Evaluation of potential oviposition deterrents for false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae)
- Authors: Dambuza, Khalipha
- Date: 2023-10-13
- Subjects: Cryptophlebia leucotreta , Pests Integrated control , Semiochemicals , Agricultural pests Control , Oviposition , Essences and essential oils
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424479 , vital:72157
- Description: There has been extensive research on the use of semiochemicals as deterrents or true repellents in insect pest management, particularly in push-pull strategies. Much of this research has focused on pests of medical and veterinary importance and has been limited for agricultural pests. This means there is an opportunity to study use of deterrents to manage pests of agricultural importance. No study has been conducted on deterrents for false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a key phytosanitary pest in citrus orchards across South Africa. This study assessed FCM oviposition deterrence in botanicals (plants (n = 11) and essential oils (n = 15)), and some commercial pesticides (n = 7) used for FCM control in South Africa. All tested botanicals were selected based on an extensive literature review of plant compounds that have been reported to deter or repel lepidopteran pests. Choice and no-choice oviposition bioassays were conducted in complete darkness in a controlled environment room. Oranges treated with solutions/suspensions of potential oviposition deterrents were placed into a cage with gravid FCM females for four hours, with oviposition being recorded every hour. Of the 33 tested compounds, only eight significantly reduced FCM oviposition (P < 0.05) compared to the control in oviposition bioassays i.e. two essential oils (lavender and peppermint), two plant crude extracts (garlic and marigold), one fruit (Mango), and three commercial FCM insecticides (Delegate, Coragen, and Warlock). All identified oviposition deterrents, except for Mango, were further investigated for their ovicidal properties in concentration response bioassays, where all botanicals were identified to have dual action (both deterrent and ovicidal properties), as they significantly (P < 0.05) reduced FCM oviposition and egg hatch. Garlic was the most efficacious botanical whilst Warlock was the only commercial insecticide that did not show ovicidal activity (F = 41.17, P = 0.0622). Larval penetration of the host fruit was less than egg hatch for all tested compounds in concentration response bioassays. Oviposition, egg hatch, and larval penetration were all affected by concentration, with the higher concentrations being the most effective. The efficacy of these deterrent compounds should be further tested in semi-field and/or field trials, and they may have potential in FCM management as allomone dispensers or sprays. They can also be implemented in push-pull strategies where they can be used in conjunction with FCM attractants. Lastly, repellence studies should be conducted in absentia of the host fruit to determine whether oviposition deterrence was a result of true repellence or odour masking. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Authors: Dambuza, Khalipha
- Date: 2023-10-13
- Subjects: Cryptophlebia leucotreta , Pests Integrated control , Semiochemicals , Agricultural pests Control , Oviposition , Essences and essential oils
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424479 , vital:72157
- Description: There has been extensive research on the use of semiochemicals as deterrents or true repellents in insect pest management, particularly in push-pull strategies. Much of this research has focused on pests of medical and veterinary importance and has been limited for agricultural pests. This means there is an opportunity to study use of deterrents to manage pests of agricultural importance. No study has been conducted on deterrents for false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a key phytosanitary pest in citrus orchards across South Africa. This study assessed FCM oviposition deterrence in botanicals (plants (n = 11) and essential oils (n = 15)), and some commercial pesticides (n = 7) used for FCM control in South Africa. All tested botanicals were selected based on an extensive literature review of plant compounds that have been reported to deter or repel lepidopteran pests. Choice and no-choice oviposition bioassays were conducted in complete darkness in a controlled environment room. Oranges treated with solutions/suspensions of potential oviposition deterrents were placed into a cage with gravid FCM females for four hours, with oviposition being recorded every hour. Of the 33 tested compounds, only eight significantly reduced FCM oviposition (P < 0.05) compared to the control in oviposition bioassays i.e. two essential oils (lavender and peppermint), two plant crude extracts (garlic and marigold), one fruit (Mango), and three commercial FCM insecticides (Delegate, Coragen, and Warlock). All identified oviposition deterrents, except for Mango, were further investigated for their ovicidal properties in concentration response bioassays, where all botanicals were identified to have dual action (both deterrent and ovicidal properties), as they significantly (P < 0.05) reduced FCM oviposition and egg hatch. Garlic was the most efficacious botanical whilst Warlock was the only commercial insecticide that did not show ovicidal activity (F = 41.17, P = 0.0622). Larval penetration of the host fruit was less than egg hatch for all tested compounds in concentration response bioassays. Oviposition, egg hatch, and larval penetration were all affected by concentration, with the higher concentrations being the most effective. The efficacy of these deterrent compounds should be further tested in semi-field and/or field trials, and they may have potential in FCM management as allomone dispensers or sprays. They can also be implemented in push-pull strategies where they can be used in conjunction with FCM attractants. Lastly, repellence studies should be conducted in absentia of the host fruit to determine whether oviposition deterrence was a result of true repellence or odour masking. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
A native weevil and an exotic planthopper: investigating potential biological control agents for nymphaea mexicana zuccarini (nymphaeaceae) and its hybrids in South Africa
- Authors: Reid, Megan Kim
- Date: 2023-03-31
- Subjects: Nymphaeaceae South Africa , Water lilies Biological control South Africa , Host specificity , Genetic variation , Bagous longulus
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422554 , vital:71957 , DOI 10.21504/10962/422554
- Description: Nymphaea mexicana Zuccarini (Nymphaeaceae) is an invasive plant originating from southern USA and Mexico that has become problematic in South Africa, invading several water bodies around the country. Manual removal of this plant is very labour intensive and is not cost efficient or effective for long term control, while the use of herbicides is damaging to the environment and expensive. Consequently, this plant is a desirable candidate for biological control, which takes advantage of enemy release of the target weed and aims to re-establish population suppression induced by host specific natural enemies. Initiating biological control requires that several steps are followed to maximise the success of the programme, and the first few of these, including overseas surveys in the native range of the plant, have already been completed. This thesis aimed to continue biological control research for this species to take further steps at effectively managing the plant. Firstly, pre-release surveys in the invaded range are necessary to: determine what factors (including enemy release) contribute to the invasiveness of the target weed; establish a baseline of information to allow for comparison after biological control agents have been released; and identify any insect herbivores that may already be present in the country. The pre-release surveys conducted in this study revealed useful information about N. mexicana invasions in South Africa and provided evidence that enemy release is applicable to this case. However, these studies determined that a native weevil, Bagous longulus Gyllenhal (Coleoptera: Curculionidae), has expanded its host range to include the exotic N. mexicana at three sites, and may thus have potential for management of the species through augmentative releases. The invasion of N. mexicana in South Africa is further complicated by the presence of several Nymphaea hybrids originating from a complex history of horticultural trade. Although previous research has shown that several hybrid groups are present in South Africa, their parentage is not known. As biological control requires the use of host specific insects adapted to overcome the unique chemical and morphological defences utilised by plant species, hybrids are notoriously difficult to manage because they possess intermediate characters inherited from parent species to which natural enemies may not have adapted. Although biological control of hybrids is challenging, other case studies have demonstrated that it is possible to find suitable agents, but the chances of success are increased if putative parents of the hybrids are known so that they can be surveyed for natural enemies. Further molecular studies including possible parents of the Nymphaea hybrids in South Africa were thus carried out in this thesis to focus future surveying efforts. Two main hybrid groups were identified with genetic similarity to two tested putative Nymphaea parents, and this will allow further investigations of these species to improve the chances of successfully managing these hybrid groups. Some of the tested hybrids showed genetic contributions from multiple groups, some of which were unidentified, so it is necessary to prioritise the most problematic hybrids for biological control. With more insight into the genetic makeup of the Nymphaea hybrids in South Africa, investigations into the host specificity of potential biological control agents can be conducted. The ideal biological control agent should have a broad enough host range to impact and survive on both N. mexicana and its hybrids, but without a host range so broad that it would pose risk to native South African species. Host specificity trials are thus necessary to determine the suitability of potential agents. The identification of B. longulus feeding on N. mexicana during pre-release surveys motivated further investigations to determine the natural distribution, field host range, and host specificity of B. longulus in experimentally controlled conditions. Further surveys were therefore conducted at native Nymphaea sites around South Africa in addition to host specificity trials using the native Nymphaea nouchali Burm. f. (Nymphaeaceae), two populations of N. mexicana, and a cultivated hybrid. Results from the surveys and host specificity tests suggest that B. longulus is widely distributed across South Africa, is specific to Nymphaea with no observed preference between N. mexicana and the native N. nouchali, and does not perform well on Nymphaea hybrids. Hence, B. longulus is promising for use in new association biological control through augmentative releases but is not suitable for management of hybrids. In addition to the potential use of the South African B. longulus, it is necessary to conduct host specificity trials for natural enemies from the native range of N. mexicana that were prioritised in previous studies. Megamelus toddi Beamer (Hemiptera: Delphacidae) is one such species that was imported into quarantined laboratory conditions from Florida, USA. Host specificity trials were conducted using the same test plants as described for the studies on B. longulus, in addition to multigeneration trials to determine how long M. toddi could survive on non-target host plants. As with the B. longulus studies, no statistically significant differences in preference were observed between N. mexicana and N. nouchali, but M. toddi could not complete development on the test hybrid, indicating that this species is also unsuitable for the management of Nymphaea hybrids. Despite suboptimal plant health, M. toddi completed development for three generations on the native N. nouchali. This lack of host specificity deems M. toddi unsafe for release in South Africa but highlights the importance of following predefined steps to develop a biological control programme. The concluding chapter of this thesis discusses the aforementioned findings in a broader context by considering the driving forces of plant invasions in general and specifically for N. mexicana in South Africa. Case studies are also consulted to provide insight into how to proceed with managing Nymphaea hybrids in South Africa, while the factors governing host specificity and host range expansion are also discussed and considered in the context of B. longulus and M. toddi. Finally, after a consideration of the limitations of these studies, recommendations are made to continue the development of biological control for N. mexicana in South Africa. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Authors: Reid, Megan Kim
- Date: 2023-03-31
- Subjects: Nymphaeaceae South Africa , Water lilies Biological control South Africa , Host specificity , Genetic variation , Bagous longulus
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422554 , vital:71957 , DOI 10.21504/10962/422554
- Description: Nymphaea mexicana Zuccarini (Nymphaeaceae) is an invasive plant originating from southern USA and Mexico that has become problematic in South Africa, invading several water bodies around the country. Manual removal of this plant is very labour intensive and is not cost efficient or effective for long term control, while the use of herbicides is damaging to the environment and expensive. Consequently, this plant is a desirable candidate for biological control, which takes advantage of enemy release of the target weed and aims to re-establish population suppression induced by host specific natural enemies. Initiating biological control requires that several steps are followed to maximise the success of the programme, and the first few of these, including overseas surveys in the native range of the plant, have already been completed. This thesis aimed to continue biological control research for this species to take further steps at effectively managing the plant. Firstly, pre-release surveys in the invaded range are necessary to: determine what factors (including enemy release) contribute to the invasiveness of the target weed; establish a baseline of information to allow for comparison after biological control agents have been released; and identify any insect herbivores that may already be present in the country. The pre-release surveys conducted in this study revealed useful information about N. mexicana invasions in South Africa and provided evidence that enemy release is applicable to this case. However, these studies determined that a native weevil, Bagous longulus Gyllenhal (Coleoptera: Curculionidae), has expanded its host range to include the exotic N. mexicana at three sites, and may thus have potential for management of the species through augmentative releases. The invasion of N. mexicana in South Africa is further complicated by the presence of several Nymphaea hybrids originating from a complex history of horticultural trade. Although previous research has shown that several hybrid groups are present in South Africa, their parentage is not known. As biological control requires the use of host specific insects adapted to overcome the unique chemical and morphological defences utilised by plant species, hybrids are notoriously difficult to manage because they possess intermediate characters inherited from parent species to which natural enemies may not have adapted. Although biological control of hybrids is challenging, other case studies have demonstrated that it is possible to find suitable agents, but the chances of success are increased if putative parents of the hybrids are known so that they can be surveyed for natural enemies. Further molecular studies including possible parents of the Nymphaea hybrids in South Africa were thus carried out in this thesis to focus future surveying efforts. Two main hybrid groups were identified with genetic similarity to two tested putative Nymphaea parents, and this will allow further investigations of these species to improve the chances of successfully managing these hybrid groups. Some of the tested hybrids showed genetic contributions from multiple groups, some of which were unidentified, so it is necessary to prioritise the most problematic hybrids for biological control. With more insight into the genetic makeup of the Nymphaea hybrids in South Africa, investigations into the host specificity of potential biological control agents can be conducted. The ideal biological control agent should have a broad enough host range to impact and survive on both N. mexicana and its hybrids, but without a host range so broad that it would pose risk to native South African species. Host specificity trials are thus necessary to determine the suitability of potential agents. The identification of B. longulus feeding on N. mexicana during pre-release surveys motivated further investigations to determine the natural distribution, field host range, and host specificity of B. longulus in experimentally controlled conditions. Further surveys were therefore conducted at native Nymphaea sites around South Africa in addition to host specificity trials using the native Nymphaea nouchali Burm. f. (Nymphaeaceae), two populations of N. mexicana, and a cultivated hybrid. Results from the surveys and host specificity tests suggest that B. longulus is widely distributed across South Africa, is specific to Nymphaea with no observed preference between N. mexicana and the native N. nouchali, and does not perform well on Nymphaea hybrids. Hence, B. longulus is promising for use in new association biological control through augmentative releases but is not suitable for management of hybrids. In addition to the potential use of the South African B. longulus, it is necessary to conduct host specificity trials for natural enemies from the native range of N. mexicana that were prioritised in previous studies. Megamelus toddi Beamer (Hemiptera: Delphacidae) is one such species that was imported into quarantined laboratory conditions from Florida, USA. Host specificity trials were conducted using the same test plants as described for the studies on B. longulus, in addition to multigeneration trials to determine how long M. toddi could survive on non-target host plants. As with the B. longulus studies, no statistically significant differences in preference were observed between N. mexicana and N. nouchali, but M. toddi could not complete development on the test hybrid, indicating that this species is also unsuitable for the management of Nymphaea hybrids. Despite suboptimal plant health, M. toddi completed development for three generations on the native N. nouchali. This lack of host specificity deems M. toddi unsafe for release in South Africa but highlights the importance of following predefined steps to develop a biological control programme. The concluding chapter of this thesis discusses the aforementioned findings in a broader context by considering the driving forces of plant invasions in general and specifically for N. mexicana in South Africa. Case studies are also consulted to provide insight into how to proceed with managing Nymphaea hybrids in South Africa, while the factors governing host specificity and host range expansion are also discussed and considered in the context of B. longulus and M. toddi. Finally, after a consideration of the limitations of these studies, recommendations are made to continue the development of biological control for N. mexicana in South Africa. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
Development and optimisation of a qPCR assay for the enumeration of Cryptophlebia leucotreta granulovirus (CrleGV) used for commercial applications
- Authors: Mela, Thuthula
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362949 , vital:65377
- Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Authors: Mela, Thuthula
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362949 , vital:65377
- Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
Effect of Helicosporidium sp. (Chlorophyta; Trebouxiophyceae) infection on Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), a biological control agent for the invasive Salvinia molesta D.S. Mitchell (Salviniaceae) in South
- Authors: Mphephu, Tshililo Emmanuel
- Date: 2022-10-14
- Subjects: Salvinia molesta South Africa , Weeds Biological control , Cyrtobagous salviniae , Ketoconazole
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365815 , vital:65792 , DOI https://doi.org/10.21504/10962/365815
- Description: The effectiveness of established biological control agents depends on biotic and abiotic interactions in the introduced range. The weevil, Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), was released as a biological control against Salvinia molesta D.S. Mitchell (Salviniaceae) in South Africa in 1985. This agent has been highly successful against S. molesta and has significantly reduced the weed’s populations around the country. However, in 2007, the parasitic alga, Helicosporidium sp. (an undescribed species), was detected in field-collected C. salviniae adults in South Africa. The distribution and impacts of this disease on the weevil and its efficacy as a control agent were not known. In this thesis, the prevalence, infection load, and impact of Helicosporidium sp. on C. salviniae was determined. In 2019, adult weevils were collected from 10 sites across the Eastern Cape, KwaZulu-Natal, Limpopo, and Western Cape provinces and screened to determine the occurrence, infection load, and geographic distribution of Helicosporidium sp. Transmission mechanisms of this disease in C. salviniae were then evaluated. The possible impact of Helicosporidium sp. was assessed by comparing the feeding rates and the reproductive output of the diseased and healthy adults of C. salviniae. An attempt was then made to eliminate the disease in C. salviniae through the application of the antibiotic, ketoconazole. Further, the role of temperature on infection load in C. salviniae was also assessed. Finally, recommendations for the long-term biological control programme against S. molesta in South Africa were made. The disease covers the entire distribution range of C. salviniae in South Africa, with the disease occurrence rate ranging from 92.15% to 100% insects infected per site. Helicosporidium sp. was found to transmit vertically within the populations of C. salviniae. Infection by the Helicosporidium sp. disease reduced the reproductive output of C. salviniae as well its impact on biomass reduction of S. molesta when a diseased culture was compared to a healthy culture from the USA. 98.44 to 98.55% of Helicosporidium sp. loads were reduced through multiple applications of ketoconazole concentrations under in vitro trials. In vivo treatments resulted in 70% control of Helicosporidium sp. in the adults of C. salviniae that were fed ketoconazole three times over a 21 day period. Adult C. salviniae feeding and survival performances were similar when fed fronds of S. molesta inoculated with ketoconazole and water. The lowest and highest disease loads of Helicosporidium sp. were recorded when the weevils were reared at 30°C and 14°C, respectively. As expected, the highest impact and reproductive output of C. salviniae were at 30°C. The evaluations discussed in this thesis highlight the role of diseases in biological control agents, and gaps in both the pre-release and post-release monitoring that should integrate screening of diseases in these studies. Although the combined application of the antibiotic and temperature will reduce Helicosporidium sp. loads and impact, this technology is most likely only applicable where the weevils are reared in small numbers in a rearing facility and not really applicable to the field situation. It is important to release healthy agents that will cause efficient control of the target weed plant species, therefore, when introducing new biological control agents, the health status of such agents needs to be understood. Therefore, long-term field monitoring and assessment of the impact of C. salviniae on S. molesta should be conducted to track all the changes that may result due to the presence of Helicosporidium sp. This long-term monitoring and assessment will give a more informative role of Helicosporidium sp. in field populations of C. salviniae. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Authors: Mphephu, Tshililo Emmanuel
- Date: 2022-10-14
- Subjects: Salvinia molesta South Africa , Weeds Biological control , Cyrtobagous salviniae , Ketoconazole
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365815 , vital:65792 , DOI https://doi.org/10.21504/10962/365815
- Description: The effectiveness of established biological control agents depends on biotic and abiotic interactions in the introduced range. The weevil, Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), was released as a biological control against Salvinia molesta D.S. Mitchell (Salviniaceae) in South Africa in 1985. This agent has been highly successful against S. molesta and has significantly reduced the weed’s populations around the country. However, in 2007, the parasitic alga, Helicosporidium sp. (an undescribed species), was detected in field-collected C. salviniae adults in South Africa. The distribution and impacts of this disease on the weevil and its efficacy as a control agent were not known. In this thesis, the prevalence, infection load, and impact of Helicosporidium sp. on C. salviniae was determined. In 2019, adult weevils were collected from 10 sites across the Eastern Cape, KwaZulu-Natal, Limpopo, and Western Cape provinces and screened to determine the occurrence, infection load, and geographic distribution of Helicosporidium sp. Transmission mechanisms of this disease in C. salviniae were then evaluated. The possible impact of Helicosporidium sp. was assessed by comparing the feeding rates and the reproductive output of the diseased and healthy adults of C. salviniae. An attempt was then made to eliminate the disease in C. salviniae through the application of the antibiotic, ketoconazole. Further, the role of temperature on infection load in C. salviniae was also assessed. Finally, recommendations for the long-term biological control programme against S. molesta in South Africa were made. The disease covers the entire distribution range of C. salviniae in South Africa, with the disease occurrence rate ranging from 92.15% to 100% insects infected per site. Helicosporidium sp. was found to transmit vertically within the populations of C. salviniae. Infection by the Helicosporidium sp. disease reduced the reproductive output of C. salviniae as well its impact on biomass reduction of S. molesta when a diseased culture was compared to a healthy culture from the USA. 98.44 to 98.55% of Helicosporidium sp. loads were reduced through multiple applications of ketoconazole concentrations under in vitro trials. In vivo treatments resulted in 70% control of Helicosporidium sp. in the adults of C. salviniae that were fed ketoconazole three times over a 21 day period. Adult C. salviniae feeding and survival performances were similar when fed fronds of S. molesta inoculated with ketoconazole and water. The lowest and highest disease loads of Helicosporidium sp. were recorded when the weevils were reared at 30°C and 14°C, respectively. As expected, the highest impact and reproductive output of C. salviniae were at 30°C. The evaluations discussed in this thesis highlight the role of diseases in biological control agents, and gaps in both the pre-release and post-release monitoring that should integrate screening of diseases in these studies. Although the combined application of the antibiotic and temperature will reduce Helicosporidium sp. loads and impact, this technology is most likely only applicable where the weevils are reared in small numbers in a rearing facility and not really applicable to the field situation. It is important to release healthy agents that will cause efficient control of the target weed plant species, therefore, when introducing new biological control agents, the health status of such agents needs to be understood. Therefore, long-term field monitoring and assessment of the impact of C. salviniae on S. molesta should be conducted to track all the changes that may result due to the presence of Helicosporidium sp. This long-term monitoring and assessment will give a more informative role of Helicosporidium sp. in field populations of C. salviniae. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
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:
- 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:
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:
- 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:
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:
- 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:
Distribution, ecological and economic impacts and competition of the invasive alien aquatic weeds (Pontederia crassipes Mart., Pistia stratiotes L., Salvinia molesta D.S. Mitch. and Azolla filiculoides Lam.) in Madagascar
- Authors: Lehavana, Adolphe
- Date: 2021-10-29
- Subjects: Pontederiaceae Madagascar , Water lettuce Madagascar , Salvinia molesta Madagascar , Azolla filiculoides Madagascar , Introduced aquatic organisms , Aquatic weeds Economic aspects , Aquatic weeds Social aspects , Aquatic weeds Geographical distribution
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191225 , vital:45072
- Description: In Madagascar, as in several countries in the world, the invasion by four aquatic weeds (Pontederia crassipes Mart. (Pontederiaceae), Pistia stratiotes L. (Araceae), Salvinia molesta D.S. Mitch Salviniaceae) and Azolla filiculoides Lam. (Azollaceae) are among the drivers of environmental and socio-economic deterioration in aquatic ecosystems. Pistia stratiotes was first recorded on the island in the 19th century, and P. crassipes from the beginning of the 20th century, while S. molesta and A. filiculoides were only documented during in the 21st century. From the 1920s, botanists such as Henri Perrier de la Bathie and Raymond Decary were already aware of the dangers caused, in particular by P. crassipes in other countries, and raised the alarm, but little attention has been paid to these species. The aim of the research conducted for this thesis was to determine the distribution, socio-economic and ecological impacts of these four invasive alien aquatic weeds in Madagascar and to make recommendations for their control. First, the distributions of these four aquatic weeds were mapped. This mapping exercise compiled data from different sources including herbarium records, online data and field visits across Madagascar. The mapping study was undertaken from August 2015 to June 2020. Except for mountainous areas above 1800 m (Tsaratanana Massif, Ankaratra Massif and Andringitra Massif) where no data were available, all of Madagascar's bioclimates were invaded by at least one of the four aquatic weeds. In total, at least one species was recorded in 18 of the 22 Regions. Pontederia crassipes was recorded in 13 Regions, S. molesta in 14 Regions, P. stratiotes in 12 Regions, and A. filiculoides in 13 Regions. Herbarium records revealed the oldest record for P. stratiotes to be 1847, 1931 for P. crassipes, 1995 for S. molesta and there were no herbarium specimens for A. filiculoides prior to the start of the current study in 2015. We now know where these four weeds occur and how abundant they are. An objective of this research was to assess the impacts of the four invasive aquatic plants on the socio-economy of the island, mainly on rice production and fishing. Between 2016 and 2019, 102 households in three regions, Soanierana Ivongo, Foulpointe and Antananarivo, were randomly selected and questioned on the impact of these weeds in their aquatic ecosystems and their livelihoods such as fishing and rice growing. Surveys revealed that the four aquatic weeds significantly threatened household activities. On the east coast of Madagascar, the invasions of these four invasive species decreased fish and freshwater shrimp production by 82%. On the high plateau of Madagascar, they reduced rice yield by 30% despite requiring an additional expense of US$ 1,107/ha for control. Although farmers surveyed only used manual control to manage these weeds, they were receptive to other control methods, including integrated control using herbicides and biological control. Another objective of this research was to determine the ecological impacts of the four weeds and specifically if freshwater ecosystem functioning would return after control. To assess the ecological impact, between February 2017 to August 2019, on Lake Antsokafina, the following abiotic and biotic factors were considered: physico-chemistry of water, succession of macrophyte community and animal diversity. With the exception of turbidity, the values of the physico-chemical parameters of the water (pH, electrical conductivity, water temperature and turbidity), were similar between the infested zone and cleared zone. A study on the invasion process of aquatic weeds showed that the plant community succession of the lake changed over time in the areas that had been cleared. The submerged species Ceratophyllum demersum was the pioneer, followed by creeping species such as Echinochloa colona and Ipomoea aquatica, before the area was recolonized by aquatic weeds. Among the aquatic weeds, S. molesta was the most aggressive, covering 92% of the area one year after the start of the experiment. For animal diversity, bird, shrimp and fish community were assessed. The cleaning of the plots in the lake allowed the resumption of fishing activity providing 50 to 200g/catch for shrimp and from 0.25 to 0.5kg/catch for fish per person per day, while no catch was obtained in the areas infested by aquatic weeds were fishermen still attempting to harvest fish/shrimp from the aquatic weed infested areas. Three species of birds, Humblot’s Heron (Ardea humbloti), the white-faced whistling duck (Dendrocygna viduata) and red-billed teal (Anas erythrorhyncha) returned once the areas had been cleared. A manipulated outdoor as descriptor for laboratory experiment was conducted to determine the level and nature of competition of four aquatic weeds species against the indigenous floating fern, Salvinia hastata Desv. (Salviniaceae), using an additive series density model. It was shown that all four invasive species outcompeted S. hastata, with P. crassipes being 24 times more dominant, followed by P. stratiotes at 12 times, S. molesta at 8 times, and finally A. filiculoides at 1.2 times more dominant. This study provided direct evidence of the biodiversity impact of these four species and thus also provided an environmental argument for their control. Based on the findings of this study, a series of recommendations was formulated to manage the invasions of alien species in Madagascar with particular attention to invasive aquatic weeds. These recommendations mainly concern the establishment of management structures and legal instruments such as the creation of a lead government agency at national level and a cross-sectorial invasive species advisory committee, which should review legislation and regulations related to invasive species. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2021
- Full Text:
- Authors: Lehavana, Adolphe
- Date: 2021-10-29
- Subjects: Pontederiaceae Madagascar , Water lettuce Madagascar , Salvinia molesta Madagascar , Azolla filiculoides Madagascar , Introduced aquatic organisms , Aquatic weeds Economic aspects , Aquatic weeds Social aspects , Aquatic weeds Geographical distribution
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191225 , vital:45072
- Description: In Madagascar, as in several countries in the world, the invasion by four aquatic weeds (Pontederia crassipes Mart. (Pontederiaceae), Pistia stratiotes L. (Araceae), Salvinia molesta D.S. Mitch Salviniaceae) and Azolla filiculoides Lam. (Azollaceae) are among the drivers of environmental and socio-economic deterioration in aquatic ecosystems. Pistia stratiotes was first recorded on the island in the 19th century, and P. crassipes from the beginning of the 20th century, while S. molesta and A. filiculoides were only documented during in the 21st century. From the 1920s, botanists such as Henri Perrier de la Bathie and Raymond Decary were already aware of the dangers caused, in particular by P. crassipes in other countries, and raised the alarm, but little attention has been paid to these species. The aim of the research conducted for this thesis was to determine the distribution, socio-economic and ecological impacts of these four invasive alien aquatic weeds in Madagascar and to make recommendations for their control. First, the distributions of these four aquatic weeds were mapped. This mapping exercise compiled data from different sources including herbarium records, online data and field visits across Madagascar. The mapping study was undertaken from August 2015 to June 2020. Except for mountainous areas above 1800 m (Tsaratanana Massif, Ankaratra Massif and Andringitra Massif) where no data were available, all of Madagascar's bioclimates were invaded by at least one of the four aquatic weeds. In total, at least one species was recorded in 18 of the 22 Regions. Pontederia crassipes was recorded in 13 Regions, S. molesta in 14 Regions, P. stratiotes in 12 Regions, and A. filiculoides in 13 Regions. Herbarium records revealed the oldest record for P. stratiotes to be 1847, 1931 for P. crassipes, 1995 for S. molesta and there were no herbarium specimens for A. filiculoides prior to the start of the current study in 2015. We now know where these four weeds occur and how abundant they are. An objective of this research was to assess the impacts of the four invasive aquatic plants on the socio-economy of the island, mainly on rice production and fishing. Between 2016 and 2019, 102 households in three regions, Soanierana Ivongo, Foulpointe and Antananarivo, were randomly selected and questioned on the impact of these weeds in their aquatic ecosystems and their livelihoods such as fishing and rice growing. Surveys revealed that the four aquatic weeds significantly threatened household activities. On the east coast of Madagascar, the invasions of these four invasive species decreased fish and freshwater shrimp production by 82%. On the high plateau of Madagascar, they reduced rice yield by 30% despite requiring an additional expense of US$ 1,107/ha for control. Although farmers surveyed only used manual control to manage these weeds, they were receptive to other control methods, including integrated control using herbicides and biological control. Another objective of this research was to determine the ecological impacts of the four weeds and specifically if freshwater ecosystem functioning would return after control. To assess the ecological impact, between February 2017 to August 2019, on Lake Antsokafina, the following abiotic and biotic factors were considered: physico-chemistry of water, succession of macrophyte community and animal diversity. With the exception of turbidity, the values of the physico-chemical parameters of the water (pH, electrical conductivity, water temperature and turbidity), were similar between the infested zone and cleared zone. A study on the invasion process of aquatic weeds showed that the plant community succession of the lake changed over time in the areas that had been cleared. The submerged species Ceratophyllum demersum was the pioneer, followed by creeping species such as Echinochloa colona and Ipomoea aquatica, before the area was recolonized by aquatic weeds. Among the aquatic weeds, S. molesta was the most aggressive, covering 92% of the area one year after the start of the experiment. For animal diversity, bird, shrimp and fish community were assessed. The cleaning of the plots in the lake allowed the resumption of fishing activity providing 50 to 200g/catch for shrimp and from 0.25 to 0.5kg/catch for fish per person per day, while no catch was obtained in the areas infested by aquatic weeds were fishermen still attempting to harvest fish/shrimp from the aquatic weed infested areas. Three species of birds, Humblot’s Heron (Ardea humbloti), the white-faced whistling duck (Dendrocygna viduata) and red-billed teal (Anas erythrorhyncha) returned once the areas had been cleared. A manipulated outdoor as descriptor for laboratory experiment was conducted to determine the level and nature of competition of four aquatic weeds species against the indigenous floating fern, Salvinia hastata Desv. (Salviniaceae), using an additive series density model. It was shown that all four invasive species outcompeted S. hastata, with P. crassipes being 24 times more dominant, followed by P. stratiotes at 12 times, S. molesta at 8 times, and finally A. filiculoides at 1.2 times more dominant. This study provided direct evidence of the biodiversity impact of these four species and thus also provided an environmental argument for their control. Based on the findings of this study, a series of recommendations was formulated to manage the invasions of alien species in Madagascar with particular attention to invasive aquatic weeds. These recommendations mainly concern the establishment of management structures and legal instruments such as the creation of a lead government agency at national level and a cross-sectorial invasive species advisory committee, which should review legislation and regulations related to invasive species. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2021
- Full Text:
The biological control of Egeria densa Planch. (Hydrocharitaceae) in South Africa
- Authors: Smith, Rosali
- Date: 2021-10-29
- Subjects: Egeria (Plant genus) Biological control South Africa , Hydrocharitaceae Biological control South Africa , Aquatic weeds Biological control South Africa , Leafminers South Africa , Plant invasions South Africa , Resilience (Ecology) South Africa , Freshwater ecology South Africa , Hydrellia South Africa , Submerged macrophyte
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191102 , vital:45060 , 10.21504/10962/191102
- Description: Over the last thirty years, biological control, the use of host-specific natural enemies, has been a huge asset in the management exotic aquatic macrophytes such as Pistia stratiotes L. (Araceae), Pontederia crassipes Mart. (Solms) (Pontederiaceae), Azolla filiculoides Lam. (Azollaceae), Salvinia molesta D.S. Mitch (Salviniaceae) and Myriophyllum aquaticum (Vell.) Verdc. (Haloragaceae), also known as the “Big Bad Five” in South Africa. Despite these successes, freshwater ecosystems in South Africa have been harder to restore to an invasive macrophyte-free space, due to chronic disturbances such eutrophication, propagule dispersal and hydrological alterations. In the Anthropocene, where human activities have profound effects on their environment, these disturbances weakens ecological resilience and drive aquatic plant invasions. Due to long periods of invasions and the presence of a new suite of exotic aquatic plant propagules, native vegetation recolonization has been slow or even absent. Instead, the release of resources, such as sunlight, nutrient and space through aquatic weed management acts as a catalyst for secondary biological invasion. New invasive aquatic weeds include submerged and rooted emergent growth types, with Egeria densa Planch. (Hydrocharitaceae) the most widely distributed submerged aquatic weed in South Africa. It can quickly form dense monoculture stands that have ecological, economic and social impacts. Because of its ability to regenerate from plant fragments with double nodes, mechanical control is inappropriate. Additionally, mechanical and chemical control not only affects E. densa but have significant non-target effects. In response to its rapid spread over the last 20 years, especially following floating invasive aquatic management, a biological control programme was initiated, and in 2018, the leaf-mining fly, Hydrellia egeriae Rodrigues (Diptera: Ephydridae) was released. This was the first release of a biological control agent against E. densa in the world, and the first agent released against a submerged aquatic weed in South Africa. This thesis comprises the subsequent step of a biological control program when permission for the release of an agent have been obtained. A brief history of macrophyte invasions in South Africa’s unique freshwater systems are given in the literature review. Contributing factors to secondary invasions within the context of ecological resilience are introduced. An argument for the benefit of biological control as nuisance control is given, especially because E. densa and its natural enemy, H. egeriae is the focus species of this thesis. The main goal after permission for the release of an agent have been obtained, is to establish and build-up field populations. Research questions in this thesis aimed to investigate factors that contribute to or negate this goal. Through laboratory and field experiments we investigated the thermal physiology of the agent, and its climatic suitability to its novel range; different release strategies on field establishment and biotic resistance through the acquisition of novel parasitoids. Considering the longevity of this biological control program, we investigated the effects of elevated CO2 on the interaction between E. densa and H. egeriae through open top chamber experiments. Laboratory thermal physiology results showed that the agent is able to survive, develop and proliferate at all E. densa sites throughout the year. This is confirmed with the establishment of the agent at two release sites, the Nahoon River in the Eastern Cape Province and the Midmar Dam in KwaZulu-Natal. Post-release surveys showed that H. egeriae requires augmentative releases to sustain field populations. Without augmentative releases, H. egeriae herbivory levels were almost negligent. However, a contributing factor to low field-populations was parasitism. The biological control agent acquired three parasitoids, which have previously been described from Hydrellia lagarosiphon Deeming (Diptera: Ephydridae), a specific herbivore to Lagarosiphon major (Ridl.) Moss (Hydrocharitaceae). These results provide information on the immediate establishment and effectiveness of the H. egeriae. Results from the elevated CO2 study suggest that E. densa will become less nutritious through a shift in leaf C/N ratio, when ambient 800ppm is bubbled into experimental growth chambers. Hydrellia egeriae feeding was affected by ambient CO2 levels and plant nutrient availability. The set levels of ambient CO2 levels used in this experiment produced dissolved inorganic carbon levels that were lower than dissolved inorganic carbon levels in E. densa invaded sites. This suggests that, submerged aquatic plant-insect interactions may be harder to predict from only laboratory experiments. Further investigations are necessary to establish system-specific characteristics i.e. dissolved inorganic carbon and target plant nutritional quality. The biological control of E. densa in South Africa is still in its infancy. This study presents results from post-release surveys up until two years after the agent was released. From this study, Hydrellia egeriae exhibits the potential to be an effective biological control agent, but release strategies should be adapted to sustain field populations and to limit field parasitism effects. Continued post-release surveys will provide a more comprehensive idea of the seasonal fluctuations of field-populations and parasitism. Surveys at multiple sites will provide information on potential site specific characteristics that contribute to or negate biological effort. Considering the high nutrient status of South African freshwater systems, a more holistic approach to E. densa management is necessary. This will require the strengthening of ecological resilience to prevent systems from shifting into an alternate invasive stable state. In addition, aquatic weed management needs to be addressed by a resilient social network, which ultimately calls for the strengthening of socio-ecological resilience. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2021
- Full Text:
- Authors: Smith, Rosali
- Date: 2021-10-29
- Subjects: Egeria (Plant genus) Biological control South Africa , Hydrocharitaceae Biological control South Africa , Aquatic weeds Biological control South Africa , Leafminers South Africa , Plant invasions South Africa , Resilience (Ecology) South Africa , Freshwater ecology South Africa , Hydrellia South Africa , Submerged macrophyte
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191102 , vital:45060 , 10.21504/10962/191102
- Description: Over the last thirty years, biological control, the use of host-specific natural enemies, has been a huge asset in the management exotic aquatic macrophytes such as Pistia stratiotes L. (Araceae), Pontederia crassipes Mart. (Solms) (Pontederiaceae), Azolla filiculoides Lam. (Azollaceae), Salvinia molesta D.S. Mitch (Salviniaceae) and Myriophyllum aquaticum (Vell.) Verdc. (Haloragaceae), also known as the “Big Bad Five” in South Africa. Despite these successes, freshwater ecosystems in South Africa have been harder to restore to an invasive macrophyte-free space, due to chronic disturbances such eutrophication, propagule dispersal and hydrological alterations. In the Anthropocene, where human activities have profound effects on their environment, these disturbances weakens ecological resilience and drive aquatic plant invasions. Due to long periods of invasions and the presence of a new suite of exotic aquatic plant propagules, native vegetation recolonization has been slow or even absent. Instead, the release of resources, such as sunlight, nutrient and space through aquatic weed management acts as a catalyst for secondary biological invasion. New invasive aquatic weeds include submerged and rooted emergent growth types, with Egeria densa Planch. (Hydrocharitaceae) the most widely distributed submerged aquatic weed in South Africa. It can quickly form dense monoculture stands that have ecological, economic and social impacts. Because of its ability to regenerate from plant fragments with double nodes, mechanical control is inappropriate. Additionally, mechanical and chemical control not only affects E. densa but have significant non-target effects. In response to its rapid spread over the last 20 years, especially following floating invasive aquatic management, a biological control programme was initiated, and in 2018, the leaf-mining fly, Hydrellia egeriae Rodrigues (Diptera: Ephydridae) was released. This was the first release of a biological control agent against E. densa in the world, and the first agent released against a submerged aquatic weed in South Africa. This thesis comprises the subsequent step of a biological control program when permission for the release of an agent have been obtained. A brief history of macrophyte invasions in South Africa’s unique freshwater systems are given in the literature review. Contributing factors to secondary invasions within the context of ecological resilience are introduced. An argument for the benefit of biological control as nuisance control is given, especially because E. densa and its natural enemy, H. egeriae is the focus species of this thesis. The main goal after permission for the release of an agent have been obtained, is to establish and build-up field populations. Research questions in this thesis aimed to investigate factors that contribute to or negate this goal. Through laboratory and field experiments we investigated the thermal physiology of the agent, and its climatic suitability to its novel range; different release strategies on field establishment and biotic resistance through the acquisition of novel parasitoids. Considering the longevity of this biological control program, we investigated the effects of elevated CO2 on the interaction between E. densa and H. egeriae through open top chamber experiments. Laboratory thermal physiology results showed that the agent is able to survive, develop and proliferate at all E. densa sites throughout the year. This is confirmed with the establishment of the agent at two release sites, the Nahoon River in the Eastern Cape Province and the Midmar Dam in KwaZulu-Natal. Post-release surveys showed that H. egeriae requires augmentative releases to sustain field populations. Without augmentative releases, H. egeriae herbivory levels were almost negligent. However, a contributing factor to low field-populations was parasitism. The biological control agent acquired three parasitoids, which have previously been described from Hydrellia lagarosiphon Deeming (Diptera: Ephydridae), a specific herbivore to Lagarosiphon major (Ridl.) Moss (Hydrocharitaceae). These results provide information on the immediate establishment and effectiveness of the H. egeriae. Results from the elevated CO2 study suggest that E. densa will become less nutritious through a shift in leaf C/N ratio, when ambient 800ppm is bubbled into experimental growth chambers. Hydrellia egeriae feeding was affected by ambient CO2 levels and plant nutrient availability. The set levels of ambient CO2 levels used in this experiment produced dissolved inorganic carbon levels that were lower than dissolved inorganic carbon levels in E. densa invaded sites. This suggests that, submerged aquatic plant-insect interactions may be harder to predict from only laboratory experiments. Further investigations are necessary to establish system-specific characteristics i.e. dissolved inorganic carbon and target plant nutritional quality. The biological control of E. densa in South Africa is still in its infancy. This study presents results from post-release surveys up until two years after the agent was released. From this study, Hydrellia egeriae exhibits the potential to be an effective biological control agent, but release strategies should be adapted to sustain field populations and to limit field parasitism effects. Continued post-release surveys will provide a more comprehensive idea of the seasonal fluctuations of field-populations and parasitism. Surveys at multiple sites will provide information on potential site specific characteristics that contribute to or negate biological effort. Considering the high nutrient status of South African freshwater systems, a more holistic approach to E. densa management is necessary. This will require the strengthening of ecological resilience to prevent systems from shifting into an alternate invasive stable state. In addition, aquatic weed management needs to be addressed by a resilient social network, which ultimately calls for the strengthening of socio-ecological resilience. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2021
- Full Text:
Evaluation of Megabruchidius tonkineus (Coleoptera: Chrysomelidae: Bruchinae), a candidate biological control agent for Gleditsia triacanthos L. (Fabaceae) in South Africa
- Salgado Astudillo, Sara Elizabeth
- Authors: Salgado Astudillo, Sara Elizabeth
- Date: 2021-10
- Subjects: Honey locust South Africa , Honey locust Biological control South Africa , Invasive plants Biological control South Africa , Biogeography South Africa , Biogeography Climatic factors South Africa , Megabruchidius tonkineus South Africa
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/188386 , vital:44749
- Description: Gleditsia triacanthos L. (Fabaceae) (honey locust) is a fast-growing, deciduous tree indigenous to the United States of America. Introduced around the world as an ornamental tree, it has become invasive in a number of countries. Where it is invasive, G. triacanthos competes and replaces indigenous species; it creates dense stands along watercourses, posing a significant environmental threat. In South Africa, G. triacanthos is regarded as one of the country’s fastest spreading weeds. Gleditsia triacanthos produces numerous seeds contained in large hanging pods. Once dislodged from the pods, the seeds are dispersed by birds and mammals, including livestock, which eat the pods. It has been suggested that the seeds should be the target for biological control programme. Some invasive alien plant species are characterised by their ability to spread and establish in new ecosystems because they tolerate a wide range of environmental conditions. In order to predict areas of likely invasion, species distribution models (SDMs) are used to identify areas climatically suitable for their invasion, so enabling better targeted control of the plant species. Gleditsia triacanthos adapts to a wide range of climates and soil types, and tolerates salinity, drought and frost. Currently primarily restricted to the Grassland Biome of South Africa, G. triacanthos has doubled its distribution area in the past 15 years, and it is not known how far the species will spread. In this study we used two different modelling programmes, CLIMEX and MaxEnt, to predict areas where G. triacanthos could find favourable growing conditions; both SDMs showed that most of the country is suitable for G. triacanthos and that it will probably continue to spread, if left unmanaged, into new bioregions, such as the Karoo. In South Africa, the Asian seed-feeding bruchid, Megabruchidius tonkineus (Pic, 1914) (Coleoptera: Chrysomelidae: Bruchinae) has been recorded in the plant’s seed pods and has been considered as a biological control agent. The insect was not released as part of a formal biological control programme and neither host-specificity nor impact studies were conducted on the species prior to its discovery. In 2017 a decision was made to re-consider its status as a Abstract biological control agent until further details of its biology, host specificity, and impact on the seeds of G. triacanthos in South Africa were available. This study shows that Megabruchidius tonkineus has established across the entire G. triacanthos population in South Africa damaging approximately 9% of seeds. Laboratory studies show that, Megabruchidius tonkineus completes its larval development in the seeds of G. triacanthos in about 66.80 ± 0.6880 SE days before eclosing. In addition, the adult females oviposit on the following Fabaceae species: Arachis hypogaea, Albizia, julibrissin, Cicer arietinum, Pisum sativum, Dipogon lignosus, Peltophorum africanum, Podalyria buxifolia Senegalia burkei, Umtiza listerina and Vachellia sieberiana. However, larval development was limited to G. triacanthos. It is concluded that the seed-feeding beetle is not a threat to native Fabaceae species in South Africa, however, it does not damage enough G. triacanthos seeds to be considered a valuable biological control agent at this stage, and additional seed-feeding biological control agents should be considered to reduce the number of G. triacanthos seeds entering the environment. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2021
- Full Text:
- Authors: Salgado Astudillo, Sara Elizabeth
- Date: 2021-10
- Subjects: Honey locust South Africa , Honey locust Biological control South Africa , Invasive plants Biological control South Africa , Biogeography South Africa , Biogeography Climatic factors South Africa , Megabruchidius tonkineus South Africa
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/188386 , vital:44749
- Description: Gleditsia triacanthos L. (Fabaceae) (honey locust) is a fast-growing, deciduous tree indigenous to the United States of America. Introduced around the world as an ornamental tree, it has become invasive in a number of countries. Where it is invasive, G. triacanthos competes and replaces indigenous species; it creates dense stands along watercourses, posing a significant environmental threat. In South Africa, G. triacanthos is regarded as one of the country’s fastest spreading weeds. Gleditsia triacanthos produces numerous seeds contained in large hanging pods. Once dislodged from the pods, the seeds are dispersed by birds and mammals, including livestock, which eat the pods. It has been suggested that the seeds should be the target for biological control programme. Some invasive alien plant species are characterised by their ability to spread and establish in new ecosystems because they tolerate a wide range of environmental conditions. In order to predict areas of likely invasion, species distribution models (SDMs) are used to identify areas climatically suitable for their invasion, so enabling better targeted control of the plant species. Gleditsia triacanthos adapts to a wide range of climates and soil types, and tolerates salinity, drought and frost. Currently primarily restricted to the Grassland Biome of South Africa, G. triacanthos has doubled its distribution area in the past 15 years, and it is not known how far the species will spread. In this study we used two different modelling programmes, CLIMEX and MaxEnt, to predict areas where G. triacanthos could find favourable growing conditions; both SDMs showed that most of the country is suitable for G. triacanthos and that it will probably continue to spread, if left unmanaged, into new bioregions, such as the Karoo. In South Africa, the Asian seed-feeding bruchid, Megabruchidius tonkineus (Pic, 1914) (Coleoptera: Chrysomelidae: Bruchinae) has been recorded in the plant’s seed pods and has been considered as a biological control agent. The insect was not released as part of a formal biological control programme and neither host-specificity nor impact studies were conducted on the species prior to its discovery. In 2017 a decision was made to re-consider its status as a Abstract biological control agent until further details of its biology, host specificity, and impact on the seeds of G. triacanthos in South Africa were available. This study shows that Megabruchidius tonkineus has established across the entire G. triacanthos population in South Africa damaging approximately 9% of seeds. Laboratory studies show that, Megabruchidius tonkineus completes its larval development in the seeds of G. triacanthos in about 66.80 ± 0.6880 SE days before eclosing. In addition, the adult females oviposit on the following Fabaceae species: Arachis hypogaea, Albizia, julibrissin, Cicer arietinum, Pisum sativum, Dipogon lignosus, Peltophorum africanum, Podalyria buxifolia Senegalia burkei, Umtiza listerina and Vachellia sieberiana. However, larval development was limited to G. triacanthos. It is concluded that the seed-feeding beetle is not a threat to native Fabaceae species in South Africa, however, it does not damage enough G. triacanthos seeds to be considered a valuable biological control agent at this stage, and additional seed-feeding biological control agents should be considered to reduce the number of G. triacanthos seeds entering the environment. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2021
- Full Text:
Potential Synergism between Entomopathogenic Fungi and Entomopathogenic Nematodes for the control of false codling moth (Thaumatotibia leucotreta)
- Authors: Prinsloo, Samantha Lee
- Date: 2021-10
- Subjects: Cryptophlebia leucotreta , Entomopathogenic fungi , Insect nematodes , Citrus Diseases and pests , Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents
- Language: English
- Type: Masters theses , text
- Identifier: http://hdl.handle.net/10962/188832 , vital:44790
- Description: False codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) (FCM), is a major phytosanitary pest of citrus in South Africa. Sufficient control measures for the soil-dwelling life stages of FCM have yet to be identified and owing to restrictions on the use of insecticides, non-chemical control options have been investigated including the use of entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPN). Laboratory and field trials on an indigenous EPF, Metarhizium anisopliae FCM Ar 23 B3, have shown that this isolate is capable of inducing mortality in FCM soil-dwelling life stages. Other agents that have been highlighted as potential controls for soil-dwelling FCM life stages are the EPN species Steinernema yirgalemense 157-C, S. jeffreyense J194 and H. noenieputensis 158-C. This study conducted laboratory bioassays to assess the virulence of these four control agents on fifth instar FCM, in 24-well plates. These results reaffirmed the virulence of the four microbial control agents at their recommended doses of 50 IJs (EPN) and 1×107 conidia/ml (EPF) against fifth instar FCM with 80 to 96% larval mortality recorded. The EPF isolate exhibited the lowest mortality whilst S. yirgalemense induced the greatest mortality. In addition, the lethal concentration (LC) values for each isolate were determined using dose response bioassays. These values were previously unknown for all EPN species and for the EPF isolate based on the methodology used in this study. The LC50 results in order from lowest to highest EPN IJ concentration requirements were 4.38 IJs (S. yirgalemense), 4.47 IJs (S. jeffreyense) and 7.11 IJs (H. noenieputensis). The EPF isolate exhibited an LC50 of 3.42×105 conidia/ml. Lastly, research has shown that the combination of two control agents may increase control of late instar lepidopteran and coleopteran larvae, through synergistic interactions. Thus, the interactions that occurred between the combination of these EPN species with the EPF isolate were determined. This study found that when all three EPN species were combined simultaneously and sequentially with the EPF isolate M. anisopliae FCM AR 23 B3, additive interactions took place with exception of the simultaneous application of S. yirgalemense and H. noenieputensis, with the EPF and S. jeffreyense applied 24 h post EPF application. For the former, a synergistic interaction was found, whilst for the latter two, an antagonistic interaction. Although no strongly synergistic interactions were observed, additive interactions have been shown to reach a synergistic level when certain parameters are changed. Moving forward, a uniform methodology for conducting EPF/EPN interaction experiments has been suggested. It has also been recommended that due to the additive interactions observed in this study, laboratory soil-bioassays and field trials should be carried out for all three EPN species in combination with the EPF isolate. This research will inevitably facilitate the constant knowledge into management strategies for the phytosanitary pest, FCM in South African citrus. , Thesis (MSc) -- Science, Zoology and Entomology, 2021
- Full Text:
- Authors: Prinsloo, Samantha Lee
- Date: 2021-10
- Subjects: Cryptophlebia leucotreta , Entomopathogenic fungi , Insect nematodes , Citrus Diseases and pests , Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents
- Language: English
- Type: Masters theses , text
- Identifier: http://hdl.handle.net/10962/188832 , vital:44790
- Description: False codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) (FCM), is a major phytosanitary pest of citrus in South Africa. Sufficient control measures for the soil-dwelling life stages of FCM have yet to be identified and owing to restrictions on the use of insecticides, non-chemical control options have been investigated including the use of entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPN). Laboratory and field trials on an indigenous EPF, Metarhizium anisopliae FCM Ar 23 B3, have shown that this isolate is capable of inducing mortality in FCM soil-dwelling life stages. Other agents that have been highlighted as potential controls for soil-dwelling FCM life stages are the EPN species Steinernema yirgalemense 157-C, S. jeffreyense J194 and H. noenieputensis 158-C. This study conducted laboratory bioassays to assess the virulence of these four control agents on fifth instar FCM, in 24-well plates. These results reaffirmed the virulence of the four microbial control agents at their recommended doses of 50 IJs (EPN) and 1×107 conidia/ml (EPF) against fifth instar FCM with 80 to 96% larval mortality recorded. The EPF isolate exhibited the lowest mortality whilst S. yirgalemense induced the greatest mortality. In addition, the lethal concentration (LC) values for each isolate were determined using dose response bioassays. These values were previously unknown for all EPN species and for the EPF isolate based on the methodology used in this study. The LC50 results in order from lowest to highest EPN IJ concentration requirements were 4.38 IJs (S. yirgalemense), 4.47 IJs (S. jeffreyense) and 7.11 IJs (H. noenieputensis). The EPF isolate exhibited an LC50 of 3.42×105 conidia/ml. Lastly, research has shown that the combination of two control agents may increase control of late instar lepidopteran and coleopteran larvae, through synergistic interactions. Thus, the interactions that occurred between the combination of these EPN species with the EPF isolate were determined. This study found that when all three EPN species were combined simultaneously and sequentially with the EPF isolate M. anisopliae FCM AR 23 B3, additive interactions took place with exception of the simultaneous application of S. yirgalemense and H. noenieputensis, with the EPF and S. jeffreyense applied 24 h post EPF application. For the former, a synergistic interaction was found, whilst for the latter two, an antagonistic interaction. Although no strongly synergistic interactions were observed, additive interactions have been shown to reach a synergistic level when certain parameters are changed. Moving forward, a uniform methodology for conducting EPF/EPN interaction experiments has been suggested. It has also been recommended that due to the additive interactions observed in this study, laboratory soil-bioassays and field trials should be carried out for all three EPN species in combination with the EPF isolate. This research will inevitably facilitate the constant knowledge into management strategies for the phytosanitary pest, FCM in South African citrus. , Thesis (MSc) -- Science, Zoology and Entomology, 2021
- Full Text:
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:
- 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:
- «
- ‹
- 1
- ›
- »