Comparison of the biology of geographically distinct populations of the citrus pest, Thaumatotibia leucotreta (Meyrick)(Lepidoptera: Tortricidae)
- Opoku-Debrah, John K, Hill, Martin P, Knox, Caroline M, Moore, Sean D
- Authors: Opoku-Debrah, John K , Hill, Martin P , Knox, Caroline M , Moore, Sean D
- Date: 2014
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/405769 , vital:70204 , xlink:href="https://hdl.handle.net/10520/EJC160246"
- Description: Baculovirus biopesticides are an important component of integrated pest management programmes worldwide. One such example is the Cryptophlebia leucotreta granulovirus (CrleGV) which is used for the control of false codling moth, Thaumatotibia (= Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae), a pest of citrus and other crops in South Africa. A potential problem associated with constant application of viral biopesticides is the differing susceptibility to the virus observed between different geographic populations of the insect host. This could be related to a number of factors, including biological performance and fitness of the target insect population. This study compared a variety of phenotypic traits between geographically distinct T. leucotreta populations collected from the Addo, Marble Hall, Citrusdal and Nelspruit regions of South Africa, and reared under laboratory conditions for several generations. Traits including pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and developmental time were used as parameters to measure biological performance and fitness. Insects from the Citrusdal region of the Western Cape exhibited significantly lower pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and the longest duration in larval and pupal development compared to the other colonies investigated. This is the first study to report differences in the performance of laboratory reared T. leucotreta from different geographic locations, and the findings may have important implications for the application of viral biopesticides for the control of this pest in South Africa.
- Full Text:
- Date Issued: 2014
- Authors: Opoku-Debrah, John K , Hill, Martin P , Knox, Caroline M , Moore, Sean D
- Date: 2014
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/405769 , vital:70204 , xlink:href="https://hdl.handle.net/10520/EJC160246"
- Description: Baculovirus biopesticides are an important component of integrated pest management programmes worldwide. One such example is the Cryptophlebia leucotreta granulovirus (CrleGV) which is used for the control of false codling moth, Thaumatotibia (= Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae), a pest of citrus and other crops in South Africa. A potential problem associated with constant application of viral biopesticides is the differing susceptibility to the virus observed between different geographic populations of the insect host. This could be related to a number of factors, including biological performance and fitness of the target insect population. This study compared a variety of phenotypic traits between geographically distinct T. leucotreta populations collected from the Addo, Marble Hall, Citrusdal and Nelspruit regions of South Africa, and reared under laboratory conditions for several generations. Traits including pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and developmental time were used as parameters to measure biological performance and fitness. Insects from the Citrusdal region of the Western Cape exhibited significantly lower pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and the longest duration in larval and pupal development compared to the other colonies investigated. This is the first study to report differences in the performance of laboratory reared T. leucotreta from different geographic locations, and the findings may have important implications for the application of viral biopesticides for the control of this pest in South Africa.
- Full Text:
- Date Issued: 2014
Overcrowding of false codling moth, Thaumatotibia leucotreta (Meyrick) leads to the isolation of five new Cryptophlebia leucotreta granulovirus (CrleGV-SA) isolates
- Opoku-Debrah, John K, Hill, Martin P, Knox, Caroline, Moore, Sean D
- Authors: Opoku-Debrah, John K , Hill, Martin P , Knox, Caroline , Moore, Sean D
- Date: 2013
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/419360 , vital:71637 , xlink:href="https://doi.org/10.1016/j.jip.2012.12.008"
- Description: False codling moth, Thaumatotibia leucotreta (Meyrick) is a serious pest of economic importance to the South African fruit industry. As part of sustainable efforts to control this pest, biological control options that involve the application of baculovirus-based biopesticides such as Cryptogran and Cryptex (both formulated with a South African isolate of Cryptophlebia leucotreta granulovirus, CrleGV-SA) are popularly used by farmers. In order to safeguard the integrity of these biopesticides as well as protect against any future development of resistance in the host, we conducted a study to bioprospect for additional CrleGV isolates as alternatives to existing ones. Using overcrowding as an induction method for latent infection, we recovered five new CrleGV isolates (CrleGV-SA Ado, CrleGV-SA Mbl, CrleGV-SA Cit, CrleGV-SA MixC and CrleGV-SA Nels). Single restriction endonuclease (REN) analysis of viral genomic DNA extracted from purified occlusion bodies showed that isolates differed in their DNA profiles. Partial sequencing of granulin and egt genes from the different isolates and multiple alignments of nucleotide sequences revealed the presence of single nucleotide polymorphisms (SNPs), some of which resulted in amino acid substitutions in the protein sequence. Based on these findings as well as comparisons with other documented CrleGV isolates, we propose two phylogenetic groups for CrleGV-SA isolates recovered in this study.
- Full Text:
- Date Issued: 2013
- Authors: Opoku-Debrah, John K , Hill, Martin P , Knox, Caroline , Moore, Sean D
- Date: 2013
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/419360 , vital:71637 , xlink:href="https://doi.org/10.1016/j.jip.2012.12.008"
- Description: False codling moth, Thaumatotibia leucotreta (Meyrick) is a serious pest of economic importance to the South African fruit industry. As part of sustainable efforts to control this pest, biological control options that involve the application of baculovirus-based biopesticides such as Cryptogran and Cryptex (both formulated with a South African isolate of Cryptophlebia leucotreta granulovirus, CrleGV-SA) are popularly used by farmers. In order to safeguard the integrity of these biopesticides as well as protect against any future development of resistance in the host, we conducted a study to bioprospect for additional CrleGV isolates as alternatives to existing ones. Using overcrowding as an induction method for latent infection, we recovered five new CrleGV isolates (CrleGV-SA Ado, CrleGV-SA Mbl, CrleGV-SA Cit, CrleGV-SA MixC and CrleGV-SA Nels). Single restriction endonuclease (REN) analysis of viral genomic DNA extracted from purified occlusion bodies showed that isolates differed in their DNA profiles. Partial sequencing of granulin and egt genes from the different isolates and multiple alignments of nucleotide sequences revealed the presence of single nucleotide polymorphisms (SNPs), some of which resulted in amino acid substitutions in the protein sequence. Based on these findings as well as comparisons with other documented CrleGV isolates, we propose two phylogenetic groups for CrleGV-SA isolates recovered in this study.
- Full Text:
- Date Issued: 2013
Heterogeneity in virulence relationships between Cryptophlebia leucotreta granulovirus isolates and geographically distinct host populations: Lessons from codling moth resistance to CpGV-M
- Opoku-Debrah, John K, Hill, Martin P, Knox, Caroline M, Moore, Sean D
- Authors: Opoku-Debrah, John K , Hill, Martin P , Knox, Caroline M , Moore, Sean D
- Date: 2016
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/417998 , vital:71500 , xlink:href="https://doi.org/10.1007/s10526-016-9728-1"
- Description: In South Africa, the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV) is used to control the citrus pest Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). However, the risk associated with continuous application of a single active ingredient is resistance. In order to manage resistance should it occur in field populations of T. leucotreta in South Africa, five new CrleGV genotypes from geographically distinct insect populations which were shown to exhibit some degree of phenotypic variation were recovered and genetically characterized. In droplet bioassays using seven CrleGV isolates against five T. leucotreta populations, some isolates were found to show higher virulence to some host populations than others. There were marked differences in the LD50 values of isolates and the number of occlusion bodies required per larva ranged between 0.79 and 3.12. The significance of these findings with respect to the application of CrleGV biopesticides and the management of resistance is discussed.
- Full Text:
- Date Issued: 2016
- Authors: Opoku-Debrah, John K , Hill, Martin P , Knox, Caroline M , Moore, Sean D
- Date: 2016
- Subjects: To be catalogued
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/417998 , vital:71500 , xlink:href="https://doi.org/10.1007/s10526-016-9728-1"
- Description: In South Africa, the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV) is used to control the citrus pest Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). However, the risk associated with continuous application of a single active ingredient is resistance. In order to manage resistance should it occur in field populations of T. leucotreta in South Africa, five new CrleGV genotypes from geographically distinct insect populations which were shown to exhibit some degree of phenotypic variation were recovered and genetically characterized. In droplet bioassays using seven CrleGV isolates against five T. leucotreta populations, some isolates were found to show higher virulence to some host populations than others. There were marked differences in the LD50 values of isolates and the number of occlusion bodies required per larva ranged between 0.79 and 3.12. The significance of these findings with respect to the application of CrleGV biopesticides and the management of resistance is discussed.
- Full Text:
- Date Issued: 2016
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
- Full Text:
- Date Issued: 2021-10-29
- 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
- Full Text:
- Date Issued: 2021-10-29
Studies on existing and new isolates of Cryptophlebia leucotreta granulovirus (CrleGV) on Thaumatotibia leucotreta populations from a range of geographic regions in South Africa
- Authors: Opoku-Debrah, John Kwadwo
- Date: 2012
- Subjects: Cryptophlebia leucotreta Cryptophlebia leucotreta -- South Africa Cryptophlebia leucotreta -- Biological control Cryptophlebia leucotreta -- Life cycles Baculoviruses Lepidoptera -- Biological control Tortricidae -- Biological control Microbial insecticides Pests -- Integrated control
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5778 , http://hdl.handle.net/10962/d1005466
- Description: Baculoviruses are arthropod-specific DNA viruses that are highly virulent to most lepidopteran insects. Their host specificity and compatibility with IPM programmes has enabled their usage as safe microbial insecticides (biopesticides). Two baculovirus-based biopesticides, Cryptogran and Cryptex, which have been formulated with Cryptophlebia leucotreta granulovirus (CrleGV) have been registered for the control of false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa and have been successfully incorporated into IPM programmes. However, several studies have indicated that insects can develop resistance to baculovirus-based biopesticide as was shown with field populations of codling moth (CM), Cydia pomonella (L.), which developed resistance to the biopesticide Cydia pomonella granulovirus (CpGV-M) in Europe. Other studies have shown that, under laboratory conditions, FCM populations differ in their susceptibility to Cryptogran and Cryptex. In order to investigate difference in susceptibility as well as protect against any future resistance by FCM to Cryptogran and Cryptex, a search for novel CrleGV-SA isolates from diseased insects from different geographic regions in South Africa was performed. Six geographic populations (Addo, Citrusdal, Marble Hall, Nelspruit, Baths and Mixed colonies) of FCM were established and maintained in the laboratory. Studies on the comparative biological performance based on pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and duration of life cycle of the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies revealed a low biological performance for the Citrusdal colony. This was attributed to the fact that FCM populations found in the Citrusdal area are not indigenous and may have been introduced from a very limited gene pool from another region. When insects from five colonies, excluding the Baths colony, were subjected to stress by overcrowding , a latent baculovirus resident in the Addo, Nelspruit, Citrusdal, Marble Hall and Mixed colonies was brought into an overt lethal state. Transmission electron micrographs revealed the presence of GV occlusion bodies (OBs) in diseased insects. DNA profiles obtained by single restriction endonuclease analysis of viral genomic DNA using BamH 1, Sa/1, Xba1 , Pst1, Xh01 , Kpn1, Hindlll and EcoR1 revealed five CrleGV-SA isolates latent within the insect populations. The new isolates were named CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl, CrleGVSA Nels and CrleGV-SA Mix isolates. The novelty of the five CrleGV-SA isolates was confirmed by the presence of unique submolar bands, indicating that each isolate was genetically different. PCR amplification and sequencing of the granulin and egt genes from the five isolates revealed several single nucleotide polymorph isms (SNPs) which, in some cases, resulted in amino acid substitutions. DNA profiles from RFLPs, as well as phylogenetic analysis based on granulin and egt sequencing showed the presence of two CrleGV-SA genome types for the CrleGV-SA isolate. Cryptex and CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl and CrleGV-SA Mix were placed as members of Group one CrleGV-SA, and Cryptogran and CrleGV-SA Nels isolate were placed into Group two CrleGV-SA. In droplet feeding bioassays, the median survival time (STso) for neonate larvae inoculated with Group one and two CrleGV-SA were determined to range from 80 - 88 hours (3.33 - 3.67 days), for all five colonies. LDso values for Group one and two CrleGV-SA against neonates from the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies varied between some populations and ranged from 0.80 - 3.12 OBs per larva, indicating some level of variation in host susceptibility. This is the first study reporting the existence of genetically distinct CrleGV baculovirus isolates infecting FCM in different geographical areas of South Africa. The results of this study have broad-ranging implications for our understanding of baculovirus-host interactions and for the application of baculovirus basedbiopesticides.
- Full Text:
- Date Issued: 2012
- Authors: Opoku-Debrah, John Kwadwo
- Date: 2012
- Subjects: Cryptophlebia leucotreta Cryptophlebia leucotreta -- South Africa Cryptophlebia leucotreta -- Biological control Cryptophlebia leucotreta -- Life cycles Baculoviruses Lepidoptera -- Biological control Tortricidae -- Biological control Microbial insecticides Pests -- Integrated control
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5778 , http://hdl.handle.net/10962/d1005466
- Description: Baculoviruses are arthropod-specific DNA viruses that are highly virulent to most lepidopteran insects. Their host specificity and compatibility with IPM programmes has enabled their usage as safe microbial insecticides (biopesticides). Two baculovirus-based biopesticides, Cryptogran and Cryptex, which have been formulated with Cryptophlebia leucotreta granulovirus (CrleGV) have been registered for the control of false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa and have been successfully incorporated into IPM programmes. However, several studies have indicated that insects can develop resistance to baculovirus-based biopesticide as was shown with field populations of codling moth (CM), Cydia pomonella (L.), which developed resistance to the biopesticide Cydia pomonella granulovirus (CpGV-M) in Europe. Other studies have shown that, under laboratory conditions, FCM populations differ in their susceptibility to Cryptogran and Cryptex. In order to investigate difference in susceptibility as well as protect against any future resistance by FCM to Cryptogran and Cryptex, a search for novel CrleGV-SA isolates from diseased insects from different geographic regions in South Africa was performed. Six geographic populations (Addo, Citrusdal, Marble Hall, Nelspruit, Baths and Mixed colonies) of FCM were established and maintained in the laboratory. Studies on the comparative biological performance based on pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and duration of life cycle of the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies revealed a low biological performance for the Citrusdal colony. This was attributed to the fact that FCM populations found in the Citrusdal area are not indigenous and may have been introduced from a very limited gene pool from another region. When insects from five colonies, excluding the Baths colony, were subjected to stress by overcrowding , a latent baculovirus resident in the Addo, Nelspruit, Citrusdal, Marble Hall and Mixed colonies was brought into an overt lethal state. Transmission electron micrographs revealed the presence of GV occlusion bodies (OBs) in diseased insects. DNA profiles obtained by single restriction endonuclease analysis of viral genomic DNA using BamH 1, Sa/1, Xba1 , Pst1, Xh01 , Kpn1, Hindlll and EcoR1 revealed five CrleGV-SA isolates latent within the insect populations. The new isolates were named CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl, CrleGVSA Nels and CrleGV-SA Mix isolates. The novelty of the five CrleGV-SA isolates was confirmed by the presence of unique submolar bands, indicating that each isolate was genetically different. PCR amplification and sequencing of the granulin and egt genes from the five isolates revealed several single nucleotide polymorph isms (SNPs) which, in some cases, resulted in amino acid substitutions. DNA profiles from RFLPs, as well as phylogenetic analysis based on granulin and egt sequencing showed the presence of two CrleGV-SA genome types for the CrleGV-SA isolate. Cryptex and CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl and CrleGV-SA Mix were placed as members of Group one CrleGV-SA, and Cryptogran and CrleGV-SA Nels isolate were placed into Group two CrleGV-SA. In droplet feeding bioassays, the median survival time (STso) for neonate larvae inoculated with Group one and two CrleGV-SA were determined to range from 80 - 88 hours (3.33 - 3.67 days), for all five colonies. LDso values for Group one and two CrleGV-SA against neonates from the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies varied between some populations and ranged from 0.80 - 3.12 OBs per larva, indicating some level of variation in host susceptibility. This is the first study reporting the existence of genetically distinct CrleGV baculovirus isolates infecting FCM in different geographical areas of South Africa. The results of this study have broad-ranging implications for our understanding of baculovirus-host interactions and for the application of baculovirus basedbiopesticides.
- Full Text:
- Date Issued: 2012
Yeast-baculovirus synergism: investigating mixed infections for improved management of the false codling moth, Thaumatotibia leucotreta
- Authors: Van der Merwe, Marcel
- 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
- Authors: Van der Merwe, Marcel
- 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
Gene expression analysis of Thaumatotibia leucotreta in response to the Cryptophlebia leucotreta granulovirus
- Authors: Ridgeway, Jaryd Antony
- Date: 2015
- Subjects: Gene expression , Insects -- Viruses , Tortricidae -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5931 , http://hdl.handle.net/10962/d1017809
- Description: Gene expression studies provide baseline information on the interactions of insects with their environment. Despite the importance of this information, limited gene expression data are available for most insect pests, including the family Tortricidae (Lepidoptera), which includes Thaumatotibia leucotreta (Meyr), an important agricultural pest in Africa. Because T. leucotreta can be controlled successfully by a granulovirus, this system is a good model for exploring insect-virus susceptibility. The main aim of this study was to investigate gene expression of T. leucotreta in responce to virus infection. However, before pursuing this aim, two objectives required completion. First, the most suitable RNA extraction method for insects needed to be determined and second, the most suitable reference genes for qPCR for Tortricidae pests needed to be identified. Once these objectives were accomplished, the response of T. leucotreta to its granulovirus was evaluated at different temperatures and points after infection.Four RNA extraction methods, the RNeasy® Mini Kit, SV Total RNA isolation system, TRIzol® reagent, and a CTAB-based method, were compared using two beetle and two moth species, including T. leucotreta. The quality of extracted RNA was similar for all four species for all extraction methods. Based on several criteria, the best RNA extraction method was the SV Total RNA isolation system. Six candidate reference genes were evaluated for qPCR using different tissue types of T. leucotreta and two other Tortricidae pests. Additionally, reference genes were evaluated for T. leucotreta with and without its granulovirus at different temperatures. Reference gene stability was found to be dependent on species and tissue type. Overall the most suitable combination of reference genes for T. leucotreta were α-actin, arginine kinase and elongation factor 1-α.Gene expression of T. leucotreta in response to granulovirus infection at different temperatures and intervals after infection was evaluated by qPCR using 13 target genes associated with the infection process. Most genes were down-regulated after 24 and 48 h.p.i. However, after 72 h.p.i most genes were up-regulated. The same trend was observed at different temperatures, where most genes were down-regulated at 15°C and 25°C but up-regulated at 35°C. These results show that there is a dynamic gene expression response in T. leucotreta due to granulovirus infection under different conditions. Not only do these findings provide insight into the control of this tortricid pest, they also contribute further to our knowledge of insect-virus interactions.
- Full Text:
- Date Issued: 2015
- Authors: Ridgeway, Jaryd Antony
- Date: 2015
- Subjects: Gene expression , Insects -- Viruses , Tortricidae -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5931 , http://hdl.handle.net/10962/d1017809
- Description: Gene expression studies provide baseline information on the interactions of insects with their environment. Despite the importance of this information, limited gene expression data are available for most insect pests, including the family Tortricidae (Lepidoptera), which includes Thaumatotibia leucotreta (Meyr), an important agricultural pest in Africa. Because T. leucotreta can be controlled successfully by a granulovirus, this system is a good model for exploring insect-virus susceptibility. The main aim of this study was to investigate gene expression of T. leucotreta in responce to virus infection. However, before pursuing this aim, two objectives required completion. First, the most suitable RNA extraction method for insects needed to be determined and second, the most suitable reference genes for qPCR for Tortricidae pests needed to be identified. Once these objectives were accomplished, the response of T. leucotreta to its granulovirus was evaluated at different temperatures and points after infection.Four RNA extraction methods, the RNeasy® Mini Kit, SV Total RNA isolation system, TRIzol® reagent, and a CTAB-based method, were compared using two beetle and two moth species, including T. leucotreta. The quality of extracted RNA was similar for all four species for all extraction methods. Based on several criteria, the best RNA extraction method was the SV Total RNA isolation system. Six candidate reference genes were evaluated for qPCR using different tissue types of T. leucotreta and two other Tortricidae pests. Additionally, reference genes were evaluated for T. leucotreta with and without its granulovirus at different temperatures. Reference gene stability was found to be dependent on species and tissue type. Overall the most suitable combination of reference genes for T. leucotreta were α-actin, arginine kinase and elongation factor 1-α.Gene expression of T. leucotreta in response to granulovirus infection at different temperatures and intervals after infection was evaluated by qPCR using 13 target genes associated with the infection process. Most genes were down-regulated after 24 and 48 h.p.i. However, after 72 h.p.i most genes were up-regulated. The same trend was observed at different temperatures, where most genes were down-regulated at 15°C and 25°C but up-regulated at 35°C. These results show that there is a dynamic gene expression response in T. leucotreta due to granulovirus infection under different conditions. Not only do these findings provide insight into the control of this tortricid pest, they also contribute further to our knowledge of insect-virus interactions.
- Full Text:
- Date Issued: 2015
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
- 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
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
- 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
Production of Cydia pomonella granulovirus (CpGV) in a heteralogous host, Thaumatotibia Leucotreta (Meyrick) (False codling moth)
- Authors: Chambers, Craig Brian
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- South Africa , Codling moth -- South Africa , Apples -- Diseases and pests -- South Africa , Codling moth -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Biological pest control agents -- South Africa , Baculoviruses -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5935 , http://hdl.handle.net/10962/d1017906
- Description: Cydia pomonella (Linnaeus) (Family: Tortricidae), the codling moth, is considered one of the most significant pests of apples and pears worldwide, causing up to 80% crop loss in orchards if no control measures are applied. Cydia pomonella is oligophagous feeding on a number of alternate hosts including quince, walnuts, apricots, peaches, plums and nectarines. Historically the control of this pest has been achieved with the use of various chemical control strategies which have maintained pest levels below the economic threshold at a relatively low cost to the grower. However, there are serious concerns surrounding the use of chemical insecticides including the development of resistance in insect populations, the banning of various insecticides, regulations for lowering of the maximum residue level and employee and consumer safety. For this reason, alternate measures of control are slowly being adopted by growers such as mating disruption, cultural methods and the use of baculovirus biopesticides as part of integrated pest management programmes. The reluctance of growers to accept baculovirus or other biological control products in the past has been due to questionable product quality and inconsistencies in their field performance. Moreover, the development and application of biological control products is more costly than the use of chemical alternatives. Baculoviruses are arthropod specific viruses that are highly virulent to a number of lepidopteran species. Due to the virulence and host specificity of baculoviruses, Cydia pomonella granulovirus has been extensively and successfully used as part of integrated pest management systems for the control of C. pomonella in Europe and around the world, including South Africa. Commercial formulations have been typically based on the Mexican strain of CpGV. However due to long-term multiple applications of CpGV and the reliance on CpGV in organic farming practices in Europe, resistance to the CpGV-M strain has developed in a number of field populations of C. pomonella. This study aimed to identify and characterize novel isolates of CpGV in South Africa and compare their virulence with the commercial standard CpGV-M. Secondly, since C. pomonella is difficult to culture on a large scale, an alternate method of CpGV production was investigated in order to determine if CpGV could be produced more efficiently and at a reduced cost without negatively impacting the quality of the product. Several isolates of CpGV were recovered either from field collected larvae or from a laboratory-reared C. pomonella colony. Characterisation of DNA profiles using a variety of restriction enzymes revealed that only a single isolate, CpGV-SA, was genetically different from the Mexican strain of the virus used in the commercially available CpGV based products in South Africa. In dose-response bioassays using CpGV-SA, LC₅₀ and LC₉₀ values for neonate C. pomonella larvae were 3.18 x 10³ OBs/ml and 7.33 x 10⁴ respectively. A comparison of these values with those of CpGV-M indicated no significant difference in the virulence of the two isolates under laboratory conditions. This is a first report of a genetically distinct CpGV isolate in South Africa. The biological activity and novelty of CpGV-SA makes this isolate a potentially important tool for CpGV resistance management in South Africa. In order to justify production of CpGV in an alternative host, studies on the comparative biological performance of C. pomonella and T. leucotreta based on oviposition, time to hatch, larval developmental times and rearing efficiency as well as production costs were performed. Thaumatotibia leucotreta was found to be more fecund and to have significantly shorter egg and larval developmental times. In addition, larval production per unit of artificial diet was significantly higher than for C. pomonella. This resulted in T. leucotreta being more cost effective to produce with implications for reduced insectary space, sanitation practices as well as the labour component of production. Virus yield data generated by inoculation both C. pomonella and T. leucotreta with nine concentrations of CpGV resulted in comparable virus yields, justifying the continuation of the research into production of CpGV in T. leucotreta. It was important to determine the LC and LT values required for mass production of CpGV in late instar T. leucotreta larvae. Dose- and time-response bioassays with CpGV-M were conducted on artificial diet to determine these values. Fourth instar LC₅₀ and LC₉₀ values were 5.96 x 10³ OBs/ml and 1.64 x 10⁵ OBs/ml respectively. LT50 and LT90 values were 81.10 hours and 88.58 hours respectively. Fifth instar LC₅₀ and LC₉₀ values were 6.88 x 10⁴ OBs/ml and 9.78 x 10⁶ OBs/ml respectively. LT₅₀ and LT₉₀ values were 111.56 hours and 137.57 hours respectively. Virus produced in fourth instar T. leucotreta larvae was bioassayed against C. pomonella neonate larvae and compared to CpGV-M to establish if production in the heterologous host negatively affected the virulence of the isolate. No significant difference in virulence was observed between virus produced in T. leucotreta and that produced in C. pomonella. The data generated in the bioassays was used in CpGV mass production trials to evaluate production. All production methods tested produced acceptable virus yields. To examine the quality of the virus product, genomic DNA was extracted from larval cadavers and subjected to REN analysis with HindIII. The resulting DNA profiles indicated that the virus product was contaminated with the homologous virus, CrleGV. Based on the above results, the use of T. leucotreta as an alternate host for the in vivo production of CpGV on a commercial basis is not at this stage viable and requires further investigation before this production methodology can be reliable used to produce CpGV. However, this study has shown that CpGV can be produced in a homologous host, T. leucotreta and significant strides have been made towards developing a set of quality control standards that are essential for further development of successful production methodology. Finally a novel isolate of CpGV has been identified with comparable virulence to the CpGV-M. This is an important finding as it has broad reaching implications for resistance management of CpGV products in South Africa.
- Full Text:
- Date Issued: 2015
- Authors: Chambers, Craig Brian
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- South Africa , Codling moth -- South Africa , Apples -- Diseases and pests -- South Africa , Codling moth -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Biological pest control agents -- South Africa , Baculoviruses -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5935 , http://hdl.handle.net/10962/d1017906
- Description: Cydia pomonella (Linnaeus) (Family: Tortricidae), the codling moth, is considered one of the most significant pests of apples and pears worldwide, causing up to 80% crop loss in orchards if no control measures are applied. Cydia pomonella is oligophagous feeding on a number of alternate hosts including quince, walnuts, apricots, peaches, plums and nectarines. Historically the control of this pest has been achieved with the use of various chemical control strategies which have maintained pest levels below the economic threshold at a relatively low cost to the grower. However, there are serious concerns surrounding the use of chemical insecticides including the development of resistance in insect populations, the banning of various insecticides, regulations for lowering of the maximum residue level and employee and consumer safety. For this reason, alternate measures of control are slowly being adopted by growers such as mating disruption, cultural methods and the use of baculovirus biopesticides as part of integrated pest management programmes. The reluctance of growers to accept baculovirus or other biological control products in the past has been due to questionable product quality and inconsistencies in their field performance. Moreover, the development and application of biological control products is more costly than the use of chemical alternatives. Baculoviruses are arthropod specific viruses that are highly virulent to a number of lepidopteran species. Due to the virulence and host specificity of baculoviruses, Cydia pomonella granulovirus has been extensively and successfully used as part of integrated pest management systems for the control of C. pomonella in Europe and around the world, including South Africa. Commercial formulations have been typically based on the Mexican strain of CpGV. However due to long-term multiple applications of CpGV and the reliance on CpGV in organic farming practices in Europe, resistance to the CpGV-M strain has developed in a number of field populations of C. pomonella. This study aimed to identify and characterize novel isolates of CpGV in South Africa and compare their virulence with the commercial standard CpGV-M. Secondly, since C. pomonella is difficult to culture on a large scale, an alternate method of CpGV production was investigated in order to determine if CpGV could be produced more efficiently and at a reduced cost without negatively impacting the quality of the product. Several isolates of CpGV were recovered either from field collected larvae or from a laboratory-reared C. pomonella colony. Characterisation of DNA profiles using a variety of restriction enzymes revealed that only a single isolate, CpGV-SA, was genetically different from the Mexican strain of the virus used in the commercially available CpGV based products in South Africa. In dose-response bioassays using CpGV-SA, LC₅₀ and LC₉₀ values for neonate C. pomonella larvae were 3.18 x 10³ OBs/ml and 7.33 x 10⁴ respectively. A comparison of these values with those of CpGV-M indicated no significant difference in the virulence of the two isolates under laboratory conditions. This is a first report of a genetically distinct CpGV isolate in South Africa. The biological activity and novelty of CpGV-SA makes this isolate a potentially important tool for CpGV resistance management in South Africa. In order to justify production of CpGV in an alternative host, studies on the comparative biological performance of C. pomonella and T. leucotreta based on oviposition, time to hatch, larval developmental times and rearing efficiency as well as production costs were performed. Thaumatotibia leucotreta was found to be more fecund and to have significantly shorter egg and larval developmental times. In addition, larval production per unit of artificial diet was significantly higher than for C. pomonella. This resulted in T. leucotreta being more cost effective to produce with implications for reduced insectary space, sanitation practices as well as the labour component of production. Virus yield data generated by inoculation both C. pomonella and T. leucotreta with nine concentrations of CpGV resulted in comparable virus yields, justifying the continuation of the research into production of CpGV in T. leucotreta. It was important to determine the LC and LT values required for mass production of CpGV in late instar T. leucotreta larvae. Dose- and time-response bioassays with CpGV-M were conducted on artificial diet to determine these values. Fourth instar LC₅₀ and LC₉₀ values were 5.96 x 10³ OBs/ml and 1.64 x 10⁵ OBs/ml respectively. LT50 and LT90 values were 81.10 hours and 88.58 hours respectively. Fifth instar LC₅₀ and LC₉₀ values were 6.88 x 10⁴ OBs/ml and 9.78 x 10⁶ OBs/ml respectively. LT₅₀ and LT₉₀ values were 111.56 hours and 137.57 hours respectively. Virus produced in fourth instar T. leucotreta larvae was bioassayed against C. pomonella neonate larvae and compared to CpGV-M to establish if production in the heterologous host negatively affected the virulence of the isolate. No significant difference in virulence was observed between virus produced in T. leucotreta and that produced in C. pomonella. The data generated in the bioassays was used in CpGV mass production trials to evaluate production. All production methods tested produced acceptable virus yields. To examine the quality of the virus product, genomic DNA was extracted from larval cadavers and subjected to REN analysis with HindIII. The resulting DNA profiles indicated that the virus product was contaminated with the homologous virus, CrleGV. Based on the above results, the use of T. leucotreta as an alternate host for the in vivo production of CpGV on a commercial basis is not at this stage viable and requires further investigation before this production methodology can be reliable used to produce CpGV. However, this study has shown that CpGV can be produced in a homologous host, T. leucotreta and significant strides have been made towards developing a set of quality control standards that are essential for further development of successful production methodology. Finally a novel isolate of CpGV has been identified with comparable virulence to the CpGV-M. This is an important finding as it has broad reaching implications for resistance management of CpGV products in South Africa.
- Full Text:
- Date Issued: 2015
Assessment of pheromone specificity in Thaumatotibia leucotreta (Meyrick) populations with focus on pest monitoring and the regional rollout of the sterile insect technique in citrus
- Authors: Joubert, Francois D
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Pheromone traps , Citrus -- Diseases and pests -- South Africa , Cryptophlebia leucotreta -- Contol , Cryptophlebia leucotreta -- Biological control
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/60665 , vital:27812
- Description: False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. It is recognized by several markets as a phytosanitary organism and the efficient control of this pest is now more important than ever. The pheromone communication between the male and female moths has been exploited in order to control FCM through the sterile insect technique (SIT). The sterilized males used for all SIT programmes across South Africa come from a colony that originates from wild material collected from the Citrusdal area of the Western Cape Province. The aim of this study was to determine if any differences in attractiveness of females to males exist between different geographical populations of FCM and if so what impact this would have on the male’s ability to locate females from other populations via the volatile sex pheromone released by the female. Laboratory trials with Y-tube olfactometers and flight tunnels tested the attraction of male moths to virgin females, but did not yield any consistent results. Field experiments were conducted with sterile male Citrusdal moths released and recaptured in yellow delta traps in two separate trials. For one trial, the traps were baited with live virgin females from five different geographical populations including Addo, Nelspruit, Marble Hall, Citrusdal and the Old colony, which is a mixture of several populations. For the other trial traps were baited with various synthetic pheromone blends including three regional blends which included South Africa, Ivory Coast and Malawi and three commercial blends including Pherolure, Isomate and Checkmate. For the virgin female trial the Citrusdal males showed a significant preference for females from their own population. There was also a significant difference in the recaptures from the different synthetic pheromones. The South African blend was the most attractive of all the regional and commercial blends. A cross-mating trial was also conducted under laboratory conditions in petri dishes with five different FCM populations including Citrusdal, Addo, Marble Hall, Nelspruit and Old (mixed origin). Females produced more eggs when mated with males from the same population for the Addo, Marble Hall, Nelspruit and Old (mixed origin) populations. The only case in which this was statistically significant was for the Marble Hall population. All the crosses produced viable eggs and the origin of the male or female did not influence egg hatch. The results from this study may lead to improvements in both the control and monitoring of FCM populations. The control methods include mating disruption, attract-and-kill and SIT. Tailoring these methods for a specific growing area with a pheromone blend originating from the area or releasing sterile moths from a colony that originates from the area may optimize the available monitoring and control options.
- Full Text:
- Date Issued: 2018
- Authors: Joubert, Francois D
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Pheromone traps , Citrus -- Diseases and pests -- South Africa , Cryptophlebia leucotreta -- Contol , Cryptophlebia leucotreta -- Biological control
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/60665 , vital:27812
- Description: False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. It is recognized by several markets as a phytosanitary organism and the efficient control of this pest is now more important than ever. The pheromone communication between the male and female moths has been exploited in order to control FCM through the sterile insect technique (SIT). The sterilized males used for all SIT programmes across South Africa come from a colony that originates from wild material collected from the Citrusdal area of the Western Cape Province. The aim of this study was to determine if any differences in attractiveness of females to males exist between different geographical populations of FCM and if so what impact this would have on the male’s ability to locate females from other populations via the volatile sex pheromone released by the female. Laboratory trials with Y-tube olfactometers and flight tunnels tested the attraction of male moths to virgin females, but did not yield any consistent results. Field experiments were conducted with sterile male Citrusdal moths released and recaptured in yellow delta traps in two separate trials. For one trial, the traps were baited with live virgin females from five different geographical populations including Addo, Nelspruit, Marble Hall, Citrusdal and the Old colony, which is a mixture of several populations. For the other trial traps were baited with various synthetic pheromone blends including three regional blends which included South Africa, Ivory Coast and Malawi and three commercial blends including Pherolure, Isomate and Checkmate. For the virgin female trial the Citrusdal males showed a significant preference for females from their own population. There was also a significant difference in the recaptures from the different synthetic pheromones. The South African blend was the most attractive of all the regional and commercial blends. A cross-mating trial was also conducted under laboratory conditions in petri dishes with five different FCM populations including Citrusdal, Addo, Marble Hall, Nelspruit and Old (mixed origin). Females produced more eggs when mated with males from the same population for the Addo, Marble Hall, Nelspruit and Old (mixed origin) populations. The only case in which this was statistically significant was for the Marble Hall population. All the crosses produced viable eggs and the origin of the male or female did not influence egg hatch. The results from this study may lead to improvements in both the control and monitoring of FCM populations. The control methods include mating disruption, attract-and-kill and SIT. Tailoring these methods for a specific growing area with a pheromone blend originating from the area or releasing sterile moths from a colony that originates from the area may optimize the available monitoring and control options.
- Full Text:
- Date Issued: 2018
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
- 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
The isolation and genetic characterisation of a novel alphabaculovirus for the microbial control of Cryptophlebia peltastica and closely related tortricid pests
- Authors: Marsberg, Tamryn
- Date: 2017
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/59292 , vital:27543
- Description: Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) is an economically damaging pest of litchis and macadamias in South Africa. Cryptophlebia peltastica causes both pre- and post-harvest damage to litchis, reducing overall yields and thus classifying the pest as a phytosanitary risk. Various control methods have been implemented against C. peltastica in an integrated pest management programme. These control methods include chemical control, cultural control and biological control. However, these methods have not yet provided satisfactory control as of yet. As a result, an alternative control option needs to be identified and implemented into the IPM programme. An alternative method of control that has proved successful in other agricultural sectors and not yet implemented in the control of C. peltastica is that of microbial control, specifically the use of baculovirus biopesticides. This study aimed to isolate and characterise a novel baculovirus from a laboratory culture of C. peltastica that could be used as a commercially available baculovirus biopesticide. In order to isolate a baculovirus a laboratory culture of C. peltastica was successfully established at Rhodes University, Grahamstown, South Africa. This is the first time a laboratory culture of C. peltastica has been established. This allowed for various biological aspects of the pest to be determined, which included: length of the life cycle, fecundity and time to oviposition, egg and larval development and percentage hatch. The results obtained from these studies found that the biology of C. peltastica was similar to that of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). Once the laboratory culture had reached high densities, larvae showing symptoms of baculovirus infection were observed. Symptomatic larvae were collected and examined for the presence of a baculovirus. An alphabaculovirus (NPV) was successfully isolated and morphologically identified using purified OBs that were sectioned and observed by transmission electron microscopy. This was then confirmed by amplifying the polyhedrin gene region using degenerate primers. A BLAST analysis found a 93% similarity to a partial polyhedrin gene sequence to be that of Epinotia granitalis (Butler) (Lepidoptera: Tortricidae). The alphabaculovirus was then genetically characterised by generating restriction profiles and sequencing the whole genome. Due to the novelty of the virus, no comparison could be made. The biological activity of the alphabaculovirus was then tested against C. peltastica and two closely related Tortricidae pests: T. leucotreta and Cydiapomonella (Linnaeous) (Lepidoptera: Tortricidae). The alphabaculovirus was highly virulent against all three species. The lethal concentrations (LC50 and LC90) for the virus against C. peltastica was 8.19 x 103 and 3.33 x 105 OBs/ml. The LC50 and LC90 for T. leucotreta was 2.29 x 103 and 9.97 x 104 OBs/ml, respectively and C. pomonella had a LC50 of 1.43 x 103 OBs/ml and LC90 1.26 x 104 OBs/ml. The virus was particularly virulent against T. leucotreta and C. pomonella as compared to C. peltastica. The biological activity of the alphabaculovirus was also tested against CpGV resistant European C. pomonella. From the results it was observed that the virus had the ability to overcome the resistance in C. pomonella and could potentially be used in the resistance management of C. pomonella. With the successful biological activity results obtained from this study, preliminary investigation were made into the mass production of the alphabaculovirus using both the in vivo and in vitro production methods. For in vivo production both the homologous host (C. peltastica) and a heterologous host (T leucotreta) were investigated. Preliminary studies focused on determining the biological activity in fifth instars of both hosts. Fifth instar LC50 and LC90 values for C. peltastica were 3.43 x 103 and 1.11 x 107 OBs/ml and for T. leucotreta the LC50 and LC90 values were 2.53 x 103 and 8.82 x 106 OBs/ml, respectively. The average yield of virus produced in each species was also determined. Cryptophlebia peltastica had the highest viral yield of 5.37 x 1010 OBs/larva and 2.93 x 1010 OBs/larva for T. leucotreta. The results obtained, from the preliminary investigation concluded that the virus could be produced in vivo in both C. peltastica and T. leucotreta, however further research is required into the mass production in both hosts. The in vitro production of the virus was also considered and the susceptibility of the virus was tested against the C. pomonella cell line, Cp14R. After infection of the Cp14R cells with budded virus collected from fifth instar C. peltastica larvae, OBs could be observed after three days. Thus, the alphabaculovirus is susceptible to the Cp14R cell line, thus has the potential to be produced in vitro and further characterised. This study is the first to report of the identification and characterisation of a novel alphabaculovirus isolated from a laboratory reared culture of C. peltastica and the potential for it to be commercially developed into a bipoesticide and used against Tortricidae pests.
- Full Text:
- Date Issued: 2017
- Authors: Marsberg, Tamryn
- Date: 2017
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/59292 , vital:27543
- Description: Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) is an economically damaging pest of litchis and macadamias in South Africa. Cryptophlebia peltastica causes both pre- and post-harvest damage to litchis, reducing overall yields and thus classifying the pest as a phytosanitary risk. Various control methods have been implemented against C. peltastica in an integrated pest management programme. These control methods include chemical control, cultural control and biological control. However, these methods have not yet provided satisfactory control as of yet. As a result, an alternative control option needs to be identified and implemented into the IPM programme. An alternative method of control that has proved successful in other agricultural sectors and not yet implemented in the control of C. peltastica is that of microbial control, specifically the use of baculovirus biopesticides. This study aimed to isolate and characterise a novel baculovirus from a laboratory culture of C. peltastica that could be used as a commercially available baculovirus biopesticide. In order to isolate a baculovirus a laboratory culture of C. peltastica was successfully established at Rhodes University, Grahamstown, South Africa. This is the first time a laboratory culture of C. peltastica has been established. This allowed for various biological aspects of the pest to be determined, which included: length of the life cycle, fecundity and time to oviposition, egg and larval development and percentage hatch. The results obtained from these studies found that the biology of C. peltastica was similar to that of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). Once the laboratory culture had reached high densities, larvae showing symptoms of baculovirus infection were observed. Symptomatic larvae were collected and examined for the presence of a baculovirus. An alphabaculovirus (NPV) was successfully isolated and morphologically identified using purified OBs that were sectioned and observed by transmission electron microscopy. This was then confirmed by amplifying the polyhedrin gene region using degenerate primers. A BLAST analysis found a 93% similarity to a partial polyhedrin gene sequence to be that of Epinotia granitalis (Butler) (Lepidoptera: Tortricidae). The alphabaculovirus was then genetically characterised by generating restriction profiles and sequencing the whole genome. Due to the novelty of the virus, no comparison could be made. The biological activity of the alphabaculovirus was then tested against C. peltastica and two closely related Tortricidae pests: T. leucotreta and Cydiapomonella (Linnaeous) (Lepidoptera: Tortricidae). The alphabaculovirus was highly virulent against all three species. The lethal concentrations (LC50 and LC90) for the virus against C. peltastica was 8.19 x 103 and 3.33 x 105 OBs/ml. The LC50 and LC90 for T. leucotreta was 2.29 x 103 and 9.97 x 104 OBs/ml, respectively and C. pomonella had a LC50 of 1.43 x 103 OBs/ml and LC90 1.26 x 104 OBs/ml. The virus was particularly virulent against T. leucotreta and C. pomonella as compared to C. peltastica. The biological activity of the alphabaculovirus was also tested against CpGV resistant European C. pomonella. From the results it was observed that the virus had the ability to overcome the resistance in C. pomonella and could potentially be used in the resistance management of C. pomonella. With the successful biological activity results obtained from this study, preliminary investigation were made into the mass production of the alphabaculovirus using both the in vivo and in vitro production methods. For in vivo production both the homologous host (C. peltastica) and a heterologous host (T leucotreta) were investigated. Preliminary studies focused on determining the biological activity in fifth instars of both hosts. Fifth instar LC50 and LC90 values for C. peltastica were 3.43 x 103 and 1.11 x 107 OBs/ml and for T. leucotreta the LC50 and LC90 values were 2.53 x 103 and 8.82 x 106 OBs/ml, respectively. The average yield of virus produced in each species was also determined. Cryptophlebia peltastica had the highest viral yield of 5.37 x 1010 OBs/larva and 2.93 x 1010 OBs/larva for T. leucotreta. The results obtained, from the preliminary investigation concluded that the virus could be produced in vivo in both C. peltastica and T. leucotreta, however further research is required into the mass production in both hosts. The in vitro production of the virus was also considered and the susceptibility of the virus was tested against the C. pomonella cell line, Cp14R. After infection of the Cp14R cells with budded virus collected from fifth instar C. peltastica larvae, OBs could be observed after three days. Thus, the alphabaculovirus is susceptible to the Cp14R cell line, thus has the potential to be produced in vitro and further characterised. This study is the first to report of the identification and characterisation of a novel alphabaculovirus isolated from a laboratory reared culture of C. peltastica and the potential for it to be commercially developed into a bipoesticide and used against Tortricidae pests.
- Full Text:
- Date Issued: 2017
Genetic analysis and field application of a UV-tolerant strain of CrleGV for improved control of Thaumatotibia leucotreta
- Authors: Bennett, Tahnee Tashia
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents , Ultraviolet radiation , Oligonucleotides
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362741 , vital:65358
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), also known as false codling moth (FCM), is indigenous to sub-Saharan Africa. Thaumatotibia leucotreta has been controlled through an integrated pest management (IPM) programme, which includes chemical control, sterile insect technique (SIT), cultural and biological control. As part of the biological control, a key component is the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA). Currently, CryptogranTM, a commercial formulation of CrleGV, is the preferred product to use in South Africa for the control of T. leucotreta. The registration of the biopesticide Cryptogran (River bioscience, South Africa) was established after conducting extensive field trials with CrleGV-SA. One of the major factors affecting the baculovirus efficacy in the field is UV irradiation. A UV-tolerant Cryptophlebia leucotreta granulovirus (CrleGV-SA-C5) isolate was isolated after consecutive cycles of UV exposure. This UV-tolerant isolate is genetically distinct from the CrleGV-SA isolate. The CrleGV-SA-C5 isolate has the potential as a biological control agent. The control of T. leucotreta in South Africa could be improved by the development of novel isolates into new biopesticide formulations. To date, there has not been any field trials conducted on the CrleGV-SA-C5 isolate. Therefore, it is important to determine the biological and genetic stability of this isolate and to conduct field trials with CrleGV-SA- C5 to test the efficacy of the isolate before possible production into a biopesticide. A de novo assembly was conducted to reassemble the genome of CrleGV-SA-C5 which was followed by a sequence comparison with the CrleGV-SA genome. The identification of SNPs, led to the design of oligonucleotides flanking the regions where the SNPs were detected. Polymerase chain reaction amplification of the target regions was conducted using the oligonucleotides. After sequence comparison, seven SNPs were detected and PCR amplification was successful using the three oligonucleotides, Pif-2, HypoP and Lef-8/HP. To differentiate between CrleGV-SA-C5 and CrleGV-SA genomes and confirm the presence of the SNPs, two methods of screening were conducted. The first was the construction of six plasmids, the plasmids contained the targeted pif-2, HypoP, and the Lef-8/HP insert regions from both the CrleGV-SA-C5 and CrleGV-SA genome region where the SNPs were identified, followed by sequencing. The Five recombinant plasmids, pC5_Pif-2, pSA_Pif-2, pC5_HypoP, pSA_HypoP, and pC5_Lef-8/HP were successfully sequenced. No amplicon was obtained for one of the plasmids used as template (pSA_Lef-8/HP) and therefore the PCR product used for cloning was sequenced instead. Sequence alignment confirmed the presence of four of the five targeted SNPs in the genome of the CrleGV-SA-C5 isolate. However, of these only one SNP (UV_7) rendered a suitable marker for the differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates as the SNPs, UV_2, UV_3 and UV_5, were also present in the CrleGV- SA sequences. The second screening method was a quantitative polymerase chain reaction (qPCR) melt curve analysis to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. qPCR melt curve analysis was done using the CrleGV-SA-C5 and CrleGV-SA HypoP PCR products. This technique was unable to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. However, this may be as a result of sequence data confirming that SNP UV_5 originally identified in the CrleGV-SA-C5 HypoP region was identical to the SNP at the same position in the CrleGV-SA HypoP region. Following the differentiation of the CrleGV-SA-C5 and CrleGV-SA isolates through two screening methods, the genetic integrity of the CrleGV-SA-C5 isolate after two virus bulk-ups was determined by PCR amplification of the target regions in the bulk-up virus followed by sequencing. Prior to virus bulk-up, surface dose bioassays were conducted on 4th instar larvae and LC50 and LC90 values of 4.01 x 106 OBs/ml and 8.75 x 109 OBs/ml respectively were obtained. The CrleGV-SA-C5 isolate was then bulked up in fourth instar T. leucotreta larvae using the LC90 value that was determined. Sequencing of the target regions from the CrleGV- SA-C5_BU2 (bulk-up 2) was conducted. Sequencing results confirmed the presence of the target SNPs in the CrleGV-SA-C5_BU2 genome. The UV-tolerance of the CrleGV-SA-C5 isolate in comparison to the CrleGV-SA isolate was evaluated by detached fruit bioassays under natural UV irradiation. Two detached fruit bioassays were set-up, a UV exposure and a non-UV exposure bioassay set-up. Three treatments were used for each bioassay set-up which were the viruses CrleGV-SA-C5 and CrleGV-SA and a ddH2O control. Statistical analysis indicated that there was no significant difference between the virus treatments in both the UV exposed detached fruit bioassay and the non-UV exposed detached fruit bioassay. This study is the second study to report on the de novo assembly of the CrleGV-SA-C5 and sequence comparison with the CrleGV-SA genome, and the first to report on the UV-tolerance of the CrleGV-SA-C5 isolate by detached fruit bioassays. Future work could involve further evaluation of intraspecific genetic variability in the CrleGV-SA-C5 isolate and to identify any additional SNPs present within the genome that can be used as suitable markers for differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates. It was recognised that it is required to conduct further detached fruit bioassays and field trials, but with improved protocols, for the efficacy and UV-tolerance of the CrleGV-SA-C5 isolate to be conclusively determined. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Bennett, Tahnee Tashia
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents , Ultraviolet radiation , Oligonucleotides
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362741 , vital:65358
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), also known as false codling moth (FCM), is indigenous to sub-Saharan Africa. Thaumatotibia leucotreta has been controlled through an integrated pest management (IPM) programme, which includes chemical control, sterile insect technique (SIT), cultural and biological control. As part of the biological control, a key component is the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA). Currently, CryptogranTM, a commercial formulation of CrleGV, is the preferred product to use in South Africa for the control of T. leucotreta. The registration of the biopesticide Cryptogran (River bioscience, South Africa) was established after conducting extensive field trials with CrleGV-SA. One of the major factors affecting the baculovirus efficacy in the field is UV irradiation. A UV-tolerant Cryptophlebia leucotreta granulovirus (CrleGV-SA-C5) isolate was isolated after consecutive cycles of UV exposure. This UV-tolerant isolate is genetically distinct from the CrleGV-SA isolate. The CrleGV-SA-C5 isolate has the potential as a biological control agent. The control of T. leucotreta in South Africa could be improved by the development of novel isolates into new biopesticide formulations. To date, there has not been any field trials conducted on the CrleGV-SA-C5 isolate. Therefore, it is important to determine the biological and genetic stability of this isolate and to conduct field trials with CrleGV-SA- C5 to test the efficacy of the isolate before possible production into a biopesticide. A de novo assembly was conducted to reassemble the genome of CrleGV-SA-C5 which was followed by a sequence comparison with the CrleGV-SA genome. The identification of SNPs, led to the design of oligonucleotides flanking the regions where the SNPs were detected. Polymerase chain reaction amplification of the target regions was conducted using the oligonucleotides. After sequence comparison, seven SNPs were detected and PCR amplification was successful using the three oligonucleotides, Pif-2, HypoP and Lef-8/HP. To differentiate between CrleGV-SA-C5 and CrleGV-SA genomes and confirm the presence of the SNPs, two methods of screening were conducted. The first was the construction of six plasmids, the plasmids contained the targeted pif-2, HypoP, and the Lef-8/HP insert regions from both the CrleGV-SA-C5 and CrleGV-SA genome region where the SNPs were identified, followed by sequencing. The Five recombinant plasmids, pC5_Pif-2, pSA_Pif-2, pC5_HypoP, pSA_HypoP, and pC5_Lef-8/HP were successfully sequenced. No amplicon was obtained for one of the plasmids used as template (pSA_Lef-8/HP) and therefore the PCR product used for cloning was sequenced instead. Sequence alignment confirmed the presence of four of the five targeted SNPs in the genome of the CrleGV-SA-C5 isolate. However, of these only one SNP (UV_7) rendered a suitable marker for the differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates as the SNPs, UV_2, UV_3 and UV_5, were also present in the CrleGV- SA sequences. The second screening method was a quantitative polymerase chain reaction (qPCR) melt curve analysis to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. qPCR melt curve analysis was done using the CrleGV-SA-C5 and CrleGV-SA HypoP PCR products. This technique was unable to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. However, this may be as a result of sequence data confirming that SNP UV_5 originally identified in the CrleGV-SA-C5 HypoP region was identical to the SNP at the same position in the CrleGV-SA HypoP region. Following the differentiation of the CrleGV-SA-C5 and CrleGV-SA isolates through two screening methods, the genetic integrity of the CrleGV-SA-C5 isolate after two virus bulk-ups was determined by PCR amplification of the target regions in the bulk-up virus followed by sequencing. Prior to virus bulk-up, surface dose bioassays were conducted on 4th instar larvae and LC50 and LC90 values of 4.01 x 106 OBs/ml and 8.75 x 109 OBs/ml respectively were obtained. The CrleGV-SA-C5 isolate was then bulked up in fourth instar T. leucotreta larvae using the LC90 value that was determined. Sequencing of the target regions from the CrleGV- SA-C5_BU2 (bulk-up 2) was conducted. Sequencing results confirmed the presence of the target SNPs in the CrleGV-SA-C5_BU2 genome. The UV-tolerance of the CrleGV-SA-C5 isolate in comparison to the CrleGV-SA isolate was evaluated by detached fruit bioassays under natural UV irradiation. Two detached fruit bioassays were set-up, a UV exposure and a non-UV exposure bioassay set-up. Three treatments were used for each bioassay set-up which were the viruses CrleGV-SA-C5 and CrleGV-SA and a ddH2O control. Statistical analysis indicated that there was no significant difference between the virus treatments in both the UV exposed detached fruit bioassay and the non-UV exposed detached fruit bioassay. This study is the second study to report on the de novo assembly of the CrleGV-SA-C5 and sequence comparison with the CrleGV-SA genome, and the first to report on the UV-tolerance of the CrleGV-SA-C5 isolate by detached fruit bioassays. Future work could involve further evaluation of intraspecific genetic variability in the CrleGV-SA-C5 isolate and to identify any additional SNPs present within the genome that can be used as suitable markers for differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates. It was recognised that it is required to conduct further detached fruit bioassays and field trials, but with improved protocols, for the efficacy and UV-tolerance of the CrleGV-SA-C5 isolate to be conclusively determined. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-10-14
The effect of orchard sanitation and predatory ants on the eclosion of the internal feeding pests and Oriental fruit fly, in South Africa
- Authors: Makitla, Tshepang
- Date: 2022-10-14
- Subjects: Orchards South Africa , Phytosanitation , Citrus Diseases and pests Biological control , Ants , Insects as biological pest control agents
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362927 , vital:65375
- Description: There are several pests of phytosanitary concern in the citrus industry in South Africa. Orchard sanitation can play an important role in suppressing the populations of these pests, however there are little data on the efficacy of sanitation techniques. Therefore, the current study investigated the effect of fruit disposal techniques and burying depths on the eclosion of the most important pests of citrus in South Africa, false codling moth Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Mediterranean fruit fly or Medfly Ceratitis capitata Wiedemann (Diptera: Tephritidae), Natal fruit fly Ceratitis rosa Karsh (Diptera: Tephritidae), and Oriental fruit fly Bactrocera dorsalis Hendel (Diptera: Tephritidae). Abscised C. sinensis fruits were inoculated with larvae of T. leucotreta, and eggs of C. capitata, C. rosa, and B. dorsalis, before being disposed as pulped, or whole, and buried at different depths (0 cm, 5 cm, 25 cm, and 50 cm). Abundance and richness of predatory ants were monitored using pitfall traps to ascertain their effect on the mortality of the immature stages of these pests. Ceratitis capitata and C. rosa failed to eclose from the inoculated fruits disposed at different depths, however, T. leucotreta and B. dorsalis adults did eclosed. Significantly fewer B. dorsalis eclosed from fruits that were pulped in comparison to eclosion where the fruit were left whole (F (3, 16) = 11.45, P < 0.01). Furthermore, depth of burial had a significant effect on the number of eclosed adults of Drosophila sp (F (3, 112) = 3.43, P < 0.01). Burying fruits at 50 cm suppressed the eclosion of all the internal feeding pests tested. Twenty-seven thousand seventy-three individual ants (Hymenoptera: Formicidae) were sampled from the same plots as used above, with at least 47% and 53% sampled from plots where pulped and whole C. sinensis fruits were disposed of, respectively. The ants were identified to morphospecies which included Pheidole1, Pheidole2, Formicinae1, Formicinae2, Formicinae3, and Myrmicinae1. The disposal of the inoculated C. sinensis fruits either as pulped or whole and burying at different depths significantly suppressed and/or delayed the eclosion of either of the tested internal feeding pests of citrus. Although, predacious ants were sampled from the same treatment plots they did not affect the survival or eclosion of the tested pests, and this could be attributed to the application of the slow toxic ant bait. Therefore, based on the observed results B. dorsalis adults showed the ability to eclose from 50 cm depth where fruit was either disposed as pulped or whole, thus, citrus farmers are advised to use hammer mill that will finely crush sanitised fruit, and/or bury fruit beyond 50 cm depth to prevent the adult od this pest from eclosing. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Makitla, Tshepang
- Date: 2022-10-14
- Subjects: Orchards South Africa , Phytosanitation , Citrus Diseases and pests Biological control , Ants , Insects as biological pest control agents
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362927 , vital:65375
- Description: There are several pests of phytosanitary concern in the citrus industry in South Africa. Orchard sanitation can play an important role in suppressing the populations of these pests, however there are little data on the efficacy of sanitation techniques. Therefore, the current study investigated the effect of fruit disposal techniques and burying depths on the eclosion of the most important pests of citrus in South Africa, false codling moth Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Mediterranean fruit fly or Medfly Ceratitis capitata Wiedemann (Diptera: Tephritidae), Natal fruit fly Ceratitis rosa Karsh (Diptera: Tephritidae), and Oriental fruit fly Bactrocera dorsalis Hendel (Diptera: Tephritidae). Abscised C. sinensis fruits were inoculated with larvae of T. leucotreta, and eggs of C. capitata, C. rosa, and B. dorsalis, before being disposed as pulped, or whole, and buried at different depths (0 cm, 5 cm, 25 cm, and 50 cm). Abundance and richness of predatory ants were monitored using pitfall traps to ascertain their effect on the mortality of the immature stages of these pests. Ceratitis capitata and C. rosa failed to eclose from the inoculated fruits disposed at different depths, however, T. leucotreta and B. dorsalis adults did eclosed. Significantly fewer B. dorsalis eclosed from fruits that were pulped in comparison to eclosion where the fruit were left whole (F (3, 16) = 11.45, P < 0.01). Furthermore, depth of burial had a significant effect on the number of eclosed adults of Drosophila sp (F (3, 112) = 3.43, P < 0.01). Burying fruits at 50 cm suppressed the eclosion of all the internal feeding pests tested. Twenty-seven thousand seventy-three individual ants (Hymenoptera: Formicidae) were sampled from the same plots as used above, with at least 47% and 53% sampled from plots where pulped and whole C. sinensis fruits were disposed of, respectively. The ants were identified to morphospecies which included Pheidole1, Pheidole2, Formicinae1, Formicinae2, Formicinae3, and Myrmicinae1. The disposal of the inoculated C. sinensis fruits either as pulped or whole and burying at different depths significantly suppressed and/or delayed the eclosion of either of the tested internal feeding pests of citrus. Although, predacious ants were sampled from the same treatment plots they did not affect the survival or eclosion of the tested pests, and this could be attributed to the application of the slow toxic ant bait. Therefore, based on the observed results B. dorsalis adults showed the ability to eclose from 50 cm depth where fruit was either disposed as pulped or whole, thus, citrus farmers are advised to use hammer mill that will finely crush sanitised fruit, and/or bury fruit beyond 50 cm depth to prevent the adult od this pest from eclosing. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-10-14
Using the larval parasitoid, Agathis bishopi (Nixon) (Hymenoptera: Braconidae), for early detection of false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) infested fruit
- Authors: Zimba, Kennedy Josaya
- Date: 2015
- Subjects: Cryptophlebia leucotreta , Citrus -- Diseases and pests -- South Africa , Pests -- Biological control -- South Africa , Cryptophlebia leucotreta -- Detection , Parasitoids -- Hosts , Braconidae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5919 , http://hdl.handle.net/10962/d1017186
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is one of the major citrus pests of economic importance for South Africa’s citrus industry. It is endemic to Africa, and therefore a phytosanitary pest with zero tolerance by most export markets. The cryptic nature of T. leucotreta makes visual inspection an inefficient method for detecting neonate larvae in fruit in the packhouse. Therefore, a more accurate method for sorting infested fruit at the packhouse, particularly for newly infested fruit could ensure market access. A recent study showed that fruit infested by T. leucotreta emit a chemical profile different from that of a healthy fruit. Several studies provide evidence that parasitoids locate their hosts feeding on fruit by exploiting the novel chemical profiles produced due to host herbivory. The aim of this study was to evaluate the potential of using the naturally occurring behaviour of a larval parasitoid Agathis bishopi (Nixon) (Hymenoptera: Braconidae) for detection of T. leucotreta infested fruit, by determining which compound in infested fruit is attractive to parasitoids. Ytube olfactometer and flight-tunnel bioassays with healthy and T. leucotreta infested fruit showed a significantly stronger response of A. bishopi female parasitoids to infested fruit. Among the volatile compounds associated with T. leucotreta infested fruit, D-limonene elicited the strongest attraction to A. bishopi female parasitoids. Attraction of mated A. bishopi female parasitoids to T. leucotreta infested fruit and D-limonene significantly increased after oviposition experience. Behavioural responses of A. bishopi female parasitoids that were associated with T. leucotreta infested fruit were investigated to determine which behaviours are distinct and interpretable. Probing and oviposition behaviours were the most noticeable and were only elicited on infested fruit when parasitoids contacted T. leucotreta frass, indicating that chemical compounds in frass are short-range cues used for final host location. Since production of D-limonene by fruit is elevated due to herbivory by different pests including mechanical injury on fruit, response of A. bishopi female parasitoids to compounds in frass offers a more specific and potentially useful mechanism for development of a detection system for T. leucotreta infested fruit. Chemical analysis of T. leucotreta frass and conditioning A. bishopi parasitoids to respond behaviourally to compounds in frass is proposed.
- Full Text:
- Date Issued: 2015
- Authors: Zimba, Kennedy Josaya
- Date: 2015
- Subjects: Cryptophlebia leucotreta , Citrus -- Diseases and pests -- South Africa , Pests -- Biological control -- South Africa , Cryptophlebia leucotreta -- Detection , Parasitoids -- Hosts , Braconidae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5919 , http://hdl.handle.net/10962/d1017186
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is one of the major citrus pests of economic importance for South Africa’s citrus industry. It is endemic to Africa, and therefore a phytosanitary pest with zero tolerance by most export markets. The cryptic nature of T. leucotreta makes visual inspection an inefficient method for detecting neonate larvae in fruit in the packhouse. Therefore, a more accurate method for sorting infested fruit at the packhouse, particularly for newly infested fruit could ensure market access. A recent study showed that fruit infested by T. leucotreta emit a chemical profile different from that of a healthy fruit. Several studies provide evidence that parasitoids locate their hosts feeding on fruit by exploiting the novel chemical profiles produced due to host herbivory. The aim of this study was to evaluate the potential of using the naturally occurring behaviour of a larval parasitoid Agathis bishopi (Nixon) (Hymenoptera: Braconidae) for detection of T. leucotreta infested fruit, by determining which compound in infested fruit is attractive to parasitoids. Ytube olfactometer and flight-tunnel bioassays with healthy and T. leucotreta infested fruit showed a significantly stronger response of A. bishopi female parasitoids to infested fruit. Among the volatile compounds associated with T. leucotreta infested fruit, D-limonene elicited the strongest attraction to A. bishopi female parasitoids. Attraction of mated A. bishopi female parasitoids to T. leucotreta infested fruit and D-limonene significantly increased after oviposition experience. Behavioural responses of A. bishopi female parasitoids that were associated with T. leucotreta infested fruit were investigated to determine which behaviours are distinct and interpretable. Probing and oviposition behaviours were the most noticeable and were only elicited on infested fruit when parasitoids contacted T. leucotreta frass, indicating that chemical compounds in frass are short-range cues used for final host location. Since production of D-limonene by fruit is elevated due to herbivory by different pests including mechanical injury on fruit, response of A. bishopi female parasitoids to compounds in frass offers a more specific and potentially useful mechanism for development of a detection system for T. leucotreta infested fruit. Chemical analysis of T. leucotreta frass and conditioning A. bishopi parasitoids to respond behaviourally to compounds in frass is proposed.
- Full Text:
- Date Issued: 2015
Geographic variation in the susceptibility of false colding Moth, Thaumatotibia Leucotreta, populations to a granulovirus (CrleGV-SA)
- Authors: Opoku-Debrah, John Kwadwo
- Date: 2008
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: vital:10586 , http://hdl.handle.net/10948/984 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Description: The false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a serious pest of citrus and other crops in Sub-Saharan Africa. The introduction of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) Cryptogran and Cryptex (biopesticides) has proven to be very effective in the control of FCM. However, markedly lower susceptibility of some codling moth (CM), Cydia pomonella (L.) populations to Cydia pomonella granulovirus (CpGV-M), another granulovirus product used in the control of CM’s in Europe have been reported. Genetic differences between FCM populations in South Africa have also been established. It is therefore possible that differences in the susceptibility of these geographically distinct FCM populations to CrleGV-SA might also exist. To investigate this phenomenon, a benchmark for pathogenecity was established. In continuation of previous work with Cryptogran against the 1st and 5th instar FCM larvae, dose-response relationships were established for all five larval instars of FCM. In surface dose-response bioassays, the LC50 values for the 2nd, 3rd and 4th instars were calculated to be 4.516 x 104, 1.662 x 105 and 2.205 x 106 occlusion bodies (OBs)/ml, respectively. The LC90 values for the 2nd, 3rd and 4th instars were calculated to be 4.287 x 106, 9.992 x 106 and 1.661 x 108 OBs/ml, respectively. Susceptibility to CrleGV-SA was found to decline with larval stage and increase with time of exposure. The protocol was used in guiding bioassays with field collected FCM larvae. Laboratory assays conducted with Cryptogran (at 1.661 x 108 OBs/ml) against field collected FCM larvae from Addo, Kirkwood, Citrusdal and Clanwilliam as well as a standard laboratory colony, showed a significant difference in pathogenecity in only one case. This significant difference was observed between 5th instars from the Addo colony and 5th instars from the other populations. Four geographically distinct FCM colonies from Addo, Citrusdal, Marble Hall and Nelspruit were also established. Since Cryptogran and Cryptex are always targeted against 1st instar FCM larvae in the field, further comparative laboratory assays were conducted with the Addo colony and an old laboratory colony. Cryptogran was significantly more pathogenic than Cryptex against both the Addo and the old colony. However, a high level of heterogeneity was observed in responses within each population.
- Full Text:
- Date Issued: 2008
- Authors: Opoku-Debrah, John Kwadwo
- Date: 2008
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: vital:10586 , http://hdl.handle.net/10948/984 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Description: The false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a serious pest of citrus and other crops in Sub-Saharan Africa. The introduction of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) Cryptogran and Cryptex (biopesticides) has proven to be very effective in the control of FCM. However, markedly lower susceptibility of some codling moth (CM), Cydia pomonella (L.) populations to Cydia pomonella granulovirus (CpGV-M), another granulovirus product used in the control of CM’s in Europe have been reported. Genetic differences between FCM populations in South Africa have also been established. It is therefore possible that differences in the susceptibility of these geographically distinct FCM populations to CrleGV-SA might also exist. To investigate this phenomenon, a benchmark for pathogenecity was established. In continuation of previous work with Cryptogran against the 1st and 5th instar FCM larvae, dose-response relationships were established for all five larval instars of FCM. In surface dose-response bioassays, the LC50 values for the 2nd, 3rd and 4th instars were calculated to be 4.516 x 104, 1.662 x 105 and 2.205 x 106 occlusion bodies (OBs)/ml, respectively. The LC90 values for the 2nd, 3rd and 4th instars were calculated to be 4.287 x 106, 9.992 x 106 and 1.661 x 108 OBs/ml, respectively. Susceptibility to CrleGV-SA was found to decline with larval stage and increase with time of exposure. The protocol was used in guiding bioassays with field collected FCM larvae. Laboratory assays conducted with Cryptogran (at 1.661 x 108 OBs/ml) against field collected FCM larvae from Addo, Kirkwood, Citrusdal and Clanwilliam as well as a standard laboratory colony, showed a significant difference in pathogenecity in only one case. This significant difference was observed between 5th instars from the Addo colony and 5th instars from the other populations. Four geographically distinct FCM colonies from Addo, Citrusdal, Marble Hall and Nelspruit were also established. Since Cryptogran and Cryptex are always targeted against 1st instar FCM larvae in the field, further comparative laboratory assays were conducted with the Addo colony and an old laboratory colony. Cryptogran was significantly more pathogenic than Cryptex against both the Addo and the old colony. However, a high level of heterogeneity was observed in responses within each population.
- Full Text:
- Date Issued: 2008
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
Agreement and coordination in XiTsonga, SeSotho and IsiXhosa: an optimality theoretic perspective
- Authors: Mitchley, Hazel
- Date: 2016
- Language: English
- Type: Thesis , Masters , MA
- Identifier: http://hdl.handle.net/10962/3423 , vital:20491
- Description: This thesis provides a unified Optimality Theoretic analysis of subject-verb agreement with coordinated preverbal subjects in three Southern Bantu languages: Xitsonga (S53), Sesotho (S33), and isiXhosa (S41). This analysis is then used to formulate a typology of agreement resolution strategies and the contexts which trigger them. Although some accounts in the Bantu literature suggest that agreement with coordinate structures is avoided by speakers (e.g. Schadeberg 1992, Voeltz 1971) especially when conjuncts are from different noun classes, I show that there is ample evidence to the contrary, and that the subject marker used is dependent on several factors, including (i) the [-HUMAN] specification on the conjuncts, (ii) whether the conjuncts are singular or plural, (iii) whether or not the conjuncts both carry the same noun class feature, and (iv) the order of the conjuncts. This thesis shows that there are various agreement resolution strategies which can beused: 1) agreement with the [+HUMAN] feature on the conjuncts, 2) agreement with the[-HUMAN] feature on the conjuncts, 3) agreement with the noun class feature on both conjuncts, 4) agreement with the noun class feature on the conjunct closest to the verb, and 5) agreement with the noun class feature on the conjunct furthest from the verb. Not all of these strategies are used by all languages, nor are these strategies interchangeable in the languages which do use them – instead, multiple factors conspire to trigger the use of a specific agreement strategy within a specific agreement featural context. I show that these effects can be captured using Optimality Theory (Prince and Smolensky 2004). The analysis makes use of seven constraints: RES#, MAX[+H], MAX[-H], DEP[-H], MAXNC, DEPNC, and AGREECLOSEST. The hierarchical ranking of these constraints not only accounts for the confinement of particular strategies to specific agreement featural contexts within a language, but also accounts for the cross-linguistic differences in the use of these strategies. I end off by examining the typological implications which follow from the OT analysis provided in this thesis.
- Full Text:
- Date Issued: 2016
- Authors: Mitchley, Hazel
- Date: 2016
- Language: English
- Type: Thesis , Masters , MA
- Identifier: http://hdl.handle.net/10962/3423 , vital:20491
- Description: This thesis provides a unified Optimality Theoretic analysis of subject-verb agreement with coordinated preverbal subjects in three Southern Bantu languages: Xitsonga (S53), Sesotho (S33), and isiXhosa (S41). This analysis is then used to formulate a typology of agreement resolution strategies and the contexts which trigger them. Although some accounts in the Bantu literature suggest that agreement with coordinate structures is avoided by speakers (e.g. Schadeberg 1992, Voeltz 1971) especially when conjuncts are from different noun classes, I show that there is ample evidence to the contrary, and that the subject marker used is dependent on several factors, including (i) the [-HUMAN] specification on the conjuncts, (ii) whether the conjuncts are singular or plural, (iii) whether or not the conjuncts both carry the same noun class feature, and (iv) the order of the conjuncts. This thesis shows that there are various agreement resolution strategies which can beused: 1) agreement with the [+HUMAN] feature on the conjuncts, 2) agreement with the[-HUMAN] feature on the conjuncts, 3) agreement with the noun class feature on both conjuncts, 4) agreement with the noun class feature on the conjunct closest to the verb, and 5) agreement with the noun class feature on the conjunct furthest from the verb. Not all of these strategies are used by all languages, nor are these strategies interchangeable in the languages which do use them – instead, multiple factors conspire to trigger the use of a specific agreement strategy within a specific agreement featural context. I show that these effects can be captured using Optimality Theory (Prince and Smolensky 2004). The analysis makes use of seven constraints: RES#, MAX[+H], MAX[-H], DEP[-H], MAXNC, DEPNC, and AGREECLOSEST. The hierarchical ranking of these constraints not only accounts for the confinement of particular strategies to specific agreement featural contexts within a language, but also accounts for the cross-linguistic differences in the use of these strategies. I end off by examining the typological implications which follow from the OT analysis provided in this thesis.
- Full Text:
- Date Issued: 2016