Determination of the effects of sunlight and UV irradiation on the structure, viability and reapplication frequency of the biopesticide cryptophlebia leucotreta granulovirus in the protection against false codling moth infestation of citrus crops
- Authors: Mwanza, Patrick
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- Effect of ultraviolet radiation on , Natural pesticides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/6346 , vital:21075
- Description: Cryptophlebia leucotreta granulovirus (CrleGV-SA) is a baculovirus specifically pathogenic to the citrus pest false codling moth, Thaumatotibia leucotreta. CrleGV- SA is formulated as a commercial biopesticide, Cryptogran® (River Bioscience, South Africa). The virus has a stable, proteinaceous crystalline occlusion body (OB) that protects the nucleocapsid. The major limitation to the use of baculoviruses is their susceptibility to the ultraviolet (UV) component of sunlight, which rapidly and greatly reduces their efficacy as biopesticides. The UVA and UVB components are the most destructive to biological organisms. To date no publication has reported the effect of UV on the structure and virulence of CrleGV, or the effectiveness of the OB as a UV protectant. In this study the effect of UV irradiation on the structure and infectivity of pure CrleGV-SA and Cryptogran® was investigated using scanning electron microscopy (SEM), Raman spectroscopy, qPCR, and bioassays. The project included laboratory and field studies. In the laboratory, CrleGV-SA and Cryptogran® were exposed to either UVA or UVB for periods of 24 hours to 7 days before analysis. In the field, Cryptogran® was applied to trees in a citrus orchard with young fruit. The fruit were collected from 24 hours to 28 days after application and bioassays conducted to assess the effect of sunlight over time on virus structure and efficacy when applied to the northern or southern sides of the trees. No surface morphological changes to the virus were detected using SEM. However, small compositional changes were detected by Raman spectroscopy. qPCR and bioassays demonstrated that UV irradiation damaged the viral DNA, greatly reducing the infectivity of pure CrleGV-SA and Cryptogran®. Exposure to UVB reduced the virulence of the virus more than UVA. The field studies revealed that the activity of CrleGV-SA decreased more on the northern side of the trees than on the southern side.
- Full Text:
- Date Issued: 2015
- Authors: Mwanza, Patrick
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- Effect of ultraviolet radiation on , Natural pesticides
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10948/6346 , vital:21075
- Description: Cryptophlebia leucotreta granulovirus (CrleGV-SA) is a baculovirus specifically pathogenic to the citrus pest false codling moth, Thaumatotibia leucotreta. CrleGV- SA is formulated as a commercial biopesticide, Cryptogran® (River Bioscience, South Africa). The virus has a stable, proteinaceous crystalline occlusion body (OB) that protects the nucleocapsid. The major limitation to the use of baculoviruses is their susceptibility to the ultraviolet (UV) component of sunlight, which rapidly and greatly reduces their efficacy as biopesticides. The UVA and UVB components are the most destructive to biological organisms. To date no publication has reported the effect of UV on the structure and virulence of CrleGV, or the effectiveness of the OB as a UV protectant. In this study the effect of UV irradiation on the structure and infectivity of pure CrleGV-SA and Cryptogran® was investigated using scanning electron microscopy (SEM), Raman spectroscopy, qPCR, and bioassays. The project included laboratory and field studies. In the laboratory, CrleGV-SA and Cryptogran® were exposed to either UVA or UVB for periods of 24 hours to 7 days before analysis. In the field, Cryptogran® was applied to trees in a citrus orchard with young fruit. The fruit were collected from 24 hours to 28 days after application and bioassays conducted to assess the effect of sunlight over time on virus structure and efficacy when applied to the northern or southern sides of the trees. No surface morphological changes to the virus were detected using SEM. However, small compositional changes were detected by Raman spectroscopy. qPCR and bioassays demonstrated that UV irradiation damaged the viral DNA, greatly reducing the infectivity of pure CrleGV-SA and Cryptogran®. Exposure to UVB reduced the virulence of the virus more than UVA. The field studies revealed that the activity of CrleGV-SA decreased more on the northern side of the trees than on the southern side.
- Full Text:
- Date Issued: 2015
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
- 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
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