Baculovirus synergism for improved management of false codling moth Thaumatotibia leucotreta Meyr. (Lepidoptera: Tortricidae)
- Authors: Taylor, David Graham
- Date: 2021-04
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Codling moth , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/176942 , vital:42774
- Description: Baculoviruses are an environmentally friendly and effective agent for managing lepidopteran pests. This includes the management of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a serious pest of citrus in Southern Africa and a major threat to the South African citrus export industry. For more than 15 years, CrleGV-SA- based biopesticides have been used as part of an integrated pest management strategy for the control of T. leucotreta in citrus orchards in South Africa, under the names Cryptogran™ and Cryptex®. While these biopesticides have been effective during this period, there are some areas in which baculovirus use could potentially be improved. Baculoviruses are notoriously slow to kill in comparison to chemical-based pesticides, and lately, pest resistance to baculoviruses has become a major concern with the development of resistance by Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) to its granulovirus occurring in the field in Europe. The consistent use of CrleGV-SA for more than 15 years in the field has raised concern that T. leucotreta could develop resistance to this virus, and has made it necessary to alter baculovirus-based management strategies to prevent this from occurring. A second baculovirus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), has recently been isolated and was shown to be effective against T. leucotreta. However, the interactions between CrleGV-SA and CrpeNPV are not yet understood and so it is important to test these interactions before both viruses are applied on the same orchards. Not only is it important to know whether these viruses could negatively impact each other, but it is also important to test whether they could interact synergistically. A synergistic interaction could not only provide a potential tool for the management of resistance, but it could also be exploited to improve baculovirus-based management of T. leucotreta. In this study, a stock of CrleGV-SA was purified by glycerol gradient centrifugation from T. leucotreta cadavers, while a stock of CrpeNPV purified from Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) cadavers was provided by River Bioscience (Pty) Ltd. These stocks were screened for purity by a multiplex polymerase chain reaction (mPCR) protocol designed to detect CrleGV-SA and CrpeNPV. The occlusion body (OB) density was then calculated using darkfield microscopy and a counting chamber. Both stocks were shown to be pure within the limits of the mPCR protocol, and the CrleGV-SA and CrpeNPV stocks were calculated to contain 3.08 × 1011 OBs/mL and 1.92 × 1011 OBs/mL respectively The first aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the dose mortality, in terms of lethal concentration. This was calculated using 7-day surface-dose biological assays for each virus and a 1:1 mixture of OBs of the two against T. leucotreta neonates. The lethal concentrations of each treatment required to kill 50 % of larvae (LC50) and 90 % of larvae (LC90) for each treatment were then calculated and compared using a probit regression. The mixed infection performed significantly better than either virus by itself, while each virus by itself did not differ significantly from the other. The LC50 for CrleGV-SA, CrpeNPV and the mixed infection were 1.53 × 104 OBs/mL, 1.15 × 104 OBs/mL and 4.38 × 103 OBs/mL respectively. The LC90 of CrleGV-SA, CrpeNPV and the mixed infection were calculated to be 4.10 × 105 OBs/mL, 1.05 × 105 OBs/mL, and 4.09 × 104 OBs/mL respectively. The second aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the speed of kill. A time-response biological assay protocol was created that allowed for effective observation of the larvae. This was then used to generate time-mortality data that were analysed by a logit regression function to calculate and compare the treatments at the time of 50 % larval mortality (LT50) and the time of 90 % mortality (LT90). Each virus by itself did not differ significantly from the other, while the mixed infection took significantly longer to kill 50 % and 90 % of the larvae, suggesting that there is competition for resources between viruses during the secondary, systemic phase of infection. The LT50 for CrleGV-SA, CrpeNPV and the mixed infection were 117.5 hours, 113.5 hours and 139.0 hours respectively. The LT90 for CrleGV-SA, CrpeNPV and the mixed infection were 153.2 hours, 159.3, and 193.4 hours respectively. Finally, the composition of OBs recovered from the cadavers produced by the time-response biological assays were investigated by mPCR. A method for extracting gDNA from OBs in neonate-sized T. leucotreta larvae is described. The presence of CrpeNPV along with CrleGV-SA was noted in 4 out of 9 larvae inoculated with only CrleGV-SA. The presence of CrleGV-SA as well as CrpeNPV was noted in all but one larva inoculated with only CrpeNPV, and both CrleGV-SA and CrpeNPV were noted in all but one larva inoculated with a 1:1 mixture of the two, with one larva only being positive for CrleGV-SA. This suggests either stock contamination or the presence of covert infections of CrleGV-SA and CrpeNPV in the T. leucotreta population used in this study. This is the second study to report an improved lethal concentration of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates, and the first study to report the slower speed of kill of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates. While the improved lethal concentration of the mixed infection is a promising step in the future improvement of baculovirus-based biopesticides, it is at the cost of a slower speed of kill. , Thesis (MSc) -- Faculty of Science, Department of Zoology and Entomology, 2021
- Full Text:
- Authors: Taylor, David Graham
- Date: 2021-04
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Codling moth , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/176942 , vital:42774
- Description: Baculoviruses are an environmentally friendly and effective agent for managing lepidopteran pests. This includes the management of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a serious pest of citrus in Southern Africa and a major threat to the South African citrus export industry. For more than 15 years, CrleGV-SA- based biopesticides have been used as part of an integrated pest management strategy for the control of T. leucotreta in citrus orchards in South Africa, under the names Cryptogran™ and Cryptex®. While these biopesticides have been effective during this period, there are some areas in which baculovirus use could potentially be improved. Baculoviruses are notoriously slow to kill in comparison to chemical-based pesticides, and lately, pest resistance to baculoviruses has become a major concern with the development of resistance by Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) to its granulovirus occurring in the field in Europe. The consistent use of CrleGV-SA for more than 15 years in the field has raised concern that T. leucotreta could develop resistance to this virus, and has made it necessary to alter baculovirus-based management strategies to prevent this from occurring. A second baculovirus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), has recently been isolated and was shown to be effective against T. leucotreta. However, the interactions between CrleGV-SA and CrpeNPV are not yet understood and so it is important to test these interactions before both viruses are applied on the same orchards. Not only is it important to know whether these viruses could negatively impact each other, but it is also important to test whether they could interact synergistically. A synergistic interaction could not only provide a potential tool for the management of resistance, but it could also be exploited to improve baculovirus-based management of T. leucotreta. In this study, a stock of CrleGV-SA was purified by glycerol gradient centrifugation from T. leucotreta cadavers, while a stock of CrpeNPV purified from Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) cadavers was provided by River Bioscience (Pty) Ltd. These stocks were screened for purity by a multiplex polymerase chain reaction (mPCR) protocol designed to detect CrleGV-SA and CrpeNPV. The occlusion body (OB) density was then calculated using darkfield microscopy and a counting chamber. Both stocks were shown to be pure within the limits of the mPCR protocol, and the CrleGV-SA and CrpeNPV stocks were calculated to contain 3.08 × 1011 OBs/mL and 1.92 × 1011 OBs/mL respectively The first aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the dose mortality, in terms of lethal concentration. This was calculated using 7-day surface-dose biological assays for each virus and a 1:1 mixture of OBs of the two against T. leucotreta neonates. The lethal concentrations of each treatment required to kill 50 % of larvae (LC50) and 90 % of larvae (LC90) for each treatment were then calculated and compared using a probit regression. The mixed infection performed significantly better than either virus by itself, while each virus by itself did not differ significantly from the other. The LC50 for CrleGV-SA, CrpeNPV and the mixed infection were 1.53 × 104 OBs/mL, 1.15 × 104 OBs/mL and 4.38 × 103 OBs/mL respectively. The LC90 of CrleGV-SA, CrpeNPV and the mixed infection were calculated to be 4.10 × 105 OBs/mL, 1.05 × 105 OBs/mL, and 4.09 × 104 OBs/mL respectively. The second aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the speed of kill. A time-response biological assay protocol was created that allowed for effective observation of the larvae. This was then used to generate time-mortality data that were analysed by a logit regression function to calculate and compare the treatments at the time of 50 % larval mortality (LT50) and the time of 90 % mortality (LT90). Each virus by itself did not differ significantly from the other, while the mixed infection took significantly longer to kill 50 % and 90 % of the larvae, suggesting that there is competition for resources between viruses during the secondary, systemic phase of infection. The LT50 for CrleGV-SA, CrpeNPV and the mixed infection were 117.5 hours, 113.5 hours and 139.0 hours respectively. The LT90 for CrleGV-SA, CrpeNPV and the mixed infection were 153.2 hours, 159.3, and 193.4 hours respectively. Finally, the composition of OBs recovered from the cadavers produced by the time-response biological assays were investigated by mPCR. A method for extracting gDNA from OBs in neonate-sized T. leucotreta larvae is described. The presence of CrpeNPV along with CrleGV-SA was noted in 4 out of 9 larvae inoculated with only CrleGV-SA. The presence of CrleGV-SA as well as CrpeNPV was noted in all but one larva inoculated with only CrpeNPV, and both CrleGV-SA and CrpeNPV were noted in all but one larva inoculated with a 1:1 mixture of the two, with one larva only being positive for CrleGV-SA. This suggests either stock contamination or the presence of covert infections of CrleGV-SA and CrpeNPV in the T. leucotreta population used in this study. This is the second study to report an improved lethal concentration of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates, and the first study to report the slower speed of kill of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates. While the improved lethal concentration of the mixed infection is a promising step in the future improvement of baculovirus-based biopesticides, it is at the cost of a slower speed of kill. , Thesis (MSc) -- Faculty of Science, Department of Zoology and Entomology, 2021
- Full Text:
Selection for improved virulence of Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) to False Codling Moth, Thaumatotibia leucotreta, by serial passage through a heterologous host
- Authors: Iita, Petrus Paulus
- Date: 2021-04
- Subjects: Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Baculoviruses , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/178180 , vital:42918
- Description: The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is endemic to southern Africa, and strongly associated with citrus. As South African citrus production is mainly for export to foreign markets, the market access risk due to the phytosanitary status of this pest is considerable and its control is therefore imperative. Various control measures as part of a rigorous integrated pest management (IPM) programme targeted against T. leucotreta have been effective at suppressing the pest in citrus, but there is still a growing need for continued improvement of the programme and augmentation of the available control options. Of these control options, biological control, particularly the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA), is a key component of IPM in citrus orchards and it has been very successful at reducing T. leucotreta populations in the field for almost two decades. There is however, a growing need for more baculovirus variants with an improved virulence against T. leucotreta for a more efficient pest management system. The newly identified insect virus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) offers a unique opportunity for an additional biopesticide in IPM for control of T. leucotreta in the field. This study aimed to conduct serial passaging of CrpeNPV through a heterologous host, T. leucotreta, in order to determine the potential for improved virulence or speed of kill against it. In order to select for a variant of CrpeNPV with improved virulence against T. leucotreta, a high dose (LC90) of the virus OBs was used to perform 12 serial passages through T. leucotreta larvae in surface-dose bioassays. Whole genome sequencing and analysis of the passaged virus, along with restriction endonuclease profiling in silico was performed to determine if the genetic identity of the virus had changed during serial passage, in relation to the original virus. These analyses indicated that the dominant genotype of CrpeNPV was maintained following 12 serial passages through the heterologous host. The biological activity of the passaged virus, along with the original virus was evaluated against neonate T. leucotreta in surface-dose bioassays and compared. Results from dose-response bioassays showed that the virulence of CrpeNPV did not improve after 12 serial passages. The LC50 values of the passaged virus and the original virus were estimated at 1.96 × 104 and 1.58 × 104 OBs/ml, respectively, whereas the LC90 values were estimated at 3.46 × 104 OBs/ml for the passaged virus and 3.68 × 104 for the original virus. Similarly, the results from time-response bioassays showed that the speed of kill of CrpeNPV did not improve after 12 serial passages. The LT50 values of the passaged virus and the original virus were 88.44 hours (3 days and 16 hours) and 83.74 hours (3 days and 12 hours), respectively, whereas the LT90 values were 115 hours (4 days 19 hours) for the passaged virus and 102 hours (4 days 6 hours) for the original virus. The virulence and speed of kill of the passaged virus decreased significantly, in relation to the original virus. When the full genome of the passaged virus was sequenced and analysed, only a few SNPs were detected in the viral genome, in comparison to the original virus. No detectable difference in REN digestion patterns were observed following REN analysis of gDNA of the passaged virus with several restriction enzymes in silico. The results for this study suggest that CrpeNPV may already be optimally suited to the heterologous host as it persists under these conditions without significant changes to the genome. These results have positive implications for the genetic integrity of CrpeNPV as a potential biocontrol agent in the field. This study is the first to report the virulence selection of CrpeNPV by serial passage through a heterologous host, and also the first to record bioassay data in terms of dose response (or lethal concentration) against T. leucotreta second instars. The data obtained have added to the knowledge about interactions between CrpeNPV and its heterologous host, and may be fundamental to continued investigation into the effect of serial passage on pathogenicity and genetic diversity of CrpeNPV. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Authors: Iita, Petrus Paulus
- Date: 2021-04
- Subjects: Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Baculoviruses , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/178180 , vital:42918
- Description: The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is endemic to southern Africa, and strongly associated with citrus. As South African citrus production is mainly for export to foreign markets, the market access risk due to the phytosanitary status of this pest is considerable and its control is therefore imperative. Various control measures as part of a rigorous integrated pest management (IPM) programme targeted against T. leucotreta have been effective at suppressing the pest in citrus, but there is still a growing need for continued improvement of the programme and augmentation of the available control options. Of these control options, biological control, particularly the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA), is a key component of IPM in citrus orchards and it has been very successful at reducing T. leucotreta populations in the field for almost two decades. There is however, a growing need for more baculovirus variants with an improved virulence against T. leucotreta for a more efficient pest management system. The newly identified insect virus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) offers a unique opportunity for an additional biopesticide in IPM for control of T. leucotreta in the field. This study aimed to conduct serial passaging of CrpeNPV through a heterologous host, T. leucotreta, in order to determine the potential for improved virulence or speed of kill against it. In order to select for a variant of CrpeNPV with improved virulence against T. leucotreta, a high dose (LC90) of the virus OBs was used to perform 12 serial passages through T. leucotreta larvae in surface-dose bioassays. Whole genome sequencing and analysis of the passaged virus, along with restriction endonuclease profiling in silico was performed to determine if the genetic identity of the virus had changed during serial passage, in relation to the original virus. These analyses indicated that the dominant genotype of CrpeNPV was maintained following 12 serial passages through the heterologous host. The biological activity of the passaged virus, along with the original virus was evaluated against neonate T. leucotreta in surface-dose bioassays and compared. Results from dose-response bioassays showed that the virulence of CrpeNPV did not improve after 12 serial passages. The LC50 values of the passaged virus and the original virus were estimated at 1.96 × 104 and 1.58 × 104 OBs/ml, respectively, whereas the LC90 values were estimated at 3.46 × 104 OBs/ml for the passaged virus and 3.68 × 104 for the original virus. Similarly, the results from time-response bioassays showed that the speed of kill of CrpeNPV did not improve after 12 serial passages. The LT50 values of the passaged virus and the original virus were 88.44 hours (3 days and 16 hours) and 83.74 hours (3 days and 12 hours), respectively, whereas the LT90 values were 115 hours (4 days 19 hours) for the passaged virus and 102 hours (4 days 6 hours) for the original virus. The virulence and speed of kill of the passaged virus decreased significantly, in relation to the original virus. When the full genome of the passaged virus was sequenced and analysed, only a few SNPs were detected in the viral genome, in comparison to the original virus. No detectable difference in REN digestion patterns were observed following REN analysis of gDNA of the passaged virus with several restriction enzymes in silico. The results for this study suggest that CrpeNPV may already be optimally suited to the heterologous host as it persists under these conditions without significant changes to the genome. These results have positive implications for the genetic integrity of CrpeNPV as a potential biocontrol agent in the field. This study is the first to report the virulence selection of CrpeNPV by serial passage through a heterologous host, and also the first to record bioassay data in terms of dose response (or lethal concentration) against T. leucotreta second instars. The data obtained have added to the knowledge about interactions between CrpeNPV and its heterologous host, and may be fundamental to continued investigation into the effect of serial passage on pathogenicity and genetic diversity of CrpeNPV. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
Post-release evaluation of Megamelus scutellaris Berg. (hemiptera: delphacidae): a biological control agent of water hyacinth Eichhornia crassipes (Mart.) Solms-Laub (Pontederiaceae) in South Africa
- Authors: Miller, Benjamin Erich
- Date: 2019
- Subjects: Megamelus scutellaris Berg. , Delphacidae , Noxious weeds -- Biological control -- South Africa , Aquatic weeds -- Biological control -- South Africa , Water hyacinth -- Biological control -- South Africa , Biological pest control agents
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/92330 , vital:30710
- Description: Water hyacinth, Eichhornia crassipes (Mart.) Solms-Laub. (Pontederiaceae) is a free-floating aquatic macrophyte from South America that was introduced to South Africa in the 1900s for its attractive ornamental flowers. The plant was classified as a serious invader in the country in the 1970s, eventually becoming the worst invasive aquatic plant in South Africa. Biological control is widely regarded as the most effective method of managing water hyacinth, as it is ecologically safe, cost-effective, and self-sustaining. To date, nine biological control agents have been released in South Africa against water hyacinth, including eight arthropods and a pathogen. Due to the cumulative effects of highly eutrophic waterbodies, which mitigate the damage caused by biological control, and the cold winters which inhibit the rate of biological control agent population build up, South Africa currently has more biological control agents released on water hyacinth than anywhere else in the world. The need for a cold-tolerant agent that can reproduce and develop quickly, while still being damaging to water hyacinth in eutrophic systems, led to the introduction of the most recently released water hyacinth biological control agent, the planthopper Megamelus scutellaris Berg (Hemiptera: Delphacidae), which was initially collected from Argentina. This thesis formed the first post-release evaluation of M. scutellaris since its release in South Africa in 2013. It included a greenhouse experiment to measure the agent’s feeding damage in relation to different nutrient levels and stocking rates, as well as a field component to evaluate both the post-winter recovery of M. scutellaris, and a nationwide survey to measure the establishment of the agent around the country in relation to climate, water quality, and plant health. In the greenhouse experiment, the feeding damage was quantified using measurements of plant growth parameters and chlorophyll fluorometry. It was found that, like other biological control agents of water hyacinth, M. scutellaris was most damaging when released in high numbers on plants grown at medium nutrient levels, and less effective on plants grown at elevated nutrient levels. A water hyacinth infestation on the Kubusi River was selected for the evaluation of the post-winter recovery of M. scutellaris. The Kubusi River is both the first site where M. scutellaris was released, and the coldest site where water hyacinth biological control agents have established successfully in South Africa. Monthly visits tracking seasonal plant health characteristics and agent population densities indicated that the populations of M. scutellaris were impacted most significantly by the season. Low temperatures led to the water hyacinth plants being of poor quality during the winter, which had a subsequent negative effect on the agent populations. The agents could only fully recover by late summer, which meant that the plants were without any significant biological control through the initial phases of the growing season, when they were most vulnerable, and a significant lag-phase occurred between the recovery of the plants and the recovery of the agent population after the winter bottleneck. A survey of all sites where M. scutellaris had been released in South Africa yielded 16 sites where the agents had successfully established, having survived at least one full winter. Among these sites were four sites where the agents were found without them having been released, indicating that they can disperse unaided to new sites. The temperature was a major factor responsible for the success or failure of establishment, with very few agents surviving in the hot areas of South Africa or in areas with a high frost incidence. The density of M. scutellaris was higher in nutrient-rich water, and on plants with more leaves, suggesting that the quality of the plants also contributed to establishment. The results of this thesis showed that M. scutellaris is able to establish successfully in South Africa, and that the agents are capable of causing significant damage to water hyacinth, making it a promising addition to the biological control programme. Novel methods of measuring subtle insect feeding damage in plants and quantifying agent populations are also discussed, along with suggestions for the future implementation of M. scutellaris in South Africa.
- Full Text:
- Authors: Miller, Benjamin Erich
- Date: 2019
- Subjects: Megamelus scutellaris Berg. , Delphacidae , Noxious weeds -- Biological control -- South Africa , Aquatic weeds -- Biological control -- South Africa , Water hyacinth -- Biological control -- South Africa , Biological pest control agents
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/92330 , vital:30710
- Description: Water hyacinth, Eichhornia crassipes (Mart.) Solms-Laub. (Pontederiaceae) is a free-floating aquatic macrophyte from South America that was introduced to South Africa in the 1900s for its attractive ornamental flowers. The plant was classified as a serious invader in the country in the 1970s, eventually becoming the worst invasive aquatic plant in South Africa. Biological control is widely regarded as the most effective method of managing water hyacinth, as it is ecologically safe, cost-effective, and self-sustaining. To date, nine biological control agents have been released in South Africa against water hyacinth, including eight arthropods and a pathogen. Due to the cumulative effects of highly eutrophic waterbodies, which mitigate the damage caused by biological control, and the cold winters which inhibit the rate of biological control agent population build up, South Africa currently has more biological control agents released on water hyacinth than anywhere else in the world. The need for a cold-tolerant agent that can reproduce and develop quickly, while still being damaging to water hyacinth in eutrophic systems, led to the introduction of the most recently released water hyacinth biological control agent, the planthopper Megamelus scutellaris Berg (Hemiptera: Delphacidae), which was initially collected from Argentina. This thesis formed the first post-release evaluation of M. scutellaris since its release in South Africa in 2013. It included a greenhouse experiment to measure the agent’s feeding damage in relation to different nutrient levels and stocking rates, as well as a field component to evaluate both the post-winter recovery of M. scutellaris, and a nationwide survey to measure the establishment of the agent around the country in relation to climate, water quality, and plant health. In the greenhouse experiment, the feeding damage was quantified using measurements of plant growth parameters and chlorophyll fluorometry. It was found that, like other biological control agents of water hyacinth, M. scutellaris was most damaging when released in high numbers on plants grown at medium nutrient levels, and less effective on plants grown at elevated nutrient levels. A water hyacinth infestation on the Kubusi River was selected for the evaluation of the post-winter recovery of M. scutellaris. The Kubusi River is both the first site where M. scutellaris was released, and the coldest site where water hyacinth biological control agents have established successfully in South Africa. Monthly visits tracking seasonal plant health characteristics and agent population densities indicated that the populations of M. scutellaris were impacted most significantly by the season. Low temperatures led to the water hyacinth plants being of poor quality during the winter, which had a subsequent negative effect on the agent populations. The agents could only fully recover by late summer, which meant that the plants were without any significant biological control through the initial phases of the growing season, when they were most vulnerable, and a significant lag-phase occurred between the recovery of the plants and the recovery of the agent population after the winter bottleneck. A survey of all sites where M. scutellaris had been released in South Africa yielded 16 sites where the agents had successfully established, having survived at least one full winter. Among these sites were four sites where the agents were found without them having been released, indicating that they can disperse unaided to new sites. The temperature was a major factor responsible for the success or failure of establishment, with very few agents surviving in the hot areas of South Africa or in areas with a high frost incidence. The density of M. scutellaris was higher in nutrient-rich water, and on plants with more leaves, suggesting that the quality of the plants also contributed to establishment. The results of this thesis showed that M. scutellaris is able to establish successfully in South Africa, and that the agents are capable of causing significant damage to water hyacinth, making it a promising addition to the biological control programme. Novel methods of measuring subtle insect feeding damage in plants and quantifying agent populations are also discussed, along with suggestions for the future implementation of M. scutellaris in South Africa.
- Full Text:
The biology, behaviour and survival of pupating false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a citrus pest in South Africa
- Authors: Love, Claire Natalie
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- South Africa , Cryptophlebia leucotreta -- Larvae -- Behavior , Citrus -- Diseases and pests , Citrus -- Diseases and pests -- Biological control , Biological pest control agents , Entomopathogenic fungi , Insect nematodes , Pupae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5941 , http://hdl.handle.net/10962/d1018907
- Description: Control of the citrus pest, false codling moth (FCM), Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is crucial for the South African citrus industry. The economic losses and phytosanitary status of this pest, coupled with increased consumer awareness and demands, has created a need for effective, IPM-compatible control measures for use against the soil-dwelling life stages of FCM. Promising developments in the field of microbial control through the use of entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPNs) have highlighted the need for research regarding pupation biology, behaviour and survival of FCM, as a good understanding of biology of the target organism is an important component of any biological control programme. The aim of this study was to improve the current understanding of FCM pupation habits through the manipulation of soil texture class, ground cover, shading, soil compaction, air temperature, and soil moisture in the laboratory. These findings would then be used to aid the biological control programmes using EPF and EPNs against FCM in the soil. Three soil texture classes (sandy loam, silt loam and silty clay loam) were obtained from orchards for use in the study. FCM larvae were allowed to drop into the soil of their own accord and the pupation behaviour that followed was then captured on film with pupae formed in the soil being kept in order to measure adult eclosion. In general, very few abiotic factors had a clear influence on FCM pupation. Larval wandering time and distance was short, but also variable between individuals. Distance did increase when soils were moist. Pupation depth was shallow, with pupal cocoons generally being formed on the soil surface. Depth of pupation was less than one centimetre for all abiotic conditions, with little burrowing into soil. Eclosion success was higher for sandier soils when these were dry and uncompacted, but the addition of both moisture and soil compaction increased FCM eclosion success. FCM was sensitive to desiccation when the soils were dry and temperature limits of 15 °C and 32 °C had a strongly negative impact on eclosion success. Preferences for particular abiotic conditions were limited to only certain moisture conditions when interacting with soil texture class and a preference for pupating in soil when it is available. Limited preference was found for particular soil textures despite this having a strong influence on eclosion success, but individuals did appear to pupate in close proximity to one another. Viable direct habitat manipulation for FCM control could not be identified. These results and all of the abiotic variables measured have important implications for EPF and EPN application, survival and persistence in the soil in order to improve the ability of these biological control agents to control FCM. These are discussed in each chapter.
- Full Text:
- Authors: Love, Claire Natalie
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- South Africa , Cryptophlebia leucotreta -- Larvae -- Behavior , Citrus -- Diseases and pests , Citrus -- Diseases and pests -- Biological control , Biological pest control agents , Entomopathogenic fungi , Insect nematodes , Pupae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5941 , http://hdl.handle.net/10962/d1018907
- Description: Control of the citrus pest, false codling moth (FCM), Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is crucial for the South African citrus industry. The economic losses and phytosanitary status of this pest, coupled with increased consumer awareness and demands, has created a need for effective, IPM-compatible control measures for use against the soil-dwelling life stages of FCM. Promising developments in the field of microbial control through the use of entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPNs) have highlighted the need for research regarding pupation biology, behaviour and survival of FCM, as a good understanding of biology of the target organism is an important component of any biological control programme. The aim of this study was to improve the current understanding of FCM pupation habits through the manipulation of soil texture class, ground cover, shading, soil compaction, air temperature, and soil moisture in the laboratory. These findings would then be used to aid the biological control programmes using EPF and EPNs against FCM in the soil. Three soil texture classes (sandy loam, silt loam and silty clay loam) were obtained from orchards for use in the study. FCM larvae were allowed to drop into the soil of their own accord and the pupation behaviour that followed was then captured on film with pupae formed in the soil being kept in order to measure adult eclosion. In general, very few abiotic factors had a clear influence on FCM pupation. Larval wandering time and distance was short, but also variable between individuals. Distance did increase when soils were moist. Pupation depth was shallow, with pupal cocoons generally being formed on the soil surface. Depth of pupation was less than one centimetre for all abiotic conditions, with little burrowing into soil. Eclosion success was higher for sandier soils when these were dry and uncompacted, but the addition of both moisture and soil compaction increased FCM eclosion success. FCM was sensitive to desiccation when the soils were dry and temperature limits of 15 °C and 32 °C had a strongly negative impact on eclosion success. Preferences for particular abiotic conditions were limited to only certain moisture conditions when interacting with soil texture class and a preference for pupating in soil when it is available. Limited preference was found for particular soil textures despite this having a strong influence on eclosion success, but individuals did appear to pupate in close proximity to one another. Viable direct habitat manipulation for FCM control could not be identified. These results and all of the abiotic variables measured have important implications for EPF and EPN application, survival and persistence in the soil in order to improve the ability of these biological control agents to control FCM. These are discussed in each chapter.
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Screening of entomopathogenic fungi against citrus mealybug (Planococcus citri (Risso)) and citrus thrips (Scirtothrips aurantii (Faure))
- FitzGerald, Véronique Chartier
- Authors: FitzGerald, Véronique Chartier
- Date: 2014
- Subjects: Entomopathogenic fungi , Citrus mealybug -- South Africa -- Eastern Cape , Citrus thrips -- South Africa -- Eastern Cape , Citrus -- Diseases and pests , Citrus mealybug -- Biological control , Citrus thrips -- Biological control , Biological pest control agents , Scanning electron microscopy , Mycoses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4166 , http://hdl.handle.net/10962/d1020887
- Description: Mealybugs (Planococcus citri) and thrips (Scirtothrips aurantii) are common and extremely damaging citrus crop pests which have proven difficult to control via conventional methods, such as chemical pesticides and insect growth regulators. The objective of this study was to determine the efficacy of entomopathogenic fungi against these pests in laboratory bioassays. Isolates of Metarhizium anisopliae and Beauveria bassiana from citrus orchards in the Eastern Cape, South Africa were maintained on Sabouraud Dextrose 4% Agar supplemented with Dodine, chloramphenicol and rifampicin at 25°C. Infectivity of the fungal isolates was initially assessed using 5th instar false codling moth, Thaumatotibia leucotreta, larvae. Mealybug bioassays were performed in 24 well plates using 1 x 107 ml-1 conidial suspensions and kept at 26°C for 5 days with a photoperiod of 12 L:12 D. A Beauveria commercial product and an un-inoculated control were also screened for comparison. Isolates GAR 17 B3 (B. bassiana) and FCM AR 23 B3 (M. anisopliae) both resulted in 67.5% mealybug crawler mortality and GB AR 23 13 3 (B. bassiana) resulted in 64% crawler mortality. These 3 isolates were further tested in dose-dependent assays. Probit analyses were conducted on the dose-dependent assays data using PROBAN to determine LC₅₀ values. For both the mealybug adult and crawlers FCM AR 23 B3 required the lowest concentration to achieve LC₅₀ at 4.96 x 10⁶ conidia ml-1 and 5.29 x 10⁵ conidia ml-1, respectively. Bioassays on adult thrips were conducted in munger cells with leaf buds inoculated with the conidial suspensions. Isolate GAR 17 B3 had the highest mortality rate at 70% on thrips while FCM AR 23 B3 resulted in 60% mortality. Identification of the isolates, FCM AR 23 B3, GAR 17 B3 and GB AR 23 13 3, were confirmed to be correct using both microscopic and molecularly techniques. ITS sequences were compared to other sequences from GenBank and confirmed phylogenetically using MEGA6. Mealybug infection was investigated using scanning electron microscopy, mycosis was confirmed but the infection process could not be followed due to the extensive waxy cuticle. These results indicate that there is potential for the isolates FCM AR 23 B3 and GAR 17 B3 to be developed as biological control agents for the control of citrus mealybug and thrips. Further research would be required to determine their ability to perform under field conditions.
- Full Text:
- Authors: FitzGerald, Véronique Chartier
- Date: 2014
- Subjects: Entomopathogenic fungi , Citrus mealybug -- South Africa -- Eastern Cape , Citrus thrips -- South Africa -- Eastern Cape , Citrus -- Diseases and pests , Citrus mealybug -- Biological control , Citrus thrips -- Biological control , Biological pest control agents , Scanning electron microscopy , Mycoses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4166 , http://hdl.handle.net/10962/d1020887
- Description: Mealybugs (Planococcus citri) and thrips (Scirtothrips aurantii) are common and extremely damaging citrus crop pests which have proven difficult to control via conventional methods, such as chemical pesticides and insect growth regulators. The objective of this study was to determine the efficacy of entomopathogenic fungi against these pests in laboratory bioassays. Isolates of Metarhizium anisopliae and Beauveria bassiana from citrus orchards in the Eastern Cape, South Africa were maintained on Sabouraud Dextrose 4% Agar supplemented with Dodine, chloramphenicol and rifampicin at 25°C. Infectivity of the fungal isolates was initially assessed using 5th instar false codling moth, Thaumatotibia leucotreta, larvae. Mealybug bioassays were performed in 24 well plates using 1 x 107 ml-1 conidial suspensions and kept at 26°C for 5 days with a photoperiod of 12 L:12 D. A Beauveria commercial product and an un-inoculated control were also screened for comparison. Isolates GAR 17 B3 (B. bassiana) and FCM AR 23 B3 (M. anisopliae) both resulted in 67.5% mealybug crawler mortality and GB AR 23 13 3 (B. bassiana) resulted in 64% crawler mortality. These 3 isolates were further tested in dose-dependent assays. Probit analyses were conducted on the dose-dependent assays data using PROBAN to determine LC₅₀ values. For both the mealybug adult and crawlers FCM AR 23 B3 required the lowest concentration to achieve LC₅₀ at 4.96 x 10⁶ conidia ml-1 and 5.29 x 10⁵ conidia ml-1, respectively. Bioassays on adult thrips were conducted in munger cells with leaf buds inoculated with the conidial suspensions. Isolate GAR 17 B3 had the highest mortality rate at 70% on thrips while FCM AR 23 B3 resulted in 60% mortality. Identification of the isolates, FCM AR 23 B3, GAR 17 B3 and GB AR 23 13 3, were confirmed to be correct using both microscopic and molecularly techniques. ITS sequences were compared to other sequences from GenBank and confirmed phylogenetically using MEGA6. Mealybug infection was investigated using scanning electron microscopy, mycosis was confirmed but the infection process could not be followed due to the extensive waxy cuticle. These results indicate that there is potential for the isolates FCM AR 23 B3 and GAR 17 B3 to be developed as biological control agents for the control of citrus mealybug and thrips. Further research would be required to determine their ability to perform under field conditions.
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Entomopathogenic fungi for control of soil-borne life stages of false codling moth, Thaumatotibia leucotreta (Meyrick) (1912) (Lepidoptera: Tortricidae)
- Authors: Coombes, Candice Anne
- Date: 2013
- Subjects: Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5607 , http://hdl.handle.net/10962/d1002057 , Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: False codling moth (FCM), Thaumatotibia leucotreta is an extremely important pest of citrus in South Africa and with the shift away from the use of chemicals, alternate control options are needed. One avenue of control which has only recently been investigated against the soil-borne life stages of FCM is the use of entomopathogenic fungi (EPF). In 2009, 12 entomopathogenic fungal isolates collected from South African citrus orchards showed good control potential during laboratory conducted bioassays. The aim of this study was to further analyse the potential of these isolates through concentration-dose and exposure-time response bioassays. After initial re-screening, concentration-dose response and exposure-time response sandconidial bioassays, three isolates were identified as exhibiting the greatest control potential against FCM in soil, Metarhizium anisopliae var. anisopliae (G 11 3 L6 and FCM Ar 23 B3) and Beauveria bassiana (G Ar 17 B3). Percentage mycosis was found to be directly related to fungal concentration as well as the amount of time FCM 5th instar larvae were exposed to the fungal conidia. LC50 values for the three isolates were not greater than 1.92 x 10⁶ conidia.ml⁻ₑ and at the LC₅₀, FCM 5th instar larvae would need to be exposed to the fungus for a maximum of 13 days to ensure a high mortality level. These isolates along with two commercially available EPF products were subjected to field persistence trials whereby net bags filled with a mixture of autoclaved sand and formulated fungal product were buried in an Eastern Cape citrus orchard. The viability of each isolate was measured on a monthly basis for a period of six months. All isolates were capable of persisting in the soil for six months with the collected isolates persisting far better than the commercially used isolates. Two of the isolates, G 11 3 L6 and G Ar 17 B3, were subjected to small scale laboratory application trials. Two formulations were investigated at two concentrations. For each isolate, each formulation and each concentration, FCM 5th instar larvae were applied and allowed to burrow into the soil to pupate before fungal application or after fungal application. Contact between fungi and FCM host is essential as, in contrast to pre-larval treatments, percentage mortality in post-larval treatments was low for both formulations and both isolates. For isolate G Ar 17 B3, a conidial suspension applied as a spray at a concentration of 1 x 10⁷ conidia.ml⁻ₑ obtained the highest percentage mortality (80 %). For isolate G 11 3 L6 however, both formulations performed equally well at a high, 1 x10⁷ conidia.ml⁻ₑ concentration (conidial suspension: 60 %; granular: 65 %) The results obtained thus far are promising for the control of FCM in citrus, but if these EPFs are to successfully integrate into current FCM control practices more research, some of which is discussed, is essential
- Full Text:
- Authors: Coombes, Candice Anne
- Date: 2013
- Subjects: Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5607 , http://hdl.handle.net/10962/d1002057 , Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: False codling moth (FCM), Thaumatotibia leucotreta is an extremely important pest of citrus in South Africa and with the shift away from the use of chemicals, alternate control options are needed. One avenue of control which has only recently been investigated against the soil-borne life stages of FCM is the use of entomopathogenic fungi (EPF). In 2009, 12 entomopathogenic fungal isolates collected from South African citrus orchards showed good control potential during laboratory conducted bioassays. The aim of this study was to further analyse the potential of these isolates through concentration-dose and exposure-time response bioassays. After initial re-screening, concentration-dose response and exposure-time response sandconidial bioassays, three isolates were identified as exhibiting the greatest control potential against FCM in soil, Metarhizium anisopliae var. anisopliae (G 11 3 L6 and FCM Ar 23 B3) and Beauveria bassiana (G Ar 17 B3). Percentage mycosis was found to be directly related to fungal concentration as well as the amount of time FCM 5th instar larvae were exposed to the fungal conidia. LC50 values for the three isolates were not greater than 1.92 x 10⁶ conidia.ml⁻ₑ and at the LC₅₀, FCM 5th instar larvae would need to be exposed to the fungus for a maximum of 13 days to ensure a high mortality level. These isolates along with two commercially available EPF products were subjected to field persistence trials whereby net bags filled with a mixture of autoclaved sand and formulated fungal product were buried in an Eastern Cape citrus orchard. The viability of each isolate was measured on a monthly basis for a period of six months. All isolates were capable of persisting in the soil for six months with the collected isolates persisting far better than the commercially used isolates. Two of the isolates, G 11 3 L6 and G Ar 17 B3, were subjected to small scale laboratory application trials. Two formulations were investigated at two concentrations. For each isolate, each formulation and each concentration, FCM 5th instar larvae were applied and allowed to burrow into the soil to pupate before fungal application or after fungal application. Contact between fungi and FCM host is essential as, in contrast to pre-larval treatments, percentage mortality in post-larval treatments was low for both formulations and both isolates. For isolate G Ar 17 B3, a conidial suspension applied as a spray at a concentration of 1 x 10⁷ conidia.ml⁻ₑ obtained the highest percentage mortality (80 %). For isolate G 11 3 L6 however, both formulations performed equally well at a high, 1 x10⁷ conidia.ml⁻ₑ concentration (conidial suspension: 60 %; granular: 65 %) The results obtained thus far are promising for the control of FCM in citrus, but if these EPFs are to successfully integrate into current FCM control practices more research, some of which is discussed, is essential
- Full Text:
Investigation of entomopathogenic fungi for control of false codling moth, Thaumatotibia leucotrata, Mediterranean fruit fly, Ceratitis capitata and Natal fruit fly, C. rosa in South African citrus
- Authors: Goble, Tarryn Anne
- Date: 2010
- Subjects: Insect pests -- Biological control , Tortricidae -- Biological control -- South Africa , Tephritidae -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5723 , http://hdl.handle.net/10962/d1005409 , Insect pests -- Biological control , Tortricidae -- Biological control -- South Africa , Tephritidae -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: The biology of key citrus pests Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Ceratitis capitata Wiedemann (Diptera: Tephritidae) and Ceratitis rosa Karsch (Diptera: Tephritidae) includes their dropping from host plants to pupate in the soil below citrus trees. Since most EP fungi are soil-borne microorganisms, the development and formulation of alternative control strategies using these fungi as subterranean control agents, targeted at larvae and pupae in the soil, can potentially benefit existing IPM management of citrus in South Africa. Thus, a survey of occurrence of entomopathogenic fungi was undertaken on soils from citrus orchards and natural vegetation (refugia) on conventionally and organically managed farms in the Eastern Cape Province in South Africa. A method for baiting soil samples with citrus pest T. leucotreta and C. capitata larvae, as well as with the standard bait insect, Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), was implemented. Sixty-two potentially useful entomopathogenic fungal isolates belonging to four genera were collected from 288 soil samples, an occurrence frequency of 21.53%. The most frequently isolated entomopathogenic fungal species was Beauveria bassiana (Balsamo) Vuillemin (15.63%), followed by Metarhizium anisopliae var. anisopliae (Metschnikoff) Sorokin (3.82%). Galleria mellonella was the most effective insect used to isolate fungal species (χ2=40.13, df=2, P≤ 0.005), with a total of 45 isolates obtained, followed by C. capitata with 11 isolates, and T. leucotreta with six isolates recovered. There was a significantly (χ2=11.65, df=1, P≤ 0.005) higher occurrence of entomopathogenic fungi in soil samples taken from refugia compared to cultivated orchards of both organically and conventionally managed farms. No significant differences were observed in the recovery of fungal isolates when soil samples from both farming systems were compared. The physiological effects and host range of 21 indigenous fungal isolates obtained in the Eastern Cape were investigated in the laboratory to establish whether these isolates could be effectively used as biological control agents against the subterranean life stages of C. rosa, C. capitata and T. leucotreta. When these pests were treated with a fungal concentration of 1 x 10⁷ conidia ml⁻¹, the percentage of T. leucotreta adults which emerged in fungal treated sand ranged from 5 to 60% (F=33.295; df=21; P=0.0001) depending on fungal isolate and the percentage of pupae with visible signs of mycosis ranged from 21 to 93% (F= 96.436; df=21; P=0.0001). Based on fungal isolates, the percentage adult survival in C. rosa and C. capitata ranged from 30 to 90% and 55 to 86% respectively. The percentage of C. rosa and C. capitata puparia with visible signs of mycosis ranged from 1 to 14% and 1 to 11% respectively. Deferred mortality due to mycosis in C. rosa and C. capitata adult flies ranged from 1 to 58% and 1 to 33% respectively, depending on fungal isolate. Entomopathogenic fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult survival or adult and pupal mycosis were considered. The relative potency of the four most virulent Beauveria isolates as well as the commercially available Beauveria bassiana product, Bb Plus® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against C. rosa, C. capitata and T. leucotreta which all exhibited a dose-dependent response. Against fruit flies the estimated LC50 values of all five Beauveria isolates ranged from 5.5 x 10¹¹ to 2.8 x 10¹² conidia/ml⁻¹. There were no significant differences between the relative potencies of these five fungal isolates. When T. leucotreta was considered, isolates: G Moss R10 and G 14 2 B5 and Bb Plus® were significantly more pathogenic than G B Ar 23 B3 and FCM 10 13 L1. The estimated LC₅₀ values of the three most pathogenic isolates ranged from 6.8 x 10⁵ to 2.1 x 10⁶ conidia/ml⁻¹, while those of the least pathogenic ranged from 1.6 x 10⁷ to 3.7 x 10⁷ conidia/ml⁻¹. Thaumatotibia leucotreta final instar larvae were exposed to two conidial concentrations, at four different exposure times (12, 48, 72 and 96 hrs) and showed an exposure time-dependant relationship (F=5.43; df=3; P=0.001). At 1 x 10⁷conidia/ml⁻¹ two Beauveria isolates: G Moss R10 and G 14 2 B5 were able to elicit a response in 50% of test insects at 72 hrs (3 days) exposure. Although a limited amount of mycosis was observed in the puparia of both fruit fly species, deferred adult mortality due to mycosis was high. The increased incidence of adult mortality suggests that post emergence mycosis in adult fruit flies may play a more significant role in field suppression than the control of fruit flies at the pupal stage. The increased incidence of pupal mortality, as well as the relatively low concentrations of conidia required to elicit meaningful responses in T. leucotreta pupae may suggest that pre-emergent control of false codling moth will play a more significant role in field suppression than the control of adult life stages using indigenous isolates of entomopathogenic fungi. Various entomopathogenic fungal application techniques targeted at key insect pests within integrated pest management (IPM) systems of citrus are discussed.
- Full Text:
- Authors: Goble, Tarryn Anne
- Date: 2010
- Subjects: Insect pests -- Biological control , Tortricidae -- Biological control -- South Africa , Tephritidae -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5723 , http://hdl.handle.net/10962/d1005409 , Insect pests -- Biological control , Tortricidae -- Biological control -- South Africa , Tephritidae -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: The biology of key citrus pests Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Ceratitis capitata Wiedemann (Diptera: Tephritidae) and Ceratitis rosa Karsch (Diptera: Tephritidae) includes their dropping from host plants to pupate in the soil below citrus trees. Since most EP fungi are soil-borne microorganisms, the development and formulation of alternative control strategies using these fungi as subterranean control agents, targeted at larvae and pupae in the soil, can potentially benefit existing IPM management of citrus in South Africa. Thus, a survey of occurrence of entomopathogenic fungi was undertaken on soils from citrus orchards and natural vegetation (refugia) on conventionally and organically managed farms in the Eastern Cape Province in South Africa. A method for baiting soil samples with citrus pest T. leucotreta and C. capitata larvae, as well as with the standard bait insect, Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), was implemented. Sixty-two potentially useful entomopathogenic fungal isolates belonging to four genera were collected from 288 soil samples, an occurrence frequency of 21.53%. The most frequently isolated entomopathogenic fungal species was Beauveria bassiana (Balsamo) Vuillemin (15.63%), followed by Metarhizium anisopliae var. anisopliae (Metschnikoff) Sorokin (3.82%). Galleria mellonella was the most effective insect used to isolate fungal species (χ2=40.13, df=2, P≤ 0.005), with a total of 45 isolates obtained, followed by C. capitata with 11 isolates, and T. leucotreta with six isolates recovered. There was a significantly (χ2=11.65, df=1, P≤ 0.005) higher occurrence of entomopathogenic fungi in soil samples taken from refugia compared to cultivated orchards of both organically and conventionally managed farms. No significant differences were observed in the recovery of fungal isolates when soil samples from both farming systems were compared. The physiological effects and host range of 21 indigenous fungal isolates obtained in the Eastern Cape were investigated in the laboratory to establish whether these isolates could be effectively used as biological control agents against the subterranean life stages of C. rosa, C. capitata and T. leucotreta. When these pests were treated with a fungal concentration of 1 x 10⁷ conidia ml⁻¹, the percentage of T. leucotreta adults which emerged in fungal treated sand ranged from 5 to 60% (F=33.295; df=21; P=0.0001) depending on fungal isolate and the percentage of pupae with visible signs of mycosis ranged from 21 to 93% (F= 96.436; df=21; P=0.0001). Based on fungal isolates, the percentage adult survival in C. rosa and C. capitata ranged from 30 to 90% and 55 to 86% respectively. The percentage of C. rosa and C. capitata puparia with visible signs of mycosis ranged from 1 to 14% and 1 to 11% respectively. Deferred mortality due to mycosis in C. rosa and C. capitata adult flies ranged from 1 to 58% and 1 to 33% respectively, depending on fungal isolate. Entomopathogenic fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult survival or adult and pupal mycosis were considered. The relative potency of the four most virulent Beauveria isolates as well as the commercially available Beauveria bassiana product, Bb Plus® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against C. rosa, C. capitata and T. leucotreta which all exhibited a dose-dependent response. Against fruit flies the estimated LC50 values of all five Beauveria isolates ranged from 5.5 x 10¹¹ to 2.8 x 10¹² conidia/ml⁻¹. There were no significant differences between the relative potencies of these five fungal isolates. When T. leucotreta was considered, isolates: G Moss R10 and G 14 2 B5 and Bb Plus® were significantly more pathogenic than G B Ar 23 B3 and FCM 10 13 L1. The estimated LC₅₀ values of the three most pathogenic isolates ranged from 6.8 x 10⁵ to 2.1 x 10⁶ conidia/ml⁻¹, while those of the least pathogenic ranged from 1.6 x 10⁷ to 3.7 x 10⁷ conidia/ml⁻¹. Thaumatotibia leucotreta final instar larvae were exposed to two conidial concentrations, at four different exposure times (12, 48, 72 and 96 hrs) and showed an exposure time-dependant relationship (F=5.43; df=3; P=0.001). At 1 x 10⁷conidia/ml⁻¹ two Beauveria isolates: G Moss R10 and G 14 2 B5 were able to elicit a response in 50% of test insects at 72 hrs (3 days) exposure. Although a limited amount of mycosis was observed in the puparia of both fruit fly species, deferred adult mortality due to mycosis was high. The increased incidence of adult mortality suggests that post emergence mycosis in adult fruit flies may play a more significant role in field suppression than the control of fruit flies at the pupal stage. The increased incidence of pupal mortality, as well as the relatively low concentrations of conidia required to elicit meaningful responses in T. leucotreta pupae may suggest that pre-emergent control of false codling moth will play a more significant role in field suppression than the control of adult life stages using indigenous isolates of entomopathogenic fungi. Various entomopathogenic fungal application techniques targeted at key insect pests within integrated pest management (IPM) systems of citrus are discussed.
- Full Text:
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