Composition and physiological roles of gut microbiota in the False Coding Moth (Thaumatotibia leucotreta)
- Authors: Richardson, Perryn Heather
- Date: 2023-10-13
- Subjects: False codling moth , Microbiomes , Insect physiology , Citrus Diseases and pests South Africa , Biological pest control , Cryptophlebia leucotreta
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424457 , vital:72155
- Description: Gut microbiota can have a profound influence on host performance, behaviour and fitness. For False Codling Moth (FCM), Thaumatotibia leucotreta (Lepidoptera: Tortricidae), a major pest of citrus in South Africa, little work has been undertaken to date on gut microbe diversity or its influence on the host. This thesis aimed to i) characterise the gut microbiome of FCM under laboratory conditions and in FCM from the field, ii) and produce moths with reduced gut microbiota through egg dechorionation, which was followed by iii) the measurement of a suite of physiological traits, namely mass, survival and thermal stress in FCM from normal laboratory, dechorionated laboratory and field collected larvae that may be indicative of overall field performance. We aimed to directly test the hypothesis that gut microbial diversity partly determines insect performance and fitness by measuring its effects on growth, development, and tolerance to cold temperatures in FCM. FCM eggs that underwent dechorionation with sodium hypochlorite had an overall effect on larval survival, egg morphology and both larval and adult moth physiological measures. Increasing concentrations of sodium hypochlorite significantly decreased insect survival, (𝜒2(1, n = 10 850) = 21.724, p-value < 0.0001), with a concentration of ≈3.69% as the concentration limit (p-value < 0.001). Successful dechorionation of FCM was achieved with a wash of sodium hypochlorite at around 3.69% concentration and was visually confirmed by reduction of FCM egg surface area, (𝜒2(25, n = 260) p-value < 0.0001) and Scanning Electron Micrographs of the egg morphology. The gut microbiome of FCM from the different focus treatments was successfully characterized. Identification of the dominant bacterial families in these microbiomes revealed Xanthobacteraceae, Beijerinckiaceae and Burkholderiaceae in both the laboratory reared and field collected larvae, which suggests their systematic association with T. leucotreta. The most abundant genera were revealed as Bradyrhizobium, Methylobacterium and Burkholderia-Caballeronia-Paraburkholderia. Comparison of larval mass showed that treatment (dechorionated or not) had a significant effect on larval mass (𝜒2(2, n = 230) = 22.703, p-value < 0.001), field larvae were heavier than both control larvae and larvae with a disrupted gut microbiome. However, adult insects with a disrupted gut microbiome had more mass than individuals from the control and field-collected larvae with intact gut microbiomes (𝜒2(2, n = 230) = 39.074, p-value < 0.001). Despite the difference in mass between larval treatments, there was no significant difference in relative protein (𝜒2(2, n = 24) = 5.680, p-value = 0.06), carbohydrate (𝜒2(2, n = 24) = 3.940, p-value = 0.14) or lipid (𝜒2(2, n = 24) = 6.032, p-value = 0.05) content between individuals from the control and dechorionated treatments and field-collected individuals. Turning to thermal physiology, insects collected from the field took significantly longer to recover from chill coma than both laboratory treatments with intact and disrupted gut microbiomes (𝜒2(2, n = 129 = 39.659, p-value < 0.001). In addition, exposure to cold stress showed that treatment had a significant effect on insect mortality (𝜒2(2, n = 272) = 9.176, p-value = 0.01), with individuals from the control and dechorionated treatment being less likely to die after experiencing cold stress compared to field-collected individuals. Differences in the mass and thermal tolerance of insects with intact and disrupted gut microbiota suggest that gut microbiota may play an important role in the cold performance of T. leucotreta, and these findings constitute the basis for future molecular work on the functions of these bacterial taxa. This research highlights the need for consideration of the effects of T. leucotreta microbiome in current pest control programs. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Richardson, Perryn Heather
- Date: 2023-10-13
- Subjects: False codling moth , Microbiomes , Insect physiology , Citrus Diseases and pests South Africa , Biological pest control , Cryptophlebia leucotreta
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424457 , vital:72155
- Description: Gut microbiota can have a profound influence on host performance, behaviour and fitness. For False Codling Moth (FCM), Thaumatotibia leucotreta (Lepidoptera: Tortricidae), a major pest of citrus in South Africa, little work has been undertaken to date on gut microbe diversity or its influence on the host. This thesis aimed to i) characterise the gut microbiome of FCM under laboratory conditions and in FCM from the field, ii) and produce moths with reduced gut microbiota through egg dechorionation, which was followed by iii) the measurement of a suite of physiological traits, namely mass, survival and thermal stress in FCM from normal laboratory, dechorionated laboratory and field collected larvae that may be indicative of overall field performance. We aimed to directly test the hypothesis that gut microbial diversity partly determines insect performance and fitness by measuring its effects on growth, development, and tolerance to cold temperatures in FCM. FCM eggs that underwent dechorionation with sodium hypochlorite had an overall effect on larval survival, egg morphology and both larval and adult moth physiological measures. Increasing concentrations of sodium hypochlorite significantly decreased insect survival, (𝜒2(1, n = 10 850) = 21.724, p-value < 0.0001), with a concentration of ≈3.69% as the concentration limit (p-value < 0.001). Successful dechorionation of FCM was achieved with a wash of sodium hypochlorite at around 3.69% concentration and was visually confirmed by reduction of FCM egg surface area, (𝜒2(25, n = 260) p-value < 0.0001) and Scanning Electron Micrographs of the egg morphology. The gut microbiome of FCM from the different focus treatments was successfully characterized. Identification of the dominant bacterial families in these microbiomes revealed Xanthobacteraceae, Beijerinckiaceae and Burkholderiaceae in both the laboratory reared and field collected larvae, which suggests their systematic association with T. leucotreta. The most abundant genera were revealed as Bradyrhizobium, Methylobacterium and Burkholderia-Caballeronia-Paraburkholderia. Comparison of larval mass showed that treatment (dechorionated or not) had a significant effect on larval mass (𝜒2(2, n = 230) = 22.703, p-value < 0.001), field larvae were heavier than both control larvae and larvae with a disrupted gut microbiome. However, adult insects with a disrupted gut microbiome had more mass than individuals from the control and field-collected larvae with intact gut microbiomes (𝜒2(2, n = 230) = 39.074, p-value < 0.001). Despite the difference in mass between larval treatments, there was no significant difference in relative protein (𝜒2(2, n = 24) = 5.680, p-value = 0.06), carbohydrate (𝜒2(2, n = 24) = 3.940, p-value = 0.14) or lipid (𝜒2(2, n = 24) = 6.032, p-value = 0.05) content between individuals from the control and dechorionated treatments and field-collected individuals. Turning to thermal physiology, insects collected from the field took significantly longer to recover from chill coma than both laboratory treatments with intact and disrupted gut microbiomes (𝜒2(2, n = 129 = 39.659, p-value < 0.001). In addition, exposure to cold stress showed that treatment had a significant effect on insect mortality (𝜒2(2, n = 272) = 9.176, p-value = 0.01), with individuals from the control and dechorionated treatment being less likely to die after experiencing cold stress compared to field-collected individuals. Differences in the mass and thermal tolerance of insects with intact and disrupted gut microbiota suggest that gut microbiota may play an important role in the cold performance of T. leucotreta, and these findings constitute the basis for future molecular work on the functions of these bacterial taxa. This research highlights the need for consideration of the effects of T. leucotreta microbiome in current pest control programs. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Date Issued: 2023-10-13
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:
- Date Issued: 2023-10-13
- 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:
- Date Issued: 2023-10-13
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:
- Date Issued: 2022-10-14
- 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:
- Date Issued: 2022-10-14
An investigation into yeast-baculovirus synergism for the improved control of Thaumatotibia leucotreta, an economically important pest of citrus
- Authors: Van der Merwe, Marcél
- Date: 2021-10-29
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Yeast , Natural pesticides , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control , Thaumatotibia leucotreta
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191236 , vital:45073
- Description: A mutualistic association between Cydia pomonella and yeasts belonging to the genus Metschnikowia has previously been demonstrated. Larval feeding galleries inoculated with M. andauensis, reduced larval mortality and enhanced larval development. Additionally, adult C. pomonella female oviposition preference was also shown to be influenced by the volatiles produced by M. andauensis. This mutualistic relationship was manipulated for biological control purposes, by combining M. pulcherrima with the baculovirus Cydia pomonella granulovirus. The combination of M. pulcherrima with brown cane sugar and CpGV in laboratory assays and field trials resulted in a significant increase in larval mortality. A similar observation was made when M. pulcherrima was substituted for Saccharomyces cerevisiae. This indicates that yeasts harbour the potential for use in biological control, especially when combined with other well-established biocontrol methods. Thaumatotibia leucotreta is a phytophagous insect endemic to southern Africa. It is highly significant to the South African citrus industry due to its classification as a phytosanitary pest by most international markets. An integrated pest management programme has been implemented to control T. leucotreta. The baculovirus Cryptophlebia leucotreta granulovirus forms one component of this programme and is highly effective. In this study, we proposed to determine which yeast species occur naturally in the gut of T. leucotreta larvae and to examine whether any of the isolated yeast species, when combined with the CrleGV-SA, enhance its effectiveness. Firstly, Navel oranges infested with T. leucotreta larvae were collected from geographically distinct citrus-producing regions across South Africa. This led to the isolation and identification of six yeast species from the gut of T. leucotreta larvae via PCR amplification and sequencing of the internal transcribed spacer region and D1/D2 domain of the large subunit. Six yeast species were identified, viz. Meyerozyma guilliermondii, Hanseniaspora uvarum, Clavispora lusitaniae, Kluyveromyces marxianus, Pichia kudriavzevii and Pichia kluyveri. Additionally, Saccharomyces cerevisiae was included as a control in all trials due to its commercial availability and use in the artificial diet used to rear T. leucotreta. Secondly, larval development and attraction assays were conducted with the isolated yeast species. Thaumatotibia leucotreta larvae that fed on Navel oranges inoculated with M. guilliermondii, P. kluyveri, H. uvarum, and S. cerevisiae had accelerated developmental periods and reduced mortality rates. Additionally, it was demonstrated that T. leucotreta neonates were attracted to YPD broth cultures inoculated with P. kluyveri, H. uvarum, P. kudriavzevii and K. marxianus for feeding. Thirdly, oviposition preference assays were conducted with adult T. leucotreta females to determine whether the isolated yeast species influence their egg-laying in two-choice and multiple-choice tests. Navel oranges were inoculated with a specific yeast isolate, and mated adult females were left to oviposit. Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to influence adult T. leucotreta female oviposition preference in two-choice tests. However, multiple-choice tests using the aforementioned yeast species did not mimic these results. Lastly, a series of detached fruit bioassays were performed to determine the optimal yeast:virus ratio, test all isolated yeast species in combination with CrleGV-SA and to further enhance yeast/virus formulation through the addition of an adjuvant and surfactant. CrleGV-SA was applied at a lethal concentration that would kill 50 % of T. leucotreta larvae. The optimal yeast concentration to use alongside CrleGV-SA was determined. Pichia kluyveri, P. kudriavzevii, K. marxianus and S. cerevisiae in combination with CrleGV-SA increased larval mortality compared to CrleGV-SA alone. The inclusion of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae plus CrleGV-SA formulations greatly enhanced their efficacy. Additionally, semi-field trials were initiated using P. kudriavzevii and S. cerevisiae, with promising preliminary results being obtained, although more replicates need to be performed. The experiments performed in this study provide a platform for further research into the application of a yeast/virus combination as a novel control and monitoring option for T. leucotreta in the field. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
- Authors: Van der Merwe, Marcél
- Date: 2021-10-29
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Yeast , Natural pesticides , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control , Thaumatotibia leucotreta
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191236 , vital:45073
- Description: A mutualistic association between Cydia pomonella and yeasts belonging to the genus Metschnikowia has previously been demonstrated. Larval feeding galleries inoculated with M. andauensis, reduced larval mortality and enhanced larval development. Additionally, adult C. pomonella female oviposition preference was also shown to be influenced by the volatiles produced by M. andauensis. This mutualistic relationship was manipulated for biological control purposes, by combining M. pulcherrima with the baculovirus Cydia pomonella granulovirus. The combination of M. pulcherrima with brown cane sugar and CpGV in laboratory assays and field trials resulted in a significant increase in larval mortality. A similar observation was made when M. pulcherrima was substituted for Saccharomyces cerevisiae. This indicates that yeasts harbour the potential for use in biological control, especially when combined with other well-established biocontrol methods. Thaumatotibia leucotreta is a phytophagous insect endemic to southern Africa. It is highly significant to the South African citrus industry due to its classification as a phytosanitary pest by most international markets. An integrated pest management programme has been implemented to control T. leucotreta. The baculovirus Cryptophlebia leucotreta granulovirus forms one component of this programme and is highly effective. In this study, we proposed to determine which yeast species occur naturally in the gut of T. leucotreta larvae and to examine whether any of the isolated yeast species, when combined with the CrleGV-SA, enhance its effectiveness. Firstly, Navel oranges infested with T. leucotreta larvae were collected from geographically distinct citrus-producing regions across South Africa. This led to the isolation and identification of six yeast species from the gut of T. leucotreta larvae via PCR amplification and sequencing of the internal transcribed spacer region and D1/D2 domain of the large subunit. Six yeast species were identified, viz. Meyerozyma guilliermondii, Hanseniaspora uvarum, Clavispora lusitaniae, Kluyveromyces marxianus, Pichia kudriavzevii and Pichia kluyveri. Additionally, Saccharomyces cerevisiae was included as a control in all trials due to its commercial availability and use in the artificial diet used to rear T. leucotreta. Secondly, larval development and attraction assays were conducted with the isolated yeast species. Thaumatotibia leucotreta larvae that fed on Navel oranges inoculated with M. guilliermondii, P. kluyveri, H. uvarum, and S. cerevisiae had accelerated developmental periods and reduced mortality rates. Additionally, it was demonstrated that T. leucotreta neonates were attracted to YPD broth cultures inoculated with P. kluyveri, H. uvarum, P. kudriavzevii and K. marxianus for feeding. Thirdly, oviposition preference assays were conducted with adult T. leucotreta females to determine whether the isolated yeast species influence their egg-laying in two-choice and multiple-choice tests. Navel oranges were inoculated with a specific yeast isolate, and mated adult females were left to oviposit. Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to influence adult T. leucotreta female oviposition preference in two-choice tests. However, multiple-choice tests using the aforementioned yeast species did not mimic these results. Lastly, a series of detached fruit bioassays were performed to determine the optimal yeast:virus ratio, test all isolated yeast species in combination with CrleGV-SA and to further enhance yeast/virus formulation through the addition of an adjuvant and surfactant. CrleGV-SA was applied at a lethal concentration that would kill 50 % of T. leucotreta larvae. The optimal yeast concentration to use alongside CrleGV-SA was determined. Pichia kluyveri, P. kudriavzevii, K. marxianus and S. cerevisiae in combination with CrleGV-SA increased larval mortality compared to CrleGV-SA alone. The inclusion of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae plus CrleGV-SA formulations greatly enhanced their efficacy. Additionally, semi-field trials were initiated using P. kudriavzevii and S. cerevisiae, with promising preliminary results being obtained, although more replicates need to be performed. The experiments performed in this study provide a platform for further research into the application of a yeast/virus combination as a novel control and monitoring option for T. leucotreta in the field. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
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:
- Date Issued: 2021-10
- 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:
- Date Issued: 2021-10
Baculovirus synergism for improved management of false codling moth Thaumatotibia leucotreta Meyr. (Lepidoptera: Tortricidae)
- Authors: Taylor, David Graham
- Date: 2021-04
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Codling moth , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/176942 , vital:42774
- Description: Baculoviruses are an environmentally friendly and effective agent for managing lepidopteran pests. This includes the management of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a serious pest of citrus in Southern Africa and a major threat to the South African citrus export industry. For more than 15 years, CrleGV-SA- based biopesticides have been used as part of an integrated pest management strategy for the control of T. leucotreta in citrus orchards in South Africa, under the names Cryptogran™ and Cryptex®. While these biopesticides have been effective during this period, there are some areas in which baculovirus use could potentially be improved. Baculoviruses are notoriously slow to kill in comparison to chemical-based pesticides, and lately, pest resistance to baculoviruses has become a major concern with the development of resistance by Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) to its granulovirus occurring in the field in Europe. The consistent use of CrleGV-SA for more than 15 years in the field has raised concern that T. leucotreta could develop resistance to this virus, and has made it necessary to alter baculovirus-based management strategies to prevent this from occurring. A second baculovirus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), has recently been isolated and was shown to be effective against T. leucotreta. However, the interactions between CrleGV-SA and CrpeNPV are not yet understood and so it is important to test these interactions before both viruses are applied on the same orchards. Not only is it important to know whether these viruses could negatively impact each other, but it is also important to test whether they could interact synergistically. A synergistic interaction could not only provide a potential tool for the management of resistance, but it could also be exploited to improve baculovirus-based management of T. leucotreta. In this study, a stock of CrleGV-SA was purified by glycerol gradient centrifugation from T. leucotreta cadavers, while a stock of CrpeNPV purified from Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) cadavers was provided by River Bioscience (Pty) Ltd. These stocks were screened for purity by a multiplex polymerase chain reaction (mPCR) protocol designed to detect CrleGV-SA and CrpeNPV. The occlusion body (OB) density was then calculated using darkfield microscopy and a counting chamber. Both stocks were shown to be pure within the limits of the mPCR protocol, and the CrleGV-SA and CrpeNPV stocks were calculated to contain 3.08 × 1011 OBs/mL and 1.92 × 1011 OBs/mL respectively The first aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the dose mortality, in terms of lethal concentration. This was calculated using 7-day surface-dose biological assays for each virus and a 1:1 mixture of OBs of the two against T. leucotreta neonates. The lethal concentrations of each treatment required to kill 50 % of larvae (LC50) and 90 % of larvae (LC90) for each treatment were then calculated and compared using a probit regression. The mixed infection performed significantly better than either virus by itself, while each virus by itself did not differ significantly from the other. The LC50 for CrleGV-SA, CrpeNPV and the mixed infection were 1.53 × 104 OBs/mL, 1.15 × 104 OBs/mL and 4.38 × 103 OBs/mL respectively. The LC90 of CrleGV-SA, CrpeNPV and the mixed infection were calculated to be 4.10 × 105 OBs/mL, 1.05 × 105 OBs/mL, and 4.09 × 104 OBs/mL respectively. The second aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the speed of kill. A time-response biological assay protocol was created that allowed for effective observation of the larvae. This was then used to generate time-mortality data that were analysed by a logit regression function to calculate and compare the treatments at the time of 50 % larval mortality (LT50) and the time of 90 % mortality (LT90). Each virus by itself did not differ significantly from the other, while the mixed infection took significantly longer to kill 50 % and 90 % of the larvae, suggesting that there is competition for resources between viruses during the secondary, systemic phase of infection. The LT50 for CrleGV-SA, CrpeNPV and the mixed infection were 117.5 hours, 113.5 hours and 139.0 hours respectively. The LT90 for CrleGV-SA, CrpeNPV and the mixed infection were 153.2 hours, 159.3, and 193.4 hours respectively. Finally, the composition of OBs recovered from the cadavers produced by the time-response biological assays were investigated by mPCR. A method for extracting gDNA from OBs in neonate-sized T. leucotreta larvae is described. The presence of CrpeNPV along with CrleGV-SA was noted in 4 out of 9 larvae inoculated with only CrleGV-SA. The presence of CrleGV-SA as well as CrpeNPV was noted in all but one larva inoculated with only CrpeNPV, and both CrleGV-SA and CrpeNPV were noted in all but one larva inoculated with a 1:1 mixture of the two, with one larva only being positive for CrleGV-SA. This suggests either stock contamination or the presence of covert infections of CrleGV-SA and CrpeNPV in the T. leucotreta population used in this study. This is the second study to report an improved lethal concentration of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates, and the first study to report the slower speed of kill of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates. While the improved lethal concentration of the mixed infection is a promising step in the future improvement of baculovirus-based biopesticides, it is at the cost of a slower speed of kill. , Thesis (MSc) -- Faculty of Science, Department of Zoology and Entomology, 2021
- Full Text:
- Date Issued: 2021-04
- Authors: Taylor, David Graham
- Date: 2021-04
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Codling moth , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/176942 , vital:42774
- Description: Baculoviruses are an environmentally friendly and effective agent for managing lepidopteran pests. This includes the management of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a serious pest of citrus in Southern Africa and a major threat to the South African citrus export industry. For more than 15 years, CrleGV-SA- based biopesticides have been used as part of an integrated pest management strategy for the control of T. leucotreta in citrus orchards in South Africa, under the names Cryptogran™ and Cryptex®. While these biopesticides have been effective during this period, there are some areas in which baculovirus use could potentially be improved. Baculoviruses are notoriously slow to kill in comparison to chemical-based pesticides, and lately, pest resistance to baculoviruses has become a major concern with the development of resistance by Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) to its granulovirus occurring in the field in Europe. The consistent use of CrleGV-SA for more than 15 years in the field has raised concern that T. leucotreta could develop resistance to this virus, and has made it necessary to alter baculovirus-based management strategies to prevent this from occurring. A second baculovirus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), has recently been isolated and was shown to be effective against T. leucotreta. However, the interactions between CrleGV-SA and CrpeNPV are not yet understood and so it is important to test these interactions before both viruses are applied on the same orchards. Not only is it important to know whether these viruses could negatively impact each other, but it is also important to test whether they could interact synergistically. A synergistic interaction could not only provide a potential tool for the management of resistance, but it could also be exploited to improve baculovirus-based management of T. leucotreta. In this study, a stock of CrleGV-SA was purified by glycerol gradient centrifugation from T. leucotreta cadavers, while a stock of CrpeNPV purified from Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) cadavers was provided by River Bioscience (Pty) Ltd. These stocks were screened for purity by a multiplex polymerase chain reaction (mPCR) protocol designed to detect CrleGV-SA and CrpeNPV. The occlusion body (OB) density was then calculated using darkfield microscopy and a counting chamber. Both stocks were shown to be pure within the limits of the mPCR protocol, and the CrleGV-SA and CrpeNPV stocks were calculated to contain 3.08 × 1011 OBs/mL and 1.92 × 1011 OBs/mL respectively The first aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the dose mortality, in terms of lethal concentration. This was calculated using 7-day surface-dose biological assays for each virus and a 1:1 mixture of OBs of the two against T. leucotreta neonates. The lethal concentrations of each treatment required to kill 50 % of larvae (LC50) and 90 % of larvae (LC90) for each treatment were then calculated and compared using a probit regression. The mixed infection performed significantly better than either virus by itself, while each virus by itself did not differ significantly from the other. The LC50 for CrleGV-SA, CrpeNPV and the mixed infection were 1.53 × 104 OBs/mL, 1.15 × 104 OBs/mL and 4.38 × 103 OBs/mL respectively. The LC90 of CrleGV-SA, CrpeNPV and the mixed infection were calculated to be 4.10 × 105 OBs/mL, 1.05 × 105 OBs/mL, and 4.09 × 104 OBs/mL respectively. The second aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the speed of kill. A time-response biological assay protocol was created that allowed for effective observation of the larvae. This was then used to generate time-mortality data that were analysed by a logit regression function to calculate and compare the treatments at the time of 50 % larval mortality (LT50) and the time of 90 % mortality (LT90). Each virus by itself did not differ significantly from the other, while the mixed infection took significantly longer to kill 50 % and 90 % of the larvae, suggesting that there is competition for resources between viruses during the secondary, systemic phase of infection. The LT50 for CrleGV-SA, CrpeNPV and the mixed infection were 117.5 hours, 113.5 hours and 139.0 hours respectively. The LT90 for CrleGV-SA, CrpeNPV and the mixed infection were 153.2 hours, 159.3, and 193.4 hours respectively. Finally, the composition of OBs recovered from the cadavers produced by the time-response biological assays were investigated by mPCR. A method for extracting gDNA from OBs in neonate-sized T. leucotreta larvae is described. The presence of CrpeNPV along with CrleGV-SA was noted in 4 out of 9 larvae inoculated with only CrleGV-SA. The presence of CrleGV-SA as well as CrpeNPV was noted in all but one larva inoculated with only CrpeNPV, and both CrleGV-SA and CrpeNPV were noted in all but one larva inoculated with a 1:1 mixture of the two, with one larva only being positive for CrleGV-SA. This suggests either stock contamination or the presence of covert infections of CrleGV-SA and CrpeNPV in the T. leucotreta population used in this study. This is the second study to report an improved lethal concentration of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates, and the first study to report the slower speed of kill of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates. While the improved lethal concentration of the mixed infection is a promising step in the future improvement of baculovirus-based biopesticides, it is at the cost of a slower speed of kill. , Thesis (MSc) -- Faculty of Science, Department of Zoology and Entomology, 2021
- Full Text:
- Date Issued: 2021-04
Assessment of pheromone specificity in Thaumatotibia leucotreta (Meyrick) populations with focus on pest monitoring and the regional rollout of the sterile insect technique in citrus
- Authors: Joubert, Francois D
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Pheromone traps , Citrus -- Diseases and pests -- South Africa , Cryptophlebia leucotreta -- Contol , Cryptophlebia leucotreta -- Biological control
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/60665 , vital:27812
- Description: False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. It is recognized by several markets as a phytosanitary organism and the efficient control of this pest is now more important than ever. The pheromone communication between the male and female moths has been exploited in order to control FCM through the sterile insect technique (SIT). The sterilized males used for all SIT programmes across South Africa come from a colony that originates from wild material collected from the Citrusdal area of the Western Cape Province. The aim of this study was to determine if any differences in attractiveness of females to males exist between different geographical populations of FCM and if so what impact this would have on the male’s ability to locate females from other populations via the volatile sex pheromone released by the female. Laboratory trials with Y-tube olfactometers and flight tunnels tested the attraction of male moths to virgin females, but did not yield any consistent results. Field experiments were conducted with sterile male Citrusdal moths released and recaptured in yellow delta traps in two separate trials. For one trial, the traps were baited with live virgin females from five different geographical populations including Addo, Nelspruit, Marble Hall, Citrusdal and the Old colony, which is a mixture of several populations. For the other trial traps were baited with various synthetic pheromone blends including three regional blends which included South Africa, Ivory Coast and Malawi and three commercial blends including Pherolure, Isomate and Checkmate. For the virgin female trial the Citrusdal males showed a significant preference for females from their own population. There was also a significant difference in the recaptures from the different synthetic pheromones. The South African blend was the most attractive of all the regional and commercial blends. A cross-mating trial was also conducted under laboratory conditions in petri dishes with five different FCM populations including Citrusdal, Addo, Marble Hall, Nelspruit and Old (mixed origin). Females produced more eggs when mated with males from the same population for the Addo, Marble Hall, Nelspruit and Old (mixed origin) populations. The only case in which this was statistically significant was for the Marble Hall population. All the crosses produced viable eggs and the origin of the male or female did not influence egg hatch. The results from this study may lead to improvements in both the control and monitoring of FCM populations. The control methods include mating disruption, attract-and-kill and SIT. Tailoring these methods for a specific growing area with a pheromone blend originating from the area or releasing sterile moths from a colony that originates from the area may optimize the available monitoring and control options.
- Full Text:
- Date Issued: 2018
- Authors: Joubert, Francois D
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Pheromone traps , Citrus -- Diseases and pests -- South Africa , Cryptophlebia leucotreta -- Contol , Cryptophlebia leucotreta -- Biological control
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/60665 , vital:27812
- Description: False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. It is recognized by several markets as a phytosanitary organism and the efficient control of this pest is now more important than ever. The pheromone communication between the male and female moths has been exploited in order to control FCM through the sterile insect technique (SIT). The sterilized males used for all SIT programmes across South Africa come from a colony that originates from wild material collected from the Citrusdal area of the Western Cape Province. The aim of this study was to determine if any differences in attractiveness of females to males exist between different geographical populations of FCM and if so what impact this would have on the male’s ability to locate females from other populations via the volatile sex pheromone released by the female. Laboratory trials with Y-tube olfactometers and flight tunnels tested the attraction of male moths to virgin females, but did not yield any consistent results. Field experiments were conducted with sterile male Citrusdal moths released and recaptured in yellow delta traps in two separate trials. For one trial, the traps were baited with live virgin females from five different geographical populations including Addo, Nelspruit, Marble Hall, Citrusdal and the Old colony, which is a mixture of several populations. For the other trial traps were baited with various synthetic pheromone blends including three regional blends which included South Africa, Ivory Coast and Malawi and three commercial blends including Pherolure, Isomate and Checkmate. For the virgin female trial the Citrusdal males showed a significant preference for females from their own population. There was also a significant difference in the recaptures from the different synthetic pheromones. The South African blend was the most attractive of all the regional and commercial blends. A cross-mating trial was also conducted under laboratory conditions in petri dishes with five different FCM populations including Citrusdal, Addo, Marble Hall, Nelspruit and Old (mixed origin). Females produced more eggs when mated with males from the same population for the Addo, Marble Hall, Nelspruit and Old (mixed origin) populations. The only case in which this was statistically significant was for the Marble Hall population. All the crosses produced viable eggs and the origin of the male or female did not influence egg hatch. The results from this study may lead to improvements in both the control and monitoring of FCM populations. The control methods include mating disruption, attract-and-kill and SIT. Tailoring these methods for a specific growing area with a pheromone blend originating from the area or releasing sterile moths from a colony that originates from the area may optimize the available monitoring and control options.
- Full Text:
- Date Issued: 2018
Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) population ecology in citrus orchards: the influence of orchard age
- Authors: Albertyn, Sonnica
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Population biology , Insect populations , Orchards , Insect nematodes , Entomopathogenic fungi
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62615 , vital:28213
- Description: Anecdotal reports in the South African citrus industry claim higher populations of false codling moth (FCM), Thaumatotibia (Cryptophlebia) leucotreta (Meyr) (Lepidoptera: Tortricidae), in orchards during the first three to five harvesting years of citrus planted in virgin soil, after which, FCM numbers seem to decrease and remain consistent. Various laboratory studies and field surveys were conducted to determine if, and why juvenile orchards (four to eight years old) experience higher FCM infestation than mature orchards (nine years and older). In laboratory trials, Washington Navel oranges and Nova Mandarins from juvenile trees were shown to be significantly more susceptible to FCM damage and significantly more attractive for oviposition in both choice and no-choice trials, than fruit from mature trees. Although fruit from juvenile Cambria Navel trees were significantly more attractive than mature orchards for oviposition, they were not more susceptible to FCM damage. In contrast, fruit from juvenile and mature Midnight Valencia orchards were equally attractive for oviposition, but fruit from juvenile trees were significantly more susceptible to FCM damage than fruit from mature trees. Artificial diets were augmented with powder from fruit from juvenile or mature Washington Navel orchards at 5%, 10%, 15% or 30%. Higher larval survival of 76%, 63%, 50% and 34%, respectively, was recorded on diets containing fruit powder from the juvenile trees than on diets containing fruit powder from the mature trees, at 69%, 57%, 44% and 27% larval survival, respectively. Bioassays were conducted to determine if differences in plant chemistry between fruit from juvenile and mature trees will have an impact on the susceptibility FCM to entomopathogenic nematodes (EPN), entomopathogenic fungi (EPF) and Cryptophlebia leucotreta granulovirus (CrleGV). No significant differences in the susceptibility of larvae reared on diets containing 15% fruit powder from juvenile and mature trees to EPN and EPF were recorded. Mortality of neonate larvae was significantly lower when placed on diets containing 15% fruit powder from mature trees (45% mortality) than diets containing 15% fruit powder from juvenile trees (61% mortality), after larvae ingested the lowest virus concentration tested, being 2 x104 OBs/ml. Data collected from field surveys showed significantly lower egg parasitism, virus infection of larvae and EPF occurrence in juvenile orchards than mature orchards. Egg parasitism was between 11% and 54% higher in mature orchards than juvenile orchards, with the exception of Mandarins during 2015, where egg parasitism was slightly higher in juvenile orchards, but not significantly so. A significantly higher proportion of larvae retrieved from mature orchards (7% of larvae) were infected with CrleGV than larvae retrieved from juvenile orchards (4% of larvae). A significantly higher occurrence of EPF was recorded in non-bearing and mature orchards, with 40% and 37% occurrence respectively, than in juvenile orchards, with 25% occurrence recorded. EPF occurrence in juvenile orchards increased significantly by 16% to 32% from the first to the third year of sampling. In contrast to results recorded in laboratory trials, similar or higher pest pressure in juvenile orchards than mature orchards did not always result in significantly higher levels of FCM damage under field conditions. FCM damage in juvenile orchards may have been lower than expected, as greater extremes of temperature and lower humidity were recorded in juvenile orchards, which would increase larval mortality. Results of this study showed that juvenile and mature orchards are significantly different and should be managed differently.
- Full Text:
- Date Issued: 2018
- Authors: Albertyn, Sonnica
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Population biology , Insect populations , Orchards , Insect nematodes , Entomopathogenic fungi
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62615 , vital:28213
- Description: Anecdotal reports in the South African citrus industry claim higher populations of false codling moth (FCM), Thaumatotibia (Cryptophlebia) leucotreta (Meyr) (Lepidoptera: Tortricidae), in orchards during the first three to five harvesting years of citrus planted in virgin soil, after which, FCM numbers seem to decrease and remain consistent. Various laboratory studies and field surveys were conducted to determine if, and why juvenile orchards (four to eight years old) experience higher FCM infestation than mature orchards (nine years and older). In laboratory trials, Washington Navel oranges and Nova Mandarins from juvenile trees were shown to be significantly more susceptible to FCM damage and significantly more attractive for oviposition in both choice and no-choice trials, than fruit from mature trees. Although fruit from juvenile Cambria Navel trees were significantly more attractive than mature orchards for oviposition, they were not more susceptible to FCM damage. In contrast, fruit from juvenile and mature Midnight Valencia orchards were equally attractive for oviposition, but fruit from juvenile trees were significantly more susceptible to FCM damage than fruit from mature trees. Artificial diets were augmented with powder from fruit from juvenile or mature Washington Navel orchards at 5%, 10%, 15% or 30%. Higher larval survival of 76%, 63%, 50% and 34%, respectively, was recorded on diets containing fruit powder from the juvenile trees than on diets containing fruit powder from the mature trees, at 69%, 57%, 44% and 27% larval survival, respectively. Bioassays were conducted to determine if differences in plant chemistry between fruit from juvenile and mature trees will have an impact on the susceptibility FCM to entomopathogenic nematodes (EPN), entomopathogenic fungi (EPF) and Cryptophlebia leucotreta granulovirus (CrleGV). No significant differences in the susceptibility of larvae reared on diets containing 15% fruit powder from juvenile and mature trees to EPN and EPF were recorded. Mortality of neonate larvae was significantly lower when placed on diets containing 15% fruit powder from mature trees (45% mortality) than diets containing 15% fruit powder from juvenile trees (61% mortality), after larvae ingested the lowest virus concentration tested, being 2 x104 OBs/ml. Data collected from field surveys showed significantly lower egg parasitism, virus infection of larvae and EPF occurrence in juvenile orchards than mature orchards. Egg parasitism was between 11% and 54% higher in mature orchards than juvenile orchards, with the exception of Mandarins during 2015, where egg parasitism was slightly higher in juvenile orchards, but not significantly so. A significantly higher proportion of larvae retrieved from mature orchards (7% of larvae) were infected with CrleGV than larvae retrieved from juvenile orchards (4% of larvae). A significantly higher occurrence of EPF was recorded in non-bearing and mature orchards, with 40% and 37% occurrence respectively, than in juvenile orchards, with 25% occurrence recorded. EPF occurrence in juvenile orchards increased significantly by 16% to 32% from the first to the third year of sampling. In contrast to results recorded in laboratory trials, similar or higher pest pressure in juvenile orchards than mature orchards did not always result in significantly higher levels of FCM damage under field conditions. FCM damage in juvenile orchards may have been lower than expected, as greater extremes of temperature and lower humidity were recorded in juvenile orchards, which would increase larval mortality. Results of this study showed that juvenile and mature orchards are significantly different and should be managed differently.
- Full Text:
- Date Issued: 2018
Yeast-baculovirus synergism: investigating mixed infections for improved management of the false codling moth, Thaumatotibia leucotreta
- Authors: Van der Merwe, Marcel
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Baculoviruses , Yeast , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62963 , vital:28347
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) or otherwise commonly known as the false codling moth is an indigenous pest of the citrus industry in southern Africa. The pest is highly significant as it impacts negatively on the export of fresh citrus fruits from South Africa to international markets. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been implemented. One component of this programme is the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV-SA) which has been formulated into the products Cryptogran™ and Cryptex®. It has previously been reported that there is a mutualistic association between Cydia pomonella (L.) (Lepidoptera: Tortricidae) also known as codling moth, and epiphytic yeasts. Cydia pomonella larval feeding galleries were colonised by yeasts and this, in turn, reduced larval mortality and enhanced larval development. It has been demonstrated in laboratory assays and field trials that combining yeast and brown cane sugar with Cydia pomonella granulovirus (CpGV) significantly increased larval mortality and lowered the proportion of injured apple fruit. This suggests that yeasts can enhance the effectiveness of an insect virus in managing pest larvae. In this study, we proposed to determine which species of yeast occur naturally in the digestive tract, frass and on the epidermis of T. leucotreta larvae and to examine whether any of these yeasts, when combined with the CrleGV-SA, have a synergistic effect in increasing mortality of T. leucotreta larvae. Firstly, Navel oranges infested with T. leucotreta larvae were collected from orchards in Sundays River Valley in Eastern Cape of South Africa. Larvae were extracted and analysed for the presence of yeast on their surface, or in their gut and frass. Four yeasts were isolated from T. leucotreta larvae and identified down to species level via PCR amplification and sequencing of internal transcribed spacer (ITS) region and D1/D2 domain of the large subunit (LSU) of rDNA region. These yeasts were isolated from the frass, epidermis and digestive tract of T. leucotreta larvae. The yeast isolates were identified as Meyerozyma caribbica, Pichia kluyveri, Pichia kudriavzevii and Hanseniaspora opuntiae. A yeast preference assay was conducted on female T. leucotreta moths to examine whether any of the isolated yeast species affected their oviposition preference. Navel oranges were inoculated with the isolated yeast species at a concentration of 6 × 108 cells.ml-1. The assay also included a Brewer’s yeast and distilled water control. Pichia kudriavzevii was shown to be the preferred yeast species for oviposition, as significantly more eggs were deposited on Navel oranges inoculated with this yeast compared to the other treatments. Lastly, a detached fruit bioassay was performed to evaluate the efficacy of mixing P. kudriavzevii with CrleGV-SA to enhance T. leucotreta larvae mortality. Pichia kudriavzevii was selected as it was demonstrated as having an effect on the oviposition preference of female T. leucotreta moths. The concentration at which P. kudriavzevii was applied remained the same as in the preference assay while CrleGV-SA was applied at lethal concentration required to kill 50 % of the population (9.31 × 107 OBs.ml-1). Although an increase in larval mortality was observed between CrleGV-SA being applied alone and the yeast/virus mixture, this result was determined not to be statistically significant. The experiments performed in this study provide a platform for further research into the application of a yeast-virus combination as a novel control option for T. leucotreta in the field. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Van der Merwe, Marcel
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Baculoviruses , Yeast , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/62963 , vital:28347
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) or otherwise commonly known as the false codling moth is an indigenous pest of the citrus industry in southern Africa. The pest is highly significant as it impacts negatively on the export of fresh citrus fruits from South Africa to international markets. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been implemented. One component of this programme is the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV-SA) which has been formulated into the products Cryptogran™ and Cryptex®. It has previously been reported that there is a mutualistic association between Cydia pomonella (L.) (Lepidoptera: Tortricidae) also known as codling moth, and epiphytic yeasts. Cydia pomonella larval feeding galleries were colonised by yeasts and this, in turn, reduced larval mortality and enhanced larval development. It has been demonstrated in laboratory assays and field trials that combining yeast and brown cane sugar with Cydia pomonella granulovirus (CpGV) significantly increased larval mortality and lowered the proportion of injured apple fruit. This suggests that yeasts can enhance the effectiveness of an insect virus in managing pest larvae. In this study, we proposed to determine which species of yeast occur naturally in the digestive tract, frass and on the epidermis of T. leucotreta larvae and to examine whether any of these yeasts, when combined with the CrleGV-SA, have a synergistic effect in increasing mortality of T. leucotreta larvae. Firstly, Navel oranges infested with T. leucotreta larvae were collected from orchards in Sundays River Valley in Eastern Cape of South Africa. Larvae were extracted and analysed for the presence of yeast on their surface, or in their gut and frass. Four yeasts were isolated from T. leucotreta larvae and identified down to species level via PCR amplification and sequencing of internal transcribed spacer (ITS) region and D1/D2 domain of the large subunit (LSU) of rDNA region. These yeasts were isolated from the frass, epidermis and digestive tract of T. leucotreta larvae. The yeast isolates were identified as Meyerozyma caribbica, Pichia kluyveri, Pichia kudriavzevii and Hanseniaspora opuntiae. A yeast preference assay was conducted on female T. leucotreta moths to examine whether any of the isolated yeast species affected their oviposition preference. Navel oranges were inoculated with the isolated yeast species at a concentration of 6 × 108 cells.ml-1. The assay also included a Brewer’s yeast and distilled water control. Pichia kudriavzevii was shown to be the preferred yeast species for oviposition, as significantly more eggs were deposited on Navel oranges inoculated with this yeast compared to the other treatments. Lastly, a detached fruit bioassay was performed to evaluate the efficacy of mixing P. kudriavzevii with CrleGV-SA to enhance T. leucotreta larvae mortality. Pichia kudriavzevii was selected as it was demonstrated as having an effect on the oviposition preference of female T. leucotreta moths. The concentration at which P. kudriavzevii was applied remained the same as in the preference assay while CrleGV-SA was applied at lethal concentration required to kill 50 % of the population (9.31 × 107 OBs.ml-1). Although an increase in larval mortality was observed between CrleGV-SA being applied alone and the yeast/virus mixture, this result was determined not to be statistically significant. The experiments performed in this study provide a platform for further research into the application of a yeast-virus combination as a novel control option for T. leucotreta in the field. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
Dispersal of sterile false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), for a sterile insect technique programme on citrus
- Authors: Wagenaar, Gideon Daniel
- Date: 2015
- Subjects: Cryptophlebia leucotreta , Insect pests -- Control -- South Africa , Citrus -- Diseases and pests -- Control -- South Africa , Insect sterilization
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: http://hdl.handle.net/10948/4977 , vital:20774
- Description: The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is an important pest of citrus in South Africa and challenging to manage due to its inconspicuous nature. An effective method currently being employed for the area-wide suppression of the FCM is the Sterile Insect Technique (SIT) and the effective dispersal of sterile moths is very important for success with SIT. This study was conducted in the Addo area of the Sundays River Valley (Eastern Cape) where the programme is commercially used. In this study, sterile male moths were released in different orchards on a citrus farm, and in nearby veld at different times of the year, and their dispersal was monitored through the use of pheromone traps. Various climatic factors were monitored. This provided insight into the local dispersal of sterile male FCM adults in response to abiotic cues (particularly climatic factors). The movement of the FCM in four citrus cultivars, namely lemons, navel and Valencia oranges and mandarins and in the nearby veld (open field), was determined at six different stages of the year. Results clearly indicated that sterile FCM movement is concentrated within citrus orchards, as very few moths were trapped beyond 30 m from the release point, particularly in navel and Valencia orchards. Of the climatic factors measured, minimum and maximum temperatures had the most significant influence on FCM dispersal, and based on the results, various recommendations are made for the releases of sterile FCM in an area-wide SIT management programmes on citrus. A better understanding of the dispersal capabilities of the FCM in an agricultural system, under different conditions and at different times of the year, is invaluable not only in improving release strategies in an SIT programme but in planning future control strategies against the FCM.
- Full Text:
- Date Issued: 2015
- Authors: Wagenaar, Gideon Daniel
- Date: 2015
- Subjects: Cryptophlebia leucotreta , Insect pests -- Control -- South Africa , Citrus -- Diseases and pests -- Control -- South Africa , Insect sterilization
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: http://hdl.handle.net/10948/4977 , vital:20774
- Description: The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is an important pest of citrus in South Africa and challenging to manage due to its inconspicuous nature. An effective method currently being employed for the area-wide suppression of the FCM is the Sterile Insect Technique (SIT) and the effective dispersal of sterile moths is very important for success with SIT. This study was conducted in the Addo area of the Sundays River Valley (Eastern Cape) where the programme is commercially used. In this study, sterile male moths were released in different orchards on a citrus farm, and in nearby veld at different times of the year, and their dispersal was monitored through the use of pheromone traps. Various climatic factors were monitored. This provided insight into the local dispersal of sterile male FCM adults in response to abiotic cues (particularly climatic factors). The movement of the FCM in four citrus cultivars, namely lemons, navel and Valencia oranges and mandarins and in the nearby veld (open field), was determined at six different stages of the year. Results clearly indicated that sterile FCM movement is concentrated within citrus orchards, as very few moths were trapped beyond 30 m from the release point, particularly in navel and Valencia orchards. Of the climatic factors measured, minimum and maximum temperatures had the most significant influence on FCM dispersal, and based on the results, various recommendations are made for the releases of sterile FCM in an area-wide SIT management programmes on citrus. A better understanding of the dispersal capabilities of the FCM in an agricultural system, under different conditions and at different times of the year, is invaluable not only in improving release strategies in an SIT programme but in planning future control strategies against the FCM.
- Full Text:
- Date Issued: 2015
Using the larval parasitoid, Agathis bishopi (Nixon) (Hymenoptera: Braconidae), for early detection of false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) infested fruit
- Authors: Zimba, Kennedy Josaya
- Date: 2015
- Subjects: Cryptophlebia leucotreta , Citrus -- Diseases and pests -- South Africa , Pests -- Biological control -- South Africa , Cryptophlebia leucotreta -- Detection , Parasitoids -- Hosts , Braconidae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5919 , http://hdl.handle.net/10962/d1017186
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is one of the major citrus pests of economic importance for South Africa’s citrus industry. It is endemic to Africa, and therefore a phytosanitary pest with zero tolerance by most export markets. The cryptic nature of T. leucotreta makes visual inspection an inefficient method for detecting neonate larvae in fruit in the packhouse. Therefore, a more accurate method for sorting infested fruit at the packhouse, particularly for newly infested fruit could ensure market access. A recent study showed that fruit infested by T. leucotreta emit a chemical profile different from that of a healthy fruit. Several studies provide evidence that parasitoids locate their hosts feeding on fruit by exploiting the novel chemical profiles produced due to host herbivory. The aim of this study was to evaluate the potential of using the naturally occurring behaviour of a larval parasitoid Agathis bishopi (Nixon) (Hymenoptera: Braconidae) for detection of T. leucotreta infested fruit, by determining which compound in infested fruit is attractive to parasitoids. Ytube olfactometer and flight-tunnel bioassays with healthy and T. leucotreta infested fruit showed a significantly stronger response of A. bishopi female parasitoids to infested fruit. Among the volatile compounds associated with T. leucotreta infested fruit, D-limonene elicited the strongest attraction to A. bishopi female parasitoids. Attraction of mated A. bishopi female parasitoids to T. leucotreta infested fruit and D-limonene significantly increased after oviposition experience. Behavioural responses of A. bishopi female parasitoids that were associated with T. leucotreta infested fruit were investigated to determine which behaviours are distinct and interpretable. Probing and oviposition behaviours were the most noticeable and were only elicited on infested fruit when parasitoids contacted T. leucotreta frass, indicating that chemical compounds in frass are short-range cues used for final host location. Since production of D-limonene by fruit is elevated due to herbivory by different pests including mechanical injury on fruit, response of A. bishopi female parasitoids to compounds in frass offers a more specific and potentially useful mechanism for development of a detection system for T. leucotreta infested fruit. Chemical analysis of T. leucotreta frass and conditioning A. bishopi parasitoids to respond behaviourally to compounds in frass is proposed.
- Full Text:
- Date Issued: 2015
- Authors: Zimba, Kennedy Josaya
- Date: 2015
- Subjects: Cryptophlebia leucotreta , Citrus -- Diseases and pests -- South Africa , Pests -- Biological control -- South Africa , Cryptophlebia leucotreta -- Detection , Parasitoids -- Hosts , Braconidae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5919 , http://hdl.handle.net/10962/d1017186
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is one of the major citrus pests of economic importance for South Africa’s citrus industry. It is endemic to Africa, and therefore a phytosanitary pest with zero tolerance by most export markets. The cryptic nature of T. leucotreta makes visual inspection an inefficient method for detecting neonate larvae in fruit in the packhouse. Therefore, a more accurate method for sorting infested fruit at the packhouse, particularly for newly infested fruit could ensure market access. A recent study showed that fruit infested by T. leucotreta emit a chemical profile different from that of a healthy fruit. Several studies provide evidence that parasitoids locate their hosts feeding on fruit by exploiting the novel chemical profiles produced due to host herbivory. The aim of this study was to evaluate the potential of using the naturally occurring behaviour of a larval parasitoid Agathis bishopi (Nixon) (Hymenoptera: Braconidae) for detection of T. leucotreta infested fruit, by determining which compound in infested fruit is attractive to parasitoids. Ytube olfactometer and flight-tunnel bioassays with healthy and T. leucotreta infested fruit showed a significantly stronger response of A. bishopi female parasitoids to infested fruit. Among the volatile compounds associated with T. leucotreta infested fruit, D-limonene elicited the strongest attraction to A. bishopi female parasitoids. Attraction of mated A. bishopi female parasitoids to T. leucotreta infested fruit and D-limonene significantly increased after oviposition experience. Behavioural responses of A. bishopi female parasitoids that were associated with T. leucotreta infested fruit were investigated to determine which behaviours are distinct and interpretable. Probing and oviposition behaviours were the most noticeable and were only elicited on infested fruit when parasitoids contacted T. leucotreta frass, indicating that chemical compounds in frass are short-range cues used for final host location. Since production of D-limonene by fruit is elevated due to herbivory by different pests including mechanical injury on fruit, response of A. bishopi female parasitoids to compounds in frass offers a more specific and potentially useful mechanism for development of a detection system for T. leucotreta infested fruit. Chemical analysis of T. leucotreta frass and conditioning A. bishopi parasitoids to respond behaviourally to compounds in frass is proposed.
- Full Text:
- Date Issued: 2015
A study on the application technology of the sterile insect technique, with focus on false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a pest of citrus in South Africa
- Authors: Nepgen, Eugene Stephan
- Date: 2014
- Subjects: Cryptophlebia leucotreta , Citrus -- Diseases and pests -- Control -- South Africa , Insect pests -- Control -- South Africa , Insect sterilization
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5879 , http://hdl.handle.net/10962/d1013199
- Description: False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. Major concerns such as progressive insecticidal resistance, the negative impact of insecticides on the environment, as well as the influence of consumers opposed to chemical residues on fruit, created opportunities for biological control methods such as Sterile Insect Technology (SIT). This technology is now established in the Western and Eastern Cape provinces of South Africa as an effective, sustainable alternative to conventional FCM control methods. Due to the prevalence of the pest in all citrus producing areas of South Africa, potential for SIT to expand is enormous. Success of an SIT programme is highly dependent on efficient application of the technology to achieve its objectives in a timeous manner. The aim of this study was to advance the application of SIT for control of FCM on citrus in South Africa, by investigating the effect of certain critical stages in the process. The effect of long-distance transportation on fitness of irradiated FCM was determined, showing reduced performance with cold-immobilized transport. A significant decrease in flight ability and longevity of irradiated FCM was found, although critically, realized fecundity was not affected. The effect of two different insecticides in the pyrethroid and organophosphate chemical groups were investigated for their residual effect on mortality of released irradiated FCM, to determine if these pest control programmes could be integrated. Both chlorpyrifos and tau-fluvalinate were effective in killing irradiated FCM for a number of days after application, after which degradation of the active ingredient rendered it harmless. This effect was found to be similar for irradiated and non-irradiated males, consequently ratios of sterile : wild male FCM should be retained regardless of whether sprays are applied or not. The modes for release of sterile FCM in an SIT programme were investigated. Efficacy of ground and aerial release platforms were tested by evaluating the recovery of released irradiated male FCM in these orchards. More irradiated FCM were recovered in orchards released from the ground compared to air. However, an economic analysis of both methods shows application of irradiated insects over a large geographical area is more cost-effective by air. Depending on the terrain and size of the target area, a combination of both methods is ideal for application of SIT for control of FCM in citrus. Development of application technology for advance of the programme is discussed and recommendations for future research and development are offered.
- Full Text:
- Date Issued: 2014
- Authors: Nepgen, Eugene Stephan
- Date: 2014
- Subjects: Cryptophlebia leucotreta , Citrus -- Diseases and pests -- Control -- South Africa , Insect pests -- Control -- South Africa , Insect sterilization
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5879 , http://hdl.handle.net/10962/d1013199
- Description: False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. Major concerns such as progressive insecticidal resistance, the negative impact of insecticides on the environment, as well as the influence of consumers opposed to chemical residues on fruit, created opportunities for biological control methods such as Sterile Insect Technology (SIT). This technology is now established in the Western and Eastern Cape provinces of South Africa as an effective, sustainable alternative to conventional FCM control methods. Due to the prevalence of the pest in all citrus producing areas of South Africa, potential for SIT to expand is enormous. Success of an SIT programme is highly dependent on efficient application of the technology to achieve its objectives in a timeous manner. The aim of this study was to advance the application of SIT for control of FCM on citrus in South Africa, by investigating the effect of certain critical stages in the process. The effect of long-distance transportation on fitness of irradiated FCM was determined, showing reduced performance with cold-immobilized transport. A significant decrease in flight ability and longevity of irradiated FCM was found, although critically, realized fecundity was not affected. The effect of two different insecticides in the pyrethroid and organophosphate chemical groups were investigated for their residual effect on mortality of released irradiated FCM, to determine if these pest control programmes could be integrated. Both chlorpyrifos and tau-fluvalinate were effective in killing irradiated FCM for a number of days after application, after which degradation of the active ingredient rendered it harmless. This effect was found to be similar for irradiated and non-irradiated males, consequently ratios of sterile : wild male FCM should be retained regardless of whether sprays are applied or not. The modes for release of sterile FCM in an SIT programme were investigated. Efficacy of ground and aerial release platforms were tested by evaluating the recovery of released irradiated male FCM in these orchards. More irradiated FCM were recovered in orchards released from the ground compared to air. However, an economic analysis of both methods shows application of irradiated insects over a large geographical area is more cost-effective by air. Depending on the terrain and size of the target area, a combination of both methods is ideal for application of SIT for control of FCM in citrus. Development of application technology for advance of the programme is discussed and recommendations for future research and development are offered.
- Full Text:
- Date Issued: 2014
Entomopathogenic fungi for control of soil-borne life stages of false codling moth, Thaumatotibia leucotreta (Meyrick) (1912) (Lepidoptera: Tortricidae)
- Authors: Coombes, Candice Anne
- Date: 2013
- Subjects: Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5607 , http://hdl.handle.net/10962/d1002057 , Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: False codling moth (FCM), Thaumatotibia leucotreta is an extremely important pest of citrus in South Africa and with the shift away from the use of chemicals, alternate control options are needed. One avenue of control which has only recently been investigated against the soil-borne life stages of FCM is the use of entomopathogenic fungi (EPF). In 2009, 12 entomopathogenic fungal isolates collected from South African citrus orchards showed good control potential during laboratory conducted bioassays. The aim of this study was to further analyse the potential of these isolates through concentration-dose and exposure-time response bioassays. After initial re-screening, concentration-dose response and exposure-time response sandconidial bioassays, three isolates were identified as exhibiting the greatest control potential against FCM in soil, Metarhizium anisopliae var. anisopliae (G 11 3 L6 and FCM Ar 23 B3) and Beauveria bassiana (G Ar 17 B3). Percentage mycosis was found to be directly related to fungal concentration as well as the amount of time FCM 5th instar larvae were exposed to the fungal conidia. LC50 values for the three isolates were not greater than 1.92 x 10⁶ conidia.ml⁻ₑ and at the LC₅₀, FCM 5th instar larvae would need to be exposed to the fungus for a maximum of 13 days to ensure a high mortality level. These isolates along with two commercially available EPF products were subjected to field persistence trials whereby net bags filled with a mixture of autoclaved sand and formulated fungal product were buried in an Eastern Cape citrus orchard. The viability of each isolate was measured on a monthly basis for a period of six months. All isolates were capable of persisting in the soil for six months with the collected isolates persisting far better than the commercially used isolates. Two of the isolates, G 11 3 L6 and G Ar 17 B3, were subjected to small scale laboratory application trials. Two formulations were investigated at two concentrations. For each isolate, each formulation and each concentration, FCM 5th instar larvae were applied and allowed to burrow into the soil to pupate before fungal application or after fungal application. Contact between fungi and FCM host is essential as, in contrast to pre-larval treatments, percentage mortality in post-larval treatments was low for both formulations and both isolates. For isolate G Ar 17 B3, a conidial suspension applied as a spray at a concentration of 1 x 10⁷ conidia.ml⁻ₑ obtained the highest percentage mortality (80 %). For isolate G 11 3 L6 however, both formulations performed equally well at a high, 1 x10⁷ conidia.ml⁻ₑ concentration (conidial suspension: 60 %; granular: 65 %) The results obtained thus far are promising for the control of FCM in citrus, but if these EPFs are to successfully integrate into current FCM control practices more research, some of which is discussed, is essential
- Full Text:
- Date Issued: 2013
- Authors: Coombes, Candice Anne
- Date: 2013
- Subjects: Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5607 , http://hdl.handle.net/10962/d1002057 , Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: False codling moth (FCM), Thaumatotibia leucotreta is an extremely important pest of citrus in South Africa and with the shift away from the use of chemicals, alternate control options are needed. One avenue of control which has only recently been investigated against the soil-borne life stages of FCM is the use of entomopathogenic fungi (EPF). In 2009, 12 entomopathogenic fungal isolates collected from South African citrus orchards showed good control potential during laboratory conducted bioassays. The aim of this study was to further analyse the potential of these isolates through concentration-dose and exposure-time response bioassays. After initial re-screening, concentration-dose response and exposure-time response sandconidial bioassays, three isolates were identified as exhibiting the greatest control potential against FCM in soil, Metarhizium anisopliae var. anisopliae (G 11 3 L6 and FCM Ar 23 B3) and Beauveria bassiana (G Ar 17 B3). Percentage mycosis was found to be directly related to fungal concentration as well as the amount of time FCM 5th instar larvae were exposed to the fungal conidia. LC50 values for the three isolates were not greater than 1.92 x 10⁶ conidia.ml⁻ₑ and at the LC₅₀, FCM 5th instar larvae would need to be exposed to the fungus for a maximum of 13 days to ensure a high mortality level. These isolates along with two commercially available EPF products were subjected to field persistence trials whereby net bags filled with a mixture of autoclaved sand and formulated fungal product were buried in an Eastern Cape citrus orchard. The viability of each isolate was measured on a monthly basis for a period of six months. All isolates were capable of persisting in the soil for six months with the collected isolates persisting far better than the commercially used isolates. Two of the isolates, G 11 3 L6 and G Ar 17 B3, were subjected to small scale laboratory application trials. Two formulations were investigated at two concentrations. For each isolate, each formulation and each concentration, FCM 5th instar larvae were applied and allowed to burrow into the soil to pupate before fungal application or after fungal application. Contact between fungi and FCM host is essential as, in contrast to pre-larval treatments, percentage mortality in post-larval treatments was low for both formulations and both isolates. For isolate G Ar 17 B3, a conidial suspension applied as a spray at a concentration of 1 x 10⁷ conidia.ml⁻ₑ obtained the highest percentage mortality (80 %). For isolate G 11 3 L6 however, both formulations performed equally well at a high, 1 x10⁷ conidia.ml⁻ₑ concentration (conidial suspension: 60 %; granular: 65 %) The results obtained thus far are promising for the control of FCM in citrus, but if these EPFs are to successfully integrate into current FCM control practices more research, some of which is discussed, is essential
- Full Text:
- Date Issued: 2013
Identifying volatile emissions associated with False Codling Moth infested citrus fruit
- Authors: Van der Walt, Rachel
- Date: 2012
- Subjects: Citrus -- Diseases and pests , Insect pests , Cryptophlebia leucotreta
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10343 , http://hdl.handle.net/10948/d1020056
- Description: False codling moth is a known pest of economic importance to many cultivated crops in South Africa and Africa south of the Sahara, and is particularly severe on citrus. If the fruit is infested just before harvest the chances of detecting signs of infestation are very low. As a result, the risk of packaging infested fruit and exporting them as healthy fruit is high. It is therefore a priority to develop a post-harvest technique for detection of False codling moth in citrus fruit at different levels of infestation in order to reduce phytosanitary risk. Compounds released and detected were indicative of infestation and were not insect produced but naturally produced fruit volatiles emitted at higher levels as a result of the insect within the fruit. Five major volatile compounds of interest were released by the infested oranges. These major volatile compounds include D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene, caryophyllene and naphthalene. Limonene was one of the most abundant volatile compounds released by the infested citrus fruit. Naphthalene, which is possibly produced due to larval feeding and development within the fruit maintained higher concentrations than controls throughout the infestation within the fruit. Naphthalene would be a good indicator of False codling moth infestation, however, not primarily for early infestation detection. A significantly higher concentration of D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene and naphthalene was detected using the SEP over the SPME technique. The application of an SPME procedure and the utilization of this method for detection of volatiles present in the headspace of intact infested fruit are evaluated and the possible volatile compounds diagnostic of Thaumatotibia leucotreta infestation of orange fruit and differences in volatile compound response in different orange varieties is discussed.
- Full Text:
- Date Issued: 2012
- Authors: Van der Walt, Rachel
- Date: 2012
- Subjects: Citrus -- Diseases and pests , Insect pests , Cryptophlebia leucotreta
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:10343 , http://hdl.handle.net/10948/d1020056
- Description: False codling moth is a known pest of economic importance to many cultivated crops in South Africa and Africa south of the Sahara, and is particularly severe on citrus. If the fruit is infested just before harvest the chances of detecting signs of infestation are very low. As a result, the risk of packaging infested fruit and exporting them as healthy fruit is high. It is therefore a priority to develop a post-harvest technique for detection of False codling moth in citrus fruit at different levels of infestation in order to reduce phytosanitary risk. Compounds released and detected were indicative of infestation and were not insect produced but naturally produced fruit volatiles emitted at higher levels as a result of the insect within the fruit. Five major volatile compounds of interest were released by the infested oranges. These major volatile compounds include D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene, caryophyllene and naphthalene. Limonene was one of the most abundant volatile compounds released by the infested citrus fruit. Naphthalene, which is possibly produced due to larval feeding and development within the fruit maintained higher concentrations than controls throughout the infestation within the fruit. Naphthalene would be a good indicator of False codling moth infestation, however, not primarily for early infestation detection. A significantly higher concentration of D-limonene, 3,7-dimethyl-1,3,6-octatriene, (E)-4,8-dimethyl-1,3,7-nonatriene and naphthalene was detected using the SEP over the SPME technique. The application of an SPME procedure and the utilization of this method for detection of volatiles present in the headspace of intact infested fruit are evaluated and the possible volatile compounds diagnostic of Thaumatotibia leucotreta infestation of orange fruit and differences in volatile compound response in different orange varieties is discussed.
- Full Text:
- Date Issued: 2012
Geographic variation in the susceptibility of false colding Moth, Thaumatotibia Leucotreta, populations to a granulovirus (CrleGV-SA)
- Authors: Opoku-Debrah, John Kwadwo
- Date: 2008
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: vital:10586 , http://hdl.handle.net/10948/984 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Description: The false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a serious pest of citrus and other crops in Sub-Saharan Africa. The introduction of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) Cryptogran and Cryptex (biopesticides) has proven to be very effective in the control of FCM. However, markedly lower susceptibility of some codling moth (CM), Cydia pomonella (L.) populations to Cydia pomonella granulovirus (CpGV-M), another granulovirus product used in the control of CM’s in Europe have been reported. Genetic differences between FCM populations in South Africa have also been established. It is therefore possible that differences in the susceptibility of these geographically distinct FCM populations to CrleGV-SA might also exist. To investigate this phenomenon, a benchmark for pathogenecity was established. In continuation of previous work with Cryptogran against the 1st and 5th instar FCM larvae, dose-response relationships were established for all five larval instars of FCM. In surface dose-response bioassays, the LC50 values for the 2nd, 3rd and 4th instars were calculated to be 4.516 x 104, 1.662 x 105 and 2.205 x 106 occlusion bodies (OBs)/ml, respectively. The LC90 values for the 2nd, 3rd and 4th instars were calculated to be 4.287 x 106, 9.992 x 106 and 1.661 x 108 OBs/ml, respectively. Susceptibility to CrleGV-SA was found to decline with larval stage and increase with time of exposure. The protocol was used in guiding bioassays with field collected FCM larvae. Laboratory assays conducted with Cryptogran (at 1.661 x 108 OBs/ml) against field collected FCM larvae from Addo, Kirkwood, Citrusdal and Clanwilliam as well as a standard laboratory colony, showed a significant difference in pathogenecity in only one case. This significant difference was observed between 5th instars from the Addo colony and 5th instars from the other populations. Four geographically distinct FCM colonies from Addo, Citrusdal, Marble Hall and Nelspruit were also established. Since Cryptogran and Cryptex are always targeted against 1st instar FCM larvae in the field, further comparative laboratory assays were conducted with the Addo colony and an old laboratory colony. Cryptogran was significantly more pathogenic than Cryptex against both the Addo and the old colony. However, a high level of heterogeneity was observed in responses within each population.
- Full Text:
- Date Issued: 2008
- Authors: Opoku-Debrah, John Kwadwo
- Date: 2008
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MTech
- Identifier: vital:10586 , http://hdl.handle.net/10948/984 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Description: The false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a serious pest of citrus and other crops in Sub-Saharan Africa. The introduction of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) Cryptogran and Cryptex (biopesticides) has proven to be very effective in the control of FCM. However, markedly lower susceptibility of some codling moth (CM), Cydia pomonella (L.) populations to Cydia pomonella granulovirus (CpGV-M), another granulovirus product used in the control of CM’s in Europe have been reported. Genetic differences between FCM populations in South Africa have also been established. It is therefore possible that differences in the susceptibility of these geographically distinct FCM populations to CrleGV-SA might also exist. To investigate this phenomenon, a benchmark for pathogenecity was established. In continuation of previous work with Cryptogran against the 1st and 5th instar FCM larvae, dose-response relationships were established for all five larval instars of FCM. In surface dose-response bioassays, the LC50 values for the 2nd, 3rd and 4th instars were calculated to be 4.516 x 104, 1.662 x 105 and 2.205 x 106 occlusion bodies (OBs)/ml, respectively. The LC90 values for the 2nd, 3rd and 4th instars were calculated to be 4.287 x 106, 9.992 x 106 and 1.661 x 108 OBs/ml, respectively. Susceptibility to CrleGV-SA was found to decline with larval stage and increase with time of exposure. The protocol was used in guiding bioassays with field collected FCM larvae. Laboratory assays conducted with Cryptogran (at 1.661 x 108 OBs/ml) against field collected FCM larvae from Addo, Kirkwood, Citrusdal and Clanwilliam as well as a standard laboratory colony, showed a significant difference in pathogenecity in only one case. This significant difference was observed between 5th instars from the Addo colony and 5th instars from the other populations. Four geographically distinct FCM colonies from Addo, Citrusdal, Marble Hall and Nelspruit were also established. Since Cryptogran and Cryptex are always targeted against 1st instar FCM larvae in the field, further comparative laboratory assays were conducted with the Addo colony and an old laboratory colony. Cryptogran was significantly more pathogenic than Cryptex against both the Addo and the old colony. However, a high level of heterogeneity was observed in responses within each population.
- Full Text:
- Date Issued: 2008
Understanding and improving the residual efficacy of the cryptophlebia leucotreta granulovirus (Cryptogran)
- Authors: Kirkman, Wayne
- Date: 2008
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5794 , http://hdl.handle.net/10962/d1005482 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Description: False codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyr) (Lepidoptera: Tortricidae), is one of the most important pests on citrus. The Cryptophlebia leucotreta granulovirus (CrleGV) has been developed into a successful biological control agent, registered under the name Cryptogran, and is currently the preferred product for the control of FCM on citrus in South Africa. A prerequisite to the continued success of Cryptogran as a means of controlling false codling moth is to understand the factors affecting field persistence of the virus, and to find ways to improve it. The aim of this study was to gain a clearer understanding of the product and the abiotic and biotic factors affecting its persistence in the field, and to investigate methods to improve this persistence. The effect of UV-irradiation on the virus was determined, and various products were tested as UV protectants in laboratory bioassays. Lignin was the most effective additive, and was tested in several field trials, where it also enhanced the efficacy of Cryptogran. Laboratory trials indicated that Cryptogran is rainfast. Cryptogran applications early in the season had a longer period of residual activity than sprays applied closer to harvest. Daytime applications were less effective that evening sprays. Sprays applied coinciding with peaks in pheromone moth trap catches were more effective than those applied between peaks. Biotic factors influencing persistence were investigated. Residual efficacy was longer when treatments were applied to blocks than as single tree treatments. Attempts were made to quantify the effect of the navel end of a navel orange on the field persistence of Cryptogran. Cryptogran was shown to be compatible with many agricultural chemicals used on citrus. Economic thresholds and various cost-benefit analyses are discussed. A list of practical recommendations to growers was drawn up, and possibilities for future research are presented.
- Full Text:
- Date Issued: 2008
- Authors: Kirkman, Wayne
- Date: 2008
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5794 , http://hdl.handle.net/10962/d1005482 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Citrus -- Diseases and pests
- Description: False codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyr) (Lepidoptera: Tortricidae), is one of the most important pests on citrus. The Cryptophlebia leucotreta granulovirus (CrleGV) has been developed into a successful biological control agent, registered under the name Cryptogran, and is currently the preferred product for the control of FCM on citrus in South Africa. A prerequisite to the continued success of Cryptogran as a means of controlling false codling moth is to understand the factors affecting field persistence of the virus, and to find ways to improve it. The aim of this study was to gain a clearer understanding of the product and the abiotic and biotic factors affecting its persistence in the field, and to investigate methods to improve this persistence. The effect of UV-irradiation on the virus was determined, and various products were tested as UV protectants in laboratory bioassays. Lignin was the most effective additive, and was tested in several field trials, where it also enhanced the efficacy of Cryptogran. Laboratory trials indicated that Cryptogran is rainfast. Cryptogran applications early in the season had a longer period of residual activity than sprays applied closer to harvest. Daytime applications were less effective that evening sprays. Sprays applied coinciding with peaks in pheromone moth trap catches were more effective than those applied between peaks. Biotic factors influencing persistence were investigated. Residual efficacy was longer when treatments were applied to blocks than as single tree treatments. Attempts were made to quantify the effect of the navel end of a navel orange on the field persistence of Cryptogran. Cryptogran was shown to be compatible with many agricultural chemicals used on citrus. Economic thresholds and various cost-benefit analyses are discussed. A list of practical recommendations to growers was drawn up, and possibilities for future research are presented.
- Full Text:
- Date Issued: 2008
Investigation of the larval parasitoids of the false codling moth, Cryptophlebia Leucotreta (Meyrick) (Lepidoptera: Tortricidae), on citrus in South Africa
- Authors: Sishuba, Nomahlubi
- Date: 2004
- Subjects: Cryptophlebia leucotreta , Tortricidae , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Parasitoids , Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5918 , http://hdl.handle.net/10962/d1016267
- Description: The study examined the larval parasitoids of Cryptophlebia leucotreta (Meyrick) on citrus in South Africa and aimed to improve the existing rearing techniques of C. leucotreta with a view to mass rearing of biological control agents. The biological characteristics of Agathis bishopi Nixon (Hymenoptera: Braconidae) were studied, with an emphasis on parasitism rates in the field, host stage preference, developmental rate, life span and offspring sex ratios. Two larval parasitoids, A. bishopi and Apophua leucotretae (Wilkinson) (Hymenoptera: Ichneumonidae), and an egg parasitoid, Trichogrammatoidea cryptophlebiae Nagaraja (Hymenoptera: Trichogrammatidae), were recorded on C. leucotreta on citrus. A. bishopi was the more abundant of the larval parasitoids and exhibited density dependent parasitism. The highest parasitism rates were observed in December with up to 38% in Sundays River Valley and 34% in Gamtoos River Valley, at a time when the highest false codling moth infestations were observed. Agathis bishopi was recorded only in the Eastern Cape Province. The sex ratio of A. bishopi was biased towards females throughout the study (77% in Gamtoos River Valley and 72% in Sundays River Valley). Agathis bishopi is a solitary, koinobiont, larval-pupal endoparasitoid. The species showed a preference for 1st and 2"d instar hosts. Females regulate the sex of their progeny according to the size and larval stage of the host, ovipositing unfertilised eggs in younger, smaller larvae (1st instars) and fertilised eggs in older, larger larvae (2nd instars). The developmental rate of A. bishopi is in synchrony with that of the moth and adults emerge when adult moths that have escaped parasitism emerge. Agathis bishopi and T. cryptophlebiae compliment each other because they have different niches and strategies of attack. Integrating A. bishopi and T. cryptophlebiae into the management of citrus orchards has potential to suppress false codling moth. Larger rearing containers seemed ideal for large-scale rearing of false codling moth. A higher percentage of adults was obtained from larvae reared in larger containers than in smaller ones. The width of the sponges used as stoppers prevented escape of the larvae. Media prepared in larger containers are easier and simpler to prepare than in smaller ones, thus eliminating many precautions otherwise necessary to prevent contamination. Moth production was greatly reduced by the high concentration of Sporekill used for egg decontamination.
- Full Text:
- Date Issued: 2004
- Authors: Sishuba, Nomahlubi
- Date: 2004
- Subjects: Cryptophlebia leucotreta , Tortricidae , Cryptophlebia leucotreta -- Control , Pests -- Biological control , Parasitoids , Citrus -- Diseases and pests
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5918 , http://hdl.handle.net/10962/d1016267
- Description: The study examined the larval parasitoids of Cryptophlebia leucotreta (Meyrick) on citrus in South Africa and aimed to improve the existing rearing techniques of C. leucotreta with a view to mass rearing of biological control agents. The biological characteristics of Agathis bishopi Nixon (Hymenoptera: Braconidae) were studied, with an emphasis on parasitism rates in the field, host stage preference, developmental rate, life span and offspring sex ratios. Two larval parasitoids, A. bishopi and Apophua leucotretae (Wilkinson) (Hymenoptera: Ichneumonidae), and an egg parasitoid, Trichogrammatoidea cryptophlebiae Nagaraja (Hymenoptera: Trichogrammatidae), were recorded on C. leucotreta on citrus. A. bishopi was the more abundant of the larval parasitoids and exhibited density dependent parasitism. The highest parasitism rates were observed in December with up to 38% in Sundays River Valley and 34% in Gamtoos River Valley, at a time when the highest false codling moth infestations were observed. Agathis bishopi was recorded only in the Eastern Cape Province. The sex ratio of A. bishopi was biased towards females throughout the study (77% in Gamtoos River Valley and 72% in Sundays River Valley). Agathis bishopi is a solitary, koinobiont, larval-pupal endoparasitoid. The species showed a preference for 1st and 2"d instar hosts. Females regulate the sex of their progeny according to the size and larval stage of the host, ovipositing unfertilised eggs in younger, smaller larvae (1st instars) and fertilised eggs in older, larger larvae (2nd instars). The developmental rate of A. bishopi is in synchrony with that of the moth and adults emerge when adult moths that have escaped parasitism emerge. Agathis bishopi and T. cryptophlebiae compliment each other because they have different niches and strategies of attack. Integrating A. bishopi and T. cryptophlebiae into the management of citrus orchards has potential to suppress false codling moth. Larger rearing containers seemed ideal for large-scale rearing of false codling moth. A higher percentage of adults was obtained from larvae reared in larger containers than in smaller ones. The width of the sponges used as stoppers prevented escape of the larvae. Media prepared in larger containers are easier and simpler to prepare than in smaller ones, thus eliminating many precautions otherwise necessary to prevent contamination. Moth production was greatly reduced by the high concentration of Sporekill used for egg decontamination.
- Full Text:
- Date Issued: 2004
The establishment of a virus free laboratory colony of Cryptophlebia leucotreta (False Codling Moth) and characterisation of Cryptophlebia leucotreta Granulovirus (CrleGV) genes
- Authors: Ludewig, Michael Hans
- Date: 2003
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
- Full Text:
- Date Issued: 2003
- Authors: Ludewig, Michael Hans
- Date: 2003
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3957 , http://hdl.handle.net/10962/d1004016 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control , DNA viruses , Agricultural pests -- Biological control , Baculoviruses
- Description: Cryptophlebia leucotreta is an economically important agricultural pest throughout Sub-Saharan Africa. CrleGV has been considered as an alternative to chemical control of this pest due to its host specificity and innocuous nature towards vertebrates. A CrleGV free laboratory colony of C. leucotreta would be useful for the isolation of genotypically pure strains of the CrleGV and for virulence comparisons between isolates. It is preferable to have a full characterisation of CrleGV prior to its registration and release into the environment as a biopesticide. A laboratory colony of C. leucotreta, set up at Rhodes University, containing a low level of infection indicated that CrleGV is vertically transmitted. To establish a virus free laboratory colony of C. leucotreta, a solution of 3.5% sodium hypochlorite and 1% Tween 20 was used to surface decontaminate C. leucotreta eggs for removal of transovum CrleGV from the laboratory colony. No apparent infection by CrleGV was induced by subjecting larvae to stress. PCR of DNA extracted from larvae using CTAB failed to detect virus in the laboratory colony. This detection protocol was able to detect down to 60 fg (480 genome copies of CrleGV). The possibility of low-level virus remaining in the colony requires monitoring of genotypic purity of virus manipulated in the colony. Sequencing of Bam HI/KpnI fragments produced a preliminary sequence of the granulin region of CrleGV. This preliminary sequence supports the trend that the gene organisation of the granulin region of the granuloviruses infecting the family Tortricidae is conserved.
- Full Text:
- Date Issued: 2003
The characterisation of a South African isolate of Cryptophlebia leucotreta Granulovirus (CIGV)
- Authors: Singh, Shalene
- Date: 2002
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control -- Africa , DNA viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4067 , http://hdl.handle.net/10962/d1004929 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control -- Africa , DNA viruses
- Description: The false codling moth (FCM), Cryptophlehia Leucatreta, causes widespread damage to economically important fruit crops throughout sub-Saharan Africa. Fruit are rendered unfit for consumption once they have been stung by FCM larvae. Larval infestation of fruit can lead to significant pre-harvest losses or post-harvest waste, posing a major problem to the citrus industry. Current control of the pest includes the use of chemical pesticides. The larval form of FCM is known to be infected by a granulovirus called Cryptophlebia leucotreta granulovirus (CIGV). Granuloviruses are highly specific against their hosts and are harmless to vertebrates, plants and the environment. The development of CIGV into a biological control agent would offer an attractive and safer alternative for the control of this pest. A full characterisation of CIGV is required prior to the virus being disseminated into the environment. In this project, the characteristics of CIGV will be examined. Viral DNA was extracted from infected larvae and the DNA analysed by restriction fragment length polymorphism (RFLP). Fragmentation profiles of the South African and Cape Verde (CV3) isolates of the virus were compared, revealing distinct differences between them. The size of the CIGV-SA genome was calculated to be 112 kbp, identical to the size of the CV3 isolate. Physical maps for five restriction enzymes were constructed for the CIGV-SA genome. The alignment of these maps with maps the CV3 isolate (for the same enzymes) further highlighted the differences between the isolates. The genetic engineering of granuloviruses could significantly improve the speed of kill of these viruses. Therefore essential genes like egt and granulin were isolated (by PCR) and their position located in the genome. Both genes were sequenced and their phylogeny with other granulin and egt genes investigated. Finally, tbe incidence of CIGV in natural populations of FCM larvae was investigated, by screening field-collected larvae for the presence of the virus. CIGV was successfully detected from dot blots of larval DNA using both radiolabelled and non-radiolabelled probes and by PCR. Trends regarding the incidence of CIGV in natural populations of larvae were also determined.
- Full Text:
- Date Issued: 2002
- Authors: Singh, Shalene
- Date: 2002
- Subjects: Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control -- Africa , DNA viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4067 , http://hdl.handle.net/10962/d1004929 , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Control , Pests -- Biological control -- Africa , DNA viruses
- Description: The false codling moth (FCM), Cryptophlehia Leucatreta, causes widespread damage to economically important fruit crops throughout sub-Saharan Africa. Fruit are rendered unfit for consumption once they have been stung by FCM larvae. Larval infestation of fruit can lead to significant pre-harvest losses or post-harvest waste, posing a major problem to the citrus industry. Current control of the pest includes the use of chemical pesticides. The larval form of FCM is known to be infected by a granulovirus called Cryptophlebia leucotreta granulovirus (CIGV). Granuloviruses are highly specific against their hosts and are harmless to vertebrates, plants and the environment. The development of CIGV into a biological control agent would offer an attractive and safer alternative for the control of this pest. A full characterisation of CIGV is required prior to the virus being disseminated into the environment. In this project, the characteristics of CIGV will be examined. Viral DNA was extracted from infected larvae and the DNA analysed by restriction fragment length polymorphism (RFLP). Fragmentation profiles of the South African and Cape Verde (CV3) isolates of the virus were compared, revealing distinct differences between them. The size of the CIGV-SA genome was calculated to be 112 kbp, identical to the size of the CV3 isolate. Physical maps for five restriction enzymes were constructed for the CIGV-SA genome. The alignment of these maps with maps the CV3 isolate (for the same enzymes) further highlighted the differences between the isolates. The genetic engineering of granuloviruses could significantly improve the speed of kill of these viruses. Therefore essential genes like egt and granulin were isolated (by PCR) and their position located in the genome. Both genes were sequenced and their phylogeny with other granulin and egt genes investigated. Finally, tbe incidence of CIGV in natural populations of FCM larvae was investigated, by screening field-collected larvae for the presence of the virus. CIGV was successfully detected from dot blots of larval DNA using both radiolabelled and non-radiolabelled probes and by PCR. Trends regarding the incidence of CIGV in natural populations of larvae were also determined.
- Full Text:
- Date Issued: 2002
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