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
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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
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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.
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Reproductive isolation mechanisms of two cryptic species of Eccritotarsus (Hemiptera: Miridae), biological control agents of water hyacinth, Eichhornia crassipes (Martius) Solms-Laubach (Pontederiaceae)
- Authors: Mnguni, Sandiso
- Date: 2019
- Subjects: Eccritotarsus , Meridae , 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/10962/68133 , vital:29202
- Description: Water hyacinth, Eichhornia crassipes (Martius) Solms-Laubach (Pontederiaceae), is one of the world’s worst alien invasive plants. It is indigenous to the Amazon basin in South America but has become a problematic alien invasive in other parts of the world. As such, several host-specific biological control agents have been sourced from the native distributions in South America and have been released to control this plant where it has become problematic. Two of these agents include the geographically and reproductively isolated cryptic species of Eccritotarsus (Hemiptera: Miridae). One of these species was collected in the upper reaches of the Amazon River in Peru, while the other was collected over 3500km away from that site, in Florianopolis, southern Brazil. These cryptic species were thought to be a single species until recently, when DNA barcoding indicated that they were likely to be two species, and the species status has now been confirmed by interbreeding experiments and detailed morphological studies. The Brazilian population remains Eccritotarsus catarinensis (Carvalho), while the Peruvian population is now known as Eccritotarsus eichhorniae (Henry). The aim of this project was to investigate the mating behaviour and other behavioural traits of the two species that have resulted in reproductive isolation, and which could have led to speciation. In addition, investigations involving analysis of chemical compound compositions of the two species aimed to determine the extent to which the compounds played a role in the development and maintenance of reproductive isolation. To achieve the aims, behavioural-observation experiments were conducted in the form of no-choice, bi-choice and multi-choice tests in 1:1, 2:1 and 3:1 sex ratio assessments, both within and between species. Chemical compound compositions of E. catarinensis and E. eichhorniae were also assessed using Nuclear Magnetic Resonance (NMR), Solid-phase micro-extraction (SPME) and Gas-Chromatography Mass-Spectrometry (GC-MS) techniques. In no-choice experiments, the highest number of single and multiple copula incidences, and average total copula duration was found within species while copulation between species was much rarer. In bi-choice experiments, E. eichhorniae females and E. catarinensis males only chose to mate with their respective conspecifics, and within species copulations continued to have higher average total copula duration. In multi-choice experiments, the highest number of single and multiple copula incidences and average total copula duration was also found within species. GC-MS analysis suggested that E. catarinensis females and E. eichhorniae males have unique chemical compounds missing in their conspecifics and same sex of the other species. Further analysis suggested that E. catarinensis females and E. eichhorniae males have similar chemical compound compositions, whereas as E. eichhorniae females and E. catarinensis males have similar chemical compound compositions. These results suggest that there are behavioural differences that led to the development and maintenance of prezygotic reproductive isolation mechanisms, and that this is probably driven by pheromones in chemical compound compositions. These two species were geographically isolated in the native range and the populations have diverged to the point that they are now reproductively incompatible and therefore, distinct species. The main driver of the speciation is most likely mate recognition and attraction, as only reproductively important traits such as pheromones, genitalia, the scent glands and antennae have changed, while other traits, including host range and morphology, have remained remarkably stable. This provides evidence that differences in sexual selection in isolated populations may be important drivers of speciation and reproductive isolation in cryptic species.
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Potential impact and host range of Pereskiophaga brasiliensis Anderson (Curculionidae): a new candidate biological control agent for the control of Pereskia aculeata Miller (Cactaceae) in South Africa
- Authors: Mdodana, Lumka Anita
- Date: 2018
- Subjects: Curculionidae -- South Africa , Cactus -- South Africa , Biological pest control agents , Alien plants-- South Africa , Pereskiophaga brasiliensis Anderson (Curculionidae) , Pereskia aculeata Miller (Cactaceae)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62350 , vital:28157
- Description: Pereskia aculeata Miller (Cactaceae) is a damaging invasive alien plant in South Africa that has negative impacts to indigenous biodiversity and ecosystem functioning. Mechanical and chemical control are not effective against P. aculeata so biological control is considered the only viable option. Two biological control agents, the leaf-feeding beetle Phenrica guerini Bechyne (Chrysomelidae) and the stem-wilting bug Catorhintha schaffneri (Coreidae), have been released in South Africa thus far. Post-release evaluations have indicated that P. guerini will not reduce P. aculeata densities to acceptable levels alone, while C. schaffneri was released very recently, so it is too soon to determine how effective that agent will be. Even if C. schaffneri is extremely damaging, it is likely that further agents will be required to reduce the densities of P. aculeata to acceptable levels within a reasonable time-scale. Additional agents should target the woody stems of P. aculeata which are not impacted by the damage of either of the released agents. Pereskiophaga brasiliensis Anderson (Curculionidae) is a promising potential candidate agent that feeds on the thick woody stems of the plant in the larval stage. Climatic matching, genetic matching and field based host specificity observations all indicated that P. brasiliensis was a promising candidate. In this study, the impact of P. brasiliensis to the target weed, P. aculeata, was quantified under quarantine conditions to determine whether it was sufficiently damaging to warrant release. This was followed by host specificity testing to determine whether P. brasiliensis was suitably host specific for release in South Africa. Impact studies indicated that P. brasiliensis was damaging to P. aculeata at insect densities that would be expected in the field. Pereskiophaga brasiliensis reduced the number of leaves of P. aculeata to a greater extent than it reduced shoot lengths, but both plant parameters were significantly reduced due to the feeding damage from the insect. This suggests that the damage from P. brasiliensis may be compatible with that of C. schaffneri which reduces shoot length to a greater degree than the number of leaves. Pereskiophaga brasiliensis is therefore sufficiently damaging to warrant release, and although interaction studies with the other agents would be required, it is expected that it should complement other existing agents. Although P. brasiliensis is sufficiently damaging, at present the host specificity data indicates that it is not suitably specific for release in South Africa because oviposition and larval development to the adult stage was recorded on both indigenous and alien plant species within the families Cactaceae and Basellaceae. This non-target feeding was recorded during no-choice tests, which are very conservative, but significant non-target damage and development to the adult stage was recorded on an indigenous plant from a different family to the target weed. Further host specificity testing, including paired and multiple choice tests, are required to confirm the broad host range of P. brasiliensis. Other biological control agents that damage the woody stems of P. aculeata should be considered. The stem-borer, Acanthodoxus machacalis (Cerambycidae) is considered the most promising of the other candidate agents as it can be sourced from a climatically matched region where genetically suitable P. aculeata plants are found, it is sufficiently damaging to the woody stems of P. aculeata and there is no evidence that the species has a broad host range. Acanthodoxus machacalis should be sourced from Rio de Janeiro, Brazil, and imported into quarantine in South Africa for host specificity testing.
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