that is being implemented against E. saccharina in South Africa. A dearth in practical knowledge regarding the implementation of push-pull was seen as a major barrier to the adoption of this strategy, as was financial instability, farmer attitudes and poor institutional support. Farmers recommended collaboration amongst stakeholders, improved education, proof of the efficacy of push-pull and incentives as tools to improve the implementation of this strategy in the coastal sugarcane growing regions of KZN. Farmers preferred direct contact with extension personnel and experiential learning opportunities when acquiring information about push- pull and other new pest management practices. If opportunities for push-pull education are increased through direct contact with extension personnel, and through on-farm demonstrations, and if inputs are provided in the form of push-pull plants, it is likely that push-pull will succeed amongst coastal LSGs, especially since farmers had an overall positive attitude towards the technology. Surveys amongst small-scale sugarcane growers (SSGs) showed that sugarcane is important in the lives of these farmers. The SSGs perceive pests to be a major constraint to their farming systems, and they identified E. saccharina as a major pest of sugarcane. The farmers also demonstrated good knowledge of sugarcane pests and vegetable pests. However, SSGs lacked knowledge regarding pest management practices and beneficial insects. Extension and advisory services should to continue concentrating on pest management practices to educate SSGs on the variety and application of pest control strategies. SSGs were found to employ complex, diverse and integrated agricultural systems that are well-suited to the implementation of IPM technologies such as push-pull. Since insect pests act were found to be a major constraint to SSG sugarcane production, push-pull was deemed a feasible pest management strategy for coastal farmers and its implementation by SSGs should be further explored. SSGs in this study were also concerned about vegetable pests, therefore if push-pull can be adapted to help protect additional crops, adoption of this technology by small-scale growers will improve.
- Full Text:
- Date Issued: 2018
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