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Evaluating baculovirus mixtures against false codling moth Thaumatotibia leucotreta Meyrick. (Lepidoptera: Tortricidae)

  • Authors: Tole, Siviwe
  • Date: 2024-10-11
  • Subjects: False codling moth Biological control , Baculoviruses , Integrated pest management , Natural pesticides , Granulovirus
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/463996 , vital:76464
  • Description: False codling moth (FCM), Thaumatotibia leucotreta, is an important pest of citrus, stone fruit, avocados, peppers, and other important agricultural crops in southern Africa. Baculovirus-based biopesticides are components in an integrated pest management (IPM) programme to manage the pest in the field. Cryptogran™ and Cryptex™ which are CrleGV-SA based-biopesticides have been effective in the control of T. leucotreta for the past 15 years. Recently, CrpeNPV-based Multimax™ and Codlmax™ have been commercialised to control T. leucotreta and other important agricultural pests. Despite these viruses being relatively host-specific and safe to humans and animals in comparison to chemical insecticides, their application is hindered by their slow speed of kill, sensitivity to UV light, and the potential for insect resistance. Research investigating the effects of mixed baculoviral interactions against target pests has been a growing field of interest due to their potential to overcome such shortcomings. Previous studies using a combination of CrleGV-SA and CrpeNPV against T. leucotreta observed a reduction in lethal concentration in laboratory bioassays, indicating that such mixtures may have the potential for application in the field. This has led to the motivation to investigate further interactions between CrleGV-SA in combination with CrpeNPV, CpGV-M, and HearNPV-Au to understand better how these viruses interact and to determine whether synergistic, additive, or antagonistic interactions can occur against T. leucotreta. The outcome of these interactions will inform researchers and farmers about best practices concerning these viruses should they be combined against T. leucotreta in the future. Prior to performing mixed baculovirus infections in laboratory bioassays, oligonucleotides targeting unique regions in the viral genomes of CrleGV-SA, CrpeNPV, CpGV-M, and HearNPV-Au were designed using Primer-BLAST. The specificity of these oligonucleotides was further tested in silico using Geneious R11 software (11.1.5). The stocks of CrpeNPV, CpGV-M, and HearNPV-Au were purified using crude OB extraction from diseased C. peltastica, C. pomonella, and H. armigera larval cadavers provided by River Bioscience (Pty) Ltd (Gqeberha, South Africa). The stock of CrleGV-SA was purified using crude OB extraction from infected T. leucotreta cadavers. Subsequently, the unique oligonucleotides were used in PCR assays to detect if the samples contained the baculoviruses of interest. Amplicons of the expected sizes were generated indicating the presence of CrleGV-SA, CrpeNPV, CpGV-M, and HearNPV-Au in each of the samples. The OBs were counted using darkfield microscopy and a counting chamber before the single and mixed infections were initiated against T. leucotreta neonate larvae. Surface-dose biological assays were used to evaluate the relative virulence in terms of lethal concentration of CrleGV-SA, CrpeNPV, and CpGV-M, alone against T. leucotreta. After 7 days, the dose mortality data was analysed using “drc” in R studio and the LC50 and LC90 were compared amongst each virus. The CrleGV-SA treatment was estimated to be the most virulent in comparison to CrpeNPV and CpGV-M. A dose discriminate assay confirmed that HearNPV does not cause mortality in T. leucotreta. Similarly, the relative virulence in terms of lethal concentration of CrleGV-SA in various ratios in combination with CrpeNPV, CpGV-M, and HearNPV-Au was determined using 7-day surface dose biological assays. The CrleGV/CrpeNPV was the most virulent mixture with lower LC50 and LC90 values measured in comparison to CrleGV/CpGV and CrleGV/HearNPV, respectively. The Tammes Bakuniak graphic method confirmed the CrleGV/CrpeNPV, CrleGV/CpGV, and CrleGV/HearNPV mixtures to be antagonistic against T. leucotreta neonate larvae in terms of lethal concentration. The last aspect of the study was to determine the probable cause of larval death. A modified CTAB protocol was used to extract genomic DNA from neonate-sized T. leucotreta cadavers collected in single and mixed infection assays. The gDNA served as templates in PCR assays using the unique oligonucleotides. In single infections, the presence of CrleGV-SA in CrpeNPV and HearNPV inoculated larvae was observed. The results suggest possible covert infections of CrleGV-SA in the T. leucotreta colony which may be caused by virus infection or an unknown stress factor. The results from the mixed infections showed the presence of each virus in all replicates except for the CrleGV/CpGV and CrleGV/HearNPV mixtures. In the CrleGV/CpGV mixture, only CrleGV-SA was present in the last replicate, suggesting a possible competition for host resources. In the CrleGV/HearNPV mixture, only CrleGV-SA was detected in all 3 replicates, suggesting that HearNPV did not have any effect and the larvae died of the CrleGV-SA infection. This is the first study to report mixtures of CrleGV-SA in combination with CpGV-M and HearNPV-Au against T. leucotreta neonate larvae. Despite the antagonistic interactions observed in the evaluated mixtures, this study has laid a foundation to further investigate how these viruses interact in dual infections for the improved control of T. leucotreta. This may be done by evaluating different ratios and combinations of baculoviruses to those used in this study. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
  • Full Text:
  • Date Issued: 2024-10-11

The expression and evaluation of CrpeNPV gp37 as a formulation additive for enhanced infectivity with CrleGV-SA and improved Thaumatotibia leucotreta control

  • Authors: Muleya, Naho
  • Date: 2024-10-11
  • Subjects: Cryptophlebia leucotreta Biological control , False Codling Moth , Cryptophlebia leucotreta granulovirus , Cryptophlebia peltastica nucleopolyhedrovirus , Citrus Diseases and pests South Africa , Baculoviruses
  • Language: English
  • Type: Academic theses , Master's theses , text
  • Identifier: http://hdl.handle.net/10962/463919 , vital:76457
  • Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a significant pest native to Africa, causing damage to citrus and posing a threat to the export of fresh citrus in South Africa. Classified as a phytosanitary risk by several South African export markets, this pest necessitates effective control measures. Baculoviruses emerge as promising biological control agents against T. leucotreta due to their inherent safety and eco-friendly characteristics. Among these, Cryptophlebia leucotreta Granulovirus (CrleGV-SA) and Cryptophlebia peltastica Nucleopolyhedrovirus (CrpeNPV) stand out, both causing larval mortality upon infecting T. leucotreta. CrleGV-SA has been formulated into the products Cryptogran™, CryptoMax™ and Cryptex®, while CrpeNPV has been formulated into the product Multimax™. Both viruses are used in integrated pest management programmes to reduce fruit damage in agricultural fields, with CrleGV-SA having been employed against T. leucotreta for nearly 20 years in South Africa. However, these control options are limited by factors such as virulence and the slow speed of kill. This limitation can be addressed by exploiting potential synergistic relationships between baculoviruses infecting the same host. Previous studies have demonstrated that the truncated CpGV gp37 can enhance the infectivity of NPVs on other lepidopteran pests, such as Spodoptera exigua (Hübner). Although the mechanism behind this phenomenon remains unclear, it presents an opportunity to enhance the effectiveness of baculovirus-based management strategies. Notably, the genome of CrpeNPV encodes gp37, while CrleGV-SA lacks this gene. The potential interaction between CrleGV-SA and CrpeNPV gp37 remains unexplored. Therefore, investigating whether they exhibit synergistic or antagonistic effects is essential for optimising baculovirus-based management of T. leucotreta. This study aims to express CrpeNPV gp37 in a bacterial system and then evaluate its effect on larval mortality when combined with CrleGV-SA in laboratory bioassays. The initial step involved extracting genomic DNA (gDNA) from occlusion bodies (OBs) of CrpeNPV. A modified Quick DNA Miniprep plus kit was utilised, which entailed pre-treatment with Na2CO3 followed by neutralisation with Tris-HCI before gDNA extraction using the kit. Subsequently, the concentration of the gDNA was estimated using a Nanodrop spectrophotometer. Oligonucleotides targeting the CrpeNPV gp37 gene were designed for PCR amplification, with the gDNA serving as a template. The gp37 amplicon was identified through agarose gel electrophoresis and then gel purified in preparation for cloning. Secondly, the purified PCR product was cloned into the intermediate vector pJET1.2/blunt and then subcloned into the bacterial expression vector pCA528 through DNA ligation. The construction of recombinant plasmids (pJET-gp37 and pCA-gp37) was conducted and verified using Colony PCR, plasmid extraction, restriction enzyme analysis, and Sanger sequencing. Thirdly, the recombinant protein (6×His-SUMO-gp37) was expressed and purified using Nickel affinity chromatography and analysed through SDS-PAGE and Western blot techniques. The expression of 6×His-SUMO-gp37 was carried out at both 25 °C and 18 °C. A time course induction study was conducted, inducing transformed cells for 0-, 3-, 5-, and 24-hours post induction (hpi). SDS-PAGE and Western blotting of samples collected at various time points revealed that 6×His-SUMO-gp37, approximately 42 kDa in size, was visible from 3 hpi, with maximal expression at 24 hpi. Solubility analysis of 6×His-SUMO-gp37 was performed at both temperatures, showing solubility at 18 °C but predominantly present in the insoluble fraction. The soluble protein was purified under native conditions, while the insoluble protein was purified under denaturing conditions. Despite being unable to elute 6×His-SUMO-gp37 under native conditions, successful elution was achieved under denaturing conditions, confirmed via Western blot analysis. No further experiments were conducted on the eluted 6×His-SUMO-gp37 under denaturing conditions. Lastly, a preliminary surface dose bioassay was conducted to evaluate the efficacy of pelleted bacteria expressing 6×His-SUMO-gp37 in combination with CrleGV-SA against T. leucotreta neonates. Two lethal concentration doses of CrleGV-SA were prepared: a low concentration (2.96×104 OBs/mL) capable of killing 40 % of the T. leucotreta population, and a high concentration (2.96×105 OBs/mL) capable of killing 90 % of the population. The target protein, 6×His-SUMO-gp37, and the control, pCA528, were obtained by lysing the cells, centrifuging the samples, and collecting the insoluble fractions in pellet form. These fractions were then resuspended in PBS and used as treatments in combination with the prepared CrleGV-SA concentration doses. The concentration of the pellets was estimated using a Nanodrop spectrophotometer by measuring the absorbance at 280 nm. The bioassay results revealed that the combination of 100 μg/mL of pelleted bacteria expressing 6×His-SUMO-gp37 with CrleGV-SA had no effect on T. leucotreta larval mortality compared to CrleGV-SA alone. A one-way ANOVA was performed to assess differences among the virus treatment groups, concluding that no statistically significant differences were observed among the groups. The experiments in this study provided valuable insights for future research, particularly in exploring the use of a protein-virus combination as a novel method for pest control. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
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  • Date Issued: 2024-10-11

An investigation into yeast-baculovirus synergism for the improved control of Thaumatotibia leucotreta, an economically important pest of citrus

  • Authors: Van der Merwe, Marcél
  • Date: 2021-10-29
  • Subjects: Baculoviruses , Cryptophlebia leucotreta , Yeast , Natural pesticides , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control , Thaumatotibia leucotreta‎
  • Language: English
  • Type: Doctoral theses , text
  • Identifier: http://hdl.handle.net/10962/191236 , vital:45073
  • Description: A mutualistic association between Cydia pomonella and yeasts belonging to the genus Metschnikowia has previously been demonstrated. Larval feeding galleries inoculated with M. andauensis, reduced larval mortality and enhanced larval development. Additionally, adult C. pomonella female oviposition preference was also shown to be influenced by the volatiles produced by M. andauensis. This mutualistic relationship was manipulated for biological control purposes, by combining M. pulcherrima with the baculovirus Cydia pomonella granulovirus. The combination of M. pulcherrima with brown cane sugar and CpGV in laboratory assays and field trials resulted in a significant increase in larval mortality. A similar observation was made when M. pulcherrima was substituted for Saccharomyces cerevisiae. This indicates that yeasts harbour the potential for use in biological control, especially when combined with other well-established biocontrol methods. Thaumatotibia leucotreta is a phytophagous insect endemic to southern Africa. It is highly significant to the South African citrus industry due to its classification as a phytosanitary pest by most international markets. An integrated pest management programme has been implemented to control T. leucotreta. The baculovirus Cryptophlebia leucotreta granulovirus forms one component of this programme and is highly effective. In this study, we proposed to determine which yeast species occur naturally in the gut of T. leucotreta larvae and to examine whether any of the isolated yeast species, when combined with the CrleGV-SA, enhance its effectiveness. Firstly, Navel oranges infested with T. leucotreta larvae were collected from geographically distinct citrus-producing regions across South Africa. This led to the isolation and identification of six yeast species from the gut of T. leucotreta larvae via PCR amplification and sequencing of the internal transcribed spacer region and D1/D2 domain of the large subunit. Six yeast species were identified, viz. Meyerozyma guilliermondii, Hanseniaspora uvarum, Clavispora lusitaniae, Kluyveromyces marxianus, Pichia kudriavzevii and Pichia kluyveri. Additionally, Saccharomyces cerevisiae was included as a control in all trials due to its commercial availability and use in the artificial diet used to rear T. leucotreta. Secondly, larval development and attraction assays were conducted with the isolated yeast species. Thaumatotibia leucotreta larvae that fed on Navel oranges inoculated with M. guilliermondii, P. kluyveri, H. uvarum, and S. cerevisiae had accelerated developmental periods and reduced mortality rates. Additionally, it was demonstrated that T. leucotreta neonates were attracted to YPD broth cultures inoculated with P. kluyveri, H. uvarum, P. kudriavzevii and K. marxianus for feeding. Thirdly, oviposition preference assays were conducted with adult T. leucotreta females to determine whether the isolated yeast species influence their egg-laying in two-choice and multiple-choice tests. Navel oranges were inoculated with a specific yeast isolate, and mated adult females were left to oviposit. Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to influence adult T. leucotreta female oviposition preference in two-choice tests. However, multiple-choice tests using the aforementioned yeast species did not mimic these results. Lastly, a series of detached fruit bioassays were performed to determine the optimal yeast:virus ratio, test all isolated yeast species in combination with CrleGV-SA and to further enhance yeast/virus formulation through the addition of an adjuvant and surfactant. CrleGV-SA was applied at a lethal concentration that would kill 50 % of T. leucotreta larvae. The optimal yeast concentration to use alongside CrleGV-SA was determined. Pichia kluyveri, P. kudriavzevii, K. marxianus and S. cerevisiae in combination with CrleGV-SA increased larval mortality compared to CrleGV-SA alone. The inclusion of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae plus CrleGV-SA formulations greatly enhanced their efficacy. Additionally, semi-field trials were initiated using P. kudriavzevii and S. cerevisiae, with promising preliminary results being obtained, although more replicates need to be performed. The experiments performed in this study provide a platform for further research into the application of a yeast/virus combination as a novel control and monitoring option for T. leucotreta in the field. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
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  • Date Issued: 2021-10-29

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:
  • Date Issued: 2021-04

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:
  • Date Issued: 2021-04

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

Yeast-baculovirus synergism: investigating mixed infections for improved management of the false codling moth, Thaumatotibia leucotreta

  • Authors: Van der Merwe, Marcél
  • Date: 2018
  • Subjects: Cryptophlebia leucotreta , Baculoviruses , Yeast , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control
  • Language: English
  • Type: Master's theses , text
  • Identifier: http://hdl.handle.net/10962/62963 , vital:28347
  • Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) or otherwise commonly known as the false codling moth is an indigenous pest of the citrus industry in southern Africa. The pest is highly significant as it impacts negatively on the export of fresh citrus fruits from South Africa to international markets. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been implemented. One component of this programme is the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV-SA) which has been formulated into the products Cryptogran™ and Cryptex®. It has previously been reported that there is a mutualistic association between Cydia pomonella (L.) (Lepidoptera: Tortricidae) also known as codling moth, and epiphytic yeasts. Cydia pomonella larval feeding galleries were colonised by yeasts and this, in turn, reduced larval mortality and enhanced larval development. It has been demonstrated in laboratory assays and field trials that combining yeast and brown cane sugar with Cydia pomonella granulovirus (CpGV) significantly increased larval mortality and lowered the proportion of injured apple fruit. This suggests that yeasts can enhance the effectiveness of an insect virus in managing pest larvae. In this study, we proposed to determine which species of yeast occur naturally in the digestive tract, frass and on the epidermis of T. leucotreta larvae and to examine whether any of these yeasts, when combined with the CrleGV-SA, have a synergistic effect in increasing mortality of T. leucotreta larvae. Firstly, Navel oranges infested with T. leucotreta larvae were collected from orchards in Sundays River Valley in Eastern Cape of South Africa. Larvae were extracted and analysed for the presence of yeast on their surface, or in their gut and frass. Four yeasts were isolated from T. leucotreta larvae and identified down to species level via PCR amplification and sequencing of internal transcribed spacer (ITS) region and D1/D2 domain of the large subunit (LSU) of rDNA region. These yeasts were isolated from the frass, epidermis and digestive tract of T. leucotreta larvae. The yeast isolates were identified as Meyerozyma caribbica, Pichia kluyveri, Pichia kudriavzevii and Hanseniaspora opuntiae. A yeast preference assay was conducted on female T. leucotreta moths to examine whether any of the isolated yeast species affected their oviposition preference. Navel oranges were inoculated with the isolated yeast species at a concentration of 6 × 108 cells.ml-1. The assay also included a Brewer’s yeast and distilled water control. Pichia kudriavzevii was shown to be the preferred yeast species for oviposition, as significantly more eggs were deposited on Navel oranges inoculated with this yeast compared to the other treatments. Lastly, a detached fruit bioassay was performed to evaluate the efficacy of mixing P. kudriavzevii with CrleGV-SA to enhance T. leucotreta larvae mortality. Pichia kudriavzevii was selected as it was demonstrated as having an effect on the oviposition preference of female T. leucotreta moths. The concentration at which P. kudriavzevii was applied remained the same as in the preference assay while CrleGV-SA was applied at lethal concentration required to kill 50 % of the population (9.31 × 107 OBs.ml-1). Although an increase in larval mortality was observed between CrleGV-SA being applied alone and the yeast/virus mixture, this result was determined not to be statistically significant. The experiments performed in this study provide a platform for further research into the application of a yeast-virus combination as a novel control option for T. leucotreta in the field. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
  • Full Text:
  • Date Issued: 2018

The isolation, genetic characterisation and biological activity of a South African Phthorimaea operculella granulovirus (PhopGV-SA) for the control of the Potato Tuber Moth, Phthorimaea operculella (Zeller)

  • Authors: Jukes, Michael David
  • Date: 2015
  • Subjects: Potato tuberworm , Potatoes -- Diseases and pests -- South Africa , Baculoviruses , Natural pesticides , Biological pest control agents , Potato tuberworm -- Biological control , Restriction enzymes, DNA
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:4147 , http://hdl.handle.net/10962/d1017908
  • Description: The potato tuber moth, Phthorimaea operculella (Zeller), is a major pest of potato crops worldwide causing significant damage to both field and stored tubers. The current control method in South Africa involves chemical insecticides, however, there is growing concern on the health and environmental risks of their use. The development of novel biopesticide based control methods may offer a potential solution for the future of insecticides. In this study a baculovirus was successfully isolated from a laboratory population of P. operculella. Transmission electron micrographs revealed granulovirus-like particles. DNA was extracted from recovered occlusion bodies and used for the PCR amplification of the lef-8, lef-9, granulin and egt genes. Sequence data was obtained and submitted to BLAST identifying the virus as a South African isolate of Phthorimaea operculella granulovirus (PhopGV-SA). Phylogenetic analysis of the lef-8, lef-9 and granulin amino acid sequences grouped the South African isolate with PhopGV-1346. Comparison of egt sequence data identified PhopGV-SA as a type II egt gene. A phylogenetic analysis of egt amino acid sequences grouped all type II genes, including PhopGV-SA, into a separate clade from types I, III, IV and V. These findings suggest that type II may represent the prototype structure for this gene with the evolution of types I, III and IV a result of large internal deletion events and subsequent divergence. PhopGV-SA was also shown to be genetically more similar to South American isolates (i.e. PhopGV-CHI or PhopGV-INDO) than it is to other African isolates, suggesting that the South African isolate originated from South America. Restriction endonuclease profiles of PhopGV-SA were similar to those of PhopGV-1346 and PhopGV-JLZ9f for the enzymes BamHI, HindIII, NruI and NdeI. A preliminary full genome sequence for PhopGV-SA was determined and compared to PhopGV-136 with some gene variation observed (i.e. odv-e66 and vp91/p95). The biological activity of PhopGV-SA against P. operculella neonate larvae was evaluated with an estimated LC₅₀ of 1.87×10⁸ OBs.ml⁻¹ being determined. This study therefore reports the characterisation of a novel South African PhopGV isolate which could potentially be developed into a biopesticide for the control of P. operculella.
  • Full Text:
  • Date Issued: 2015

Genetic and biological characterisation of a novel South African Plutella xylostella granulovirus (PlxyGV) isolate

  • Authors: Abdulkadir, Fatima
  • Date: 2014
  • Subjects: Diamondback moth , Diamondback moth -- Control -- South Africa , Plutellidae -- Control -- South Africa , Baculoviruses , Cruciferae -- Diseases and pests -- South Africa
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:4113 , http://hdl.handle.net/10962/d1013059
  • Description: The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is an important pest of cruciferous crops worldwide. The prolonged use of synthetic chemical insecticides as a primary means of control has resulted in the development of resistance in pest populations. In addition, the pest has also evolved resistance to the bacterial insecticidal protein of Bacillus thuringiensis which is also widely used as a method of control. Baculoviruses are considered as effective alternatives to conventional methods of control when incorporated into integrated pest management (IPM) programmes. These viruses target the larval stages of insects, are generally host-specific and are safe for use in the environment. This study aimed to isolate a baculovirus from a laboratory-reared P. xylostella colony, characterise it genetically and then evaluate its virulence against neonate and fourth instar larvae. A laboratory colony of P. xylostella was established using pupae and asymptomatic larvae collected from a cabbage plantation outside Grahamstown in the Eastern Cape province of South Africa. The colony flourished in the laboratory due to prime conditions and availability of food. The duration of development from egg to adult was determined by observation and imaging of the various life stages. The mean developmental time from egg to adult was observed to be 14.59 ± 0.21 days. The population of the insects increased rapidly in number leading to overcrowding of the insect colony, and hence appearance of larvae with viral symptoms. Occlusion bodies (OBs) were extracted from symptomatic larval cadavers and purified by glycerol gradient centrifugation. Analysis of the purified OBs by transmission electron microscopy revealed the presence of a granulovirus which was named PlxyGV-SA. The virus isolate was genetically characterised by restriction endonuclease analysis of the genomic DNA, and PCR amplification and sequencing of selected viral genes. The complete genome sequence of a Japanese P. xylostella granulovirus isolate, PlxyGV-Japan, has been deposited on the GenBank database providing a reference strain for comparison with DNA profiles and selected gene sequences of PlxyGV-SA. BLAST analysis of the granulin gene confirmed the isolation of a novel South African PlxyGV isolate. Comparison of the restriction profiles of PlxyGV-SA with profiles of PlxyGV-Japan and other documented PlxyGV profiles obtained by agarose gel electrophoresis revealed that PlxyGV-SA is a genetically distinct isolate. The data obtained from the sequencing and alignment of ecdysteroid UDP-glucosyltransferase (egt), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes with those of PlxyGV-Japan also showed that PlxyGV-SA is a genetically different isolate. In order to determine the biological activity of PlxyGV-SA against neonate and fourth instar P. xylostella larvae, surface dose bioassays were conducted. The median lethal concentration of the virus required to kill 50% (LC₅₀) and 90% (LC₉₀) of the larvae was estimated by feeding insects with a range of doses. In addition, the time to kill 50% of the larvae (LT₅₀) was determined by feeding insects with the LC₉₀ concentration. Larval mortality was monitored daily until pupation. The data obtained from the dose response assays were subjected to probit analysis using Proban statistical software. The time response was determined using GraphPad Prism software (version 6.0). The LC₅₀ and LC₉₀ values for the neonate larvae were 3.56 × 10⁵ and 1.14 × 10⁷ OBs/ml respectively. The LT₅₀ was determined to be 104 hours. The neonate larvae were found to be more susceptible to infection than the fourth instar larvae with the same virus concentration. The concentrations used for the neonate larvae assay did not have a significant effect on the fourth instar as no mortality was recorded. This is the first study to describe a novel South African PlxyGV isolate and the results suggest that PlxyGV-SA has significant potential for development as an effective biopesticide for the control of P. xylostella in the field.
  • Full Text:
  • Date Issued: 2014

A baculovirus-mediated expression system for the analysis of HaSV RNA packaging

  • Authors: Mendes, Adriano
  • Date: 2012
  • Subjects: RNA , Baculoviruses , Helicoverpa armigera , Plasmids
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:4025 , http://hdl.handle.net/10962/d1004085 , RNA , Baculoviruses , Helicoverpa armigera , Plasmids
  • Description: The Helicoverpa armigera stunt virus (HaSV) is a member of a family of small nonenveloped (+) ssRNA insect viruses currently known as the Tetraviridae. This family is unique in terms of the T=4 quasi-symmetry of its capsid particles and the unusually narrow host range and tissue tropism. Assembly of tetraviral particles has been well characterised and involves the combination of 240 copies of a single capsid precursor protein (VCap) into a procapsid followed by autoproteolytic cleavage to yield the major (β) and minor (γ) capsid subunits within the mature particle. HaSV has two genomic RNAs, RNA 1 encoding the replicase and RNA 2 encoding VCap and p17, the ORF of which lies upstream of and overlaping with the 5’ end of the VCap ORF. Prior to this study, Vlok (2009) used a plasmid expression system to study RNA packaging in HaSV VLPs assembled in Spodoptera frugiperda 9 (Sf9) cells co-expressing p17 and VCap. The study showed that the p17 ORF was required for the packaging of RNA 2 during capsid assembly but it was unclear whether p17 expression was required for packaging. In addition, expression from the transfected plasmids was sub-optimal affecting both the yield of VLPs and the detection of p17. The aim of this study was to use the plasmid system to test whether p17 expression was required for plasmid-derived VLP RNA packaging and then develop a baculovirus-mediated system to test this hypothesis. By using a plasmid in which the start codon of p17 was mutated, it was shown that p17 expression was required for RNA 2 packaging into plasmid-VLPs. For the baculovirus system, four recombinant baculoviruses based upon the pFastBac Dual expression system, were constructed. These included Bac20, expressing wild type RNA 2, Bac21, RNA 2 with p17 silenced, Bac23, RNA 2 and p17 expressed on a separate transcript and Bac24, RNA 2 with p17 silenced plus p17 expressed on a separate transcript. Assembly of VLPs was more efficient using the baculovirus expression system and p17 expression was observed in cells infected with Bac20, Bac23 and Bac24, but not Bac21. In contrast to the plasmid-VLPs, bac-VLPs did not require p17 for the encapsidation of RNA 2. In addition to RNA 2, Bac23 and Bac24 packaged the p17 mRNA transcribed separately from RNA 2. This insinuated that bac-VLPs may be packaging RNA non-selectively. It was proposed that p17 may play a role in packaging in an RNA-limiting environment (plasmid system) but functioned differently when viral RNA was in excess (baculovirus system). This data points to the importance of developing a replication system for the analysis of the packaging pathways of these viruses and this study has laid down the foundations for such a system in which RNA 1 and RNA 2 can be introduced into a single cell by means of a single recombinant virus.
  • Full Text:
  • Date Issued: 2012

Enumeration of insect viruses using microscopic and molecular analyses: South African isolate of cryotophlebia leucotreta granulovirus as a case study

  • Authors: Dhladhla, Busisiwe I R
  • Date: 2012
  • Subjects: Baculoviruses , Insects -- Viruses , Molecular genetics , Microbial genomics
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:10318 , http://hdl.handle.net/10948/d1008395 , Baculoviruses , Insects -- Viruses , Molecular genetics , Microbial genomics
  • Description: Baculoviruses have been used as biocontrol agents to control insect pests in agriculture since the 1970s. Out of the fifteen virus families known to infect insects, baculoviruses offer the greatest potential as insect biopesticides, due to their high host specificity which makes them extremely safe to humans, other vertebrates, plants and non-target microorganisms. They comprise of two genera: nucleopolyhedroviruses (NPVs) and granuloviruses (GVs). The South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA) which is infectious for the false codling moth (FCM), Thaumatotibia leucotreta, (Meyrick) (Lepidoptera: Tortricidae), has been successfully developed into two commercial biopesticides; Cryptogran® and Cryptex®, for the control of FCM in citrus crops. The current method of enumeration used for CrleGV-SA virus particles in routine experiments during the production of the GV as biopesticides, is dark field microscopy. However, due to the small size of GVs (300-500 nm in length), the technique is not easy to perform on these viruses, and no systemic comparison has been made of potential alternative methods. Therefore, the main objective of this study was to develop a quantitative enumeration method for CrleGV-SA occlusion bodies (OBs) which is accurate, reliable, and feasible, and compare the developed methods of enumeration to the current method. Purified and semi-purified CrleGV-SA viral stocks were prepared for enumeration studies using spectrophotometry, dark field microscopy, scanning electron microscopy (SEM) and real time qPCR. Spectrophotometry was found to be an unreliable method for enumeration of GVs in the production, standardisation, and quality control of biopesticides. Dark field microscopy and SEM were found to be accurate, and statistically comparable (p = 0.064) enumeration techniques. qPCR is currently being optimised for the enumeration of GVs. This technique was demonstrated to generate accurate standard curves for absolute quantification of virus particles for pure and semi-pure virus preparations. qPCR offers the greatest potential as an accurate enumeration method because it is not affected by contamination with non-biological contaminating debris, nor by other biological material due to the specificity of PCR primers. Further work is required to fully develop qPCR as an enumeration method for GVs. However, dark field microscopy has been successfully validated as an enumeration method. SEM, which has a high resolution compared to light microscopy, has an added advantage over dark field microscopy, which is to distinguish virus particles in semi-pure viral stock preparations during counting. Therefore, SEM currently provides the most unambiguous and feasible enumeration method for GVs in both purified and semi-purified virus samples.
  • Full Text:
  • Date Issued: 2012

Development of techniques for the isolation of a granulovirus from potato tuber moth, phthorimaea operculella (Zeller)

  • Authors: King, Shirley Anne
  • Date: 2011
  • Subjects: Potato tuberworm -- Larvae , Agricultural pests -- Biological control , Potato tuberworm , Baculoviruses
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:5910 , http://hdl.handle.net/10962/d1015202
  • Description: Phthorimaea operculella, commonly known as the Potato Tuber Moth, is an economically important agricultural pest worldwide. The baculovirus, Phthorimaea operculella granulovirus (PhoGV) has been considered as a means of control alternative to chemical control because of its host specificity and harmless impact on other organisms and ecosystems. An isolate of PhoGV obtained from a South African PTM population would be beneficial in the production of a biopesticide, which is not yet available. An efficient and cost-effective rearing method would be advantageous for potential commercial production. Commercial table and seed potato plantations and storage facilities located in Patensie, Bathurst, Howick and Ivanhoe were surveyed for PTM infestations. Patensie was the only site where milky discoloured larvae were found, a potential symptom of PhoGV infection. TEM analysis revealed no virus in these samples. Since no virus was found in the field-collected samples, PTM insects were collected to initiate rearing in the laboratory. PTM was raised by three different methods in the laboratory. A cost/benefit analysis, survival rate, fertility and sex ratio were recorded for each rearing method. Rearing method one was deemed unsuccessful for efficient commercial rearing, as survival percentage and fertility were low. Rearing methods two and three had high survival rates and high fertility, and were efficient and less labour intensive than rearing method one. Rearing method three was the most productive technique, but for commercial production rearing method two was considered the most manageable and efficient. The sex ratio was 1:1 for all three cultures. The cost analysis revealed that rearing methods two and three were less expensive than rearing method one because less labour was required to monitor insects. The success of rearing PTM for 19 months will enable these cultures to be up-scaled to a large production facility for mass rearing. Virus was not found in the field surveys or in laboratory cultures, therefore chemical, temperature, humidity and carbon dioxide stressors were used in an attempt to initiate a baculoviral infection. Symptoms were exhibited in larvae subjected to chemical, temperature and humidity treatments, but these were confirmed by TEM analysis not to be a result of PhoGV infection. The success of rearing PTM in the laboratory suggests that the method could be used in the commercial rearing of the insects in a large mass-rearing facility. The data obtained from induction protocols have allowed for better understanding for future induction for PhoGV and other baculoviruses in other insect species. The failure to isolate a South African PhoGV strain for developing a biopesticide against PTM has motivated further studies in obtaining a baculovirus in order for South Africa to develop a commercial product against this pest.
  • Full Text:
  • Date Issued: 2011

The development of a baculovirus expression system for the production of Helicoverpa armigera stunt virus capsids for use in the encapsidation of foreign molecules

  • Authors: Mosisili, Kekeletso Mpho Thakane
  • Date: 2003
  • Subjects: Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:4088 , http://hdl.handle.net/10962/d1007700 , Helicoverpa armigera , Fall armyworm , Baculoviruses , Insects -- Viruses , RNA -- Analysis
  • Description: The capsid protein of Helicoverpa armigera stunt virus (HaSV) a T=4 insect virus was expressed in Spodoptera frugiperda 9 cells using a baculovirus vector. When the insect cells were infected at a high MOl the expressed coat protein assembled into virus-like particles (VLPs) that spontaneously underwent maturation and were morphologically indistinguishable from wild-type HaSV. The VLPs were electron dense when viewed under EM and encapsidated their coat protein mRNA. When Sf9 cells were infected at a low multiplicity of infection (MOl) the expressed capsid protein assembled into procapsids that did not spontaneously undergo maturation. These procapsids underwent autoproteolytic maturation cleavage when they were treated with an acidic buffer. The procapsids were used in the encapsidation of a FITC labelled peptide. The peptide encapsidating VLPs showed an increase in their buoyant density that was not collaborated by an increase in the concentration of the FITC labelled peptide detected when these samples were compared to control samples with similar buoyant densities.
  • Full Text:
  • Date Issued: 2003

The establishment of a virus free laboratory colony of Cryptophlebia leucotreta (False Codling Moth) and characterisation of Cryptophlebia leucotreta Granulovirus (CrleGV) genes

  • Authors: Ludewig, Michael Hans
  • Date: 2003
  • Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
  • Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
  • Full Text:
  • Date Issued: 2003
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