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
- Full Text:
- Date Issued: 2024-10-11
- 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
- Full Text:
- Date Issued: 2024-10-11
An assessment of the status of psylloid species (Hemiptera: Psylloidea) as potential pests of commercial citrus in southern Africa: implications for pest management
- Authors: Moagi, Raynold
- Date: 2024-10-11
- Subjects: Citrus Diseases and pests South Africa , Candidatus Liberibacter , Psylloidea , Polymerase chain reaction , Insect trapping Equipment and supplies , Pests Control
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/464417 , vital:76509
- Description: Psylloids (Hemiptera: Psylloidea), constitute a group of plant sap-sucking insects, some of which are economically significant pests in different ecosystems due to their potential to transmit Gram-negative bacteria, such as the Candidatus Liberibacter species. The African citrus triozid (ACT), Trioza erytreae (Del Guercio), which transmits African citrus greening and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, which transmits Asian citrus greening are significant threats to citrus. Asian citrus psyllid poses a global economic threat due to its ability to vector “Candidatus Liberibacter asiaticus” (CLas), which can rapidly kill citrus trees. However, both ACP and CLas are currently not present in southern Africa but are present in East and West Africa. In the Afrotropical region, 71 triozid species are known to occur and approximately 41 described Diaphorina species in southern Africa. Currently, two indigenous Diaphorina species, Diaphorina punctulata and Diaphorina zebrana have been documented to feed on citrus. There is a significant knowledge gap regarding the ecological roles of other indigenous psylloid species occurring within the citrus environments. Therefore, this study aimed to: (i) determine the diversity and community structure of psylloid species in citrus environments, and (ii) their host ranges through DNA analysis of gut contents to determine if they fed on citrus. Field surveys were carried out across 12 distinct commercial citrus environments across Limpopo and Mpumalanga provinces between 2022 and 2023. Psylloids were collected using yellow sticky traps and an insect sweep-net. Collected psylloid specimens were preserved in 70% ethanol vials and identified to the lowest possible taxonomic level (i.e. genus or species) using both published and unpublished dichotomous identification keys. Furthermore, citrus leaf samples were collected from the same plants on which psylloids were found in the orchards. Genomic DNA (gDNA) was extracted from both leaf and psylloid samples using two different DNA extraction methods. To confirm if citrus DNA could be detected in the psylloid guts, all leaf gDNA samples were initially amplified using the rbcLaF/R primer pair, targeting a 530-bp region of the chloroplast rbcL gene through the polymerase chain reaction (PCR). Lastly, gut content analysis was performed on 11 psylloid species using the same primer pair through PCR to detect citrus DNA. A total of 4,900 psylloids belonging to five families (i.e. Aphalaridae, Carsidaridae, Liviidae, Psyllidae and Triozidae), 19 genera and 47 species, were collected in citrus environments. More psylloids were recorded in Limpopo (3,754) than in Mpumalanga (1,146). The most abundant species were Pauropsylla trichaeta (1,680), followed by Diaphorina punctulata (466), Trioza erytreae (426), Diaphorina virgata (371), Euryconus sp. (358), Cacopsylla sp. (311), Retroacizzia mopanei (263), Acizzia russellae-group (240), Acizzia sp.3 (216) and Acizzia sp.2 (140). Yellow sticky traps captured 3,265 psylloids in citrus orchards, while an insect sweep-net collected 1,635 psylloids (477 from citrus orchards and 1,158 from adjacent natural vegetation). Data from the insect sweep-net revealed that 22 psylloid species were recorded on citrus. In comparison, nine psylloid species were found on Vachellia spp. and unidentified plant species separately, whereas six, three and two psylloid species were recorded on marula, Ficus sp. and mopane, respectively. The abundance, richness and community structure of psylloids differed significantly between the collection methods, provinces and among plant species. The rbcLaF/R primer pair amplified all citrus leaf gDNA samples, producing amplicons of the targeted 530-bp size. The PCR analysis of 11 psylloid species showed that the rbcLaF/R primer pair amplified plant DNA, with PCR-amplified plant DNA samples producing amplicons between 500-bp and 750-bp in the gut contents of five psyllid species: Diaphorina punctulata, Diaphorina virgata, Diaphorina zebrana, Euryconus sp. and Trioza erytreae. However, the targeted 530-bp plant DNA region was only amplified from the gut contents of Euryconus sp. and Diaphorina punctulata. This study documented psylloid diversity and community structure within commercial citrus environments. The findings indicate that the community of psylloids was diverse in citrus environments, with yellow sticky traps being more effective in monitoring different psyllid species within these environments. Furthermore, the PCR analysis detected citrus DNA in the gut contents of Euryconus sp. and Diaphorina punctulata, suggesting that they could be nibbling on citrus when their specific or main host-plants adjacent to citrus orchards are depleted. However, these insects do not lay their eggs or complete their life cycle on citrus, further confirming that citrus is not their host-plant. Thus, further studies, including Sanger sequencing of PCR-amplified plant DNA, are recommended to confirm the ingested plant species, and host-specific testing including infection trials needs to be conducted. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Date Issued: 2024-10-11
- Authors: Moagi, Raynold
- Date: 2024-10-11
- Subjects: Citrus Diseases and pests South Africa , Candidatus Liberibacter , Psylloidea , Polymerase chain reaction , Insect trapping Equipment and supplies , Pests Control
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/464417 , vital:76509
- Description: Psylloids (Hemiptera: Psylloidea), constitute a group of plant sap-sucking insects, some of which are economically significant pests in different ecosystems due to their potential to transmit Gram-negative bacteria, such as the Candidatus Liberibacter species. The African citrus triozid (ACT), Trioza erytreae (Del Guercio), which transmits African citrus greening and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, which transmits Asian citrus greening are significant threats to citrus. Asian citrus psyllid poses a global economic threat due to its ability to vector “Candidatus Liberibacter asiaticus” (CLas), which can rapidly kill citrus trees. However, both ACP and CLas are currently not present in southern Africa but are present in East and West Africa. In the Afrotropical region, 71 triozid species are known to occur and approximately 41 described Diaphorina species in southern Africa. Currently, two indigenous Diaphorina species, Diaphorina punctulata and Diaphorina zebrana have been documented to feed on citrus. There is a significant knowledge gap regarding the ecological roles of other indigenous psylloid species occurring within the citrus environments. Therefore, this study aimed to: (i) determine the diversity and community structure of psylloid species in citrus environments, and (ii) their host ranges through DNA analysis of gut contents to determine if they fed on citrus. Field surveys were carried out across 12 distinct commercial citrus environments across Limpopo and Mpumalanga provinces between 2022 and 2023. Psylloids were collected using yellow sticky traps and an insect sweep-net. Collected psylloid specimens were preserved in 70% ethanol vials and identified to the lowest possible taxonomic level (i.e. genus or species) using both published and unpublished dichotomous identification keys. Furthermore, citrus leaf samples were collected from the same plants on which psylloids were found in the orchards. Genomic DNA (gDNA) was extracted from both leaf and psylloid samples using two different DNA extraction methods. To confirm if citrus DNA could be detected in the psylloid guts, all leaf gDNA samples were initially amplified using the rbcLaF/R primer pair, targeting a 530-bp region of the chloroplast rbcL gene through the polymerase chain reaction (PCR). Lastly, gut content analysis was performed on 11 psylloid species using the same primer pair through PCR to detect citrus DNA. A total of 4,900 psylloids belonging to five families (i.e. Aphalaridae, Carsidaridae, Liviidae, Psyllidae and Triozidae), 19 genera and 47 species, were collected in citrus environments. More psylloids were recorded in Limpopo (3,754) than in Mpumalanga (1,146). The most abundant species were Pauropsylla trichaeta (1,680), followed by Diaphorina punctulata (466), Trioza erytreae (426), Diaphorina virgata (371), Euryconus sp. (358), Cacopsylla sp. (311), Retroacizzia mopanei (263), Acizzia russellae-group (240), Acizzia sp.3 (216) and Acizzia sp.2 (140). Yellow sticky traps captured 3,265 psylloids in citrus orchards, while an insect sweep-net collected 1,635 psylloids (477 from citrus orchards and 1,158 from adjacent natural vegetation). Data from the insect sweep-net revealed that 22 psylloid species were recorded on citrus. In comparison, nine psylloid species were found on Vachellia spp. and unidentified plant species separately, whereas six, three and two psylloid species were recorded on marula, Ficus sp. and mopane, respectively. The abundance, richness and community structure of psylloids differed significantly between the collection methods, provinces and among plant species. The rbcLaF/R primer pair amplified all citrus leaf gDNA samples, producing amplicons of the targeted 530-bp size. The PCR analysis of 11 psylloid species showed that the rbcLaF/R primer pair amplified plant DNA, with PCR-amplified plant DNA samples producing amplicons between 500-bp and 750-bp in the gut contents of five psyllid species: Diaphorina punctulata, Diaphorina virgata, Diaphorina zebrana, Euryconus sp. and Trioza erytreae. However, the targeted 530-bp plant DNA region was only amplified from the gut contents of Euryconus sp. and Diaphorina punctulata. This study documented psylloid diversity and community structure within commercial citrus environments. The findings indicate that the community of psylloids was diverse in citrus environments, with yellow sticky traps being more effective in monitoring different psyllid species within these environments. Furthermore, the PCR analysis detected citrus DNA in the gut contents of Euryconus sp. and Diaphorina punctulata, suggesting that they could be nibbling on citrus when their specific or main host-plants adjacent to citrus orchards are depleted. However, these insects do not lay their eggs or complete their life cycle on citrus, further confirming that citrus is not their host-plant. Thus, further studies, including Sanger sequencing of PCR-amplified plant DNA, are recommended to confirm the ingested plant species, and host-specific testing including infection trials needs to be conducted. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Date Issued: 2024-10-11
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