Augmentation of Aphytis melinus DeBach (Hymenoptera: Aphelinidae) for the control of California red scale Aonidiella aurantii Maskell (Hemiptera: Diaspididae) on citrus
- De Beer, Ernst Friedrich Ludwig
- Authors: De Beer, Ernst Friedrich Ludwig
- Date: 2024-04-05
- Subjects: Aphytis melinus , Aphytis , Aonidiella aurantii , Citrus Diseases and pests South Africa , Pests Biological control
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/435638 , vital:73175 , DOI 10.21504/10962/435638
- Description: Aphytis lingnanensis was reared and tested in South Africa in the early 2000s for augmentation against red scale on citrus and was found to be ineffective. Aphytis melinus is now commercially available and it is important that the efficacy of augmentation thereof on red scale is determined locally. Field trials, fitness assessments and molecular identification on A. melinus from two insectaries were done. Field trials was done in seven, five and six pairs of comparable release and control orchards across the Eastern and Western Cape during the seasons of 2019/2020, 2020/2021 and 2021/2022 respectively. Red scale infestation was monitored and a sample of 20 infested fruit from each orchard was randomly collected every four weeks. Aphytis spp. responsible for parasitism were identified and the percentage parasitism recorded. Results of this study of field trials suggest that the augmentation of A. melinus did not significantly increase the level of parasitism above that of the untreated control. Five repetitions with six replicates of flight and longevity tests were performed with wasps from each insectary. Wasps in the longevity test from two insectaries were kept at 23 °C and 65% RH with honey. Flight tests were performed in tubes of 16 by 30 cm, with a light above a clear, sticky ceiling at 23 °C and 65% RH. On average in five replicates, 65%, 33% and 17% A. melinus wasps were alive on day one, five and 10 respectively. The overall sex ratio was 1.58 for females to males, but 1.05, 2.19 and 2.66 for non-flyers, non-crawlers, crawlers, and flyers respectively. In flight tests for both insectaries combined, only 36.97% of wasps could initiate flight in 24 h while 56.96% remained on the tube floor, and 6.05% attempted to crawl upwards. No significant differences in flight performance were recorded between the two insectaries. Wasps from the local insectary lived significantly longer during the longevity tests but were shorter in transit than wasps from the overseas insectary. COI genes were sequenced and compared against Genbank sequences using BLAST. Molecular identifications did not confirm morphological identifications for all species, indicating unexpected genetic complexity. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Date Issued: 2024-04-05
- Authors: De Beer, Ernst Friedrich Ludwig
- Date: 2024-04-05
- Subjects: Aphytis melinus , Aphytis , Aonidiella aurantii , Citrus Diseases and pests South Africa , Pests Biological control
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/435638 , vital:73175 , DOI 10.21504/10962/435638
- Description: Aphytis lingnanensis was reared and tested in South Africa in the early 2000s for augmentation against red scale on citrus and was found to be ineffective. Aphytis melinus is now commercially available and it is important that the efficacy of augmentation thereof on red scale is determined locally. Field trials, fitness assessments and molecular identification on A. melinus from two insectaries were done. Field trials was done in seven, five and six pairs of comparable release and control orchards across the Eastern and Western Cape during the seasons of 2019/2020, 2020/2021 and 2021/2022 respectively. Red scale infestation was monitored and a sample of 20 infested fruit from each orchard was randomly collected every four weeks. Aphytis spp. responsible for parasitism were identified and the percentage parasitism recorded. Results of this study of field trials suggest that the augmentation of A. melinus did not significantly increase the level of parasitism above that of the untreated control. Five repetitions with six replicates of flight and longevity tests were performed with wasps from each insectary. Wasps in the longevity test from two insectaries were kept at 23 °C and 65% RH with honey. Flight tests were performed in tubes of 16 by 30 cm, with a light above a clear, sticky ceiling at 23 °C and 65% RH. On average in five replicates, 65%, 33% and 17% A. melinus wasps were alive on day one, five and 10 respectively. The overall sex ratio was 1.58 for females to males, but 1.05, 2.19 and 2.66 for non-flyers, non-crawlers, crawlers, and flyers respectively. In flight tests for both insectaries combined, only 36.97% of wasps could initiate flight in 24 h while 56.96% remained on the tube floor, and 6.05% attempted to crawl upwards. No significant differences in flight performance were recorded between the two insectaries. Wasps from the local insectary lived significantly longer during the longevity tests but were shorter in transit than wasps from the overseas insectary. COI genes were sequenced and compared against Genbank sequences using BLAST. Molecular identifications did not confirm morphological identifications for all species, indicating unexpected genetic complexity. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Date Issued: 2024-04-05
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
Genetic analysis and field application of a UV-tolerant strain of CrleGV for improved control of Thaumatotibia leucotreta
- Authors: Bennett, Tahnee Tashia
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents , Ultraviolet radiation , Oligonucleotides
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362741 , vital:65358
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), also known as false codling moth (FCM), is indigenous to sub-Saharan Africa. Thaumatotibia leucotreta has been controlled through an integrated pest management (IPM) programme, which includes chemical control, sterile insect technique (SIT), cultural and biological control. As part of the biological control, a key component is the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA). Currently, CryptogranTM, a commercial formulation of CrleGV, is the preferred product to use in South Africa for the control of T. leucotreta. The registration of the biopesticide Cryptogran (River bioscience, South Africa) was established after conducting extensive field trials with CrleGV-SA. One of the major factors affecting the baculovirus efficacy in the field is UV irradiation. A UV-tolerant Cryptophlebia leucotreta granulovirus (CrleGV-SA-C5) isolate was isolated after consecutive cycles of UV exposure. This UV-tolerant isolate is genetically distinct from the CrleGV-SA isolate. The CrleGV-SA-C5 isolate has the potential as a biological control agent. The control of T. leucotreta in South Africa could be improved by the development of novel isolates into new biopesticide formulations. To date, there has not been any field trials conducted on the CrleGV-SA-C5 isolate. Therefore, it is important to determine the biological and genetic stability of this isolate and to conduct field trials with CrleGV-SA- C5 to test the efficacy of the isolate before possible production into a biopesticide. A de novo assembly was conducted to reassemble the genome of CrleGV-SA-C5 which was followed by a sequence comparison with the CrleGV-SA genome. The identification of SNPs, led to the design of oligonucleotides flanking the regions where the SNPs were detected. Polymerase chain reaction amplification of the target regions was conducted using the oligonucleotides. After sequence comparison, seven SNPs were detected and PCR amplification was successful using the three oligonucleotides, Pif-2, HypoP and Lef-8/HP. To differentiate between CrleGV-SA-C5 and CrleGV-SA genomes and confirm the presence of the SNPs, two methods of screening were conducted. The first was the construction of six plasmids, the plasmids contained the targeted pif-2, HypoP, and the Lef-8/HP insert regions from both the CrleGV-SA-C5 and CrleGV-SA genome region where the SNPs were identified, followed by sequencing. The Five recombinant plasmids, pC5_Pif-2, pSA_Pif-2, pC5_HypoP, pSA_HypoP, and pC5_Lef-8/HP were successfully sequenced. No amplicon was obtained for one of the plasmids used as template (pSA_Lef-8/HP) and therefore the PCR product used for cloning was sequenced instead. Sequence alignment confirmed the presence of four of the five targeted SNPs in the genome of the CrleGV-SA-C5 isolate. However, of these only one SNP (UV_7) rendered a suitable marker for the differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates as the SNPs, UV_2, UV_3 and UV_5, were also present in the CrleGV- SA sequences. The second screening method was a quantitative polymerase chain reaction (qPCR) melt curve analysis to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. qPCR melt curve analysis was done using the CrleGV-SA-C5 and CrleGV-SA HypoP PCR products. This technique was unable to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. However, this may be as a result of sequence data confirming that SNP UV_5 originally identified in the CrleGV-SA-C5 HypoP region was identical to the SNP at the same position in the CrleGV-SA HypoP region. Following the differentiation of the CrleGV-SA-C5 and CrleGV-SA isolates through two screening methods, the genetic integrity of the CrleGV-SA-C5 isolate after two virus bulk-ups was determined by PCR amplification of the target regions in the bulk-up virus followed by sequencing. Prior to virus bulk-up, surface dose bioassays were conducted on 4th instar larvae and LC50 and LC90 values of 4.01 x 106 OBs/ml and 8.75 x 109 OBs/ml respectively were obtained. The CrleGV-SA-C5 isolate was then bulked up in fourth instar T. leucotreta larvae using the LC90 value that was determined. Sequencing of the target regions from the CrleGV- SA-C5_BU2 (bulk-up 2) was conducted. Sequencing results confirmed the presence of the target SNPs in the CrleGV-SA-C5_BU2 genome. The UV-tolerance of the CrleGV-SA-C5 isolate in comparison to the CrleGV-SA isolate was evaluated by detached fruit bioassays under natural UV irradiation. Two detached fruit bioassays were set-up, a UV exposure and a non-UV exposure bioassay set-up. Three treatments were used for each bioassay set-up which were the viruses CrleGV-SA-C5 and CrleGV-SA and a ddH2O control. Statistical analysis indicated that there was no significant difference between the virus treatments in both the UV exposed detached fruit bioassay and the non-UV exposed detached fruit bioassay. This study is the second study to report on the de novo assembly of the CrleGV-SA-C5 and sequence comparison with the CrleGV-SA genome, and the first to report on the UV-tolerance of the CrleGV-SA-C5 isolate by detached fruit bioassays. Future work could involve further evaluation of intraspecific genetic variability in the CrleGV-SA-C5 isolate and to identify any additional SNPs present within the genome that can be used as suitable markers for differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates. It was recognised that it is required to conduct further detached fruit bioassays and field trials, but with improved protocols, for the efficacy and UV-tolerance of the CrleGV-SA-C5 isolate to be conclusively determined. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Bennett, Tahnee Tashia
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents , Ultraviolet radiation , Oligonucleotides
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362741 , vital:65358
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), also known as false codling moth (FCM), is indigenous to sub-Saharan Africa. Thaumatotibia leucotreta has been controlled through an integrated pest management (IPM) programme, which includes chemical control, sterile insect technique (SIT), cultural and biological control. As part of the biological control, a key component is the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA). Currently, CryptogranTM, a commercial formulation of CrleGV, is the preferred product to use in South Africa for the control of T. leucotreta. The registration of the biopesticide Cryptogran (River bioscience, South Africa) was established after conducting extensive field trials with CrleGV-SA. One of the major factors affecting the baculovirus efficacy in the field is UV irradiation. A UV-tolerant Cryptophlebia leucotreta granulovirus (CrleGV-SA-C5) isolate was isolated after consecutive cycles of UV exposure. This UV-tolerant isolate is genetically distinct from the CrleGV-SA isolate. The CrleGV-SA-C5 isolate has the potential as a biological control agent. The control of T. leucotreta in South Africa could be improved by the development of novel isolates into new biopesticide formulations. To date, there has not been any field trials conducted on the CrleGV-SA-C5 isolate. Therefore, it is important to determine the biological and genetic stability of this isolate and to conduct field trials with CrleGV-SA- C5 to test the efficacy of the isolate before possible production into a biopesticide. A de novo assembly was conducted to reassemble the genome of CrleGV-SA-C5 which was followed by a sequence comparison with the CrleGV-SA genome. The identification of SNPs, led to the design of oligonucleotides flanking the regions where the SNPs were detected. Polymerase chain reaction amplification of the target regions was conducted using the oligonucleotides. After sequence comparison, seven SNPs were detected and PCR amplification was successful using the three oligonucleotides, Pif-2, HypoP and Lef-8/HP. To differentiate between CrleGV-SA-C5 and CrleGV-SA genomes and confirm the presence of the SNPs, two methods of screening were conducted. The first was the construction of six plasmids, the plasmids contained the targeted pif-2, HypoP, and the Lef-8/HP insert regions from both the CrleGV-SA-C5 and CrleGV-SA genome region where the SNPs were identified, followed by sequencing. The Five recombinant plasmids, pC5_Pif-2, pSA_Pif-2, pC5_HypoP, pSA_HypoP, and pC5_Lef-8/HP were successfully sequenced. No amplicon was obtained for one of the plasmids used as template (pSA_Lef-8/HP) and therefore the PCR product used for cloning was sequenced instead. Sequence alignment confirmed the presence of four of the five targeted SNPs in the genome of the CrleGV-SA-C5 isolate. However, of these only one SNP (UV_7) rendered a suitable marker for the differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates as the SNPs, UV_2, UV_3 and UV_5, were also present in the CrleGV- SA sequences. The second screening method was a quantitative polymerase chain reaction (qPCR) melt curve analysis to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. qPCR melt curve analysis was done using the CrleGV-SA-C5 and CrleGV-SA HypoP PCR products. This technique was unable to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. However, this may be as a result of sequence data confirming that SNP UV_5 originally identified in the CrleGV-SA-C5 HypoP region was identical to the SNP at the same position in the CrleGV-SA HypoP region. Following the differentiation of the CrleGV-SA-C5 and CrleGV-SA isolates through two screening methods, the genetic integrity of the CrleGV-SA-C5 isolate after two virus bulk-ups was determined by PCR amplification of the target regions in the bulk-up virus followed by sequencing. Prior to virus bulk-up, surface dose bioassays were conducted on 4th instar larvae and LC50 and LC90 values of 4.01 x 106 OBs/ml and 8.75 x 109 OBs/ml respectively were obtained. The CrleGV-SA-C5 isolate was then bulked up in fourth instar T. leucotreta larvae using the LC90 value that was determined. Sequencing of the target regions from the CrleGV- SA-C5_BU2 (bulk-up 2) was conducted. Sequencing results confirmed the presence of the target SNPs in the CrleGV-SA-C5_BU2 genome. The UV-tolerance of the CrleGV-SA-C5 isolate in comparison to the CrleGV-SA isolate was evaluated by detached fruit bioassays under natural UV irradiation. Two detached fruit bioassays were set-up, a UV exposure and a non-UV exposure bioassay set-up. Three treatments were used for each bioassay set-up which were the viruses CrleGV-SA-C5 and CrleGV-SA and a ddH2O control. Statistical analysis indicated that there was no significant difference between the virus treatments in both the UV exposed detached fruit bioassay and the non-UV exposed detached fruit bioassay. This study is the second study to report on the de novo assembly of the CrleGV-SA-C5 and sequence comparison with the CrleGV-SA genome, and the first to report on the UV-tolerance of the CrleGV-SA-C5 isolate by detached fruit bioassays. Future work could involve further evaluation of intraspecific genetic variability in the CrleGV-SA-C5 isolate and to identify any additional SNPs present within the genome that can be used as suitable markers for differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates. It was recognised that it is required to conduct further detached fruit bioassays and field trials, but with improved protocols, for the efficacy and UV-tolerance of the CrleGV-SA-C5 isolate to be conclusively determined. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-10-14
Selection for improved virulence of Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) to False Codling Moth, Thaumatotibia leucotreta, by serial passage through a heterologous host
- Authors: Iita, Petrus Paulus
- Date: 2021-04
- Subjects: Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Baculoviruses , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/178180 , vital:42918
- Description: The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is endemic to southern Africa, and strongly associated with citrus. As South African citrus production is mainly for export to foreign markets, the market access risk due to the phytosanitary status of this pest is considerable and its control is therefore imperative. Various control measures as part of a rigorous integrated pest management (IPM) programme targeted against T. leucotreta have been effective at suppressing the pest in citrus, but there is still a growing need for continued improvement of the programme and augmentation of the available control options. Of these control options, biological control, particularly the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA), is a key component of IPM in citrus orchards and it has been very successful at reducing T. leucotreta populations in the field for almost two decades. There is however, a growing need for more baculovirus variants with an improved virulence against T. leucotreta for a more efficient pest management system. The newly identified insect virus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) offers a unique opportunity for an additional biopesticide in IPM for control of T. leucotreta in the field. This study aimed to conduct serial passaging of CrpeNPV through a heterologous host, T. leucotreta, in order to determine the potential for improved virulence or speed of kill against it. In order to select for a variant of CrpeNPV with improved virulence against T. leucotreta, a high dose (LC90) of the virus OBs was used to perform 12 serial passages through T. leucotreta larvae in surface-dose bioassays. Whole genome sequencing and analysis of the passaged virus, along with restriction endonuclease profiling in silico was performed to determine if the genetic identity of the virus had changed during serial passage, in relation to the original virus. These analyses indicated that the dominant genotype of CrpeNPV was maintained following 12 serial passages through the heterologous host. The biological activity of the passaged virus, along with the original virus was evaluated against neonate T. leucotreta in surface-dose bioassays and compared. Results from dose-response bioassays showed that the virulence of CrpeNPV did not improve after 12 serial passages. The LC50 values of the passaged virus and the original virus were estimated at 1.96 × 104 and 1.58 × 104 OBs/ml, respectively, whereas the LC90 values were estimated at 3.46 × 104 OBs/ml for the passaged virus and 3.68 × 104 for the original virus. Similarly, the results from time-response bioassays showed that the speed of kill of CrpeNPV did not improve after 12 serial passages. The LT50 values of the passaged virus and the original virus were 88.44 hours (3 days and 16 hours) and 83.74 hours (3 days and 12 hours), respectively, whereas the LT90 values were 115 hours (4 days 19 hours) for the passaged virus and 102 hours (4 days 6 hours) for the original virus. The virulence and speed of kill of the passaged virus decreased significantly, in relation to the original virus. When the full genome of the passaged virus was sequenced and analysed, only a few SNPs were detected in the viral genome, in comparison to the original virus. No detectable difference in REN digestion patterns were observed following REN analysis of gDNA of the passaged virus with several restriction enzymes in silico. The results for this study suggest that CrpeNPV may already be optimally suited to the heterologous host as it persists under these conditions without significant changes to the genome. These results have positive implications for the genetic integrity of CrpeNPV as a potential biocontrol agent in the field. This study is the first to report the virulence selection of CrpeNPV by serial passage through a heterologous host, and also the first to record bioassay data in terms of dose response (or lethal concentration) against T. leucotreta second instars. The data obtained have added to the knowledge about interactions between CrpeNPV and its heterologous host, and may be fundamental to continued investigation into the effect of serial passage on pathogenicity and genetic diversity of CrpeNPV. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
- Authors: Iita, Petrus Paulus
- Date: 2021-04
- Subjects: Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Baculoviruses , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/178180 , vital:42918
- Description: The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is endemic to southern Africa, and strongly associated with citrus. As South African citrus production is mainly for export to foreign markets, the market access risk due to the phytosanitary status of this pest is considerable and its control is therefore imperative. Various control measures as part of a rigorous integrated pest management (IPM) programme targeted against T. leucotreta have been effective at suppressing the pest in citrus, but there is still a growing need for continued improvement of the programme and augmentation of the available control options. Of these control options, biological control, particularly the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA), is a key component of IPM in citrus orchards and it has been very successful at reducing T. leucotreta populations in the field for almost two decades. There is however, a growing need for more baculovirus variants with an improved virulence against T. leucotreta for a more efficient pest management system. The newly identified insect virus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) offers a unique opportunity for an additional biopesticide in IPM for control of T. leucotreta in the field. This study aimed to conduct serial passaging of CrpeNPV through a heterologous host, T. leucotreta, in order to determine the potential for improved virulence or speed of kill against it. In order to select for a variant of CrpeNPV with improved virulence against T. leucotreta, a high dose (LC90) of the virus OBs was used to perform 12 serial passages through T. leucotreta larvae in surface-dose bioassays. Whole genome sequencing and analysis of the passaged virus, along with restriction endonuclease profiling in silico was performed to determine if the genetic identity of the virus had changed during serial passage, in relation to the original virus. These analyses indicated that the dominant genotype of CrpeNPV was maintained following 12 serial passages through the heterologous host. The biological activity of the passaged virus, along with the original virus was evaluated against neonate T. leucotreta in surface-dose bioassays and compared. Results from dose-response bioassays showed that the virulence of CrpeNPV did not improve after 12 serial passages. The LC50 values of the passaged virus and the original virus were estimated at 1.96 × 104 and 1.58 × 104 OBs/ml, respectively, whereas the LC90 values were estimated at 3.46 × 104 OBs/ml for the passaged virus and 3.68 × 104 for the original virus. Similarly, the results from time-response bioassays showed that the speed of kill of CrpeNPV did not improve after 12 serial passages. The LT50 values of the passaged virus and the original virus were 88.44 hours (3 days and 16 hours) and 83.74 hours (3 days and 12 hours), respectively, whereas the LT90 values were 115 hours (4 days 19 hours) for the passaged virus and 102 hours (4 days 6 hours) for the original virus. The virulence and speed of kill of the passaged virus decreased significantly, in relation to the original virus. When the full genome of the passaged virus was sequenced and analysed, only a few SNPs were detected in the viral genome, in comparison to the original virus. No detectable difference in REN digestion patterns were observed following REN analysis of gDNA of the passaged virus with several restriction enzymes in silico. The results for this study suggest that CrpeNPV may already be optimally suited to the heterologous host as it persists under these conditions without significant changes to the genome. These results have positive implications for the genetic integrity of CrpeNPV as a potential biocontrol agent in the field. This study is the first to report the virulence selection of CrpeNPV by serial passage through a heterologous host, and also the first to record bioassay data in terms of dose response (or lethal concentration) against T. leucotreta second instars. The data obtained have added to the knowledge about interactions between CrpeNPV and its heterologous host, and may be fundamental to continued investigation into the effect of serial passage on pathogenicity and genetic diversity of CrpeNPV. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-04
An Integrated Management System to reduce False Codling Moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) infested citrus fruit from being packed for export
- Authors: Mac Aleer, Clint
- Date: 2019
- Subjects: Cryptophlebia leucotreta -- South Africa , Cryptophlebia leucotreta -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Insecticides , Citrus fruit industry -- South Africa , South Africa -- Commerce -- European Economic Community Countries
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/92219 , vital:30691
- Description: False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is indigenous to southern Africa and is an important pest of citrus in this region. As a result of its endemism to sub-Saharan Africa, several countries to which South Africa exports citrus, regulate it as a phytosanitary pest. Consequently, it is necessary to ship fruit to these markets under cold-disinfestation protocols. This has been possible, as until recently, all of these markets could be considered relatively small niche markets. The South African citrus industry exports approximately 130 million cartons of fruit (15 kg equivalent) annually. During the 2017 season, a total of 48 million cartons were exported to the European Union (EU), which is the equivalent of 41% of South Africa’s total export volume, thus making the EU South Africa’s most important export market. In 2013 the European and Mediterranean Plant Protection Organisation (EPPO) conducted a pest risk analysis (PRA) on FCM, leading to the EU declaring it an officially regulated pest for this region, effective of 1 January 2018. Citrus is regarded as a preferred non-native host of FCM and South African citrus was identified as a primary focus due to large volumes being exported to Europe. Shipping under cold disinfestation is not possible with such large volumes of fruit. Additionally, several cultivars would suffer high levels of chilling injury under such conditions. In this study, an Integrated Management System was tested with pre- and postharvest controls to test the hypothesis that pre-harvest interventions resulted in lower post-harvest infection. Thirty orchards ranging from soft citrus cultivars such as Nule and Nova Mandarins, to Navel orange cultivars such as Newhall, Palmer and Late Navel and ending with Valencia cultivars such as Midknight and Delta, were identified for this study. This system relies on pre-harvest inspections such as FCM trap counts and fruit infestation on data trees in every orchard, with associated thresholds for action or continued compliance. Inspections were conducted on a weekly basis. There was a significant relationship between the moth catches and FCM infestation for the full monitoring period, using a two-week lag period for infestation. Inspections of harvested fruit were conducted at the packhouse to determine FCM infestation. This included inspection of the fruit on delivery to the packhouse, on the packing line, and a final fruit sample taken from the packed product and inspected for FCM. The highest levels of infestation were recorded on the Navel cultivars, thus confirming that Navels cultivars are a preferred host for FCM. Significant positive relationships were recorded between FCM infestation during the last 4 weeks before harvest and the level of infestation in the fruit delivered to the packhouse and between the fruit delivered to the packhouse and in the fruit packed in a carton for export. There was a substantial reduction in infestation between the fruit delivered to the packhouse and the fruit packed in a carton for export, with certain orchards recording as much as a 93% reduction in the fruit packed in a carton, which indicated that the packhouse could effectively identify and remove FCM infested fruit. The outcome of the study is that a holistic management approach minimizes the risk of FCM in citrus fruit destined for export and therefore mitigate the risk associated with FCM.
- Full Text:
- Date Issued: 2019
- Authors: Mac Aleer, Clint
- Date: 2019
- Subjects: Cryptophlebia leucotreta -- South Africa , Cryptophlebia leucotreta -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Insecticides , Citrus fruit industry -- South Africa , South Africa -- Commerce -- European Economic Community Countries
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/92219 , vital:30691
- Description: False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is indigenous to southern Africa and is an important pest of citrus in this region. As a result of its endemism to sub-Saharan Africa, several countries to which South Africa exports citrus, regulate it as a phytosanitary pest. Consequently, it is necessary to ship fruit to these markets under cold-disinfestation protocols. This has been possible, as until recently, all of these markets could be considered relatively small niche markets. The South African citrus industry exports approximately 130 million cartons of fruit (15 kg equivalent) annually. During the 2017 season, a total of 48 million cartons were exported to the European Union (EU), which is the equivalent of 41% of South Africa’s total export volume, thus making the EU South Africa’s most important export market. In 2013 the European and Mediterranean Plant Protection Organisation (EPPO) conducted a pest risk analysis (PRA) on FCM, leading to the EU declaring it an officially regulated pest for this region, effective of 1 January 2018. Citrus is regarded as a preferred non-native host of FCM and South African citrus was identified as a primary focus due to large volumes being exported to Europe. Shipping under cold disinfestation is not possible with such large volumes of fruit. Additionally, several cultivars would suffer high levels of chilling injury under such conditions. In this study, an Integrated Management System was tested with pre- and postharvest controls to test the hypothesis that pre-harvest interventions resulted in lower post-harvest infection. Thirty orchards ranging from soft citrus cultivars such as Nule and Nova Mandarins, to Navel orange cultivars such as Newhall, Palmer and Late Navel and ending with Valencia cultivars such as Midknight and Delta, were identified for this study. This system relies on pre-harvest inspections such as FCM trap counts and fruit infestation on data trees in every orchard, with associated thresholds for action or continued compliance. Inspections were conducted on a weekly basis. There was a significant relationship between the moth catches and FCM infestation for the full monitoring period, using a two-week lag period for infestation. Inspections of harvested fruit were conducted at the packhouse to determine FCM infestation. This included inspection of the fruit on delivery to the packhouse, on the packing line, and a final fruit sample taken from the packed product and inspected for FCM. The highest levels of infestation were recorded on the Navel cultivars, thus confirming that Navels cultivars are a preferred host for FCM. Significant positive relationships were recorded between FCM infestation during the last 4 weeks before harvest and the level of infestation in the fruit delivered to the packhouse and between the fruit delivered to the packhouse and in the fruit packed in a carton for export. There was a substantial reduction in infestation between the fruit delivered to the packhouse and the fruit packed in a carton for export, with certain orchards recording as much as a 93% reduction in the fruit packed in a carton, which indicated that the packhouse could effectively identify and remove FCM infested fruit. The outcome of the study is that a holistic management approach minimizes the risk of FCM in citrus fruit destined for export and therefore mitigate the risk associated with FCM.
- Full Text:
- Date Issued: 2019
Assessment of pheromone specificity in Thaumatotibia leucotreta (Meyrick) populations with focus on pest monitoring and the regional rollout of the sterile insect technique in citrus
- Authors: Joubert, Francois D
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Pheromone traps , Citrus -- Diseases and pests -- South Africa , Cryptophlebia leucotreta -- Contol , Cryptophlebia leucotreta -- Biological control
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/60665 , vital:27812
- Description: False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. It is recognized by several markets as a phytosanitary organism and the efficient control of this pest is now more important than ever. The pheromone communication between the male and female moths has been exploited in order to control FCM through the sterile insect technique (SIT). The sterilized males used for all SIT programmes across South Africa come from a colony that originates from wild material collected from the Citrusdal area of the Western Cape Province. The aim of this study was to determine if any differences in attractiveness of females to males exist between different geographical populations of FCM and if so what impact this would have on the male’s ability to locate females from other populations via the volatile sex pheromone released by the female. Laboratory trials with Y-tube olfactometers and flight tunnels tested the attraction of male moths to virgin females, but did not yield any consistent results. Field experiments were conducted with sterile male Citrusdal moths released and recaptured in yellow delta traps in two separate trials. For one trial, the traps were baited with live virgin females from five different geographical populations including Addo, Nelspruit, Marble Hall, Citrusdal and the Old colony, which is a mixture of several populations. For the other trial traps were baited with various synthetic pheromone blends including three regional blends which included South Africa, Ivory Coast and Malawi and three commercial blends including Pherolure, Isomate and Checkmate. For the virgin female trial the Citrusdal males showed a significant preference for females from their own population. There was also a significant difference in the recaptures from the different synthetic pheromones. The South African blend was the most attractive of all the regional and commercial blends. A cross-mating trial was also conducted under laboratory conditions in petri dishes with five different FCM populations including Citrusdal, Addo, Marble Hall, Nelspruit and Old (mixed origin). Females produced more eggs when mated with males from the same population for the Addo, Marble Hall, Nelspruit and Old (mixed origin) populations. The only case in which this was statistically significant was for the Marble Hall population. All the crosses produced viable eggs and the origin of the male or female did not influence egg hatch. The results from this study may lead to improvements in both the control and monitoring of FCM populations. The control methods include mating disruption, attract-and-kill and SIT. Tailoring these methods for a specific growing area with a pheromone blend originating from the area or releasing sterile moths from a colony that originates from the area may optimize the available monitoring and control options.
- Full Text:
- Date Issued: 2018
- Authors: Joubert, Francois D
- Date: 2018
- Subjects: Cryptophlebia leucotreta , Pheromone traps , Citrus -- Diseases and pests -- South Africa , Cryptophlebia leucotreta -- Contol , Cryptophlebia leucotreta -- Biological control
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/60665 , vital:27812
- Description: False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. It is recognized by several markets as a phytosanitary organism and the efficient control of this pest is now more important than ever. The pheromone communication between the male and female moths has been exploited in order to control FCM through the sterile insect technique (SIT). The sterilized males used for all SIT programmes across South Africa come from a colony that originates from wild material collected from the Citrusdal area of the Western Cape Province. The aim of this study was to determine if any differences in attractiveness of females to males exist between different geographical populations of FCM and if so what impact this would have on the male’s ability to locate females from other populations via the volatile sex pheromone released by the female. Laboratory trials with Y-tube olfactometers and flight tunnels tested the attraction of male moths to virgin females, but did not yield any consistent results. Field experiments were conducted with sterile male Citrusdal moths released and recaptured in yellow delta traps in two separate trials. For one trial, the traps were baited with live virgin females from five different geographical populations including Addo, Nelspruit, Marble Hall, Citrusdal and the Old colony, which is a mixture of several populations. For the other trial traps were baited with various synthetic pheromone blends including three regional blends which included South Africa, Ivory Coast and Malawi and three commercial blends including Pherolure, Isomate and Checkmate. For the virgin female trial the Citrusdal males showed a significant preference for females from their own population. There was also a significant difference in the recaptures from the different synthetic pheromones. The South African blend was the most attractive of all the regional and commercial blends. A cross-mating trial was also conducted under laboratory conditions in petri dishes with five different FCM populations including Citrusdal, Addo, Marble Hall, Nelspruit and Old (mixed origin). Females produced more eggs when mated with males from the same population for the Addo, Marble Hall, Nelspruit and Old (mixed origin) populations. The only case in which this was statistically significant was for the Marble Hall population. All the crosses produced viable eggs and the origin of the male or female did not influence egg hatch. The results from this study may lead to improvements in both the control and monitoring of FCM populations. The control methods include mating disruption, attract-and-kill and SIT. Tailoring these methods for a specific growing area with a pheromone blend originating from the area or releasing sterile moths from a colony that originates from the area may optimize the available monitoring and control options.
- Full Text:
- Date Issued: 2018
Baculovirus synergism: investigating mixed alphabaculovirus and betabaculovirus infections in the false codling moth, thaumatotibia leucotreta, for improved pest control
- Authors: Jukes, Michael David
- Date: 2018
- Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61797 , vital:28061
- Description: Baculovirus based biopesticides are an effective and environmentally friendly approach for the control of agriculturally important insect pests. The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is indigenous to southern Africa and is a major pest of citrus crops. This moth poses a serious risk to export of fruit to foreign markets and the control of this pest is therefore imperative. The Cryptophlebia leucotreta granulovirus (CrleGV) has been commercially formulated into the products Cryptogran™ and Cryptex®. These products have been used successfully for over a decade as part of a rigorous integrated pest management (IPM) programme to control T. leucotreta in South Africa. There is however, a continuous need to improve this programme while also addressing new challenges as they arise. An example of a rising concern is the possibility of resistance developing towards CrleGV. This was seen in Europe with field populations of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), which developed resistance to the Mexican isolate of the Cydia pomonella granulovirus (CpGV-M). To prevent such a scenario occurring in South Africa, there is a need to improve existing methods of control. For example, additional baculovirus variants can be isolated and characterised for determining virulence, which can then be developed as new biopesticides. Additionally, the potential for synergistic effects between different baculoviruses infecting the same host can be explored for improved virulence. A novel nucleopolyhedrovirus was recently identified in T. leucotreta larval homogenates which were also infected with CrleGV. This provided unique opportunities for continued research and development. In this study, a method using C. pomonella larvae, which can be infected by the NPV but not by CrleGV, was developed to separate the NPV from GV-NPV mixtures in an in vivo system. Examination of NPV OBs by transmission electron microscopy showed purified occlusion bodies with a single nucleopolyhedrovirus morphology (SNPV). Genetic characterisation identified the novel NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which was recently isolated from the litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae). To begin examining the potential for synergism between the two viruses, a multiplex PCR assay was developed to accurately detect CrleGV and/or CrpeNPV in mixed infections. This assay was applied to various samples to screen for the presence of CrpeNPV and CrleGV. Additionally, a validation experiment was performed using different combinations of CrpeNPV and/or CrleGV to evaluate the effectiveness of the mPCR assay. The results obtained indicated a high degree of specificity with the correct amplicons generated for each test sample. The biological activity of CrpeNPV and CrleGV were evaluated using surface dose bioassays, both individually and in various combinations, against T. leucotreta neonate larvae in a laboratory setting. A synergistic effect was recorded in the combination treatments, showing improved virulence when compared against each virus in isolation. The LC90 for CrpeNPV and CrleGV when applied alone against T. leucotreta was calculated to be 2.75*106 and 3.00*106 OBs.ml"1 respectively. These values decreased to 1.07*106 and 7.18*105 OBs.ml"1 when combinations of CrleGV and CrpeNPV were applied at ratios of 3:1 and 1:3 respectively. These results indicate a potential for developing improved biopesticides for the control of T. leucotreta in the field. To better understand the interactions between CrleGV and CrpeNPV, experiments involving the serial passage of these viruses through T. leucotreta larvae were performed. This was done using each virus in isolation as well as both viruses in different combinations. Genomic DNA was extracted from recovered occlusion bodies after each passage and examined by multiplex and quantitative PCR. This analysis enabled the detection of each virus present throughout this assay, as well as recording shifts in the ratio of CrleGV and CrpeNPV at each passage. CrleGV rapidly became the dominant virus in all treatments, indicating a potentially antagonistic interaction during serial passage. Additionally, CrpeNPV and CrleGV were detected in treatments which were not originally inoculated with one or either virus, indicating potential covert infections in T. leucotreta. Occlusion bodies recovered from the final passage were used to inoculate C. pomonella larvae to isolate CrpeNPV from CrleGV. Genomic DNA was extracted from these CrpeNPV OBs and examined by restriction endonuclease assays and next generation sequencing. This enabled the identification of potential recombination events which may have occurred during the dual GV and NPV infections throughout the passage assay. No recombination events were identified in the CrpeNPV genome sequences assembled from virus collected at the end of the passage assay. Lastly, the efficacy of CrpeNPV and CrleGV, both alone and in various combinations, was evaluated in the field. In two separate trials conducted on citrus, unfavorable field conditions resulted in no significant reduction in fruit infestation for both the virus and chemical treatments. While not statistically significant, virus treatments were recorded to have the lowest levels of fruit infestation with a measured reduction of up to 64 %. This study is the first to report a synergistic effect between CrleGV and CrpeNPV in T. leucotreta. The discovery of beneficial interactions creates an opportunity for the development of novel biopesticides for improved control of this pest in South Africa.
- Full Text:
- Date Issued: 2018
- Authors: Jukes, Michael David
- Date: 2018
- Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/61797 , vital:28061
- Description: Baculovirus based biopesticides are an effective and environmentally friendly approach for the control of agriculturally important insect pests. The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is indigenous to southern Africa and is a major pest of citrus crops. This moth poses a serious risk to export of fruit to foreign markets and the control of this pest is therefore imperative. The Cryptophlebia leucotreta granulovirus (CrleGV) has been commercially formulated into the products Cryptogran™ and Cryptex®. These products have been used successfully for over a decade as part of a rigorous integrated pest management (IPM) programme to control T. leucotreta in South Africa. There is however, a continuous need to improve this programme while also addressing new challenges as they arise. An example of a rising concern is the possibility of resistance developing towards CrleGV. This was seen in Europe with field populations of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), which developed resistance to the Mexican isolate of the Cydia pomonella granulovirus (CpGV-M). To prevent such a scenario occurring in South Africa, there is a need to improve existing methods of control. For example, additional baculovirus variants can be isolated and characterised for determining virulence, which can then be developed as new biopesticides. Additionally, the potential for synergistic effects between different baculoviruses infecting the same host can be explored for improved virulence. A novel nucleopolyhedrovirus was recently identified in T. leucotreta larval homogenates which were also infected with CrleGV. This provided unique opportunities for continued research and development. In this study, a method using C. pomonella larvae, which can be infected by the NPV but not by CrleGV, was developed to separate the NPV from GV-NPV mixtures in an in vivo system. Examination of NPV OBs by transmission electron microscopy showed purified occlusion bodies with a single nucleopolyhedrovirus morphology (SNPV). Genetic characterisation identified the novel NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which was recently isolated from the litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae). To begin examining the potential for synergism between the two viruses, a multiplex PCR assay was developed to accurately detect CrleGV and/or CrpeNPV in mixed infections. This assay was applied to various samples to screen for the presence of CrpeNPV and CrleGV. Additionally, a validation experiment was performed using different combinations of CrpeNPV and/or CrleGV to evaluate the effectiveness of the mPCR assay. The results obtained indicated a high degree of specificity with the correct amplicons generated for each test sample. The biological activity of CrpeNPV and CrleGV were evaluated using surface dose bioassays, both individually and in various combinations, against T. leucotreta neonate larvae in a laboratory setting. A synergistic effect was recorded in the combination treatments, showing improved virulence when compared against each virus in isolation. The LC90 for CrpeNPV and CrleGV when applied alone against T. leucotreta was calculated to be 2.75*106 and 3.00*106 OBs.ml"1 respectively. These values decreased to 1.07*106 and 7.18*105 OBs.ml"1 when combinations of CrleGV and CrpeNPV were applied at ratios of 3:1 and 1:3 respectively. These results indicate a potential for developing improved biopesticides for the control of T. leucotreta in the field. To better understand the interactions between CrleGV and CrpeNPV, experiments involving the serial passage of these viruses through T. leucotreta larvae were performed. This was done using each virus in isolation as well as both viruses in different combinations. Genomic DNA was extracted from recovered occlusion bodies after each passage and examined by multiplex and quantitative PCR. This analysis enabled the detection of each virus present throughout this assay, as well as recording shifts in the ratio of CrleGV and CrpeNPV at each passage. CrleGV rapidly became the dominant virus in all treatments, indicating a potentially antagonistic interaction during serial passage. Additionally, CrpeNPV and CrleGV were detected in treatments which were not originally inoculated with one or either virus, indicating potential covert infections in T. leucotreta. Occlusion bodies recovered from the final passage were used to inoculate C. pomonella larvae to isolate CrpeNPV from CrleGV. Genomic DNA was extracted from these CrpeNPV OBs and examined by restriction endonuclease assays and next generation sequencing. This enabled the identification of potential recombination events which may have occurred during the dual GV and NPV infections throughout the passage assay. No recombination events were identified in the CrpeNPV genome sequences assembled from virus collected at the end of the passage assay. Lastly, the efficacy of CrpeNPV and CrleGV, both alone and in various combinations, was evaluated in the field. In two separate trials conducted on citrus, unfavorable field conditions resulted in no significant reduction in fruit infestation for both the virus and chemical treatments. While not statistically significant, virus treatments were recorded to have the lowest levels of fruit infestation with a measured reduction of up to 64 %. This study is the first to report a synergistic effect between CrleGV and CrpeNPV in T. leucotreta. The discovery of beneficial interactions creates an opportunity for the development of novel biopesticides for improved control of this pest in South Africa.
- Full Text:
- Date Issued: 2018
Yeast-baculovirus synergism: investigating mixed infections for improved management of the false codling moth, Thaumatotibia leucotreta
- Authors: Van der Merwe, Marcél
- 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, Marcél
- 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
Improving the cold tolerance of false codling moth, thaumatotibia leucotreta, for better performance in a sterile insect release programme
- Authors: Daniel, Claire Ashleigh
- Date: 2017
- Subjects: Cryptophlebia leucotreta -- South Africa , Cryptophlebia leucotreta -- Reproduction -- Effect of temperature on , Cryptophlebia leucotreta -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Insecticides , Citrus fruit industry -- South Africa
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/5271 , vital:20803
- Description: The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a major pest of citrus and other important crops in Sub-Saharan Africa. The introduction of a sterile insect technique (SIT) programme for FCM in South Africa has proven to be very effective in the control of FCM. The objective was to flood citrus orchards with large numbers of sterile males resulting in a ratio of at least 10 sterile to 1 wild moth, increasing the probability of a female moth mating with a sterile male. This target is often achieved and the programme is generally successful, however there are some challenges regarding this programme. The mass rearing environment, artificial diet, handling methods and irradiation have an impact on sterile insect quality as environmental differences between the rearing facility and field, influences the insect phenotype and competitiveness. This is evident as wild male moths can theoretically actively fly at a temperature of 12°C and laboratory-reared sterilized moths, due to the radiation treatment, appear unable to fly below 20°C. As a consequence, sterile males are out-competed by wild males during the cooler months of the year. This is detrimental to the SIT programme as FCM do not undergo diapause, meaning they are active during winter and will still reproduce. Therefore, to maximize the effect of the SIT programme, it is vital to increase the flight ability of mass reared sterile males at lower temperatures. Various studies have shown that by adding cryoprotectants to the basic laboratory diets increases the cold tolerance of certain insects and thus may allow them to be mobile at lower temperatures, however it imperative that any chemical used to augment the commercial diet of the insect has no negative effects on the insect physiology and development. To investigate this detail for FCM, five generations of FCM were reared on diets augmented with various known insect cryoprotectants. These augmented FCM were subsequently used in experiments designed to determine firstly, if the cryoprotectants had a positive result on the cold tolerance of the FCM, and secondly, if they had any adverse effects on other physiological aspects such as duration of development. Laboratory trials indicated that the flight ability of male FCM was improved when larvae were reared on diets augmented with trehalose and cholesterol (with an average of 40 % of cholesterol and trehalose augmented males that flew at 15 °C where 0 % of the control flew). Results obtained during the field trials support the laboratory results as there was a significant increase in the number of trehalose augmented moths caught in the field during March and July (winter). Results also showed potential for cholesterol to be used as an additive. Other important findings show that both cholesterol and trehalose have no negative impacts on developmental rate, pupal size, and egg production and viability. Trehalose was found to increase the pupal mass of male and female FCM, as well as the number of eggs laid per female. Cholesterol was found to increase developmental rate and the number of eggs laid. The main findings of this study were that diet additives could improve the massrearing of FCM for SIT and the competitiveness of the males, especially at lower temperatures. However, the additives were expensive and cost could well be a constraint to the wide scale implementation of the new technology.
- Full Text:
- Date Issued: 2017
- Authors: Daniel, Claire Ashleigh
- Date: 2017
- Subjects: Cryptophlebia leucotreta -- South Africa , Cryptophlebia leucotreta -- Reproduction -- Effect of temperature on , Cryptophlebia leucotreta -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Insecticides , Citrus fruit industry -- South Africa
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/5271 , vital:20803
- Description: The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a major pest of citrus and other important crops in Sub-Saharan Africa. The introduction of a sterile insect technique (SIT) programme for FCM in South Africa has proven to be very effective in the control of FCM. The objective was to flood citrus orchards with large numbers of sterile males resulting in a ratio of at least 10 sterile to 1 wild moth, increasing the probability of a female moth mating with a sterile male. This target is often achieved and the programme is generally successful, however there are some challenges regarding this programme. The mass rearing environment, artificial diet, handling methods and irradiation have an impact on sterile insect quality as environmental differences between the rearing facility and field, influences the insect phenotype and competitiveness. This is evident as wild male moths can theoretically actively fly at a temperature of 12°C and laboratory-reared sterilized moths, due to the radiation treatment, appear unable to fly below 20°C. As a consequence, sterile males are out-competed by wild males during the cooler months of the year. This is detrimental to the SIT programme as FCM do not undergo diapause, meaning they are active during winter and will still reproduce. Therefore, to maximize the effect of the SIT programme, it is vital to increase the flight ability of mass reared sterile males at lower temperatures. Various studies have shown that by adding cryoprotectants to the basic laboratory diets increases the cold tolerance of certain insects and thus may allow them to be mobile at lower temperatures, however it imperative that any chemical used to augment the commercial diet of the insect has no negative effects on the insect physiology and development. To investigate this detail for FCM, five generations of FCM were reared on diets augmented with various known insect cryoprotectants. These augmented FCM were subsequently used in experiments designed to determine firstly, if the cryoprotectants had a positive result on the cold tolerance of the FCM, and secondly, if they had any adverse effects on other physiological aspects such as duration of development. Laboratory trials indicated that the flight ability of male FCM was improved when larvae were reared on diets augmented with trehalose and cholesterol (with an average of 40 % of cholesterol and trehalose augmented males that flew at 15 °C where 0 % of the control flew). Results obtained during the field trials support the laboratory results as there was a significant increase in the number of trehalose augmented moths caught in the field during March and July (winter). Results also showed potential for cholesterol to be used as an additive. Other important findings show that both cholesterol and trehalose have no negative impacts on developmental rate, pupal size, and egg production and viability. Trehalose was found to increase the pupal mass of male and female FCM, as well as the number of eggs laid per female. Cholesterol was found to increase developmental rate and the number of eggs laid. The main findings of this study were that diet additives could improve the massrearing of FCM for SIT and the competitiveness of the males, especially at lower temperatures. However, the additives were expensive and cost could well be a constraint to the wide scale implementation of the new technology.
- Full Text:
- Date Issued: 2017
The pest status and integrated management programme of carob moth, Ectomyelois ceratoniae Zeller, attacking citrus in South Africa
- Authors: Thackeray, Sean Robin
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/7758 , vital:21293
- Description: The carob moth, Ectomyelois ceratoniae Zeller, is a pest of agricultural commodities and stored products around the world. Carob moth is known to infest citrus in the Mediterranean region and in southern Africa. In grapefruit cultivars, carob moth infestations are associated with high levels of mealybug. However, although this relationship has been observed in other citrus types such as Navel oranges, this has never been quantified. A recent survey of infested fruit from various production areas in South Africa indicated that the pest status of carob moth on Navel oranges may have been underestimated. As a result of the incidental pest status of carob moth on citrus in South Africa in the past, a species specific integrated pest management (IPM) programme does not exist. Therefore, the overriding aim of this theses was to evaluate the pest status of carob moth in citrus and establish a species specific IPM programme by determine the autecology of carob moth in citrus. Reliable methods for monitoring carob moth in citrus orchards both for producers and for research purposes were developed. A user-friendly monitoring method for determining weekly carob moth infestation through dropped fruit was suitable for producers. A timed scouting method was also developed; although the accuracy of this method varied with the experience of the scout. The pest status of carob moth was highest in the Loskop Valley, Nelspruit and the Vaalharts production areas and economic injury to growers ranged from R512.35 to R3 719.80 per hectare as a direct result of infestation. No infestation was recorded in the Sundays River Valley and Citrusdal production areas over both the 2014-15 and 201516 growing seasons. A laboratory study showed the survival of carob moth larvae infesting citrus is less than 10% in the absence of mealybug. However, this increases to almost 40% in the presence of mealybug residues and sooty mould. There was a significant relationship between carob infestation at harvest and mealybug infestation in the middle months of the growing season. The relationship between carob moth and mealybug indicates that current production guidelines for the management of mealybug in citrus may need to be amended. Consequently, it is proposed that an orchard with a history of carob moth infestation and a high mealybug infestation in the previous season should be subjected to an early season preventative application of a registered control product. Also, if mealybug infestation in December is higher than a 5% of fruit per tree, then a corrective application of a registered product is recommended. The application of 2,4-D at petal drop reduced the size of the navel-end opening, decreasing the proportion of mealybug found in the navel-end, subsequently reducing carob moth infestation, resulting in a direct benefit for producers. Products registered for the control of false codling moth (FCM), Thaumatotibia leucotreta Meyrick, were effective in reducing carob moth infestation. In a spray trial conducted over two seasons, Delegate® and Runner® reduced infestation significantly in the 2014-15 season (over 80%), while only Delegate® was effective in the 2015-16 season (over 80%). If a late season corrective chemical application is targeted at both FCM and carob moth, this application should take place between 6-7 weeks prior to harvest. The mating disruption product, SPLAT® EC, reduced carob moth infestation by 70% compared to the untreated control. A laboratory culture was established and head-capsule size categories were determined for all five carob moth instars. A parasitoid survey indicated that parasitism of carob moth larvae is generally less than 5% in citrus orchards and a new species of Braconidae was described as Phanterotoma carobivora van Achterberg and Thackeray. Carob moth fifth instar were found to be the most cold-tolerant larval stage, and were shown to be more cold susceptible than the most cold-tolerant FCM instars at -0.55ºC for eighteen days. This cold treatment resulted in a mortality of 94.6% fifth instar carob moth compared to a combined fourth and fifth instar mortality of 87.8% for FCM after eighteen days. These results indicate that post-harvest cold treatments targeting FCM will be as, if not more, effective against carob moth, suggesting that current phytosanitary legislation for carob moth should be amended to incorporate this study’s findings.
- Full Text:
- Date Issued: 2017
- Authors: Thackeray, Sean Robin
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/7758 , vital:21293
- Description: The carob moth, Ectomyelois ceratoniae Zeller, is a pest of agricultural commodities and stored products around the world. Carob moth is known to infest citrus in the Mediterranean region and in southern Africa. In grapefruit cultivars, carob moth infestations are associated with high levels of mealybug. However, although this relationship has been observed in other citrus types such as Navel oranges, this has never been quantified. A recent survey of infested fruit from various production areas in South Africa indicated that the pest status of carob moth on Navel oranges may have been underestimated. As a result of the incidental pest status of carob moth on citrus in South Africa in the past, a species specific integrated pest management (IPM) programme does not exist. Therefore, the overriding aim of this theses was to evaluate the pest status of carob moth in citrus and establish a species specific IPM programme by determine the autecology of carob moth in citrus. Reliable methods for monitoring carob moth in citrus orchards both for producers and for research purposes were developed. A user-friendly monitoring method for determining weekly carob moth infestation through dropped fruit was suitable for producers. A timed scouting method was also developed; although the accuracy of this method varied with the experience of the scout. The pest status of carob moth was highest in the Loskop Valley, Nelspruit and the Vaalharts production areas and economic injury to growers ranged from R512.35 to R3 719.80 per hectare as a direct result of infestation. No infestation was recorded in the Sundays River Valley and Citrusdal production areas over both the 2014-15 and 201516 growing seasons. A laboratory study showed the survival of carob moth larvae infesting citrus is less than 10% in the absence of mealybug. However, this increases to almost 40% in the presence of mealybug residues and sooty mould. There was a significant relationship between carob infestation at harvest and mealybug infestation in the middle months of the growing season. The relationship between carob moth and mealybug indicates that current production guidelines for the management of mealybug in citrus may need to be amended. Consequently, it is proposed that an orchard with a history of carob moth infestation and a high mealybug infestation in the previous season should be subjected to an early season preventative application of a registered control product. Also, if mealybug infestation in December is higher than a 5% of fruit per tree, then a corrective application of a registered product is recommended. The application of 2,4-D at petal drop reduced the size of the navel-end opening, decreasing the proportion of mealybug found in the navel-end, subsequently reducing carob moth infestation, resulting in a direct benefit for producers. Products registered for the control of false codling moth (FCM), Thaumatotibia leucotreta Meyrick, were effective in reducing carob moth infestation. In a spray trial conducted over two seasons, Delegate® and Runner® reduced infestation significantly in the 2014-15 season (over 80%), while only Delegate® was effective in the 2015-16 season (over 80%). If a late season corrective chemical application is targeted at both FCM and carob moth, this application should take place between 6-7 weeks prior to harvest. The mating disruption product, SPLAT® EC, reduced carob moth infestation by 70% compared to the untreated control. A laboratory culture was established and head-capsule size categories were determined for all five carob moth instars. A parasitoid survey indicated that parasitism of carob moth larvae is generally less than 5% in citrus orchards and a new species of Braconidae was described as Phanterotoma carobivora van Achterberg and Thackeray. Carob moth fifth instar were found to be the most cold-tolerant larval stage, and were shown to be more cold susceptible than the most cold-tolerant FCM instars at -0.55ºC for eighteen days. This cold treatment resulted in a mortality of 94.6% fifth instar carob moth compared to a combined fourth and fifth instar mortality of 87.8% for FCM after eighteen days. These results indicate that post-harvest cold treatments targeting FCM will be as, if not more, effective against carob moth, suggesting that current phytosanitary legislation for carob moth should be amended to incorporate this study’s findings.
- Full Text:
- Date Issued: 2017
Developing an attractant for monitoring fruit-feeding moths in citrus orchards
- Authors: Goddard, Mathew Keith
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2981 , vital:20349
- Description: Fruit-piercing moths are a sporadic pest of citrus, especially in the Eastern Cape Province of South Africa, where the adults can cause significant damage in outbreak years. Currently the only way in which to successfully control fruit-feeding moths within the orchards is the use of repellent lights. However, growers confuse fruit-piercing moths with fruit-sucking moths that don‘t cause primary damage, and there is no way of monitoring which moth species are attacking the fruit in the orchards during the night. In a previous study, banana was shown to be the most attractive bait for a variety of fruit-feeding moth species. Therefore the aim of this study was to determine the population dynamics of fruit-feeding moths develop a cost-effective alternative to the use of fresh banana as a bait for fruit-piercing moths. Fresh banana was compared to nine alternative synthetic attractants, frozen banana and a control under field conditions in several orchards in the Eastern Cape Province. Once again, banana was shown to be the most attractive bait. Some 23 species of fruit-feeding moth species were sampled in the traps, but there was only two fruit-piercing species, Serrodes partita (Fabricius) (Lepidoptera: Noctuidae) and Eudocima sp. Surprisingly S. partita, which was thought to be the main pest, comprised only 6.9% of trap catches. Serrodes partita, is a sporadic pest, only becoming problematic every five to 10 years after good rainfall in the Little Karoo region that causes flushes of their larval host, wild plum, Pappea capensis (Ecklon and Zeyher). During these outbreaks, damage to fruit can range from 70 to 90% and this is especially so for soft skinned citrus. A study on the morphology of the proboscis confirmed that only two species of fruit-piercing moths were present. Trap catches over three citrus growing seasons was linked to fruit damage found within several orchards. Once again fruit-piercing moth damage was relatively low in comparison to other types of damage such as mechanical and undefined damage. There was a very weak correlation between S. partita trap catches and damage, but generally damage was recorded two to three weeks after a peak in S. partita trap catches. Climatic conditions were also recorded and compared to weekly trap catches of S. partita, and while temperature and wind direction had no influence on moth populations, precipitation in the orchards was weakly correlated with trap catches. This study has shown that in non-outbreak seasons, the main fruit-piercing moth, S. partita comprises a small percentage of fruit-feeding moths in citrus orchards, but that growers are unable to determine the difference between fruit-piercing species and the harmless fruit-sucking species. Further fresh banana remains the best method for attracting fruit-piecing moths to traps, but this is not cost effective and thus a commercially viable protocol for monitoring these species remains elusive.
- Full Text:
- Date Issued: 2016
- Authors: Goddard, Mathew Keith
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2981 , vital:20349
- Description: Fruit-piercing moths are a sporadic pest of citrus, especially in the Eastern Cape Province of South Africa, where the adults can cause significant damage in outbreak years. Currently the only way in which to successfully control fruit-feeding moths within the orchards is the use of repellent lights. However, growers confuse fruit-piercing moths with fruit-sucking moths that don‘t cause primary damage, and there is no way of monitoring which moth species are attacking the fruit in the orchards during the night. In a previous study, banana was shown to be the most attractive bait for a variety of fruit-feeding moth species. Therefore the aim of this study was to determine the population dynamics of fruit-feeding moths develop a cost-effective alternative to the use of fresh banana as a bait for fruit-piercing moths. Fresh banana was compared to nine alternative synthetic attractants, frozen banana and a control under field conditions in several orchards in the Eastern Cape Province. Once again, banana was shown to be the most attractive bait. Some 23 species of fruit-feeding moth species were sampled in the traps, but there was only two fruit-piercing species, Serrodes partita (Fabricius) (Lepidoptera: Noctuidae) and Eudocima sp. Surprisingly S. partita, which was thought to be the main pest, comprised only 6.9% of trap catches. Serrodes partita, is a sporadic pest, only becoming problematic every five to 10 years after good rainfall in the Little Karoo region that causes flushes of their larval host, wild plum, Pappea capensis (Ecklon and Zeyher). During these outbreaks, damage to fruit can range from 70 to 90% and this is especially so for soft skinned citrus. A study on the morphology of the proboscis confirmed that only two species of fruit-piercing moths were present. Trap catches over three citrus growing seasons was linked to fruit damage found within several orchards. Once again fruit-piercing moth damage was relatively low in comparison to other types of damage such as mechanical and undefined damage. There was a very weak correlation between S. partita trap catches and damage, but generally damage was recorded two to three weeks after a peak in S. partita trap catches. Climatic conditions were also recorded and compared to weekly trap catches of S. partita, and while temperature and wind direction had no influence on moth populations, precipitation in the orchards was weakly correlated with trap catches. This study has shown that in non-outbreak seasons, the main fruit-piercing moth, S. partita comprises a small percentage of fruit-feeding moths in citrus orchards, but that growers are unable to determine the difference between fruit-piercing species and the harmless fruit-sucking species. Further fresh banana remains the best method for attracting fruit-piecing moths to traps, but this is not cost effective and thus a commercially viable protocol for monitoring these species remains elusive.
- Full Text:
- Date Issued: 2016
Field evaluation of the use of select entomopathogenic fungal isolates as microbial control agents of the soil-dwelling life stages of a key South African citrus pest, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae)
- Authors: Coombes, Candice Anne
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/507 , vital:19965
- Description: The control of false codling moth (FCM), Thaumatotibia leucotreta (Meyrick, 1912) (Lepidoptera: Tortricidae), in citrus orchards is strongly reliant on the use of integrated pest management as key export markets impose stringent chemical restrictions on exported fruit and have a strict no entry policy towards this phytosanitary pest. Most current, registered control methods target the above-ground life stages of FCM, not the soil-dwelling life stages. As such, entomopathogenic fungi which are ubiquitous, percutaneously infective soil-borne microbes that have been used successfully as control agents worldwide, present ideal candidates as additional control agents. Following an initial identification of 62 fungal entomopathogens isolated from soil collected from citrus orchards in the Eastern Cape Province, South Africa, further laboratory research has highlighted three isolates as having the greatest control potential against FCM subterranean life stages: Metarhizium anisopliae G 11 3 L6 (Ma1), M. anisopliae FCM Ar 23 B3 (Ma2) and Beauveria bassiana G Ar 17 B3 (Bb1). These isolates are capable of causing above 80% laboratory-induced mycosis of FCM fifth instars. Whether this level of efficacy was obtainable under sub-optimal and fluctuating field conditions was unknown. Thus, this thesis aimed to address the following issues with regards to the three most laboratory-virulent fungal isolates: field efficacy, field persistence, optimal application rate, application timing, environmental dependency, compatibility with fungicides and the use of different wetting agents to promote field efficacy. Following fungal application to one hectare treatment blocks in the field, FCM infestation within fruit was reduced by 28.3% to 81.7%. Isolate Bb1 performed best under moderate to high soil moisture whilst Ma2 was more effective under low soil moisture conditions. All isolates, with the exception of Ma2 at one site, were recorded in the soil five months post-application. None of the wetting agents tested were found to be highly toxic to fungal germination and similar physical suspension characteristics were observed. Fungicide toxicity varied amongst isolates and test conditions. However, only Dithane (a.i. mancozeb) was considered incompatible with isolate Ma2. The implication of these results and the way forward is discussed. This study is the first report of the field efficacy of three laboratory-virulent fungal isolates applied to the soil of conventional citrus orchards against FCM soil-dwelling life stages. As such, it provides a foundation on which future research can build to ensure the development and commercialisation of a cost-effective and consistently reliable product.
- Full Text:
- Date Issued: 2016
- Authors: Coombes, Candice Anne
- Date: 2016
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/507 , vital:19965
- Description: The control of false codling moth (FCM), Thaumatotibia leucotreta (Meyrick, 1912) (Lepidoptera: Tortricidae), in citrus orchards is strongly reliant on the use of integrated pest management as key export markets impose stringent chemical restrictions on exported fruit and have a strict no entry policy towards this phytosanitary pest. Most current, registered control methods target the above-ground life stages of FCM, not the soil-dwelling life stages. As such, entomopathogenic fungi which are ubiquitous, percutaneously infective soil-borne microbes that have been used successfully as control agents worldwide, present ideal candidates as additional control agents. Following an initial identification of 62 fungal entomopathogens isolated from soil collected from citrus orchards in the Eastern Cape Province, South Africa, further laboratory research has highlighted three isolates as having the greatest control potential against FCM subterranean life stages: Metarhizium anisopliae G 11 3 L6 (Ma1), M. anisopliae FCM Ar 23 B3 (Ma2) and Beauveria bassiana G Ar 17 B3 (Bb1). These isolates are capable of causing above 80% laboratory-induced mycosis of FCM fifth instars. Whether this level of efficacy was obtainable under sub-optimal and fluctuating field conditions was unknown. Thus, this thesis aimed to address the following issues with regards to the three most laboratory-virulent fungal isolates: field efficacy, field persistence, optimal application rate, application timing, environmental dependency, compatibility with fungicides and the use of different wetting agents to promote field efficacy. Following fungal application to one hectare treatment blocks in the field, FCM infestation within fruit was reduced by 28.3% to 81.7%. Isolate Bb1 performed best under moderate to high soil moisture whilst Ma2 was more effective under low soil moisture conditions. All isolates, with the exception of Ma2 at one site, were recorded in the soil five months post-application. None of the wetting agents tested were found to be highly toxic to fungal germination and similar physical suspension characteristics were observed. Fungicide toxicity varied amongst isolates and test conditions. However, only Dithane (a.i. mancozeb) was considered incompatible with isolate Ma2. The implication of these results and the way forward is discussed. This study is the first report of the field efficacy of three laboratory-virulent fungal isolates applied to the soil of conventional citrus orchards against FCM soil-dwelling life stages. As such, it provides a foundation on which future research can build to ensure the development and commercialisation of a cost-effective and consistently reliable product.
- Full Text:
- Date Issued: 2016
Genetic and biological characterisation of a novel South African Cydia pomonella granulovirus (CpGV-SA) isolate
- Motsoeneng, Boitumelo Madika
- Authors: Motsoeneng, Boitumelo Madika
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:20503 , http://hdl.handle.net/10962/d1021266
- Description: The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the primary pest of pome fruit cultivated worldwide. The control of this insect pest has been dependent on the frequent use of broad-spectrum chemical pesticides, which has led to the development of resistance in pest populations and negative effects on human health and the environment. The Betabaculovirus of C. pomonella has successfully been applied as a biological control agent in integrated pest management (IPM) programmes for the suppression of pest populations worldwide. Previously, all Cydia pomonella granulovirus (CpGV) biopesticides were based on a Mexican isolate (CpGV-M) and although these products are highly efficient at controlling C. pomonella, resistance cases have been reported across Europe. The identification of novel CpGV isolates as additional or alternative control agents to manage resistance is therefore necessary. This study aimed to genetically and biologically characterise a novel South African C. pomonella granulovirus isolate and to test its virulence against neonate larvae. Based on the morphology of the occlusion bodies observed using transmission electron microscopy, granuloviruses were recovered from diseased and dead larvae collected from an orchard in South Africa where no virus applications had been made. DNA was extracted and the identification of the isolated granulovirus was achieved through the PCR amplification and sequencing of the lef-8, lef-9, granulin and egt genes. Submission of the gene sequences to BLAST revealed high percentage identities to sequences from various CpGV isolates, resulting in the naming of the isolate in this study as the South African Cydia pomonella granulovirus (CpGV-SA) isolate. Phylogenetic analysis based on the single nucleotide polymorphisms (SNPs) detected in the lef-8, lef-9 and granulin nucleotide sequences grouped the South African isolate with CpGV-E2 (genome type B) and CpGV-S (genome type E). The CpGV-SA isolate was further genetically characterised by restriction endonuclease analysis and complete sequencing of the genomic DNA. Differences were observed for the BamHI, EcoRI, PstI and XhoI profiles of CpGV-SA in comparison to the respective profiles generated for CpGV-M extracted from a biopesticide, Carpovirusine® (Arysta Lifescience, France). Several genetic variations between the complete genome sequence of CpGV-SA and the reference isolate, CpGV-M1, as well as a recent genome submission of CpGV-M, both representing genome type A were observed. The complete genome analysis confirmed that CpGV-SA is genetically different from the Mexican CpGV isolate, used in thedevelopment of most biopesticides. In silico restriction profiles of the genome sequence obtained for CpGV-SA and genome sequences of genetically different CpGV isolates originating from Mexico (M1 and M), England (E2), Canada (S) and Iran (I12 and I07), available on the NCBI’s GenBank database confirmed that CpGV-SA is of mixed genotypes. Furthermore, the South African isolate shared the single common difference found in the pe38 gene of resistance overcoming isolates, which was the absence of an internal 24 nucleotide repeat present in CpGV-M1. In addition to the common difference, SNPs detected in the pe38 gene grouped the isolate with the CpGV-S isolate, suggesting that the CpGV-SA isolate is predominantly of genome type E. To determine the biological activity of CpGV-SA against neonate C. pomonella larvae, surface bioassays were conducted alongside CpGV-M (Carpovirusine®) bioassays. The LC50 and LC90 values for the South African isolate were 1.6 × 103 and 1.2 × 105 OBs/ml respectively. The LT50 was determined to be 135 hours. These values were similar to the values obtained for CpGV-M (Carpovirusine®). The results in this study suggest that a novel South African CpGV isolate of mixed genotypes, potentially able to overcome resistance in C. pomonella, with biological activity similar to CpGV-M (Carpovirusine®) and important for the control of C. pomonella was recovered. The CpGV-SA isolate could therefore potentially be developed into a biopesticide for use in resistance management strategies against C. pomonella populations in South Africa.
- Full Text:
- Date Issued: 2016
- Authors: Motsoeneng, Boitumelo Madika
- Date: 2016
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:20503 , http://hdl.handle.net/10962/d1021266
- Description: The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the primary pest of pome fruit cultivated worldwide. The control of this insect pest has been dependent on the frequent use of broad-spectrum chemical pesticides, which has led to the development of resistance in pest populations and negative effects on human health and the environment. The Betabaculovirus of C. pomonella has successfully been applied as a biological control agent in integrated pest management (IPM) programmes for the suppression of pest populations worldwide. Previously, all Cydia pomonella granulovirus (CpGV) biopesticides were based on a Mexican isolate (CpGV-M) and although these products are highly efficient at controlling C. pomonella, resistance cases have been reported across Europe. The identification of novel CpGV isolates as additional or alternative control agents to manage resistance is therefore necessary. This study aimed to genetically and biologically characterise a novel South African C. pomonella granulovirus isolate and to test its virulence against neonate larvae. Based on the morphology of the occlusion bodies observed using transmission electron microscopy, granuloviruses were recovered from diseased and dead larvae collected from an orchard in South Africa where no virus applications had been made. DNA was extracted and the identification of the isolated granulovirus was achieved through the PCR amplification and sequencing of the lef-8, lef-9, granulin and egt genes. Submission of the gene sequences to BLAST revealed high percentage identities to sequences from various CpGV isolates, resulting in the naming of the isolate in this study as the South African Cydia pomonella granulovirus (CpGV-SA) isolate. Phylogenetic analysis based on the single nucleotide polymorphisms (SNPs) detected in the lef-8, lef-9 and granulin nucleotide sequences grouped the South African isolate with CpGV-E2 (genome type B) and CpGV-S (genome type E). The CpGV-SA isolate was further genetically characterised by restriction endonuclease analysis and complete sequencing of the genomic DNA. Differences were observed for the BamHI, EcoRI, PstI and XhoI profiles of CpGV-SA in comparison to the respective profiles generated for CpGV-M extracted from a biopesticide, Carpovirusine® (Arysta Lifescience, France). Several genetic variations between the complete genome sequence of CpGV-SA and the reference isolate, CpGV-M1, as well as a recent genome submission of CpGV-M, both representing genome type A were observed. The complete genome analysis confirmed that CpGV-SA is genetically different from the Mexican CpGV isolate, used in thedevelopment of most biopesticides. In silico restriction profiles of the genome sequence obtained for CpGV-SA and genome sequences of genetically different CpGV isolates originating from Mexico (M1 and M), England (E2), Canada (S) and Iran (I12 and I07), available on the NCBI’s GenBank database confirmed that CpGV-SA is of mixed genotypes. Furthermore, the South African isolate shared the single common difference found in the pe38 gene of resistance overcoming isolates, which was the absence of an internal 24 nucleotide repeat present in CpGV-M1. In addition to the common difference, SNPs detected in the pe38 gene grouped the isolate with the CpGV-S isolate, suggesting that the CpGV-SA isolate is predominantly of genome type E. To determine the biological activity of CpGV-SA against neonate C. pomonella larvae, surface bioassays were conducted alongside CpGV-M (Carpovirusine®) bioassays. The LC50 and LC90 values for the South African isolate were 1.6 × 103 and 1.2 × 105 OBs/ml respectively. The LT50 was determined to be 135 hours. These values were similar to the values obtained for CpGV-M (Carpovirusine®). The results in this study suggest that a novel South African CpGV isolate of mixed genotypes, potentially able to overcome resistance in C. pomonella, with biological activity similar to CpGV-M (Carpovirusine®) and important for the control of C. pomonella was recovered. The CpGV-SA isolate could therefore potentially be developed into a biopesticide for use in resistance management strategies against C. pomonella populations in South Africa.
- Full Text:
- Date Issued: 2016
Production of Cydia pomonella granulovirus (CpGV) in a heteralogous host, Thaumatotibia Leucotreta (Meyrick) (False codling moth)
- Authors: Chambers, Craig Brian
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- South Africa , Codling moth -- South Africa , Apples -- Diseases and pests -- South Africa , Codling moth -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Biological pest control agents -- South Africa , Baculoviruses -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5935 , http://hdl.handle.net/10962/d1017906
- Description: Cydia pomonella (Linnaeus) (Family: Tortricidae), the codling moth, is considered one of the most significant pests of apples and pears worldwide, causing up to 80% crop loss in orchards if no control measures are applied. Cydia pomonella is oligophagous feeding on a number of alternate hosts including quince, walnuts, apricots, peaches, plums and nectarines. Historically the control of this pest has been achieved with the use of various chemical control strategies which have maintained pest levels below the economic threshold at a relatively low cost to the grower. However, there are serious concerns surrounding the use of chemical insecticides including the development of resistance in insect populations, the banning of various insecticides, regulations for lowering of the maximum residue level and employee and consumer safety. For this reason, alternate measures of control are slowly being adopted by growers such as mating disruption, cultural methods and the use of baculovirus biopesticides as part of integrated pest management programmes. The reluctance of growers to accept baculovirus or other biological control products in the past has been due to questionable product quality and inconsistencies in their field performance. Moreover, the development and application of biological control products is more costly than the use of chemical alternatives. Baculoviruses are arthropod specific viruses that are highly virulent to a number of lepidopteran species. Due to the virulence and host specificity of baculoviruses, Cydia pomonella granulovirus has been extensively and successfully used as part of integrated pest management systems for the control of C. pomonella in Europe and around the world, including South Africa. Commercial formulations have been typically based on the Mexican strain of CpGV. However due to long-term multiple applications of CpGV and the reliance on CpGV in organic farming practices in Europe, resistance to the CpGV-M strain has developed in a number of field populations of C. pomonella. This study aimed to identify and characterize novel isolates of CpGV in South Africa and compare their virulence with the commercial standard CpGV-M. Secondly, since C. pomonella is difficult to culture on a large scale, an alternate method of CpGV production was investigated in order to determine if CpGV could be produced more efficiently and at a reduced cost without negatively impacting the quality of the product. Several isolates of CpGV were recovered either from field collected larvae or from a laboratory-reared C. pomonella colony. Characterisation of DNA profiles using a variety of restriction enzymes revealed that only a single isolate, CpGV-SA, was genetically different from the Mexican strain of the virus used in the commercially available CpGV based products in South Africa. In dose-response bioassays using CpGV-SA, LC₅₀ and LC₉₀ values for neonate C. pomonella larvae were 3.18 x 10³ OBs/ml and 7.33 x 10⁴ respectively. A comparison of these values with those of CpGV-M indicated no significant difference in the virulence of the two isolates under laboratory conditions. This is a first report of a genetically distinct CpGV isolate in South Africa. The biological activity and novelty of CpGV-SA makes this isolate a potentially important tool for CpGV resistance management in South Africa. In order to justify production of CpGV in an alternative host, studies on the comparative biological performance of C. pomonella and T. leucotreta based on oviposition, time to hatch, larval developmental times and rearing efficiency as well as production costs were performed. Thaumatotibia leucotreta was found to be more fecund and to have significantly shorter egg and larval developmental times. In addition, larval production per unit of artificial diet was significantly higher than for C. pomonella. This resulted in T. leucotreta being more cost effective to produce with implications for reduced insectary space, sanitation practices as well as the labour component of production. Virus yield data generated by inoculation both C. pomonella and T. leucotreta with nine concentrations of CpGV resulted in comparable virus yields, justifying the continuation of the research into production of CpGV in T. leucotreta. It was important to determine the LC and LT values required for mass production of CpGV in late instar T. leucotreta larvae. Dose- and time-response bioassays with CpGV-M were conducted on artificial diet to determine these values. Fourth instar LC₅₀ and LC₉₀ values were 5.96 x 10³ OBs/ml and 1.64 x 10⁵ OBs/ml respectively. LT50 and LT90 values were 81.10 hours and 88.58 hours respectively. Fifth instar LC₅₀ and LC₉₀ values were 6.88 x 10⁴ OBs/ml and 9.78 x 10⁶ OBs/ml respectively. LT₅₀ and LT₉₀ values were 111.56 hours and 137.57 hours respectively. Virus produced in fourth instar T. leucotreta larvae was bioassayed against C. pomonella neonate larvae and compared to CpGV-M to establish if production in the heterologous host negatively affected the virulence of the isolate. No significant difference in virulence was observed between virus produced in T. leucotreta and that produced in C. pomonella. The data generated in the bioassays was used in CpGV mass production trials to evaluate production. All production methods tested produced acceptable virus yields. To examine the quality of the virus product, genomic DNA was extracted from larval cadavers and subjected to REN analysis with HindIII. The resulting DNA profiles indicated that the virus product was contaminated with the homologous virus, CrleGV. Based on the above results, the use of T. leucotreta as an alternate host for the in vivo production of CpGV on a commercial basis is not at this stage viable and requires further investigation before this production methodology can be reliable used to produce CpGV. However, this study has shown that CpGV can be produced in a homologous host, T. leucotreta and significant strides have been made towards developing a set of quality control standards that are essential for further development of successful production methodology. Finally a novel isolate of CpGV has been identified with comparable virulence to the CpGV-M. This is an important finding as it has broad reaching implications for resistance management of CpGV products in South Africa.
- Full Text:
- Date Issued: 2015
- Authors: Chambers, Craig Brian
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- South Africa , Codling moth -- South Africa , Apples -- Diseases and pests -- South Africa , Codling moth -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Biological pest control agents -- South Africa , Baculoviruses -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:5935 , http://hdl.handle.net/10962/d1017906
- Description: Cydia pomonella (Linnaeus) (Family: Tortricidae), the codling moth, is considered one of the most significant pests of apples and pears worldwide, causing up to 80% crop loss in orchards if no control measures are applied. Cydia pomonella is oligophagous feeding on a number of alternate hosts including quince, walnuts, apricots, peaches, plums and nectarines. Historically the control of this pest has been achieved with the use of various chemical control strategies which have maintained pest levels below the economic threshold at a relatively low cost to the grower. However, there are serious concerns surrounding the use of chemical insecticides including the development of resistance in insect populations, the banning of various insecticides, regulations for lowering of the maximum residue level and employee and consumer safety. For this reason, alternate measures of control are slowly being adopted by growers such as mating disruption, cultural methods and the use of baculovirus biopesticides as part of integrated pest management programmes. The reluctance of growers to accept baculovirus or other biological control products in the past has been due to questionable product quality and inconsistencies in their field performance. Moreover, the development and application of biological control products is more costly than the use of chemical alternatives. Baculoviruses are arthropod specific viruses that are highly virulent to a number of lepidopteran species. Due to the virulence and host specificity of baculoviruses, Cydia pomonella granulovirus has been extensively and successfully used as part of integrated pest management systems for the control of C. pomonella in Europe and around the world, including South Africa. Commercial formulations have been typically based on the Mexican strain of CpGV. However due to long-term multiple applications of CpGV and the reliance on CpGV in organic farming practices in Europe, resistance to the CpGV-M strain has developed in a number of field populations of C. pomonella. This study aimed to identify and characterize novel isolates of CpGV in South Africa and compare their virulence with the commercial standard CpGV-M. Secondly, since C. pomonella is difficult to culture on a large scale, an alternate method of CpGV production was investigated in order to determine if CpGV could be produced more efficiently and at a reduced cost without negatively impacting the quality of the product. Several isolates of CpGV were recovered either from field collected larvae or from a laboratory-reared C. pomonella colony. Characterisation of DNA profiles using a variety of restriction enzymes revealed that only a single isolate, CpGV-SA, was genetically different from the Mexican strain of the virus used in the commercially available CpGV based products in South Africa. In dose-response bioassays using CpGV-SA, LC₅₀ and LC₉₀ values for neonate C. pomonella larvae were 3.18 x 10³ OBs/ml and 7.33 x 10⁴ respectively. A comparison of these values with those of CpGV-M indicated no significant difference in the virulence of the two isolates under laboratory conditions. This is a first report of a genetically distinct CpGV isolate in South Africa. The biological activity and novelty of CpGV-SA makes this isolate a potentially important tool for CpGV resistance management in South Africa. In order to justify production of CpGV in an alternative host, studies on the comparative biological performance of C. pomonella and T. leucotreta based on oviposition, time to hatch, larval developmental times and rearing efficiency as well as production costs were performed. Thaumatotibia leucotreta was found to be more fecund and to have significantly shorter egg and larval developmental times. In addition, larval production per unit of artificial diet was significantly higher than for C. pomonella. This resulted in T. leucotreta being more cost effective to produce with implications for reduced insectary space, sanitation practices as well as the labour component of production. Virus yield data generated by inoculation both C. pomonella and T. leucotreta with nine concentrations of CpGV resulted in comparable virus yields, justifying the continuation of the research into production of CpGV in T. leucotreta. It was important to determine the LC and LT values required for mass production of CpGV in late instar T. leucotreta larvae. Dose- and time-response bioassays with CpGV-M were conducted on artificial diet to determine these values. Fourth instar LC₅₀ and LC₉₀ values were 5.96 x 10³ OBs/ml and 1.64 x 10⁵ OBs/ml respectively. LT50 and LT90 values were 81.10 hours and 88.58 hours respectively. Fifth instar LC₅₀ and LC₉₀ values were 6.88 x 10⁴ OBs/ml and 9.78 x 10⁶ OBs/ml respectively. LT₅₀ and LT₉₀ values were 111.56 hours and 137.57 hours respectively. Virus produced in fourth instar T. leucotreta larvae was bioassayed against C. pomonella neonate larvae and compared to CpGV-M to establish if production in the heterologous host negatively affected the virulence of the isolate. No significant difference in virulence was observed between virus produced in T. leucotreta and that produced in C. pomonella. The data generated in the bioassays was used in CpGV mass production trials to evaluate production. All production methods tested produced acceptable virus yields. To examine the quality of the virus product, genomic DNA was extracted from larval cadavers and subjected to REN analysis with HindIII. The resulting DNA profiles indicated that the virus product was contaminated with the homologous virus, CrleGV. Based on the above results, the use of T. leucotreta as an alternate host for the in vivo production of CpGV on a commercial basis is not at this stage viable and requires further investigation before this production methodology can be reliable used to produce CpGV. However, this study has shown that CpGV can be produced in a homologous host, T. leucotreta and significant strides have been made towards developing a set of quality control standards that are essential for further development of successful production methodology. Finally a novel isolate of CpGV has been identified with comparable virulence to the CpGV-M. This is an important finding as it has broad reaching implications for resistance management of CpGV products in South Africa.
- Full Text:
- Date Issued: 2015
The biology, behaviour and survival of pupating false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a citrus pest in South Africa
- Authors: Love, Claire Natalie
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- South Africa , Cryptophlebia leucotreta -- Larvae -- Behavior , Citrus -- Diseases and pests , Citrus -- Diseases and pests -- Biological control , Biological pest control agents , Entomopathogenic fungi , Insect nematodes , Pupae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5941 , http://hdl.handle.net/10962/d1018907
- Description: Control of the citrus pest, false codling moth (FCM), Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is crucial for the South African citrus industry. The economic losses and phytosanitary status of this pest, coupled with increased consumer awareness and demands, has created a need for effective, IPM-compatible control measures for use against the soil-dwelling life stages of FCM. Promising developments in the field of microbial control through the use of entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPNs) have highlighted the need for research regarding pupation biology, behaviour and survival of FCM, as a good understanding of biology of the target organism is an important component of any biological control programme. The aim of this study was to improve the current understanding of FCM pupation habits through the manipulation of soil texture class, ground cover, shading, soil compaction, air temperature, and soil moisture in the laboratory. These findings would then be used to aid the biological control programmes using EPF and EPNs against FCM in the soil. Three soil texture classes (sandy loam, silt loam and silty clay loam) were obtained from orchards for use in the study. FCM larvae were allowed to drop into the soil of their own accord and the pupation behaviour that followed was then captured on film with pupae formed in the soil being kept in order to measure adult eclosion. In general, very few abiotic factors had a clear influence on FCM pupation. Larval wandering time and distance was short, but also variable between individuals. Distance did increase when soils were moist. Pupation depth was shallow, with pupal cocoons generally being formed on the soil surface. Depth of pupation was less than one centimetre for all abiotic conditions, with little burrowing into soil. Eclosion success was higher for sandier soils when these were dry and uncompacted, but the addition of both moisture and soil compaction increased FCM eclosion success. FCM was sensitive to desiccation when the soils were dry and temperature limits of 15 °C and 32 °C had a strongly negative impact on eclosion success. Preferences for particular abiotic conditions were limited to only certain moisture conditions when interacting with soil texture class and a preference for pupating in soil when it is available. Limited preference was found for particular soil textures despite this having a strong influence on eclosion success, but individuals did appear to pupate in close proximity to one another. Viable direct habitat manipulation for FCM control could not be identified. These results and all of the abiotic variables measured have important implications for EPF and EPN application, survival and persistence in the soil in order to improve the ability of these biological control agents to control FCM. These are discussed in each chapter.
- Full Text:
- Date Issued: 2015
- Authors: Love, Claire Natalie
- Date: 2015
- Subjects: Cryptophlebia leucotreta -- South Africa , Cryptophlebia leucotreta -- Larvae -- Behavior , Citrus -- Diseases and pests , Citrus -- Diseases and pests -- Biological control , Biological pest control agents , Entomopathogenic fungi , Insect nematodes , Pupae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5941 , http://hdl.handle.net/10962/d1018907
- Description: Control of the citrus pest, false codling moth (FCM), Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is crucial for the South African citrus industry. The economic losses and phytosanitary status of this pest, coupled with increased consumer awareness and demands, has created a need for effective, IPM-compatible control measures for use against the soil-dwelling life stages of FCM. Promising developments in the field of microbial control through the use of entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPNs) have highlighted the need for research regarding pupation biology, behaviour and survival of FCM, as a good understanding of biology of the target organism is an important component of any biological control programme. The aim of this study was to improve the current understanding of FCM pupation habits through the manipulation of soil texture class, ground cover, shading, soil compaction, air temperature, and soil moisture in the laboratory. These findings would then be used to aid the biological control programmes using EPF and EPNs against FCM in the soil. Three soil texture classes (sandy loam, silt loam and silty clay loam) were obtained from orchards for use in the study. FCM larvae were allowed to drop into the soil of their own accord and the pupation behaviour that followed was then captured on film with pupae formed in the soil being kept in order to measure adult eclosion. In general, very few abiotic factors had a clear influence on FCM pupation. Larval wandering time and distance was short, but also variable between individuals. Distance did increase when soils were moist. Pupation depth was shallow, with pupal cocoons generally being formed on the soil surface. Depth of pupation was less than one centimetre for all abiotic conditions, with little burrowing into soil. Eclosion success was higher for sandier soils when these were dry and uncompacted, but the addition of both moisture and soil compaction increased FCM eclosion success. FCM was sensitive to desiccation when the soils were dry and temperature limits of 15 °C and 32 °C had a strongly negative impact on eclosion success. Preferences for particular abiotic conditions were limited to only certain moisture conditions when interacting with soil texture class and a preference for pupating in soil when it is available. Limited preference was found for particular soil textures despite this having a strong influence on eclosion success, but individuals did appear to pupate in close proximity to one another. Viable direct habitat manipulation for FCM control could not be identified. These results and all of the abiotic variables measured have important implications for EPF and EPN application, survival and persistence in the soil in order to improve the ability of these biological control agents to control FCM. These are discussed in each chapter.
- Full Text:
- Date Issued: 2015
The isolation, genetic characterisation and biological activity of a South African Phthorimaea operculella granulovirus (PhopGV-SA) for the control of the Potato Tuber Moth, Phthorimaea operculella (Zeller)
- Authors: Jukes, Michael David
- Date: 2015
- Subjects: Potato tuberworm , Potatoes -- Diseases and pests -- South Africa , Baculoviruses , Natural pesticides , Biological pest control agents , Potato tuberworm -- Biological control , Restriction enzymes, DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4147 , http://hdl.handle.net/10962/d1017908
- Description: The potato tuber moth, Phthorimaea operculella (Zeller), is a major pest of potato crops worldwide causing significant damage to both field and stored tubers. The current control method in South Africa involves chemical insecticides, however, there is growing concern on the health and environmental risks of their use. The development of novel biopesticide based control methods may offer a potential solution for the future of insecticides. In this study a baculovirus was successfully isolated from a laboratory population of P. operculella. Transmission electron micrographs revealed granulovirus-like particles. DNA was extracted from recovered occlusion bodies and used for the PCR amplification of the lef-8, lef-9, granulin and egt genes. Sequence data was obtained and submitted to BLAST identifying the virus as a South African isolate of Phthorimaea operculella granulovirus (PhopGV-SA). Phylogenetic analysis of the lef-8, lef-9 and granulin amino acid sequences grouped the South African isolate with PhopGV-1346. Comparison of egt sequence data identified PhopGV-SA as a type II egt gene. A phylogenetic analysis of egt amino acid sequences grouped all type II genes, including PhopGV-SA, into a separate clade from types I, III, IV and V. These findings suggest that type II may represent the prototype structure for this gene with the evolution of types I, III and IV a result of large internal deletion events and subsequent divergence. PhopGV-SA was also shown to be genetically more similar to South American isolates (i.e. PhopGV-CHI or PhopGV-INDO) than it is to other African isolates, suggesting that the South African isolate originated from South America. Restriction endonuclease profiles of PhopGV-SA were similar to those of PhopGV-1346 and PhopGV-JLZ9f for the enzymes BamHI, HindIII, NruI and NdeI. A preliminary full genome sequence for PhopGV-SA was determined and compared to PhopGV-136 with some gene variation observed (i.e. odv-e66 and vp91/p95). The biological activity of PhopGV-SA against P. operculella neonate larvae was evaluated with an estimated LC₅₀ of 1.87×10⁸ OBs.ml⁻¹ being determined. This study therefore reports the characterisation of a novel South African PhopGV isolate which could potentially be developed into a biopesticide for the control of P. operculella.
- Full Text:
- Date Issued: 2015
- Authors: Jukes, Michael David
- Date: 2015
- Subjects: Potato tuberworm , Potatoes -- Diseases and pests -- South Africa , Baculoviruses , Natural pesticides , Biological pest control agents , Potato tuberworm -- Biological control , Restriction enzymes, DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4147 , http://hdl.handle.net/10962/d1017908
- Description: The potato tuber moth, Phthorimaea operculella (Zeller), is a major pest of potato crops worldwide causing significant damage to both field and stored tubers. The current control method in South Africa involves chemical insecticides, however, there is growing concern on the health and environmental risks of their use. The development of novel biopesticide based control methods may offer a potential solution for the future of insecticides. In this study a baculovirus was successfully isolated from a laboratory population of P. operculella. Transmission electron micrographs revealed granulovirus-like particles. DNA was extracted from recovered occlusion bodies and used for the PCR amplification of the lef-8, lef-9, granulin and egt genes. Sequence data was obtained and submitted to BLAST identifying the virus as a South African isolate of Phthorimaea operculella granulovirus (PhopGV-SA). Phylogenetic analysis of the lef-8, lef-9 and granulin amino acid sequences grouped the South African isolate with PhopGV-1346. Comparison of egt sequence data identified PhopGV-SA as a type II egt gene. A phylogenetic analysis of egt amino acid sequences grouped all type II genes, including PhopGV-SA, into a separate clade from types I, III, IV and V. These findings suggest that type II may represent the prototype structure for this gene with the evolution of types I, III and IV a result of large internal deletion events and subsequent divergence. PhopGV-SA was also shown to be genetically more similar to South American isolates (i.e. PhopGV-CHI or PhopGV-INDO) than it is to other African isolates, suggesting that the South African isolate originated from South America. Restriction endonuclease profiles of PhopGV-SA were similar to those of PhopGV-1346 and PhopGV-JLZ9f for the enzymes BamHI, HindIII, NruI and NdeI. A preliminary full genome sequence for PhopGV-SA was determined and compared to PhopGV-136 with some gene variation observed (i.e. odv-e66 and vp91/p95). The biological activity of PhopGV-SA against P. operculella neonate larvae was evaluated with an estimated LC₅₀ of 1.87×10⁸ OBs.ml⁻¹ being determined. This study therefore reports the characterisation of a novel South African PhopGV isolate which could potentially be developed into a biopesticide for the control of P. operculella.
- Full Text:
- Date Issued: 2015
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
Genetic and biological characterisation of a novel South African Plutella xylostella granulovirus (PlxyGV) isolate
- Authors: Abdulkadir, Fatima
- Date: 2014
- Subjects: Diamondback moth , Diamondback moth -- Control -- South Africa , Plutellidae -- Control -- South Africa , Baculoviruses , Cruciferae -- Diseases and pests -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4113 , http://hdl.handle.net/10962/d1013059
- Description: The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is an important pest of cruciferous crops worldwide. The prolonged use of synthetic chemical insecticides as a primary means of control has resulted in the development of resistance in pest populations. In addition, the pest has also evolved resistance to the bacterial insecticidal protein of Bacillus thuringiensis which is also widely used as a method of control. Baculoviruses are considered as effective alternatives to conventional methods of control when incorporated into integrated pest management (IPM) programmes. These viruses target the larval stages of insects, are generally host-specific and are safe for use in the environment. This study aimed to isolate a baculovirus from a laboratory-reared P. xylostella colony, characterise it genetically and then evaluate its virulence against neonate and fourth instar larvae. A laboratory colony of P. xylostella was established using pupae and asymptomatic larvae collected from a cabbage plantation outside Grahamstown in the Eastern Cape province of South Africa. The colony flourished in the laboratory due to prime conditions and availability of food. The duration of development from egg to adult was determined by observation and imaging of the various life stages. The mean developmental time from egg to adult was observed to be 14.59 ± 0.21 days. The population of the insects increased rapidly in number leading to overcrowding of the insect colony, and hence appearance of larvae with viral symptoms. Occlusion bodies (OBs) were extracted from symptomatic larval cadavers and purified by glycerol gradient centrifugation. Analysis of the purified OBs by transmission electron microscopy revealed the presence of a granulovirus which was named PlxyGV-SA. The virus isolate was genetically characterised by restriction endonuclease analysis of the genomic DNA, and PCR amplification and sequencing of selected viral genes. The complete genome sequence of a Japanese P. xylostella granulovirus isolate, PlxyGV-Japan, has been deposited on the GenBank database providing a reference strain for comparison with DNA profiles and selected gene sequences of PlxyGV-SA. BLAST analysis of the granulin gene confirmed the isolation of a novel South African PlxyGV isolate. Comparison of the restriction profiles of PlxyGV-SA with profiles of PlxyGV-Japan and other documented PlxyGV profiles obtained by agarose gel electrophoresis revealed that PlxyGV-SA is a genetically distinct isolate. The data obtained from the sequencing and alignment of ecdysteroid UDP-glucosyltransferase (egt), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes with those of PlxyGV-Japan also showed that PlxyGV-SA is a genetically different isolate. In order to determine the biological activity of PlxyGV-SA against neonate and fourth instar P. xylostella larvae, surface dose bioassays were conducted. The median lethal concentration of the virus required to kill 50% (LC₅₀) and 90% (LC₉₀) of the larvae was estimated by feeding insects with a range of doses. In addition, the time to kill 50% of the larvae (LT₅₀) was determined by feeding insects with the LC₉₀ concentration. Larval mortality was monitored daily until pupation. The data obtained from the dose response assays were subjected to probit analysis using Proban statistical software. The time response was determined using GraphPad Prism software (version 6.0). The LC₅₀ and LC₉₀ values for the neonate larvae were 3.56 × 10⁵ and 1.14 × 10⁷ OBs/ml respectively. The LT₅₀ was determined to be 104 hours. The neonate larvae were found to be more susceptible to infection than the fourth instar larvae with the same virus concentration. The concentrations used for the neonate larvae assay did not have a significant effect on the fourth instar as no mortality was recorded. This is the first study to describe a novel South African PlxyGV isolate and the results suggest that PlxyGV-SA has significant potential for development as an effective biopesticide for the control of P. xylostella in the field.
- Full Text:
- Date Issued: 2014
- Authors: Abdulkadir, Fatima
- Date: 2014
- Subjects: Diamondback moth , Diamondback moth -- Control -- South Africa , Plutellidae -- Control -- South Africa , Baculoviruses , Cruciferae -- Diseases and pests -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4113 , http://hdl.handle.net/10962/d1013059
- Description: The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is an important pest of cruciferous crops worldwide. The prolonged use of synthetic chemical insecticides as a primary means of control has resulted in the development of resistance in pest populations. In addition, the pest has also evolved resistance to the bacterial insecticidal protein of Bacillus thuringiensis which is also widely used as a method of control. Baculoviruses are considered as effective alternatives to conventional methods of control when incorporated into integrated pest management (IPM) programmes. These viruses target the larval stages of insects, are generally host-specific and are safe for use in the environment. This study aimed to isolate a baculovirus from a laboratory-reared P. xylostella colony, characterise it genetically and then evaluate its virulence against neonate and fourth instar larvae. A laboratory colony of P. xylostella was established using pupae and asymptomatic larvae collected from a cabbage plantation outside Grahamstown in the Eastern Cape province of South Africa. The colony flourished in the laboratory due to prime conditions and availability of food. The duration of development from egg to adult was determined by observation and imaging of the various life stages. The mean developmental time from egg to adult was observed to be 14.59 ± 0.21 days. The population of the insects increased rapidly in number leading to overcrowding of the insect colony, and hence appearance of larvae with viral symptoms. Occlusion bodies (OBs) were extracted from symptomatic larval cadavers and purified by glycerol gradient centrifugation. Analysis of the purified OBs by transmission electron microscopy revealed the presence of a granulovirus which was named PlxyGV-SA. The virus isolate was genetically characterised by restriction endonuclease analysis of the genomic DNA, and PCR amplification and sequencing of selected viral genes. The complete genome sequence of a Japanese P. xylostella granulovirus isolate, PlxyGV-Japan, has been deposited on the GenBank database providing a reference strain for comparison with DNA profiles and selected gene sequences of PlxyGV-SA. BLAST analysis of the granulin gene confirmed the isolation of a novel South African PlxyGV isolate. Comparison of the restriction profiles of PlxyGV-SA with profiles of PlxyGV-Japan and other documented PlxyGV profiles obtained by agarose gel electrophoresis revealed that PlxyGV-SA is a genetically distinct isolate. The data obtained from the sequencing and alignment of ecdysteroid UDP-glucosyltransferase (egt), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes with those of PlxyGV-Japan also showed that PlxyGV-SA is a genetically different isolate. In order to determine the biological activity of PlxyGV-SA against neonate and fourth instar P. xylostella larvae, surface dose bioassays were conducted. The median lethal concentration of the virus required to kill 50% (LC₅₀) and 90% (LC₉₀) of the larvae was estimated by feeding insects with a range of doses. In addition, the time to kill 50% of the larvae (LT₅₀) was determined by feeding insects with the LC₉₀ concentration. Larval mortality was monitored daily until pupation. The data obtained from the dose response assays were subjected to probit analysis using Proban statistical software. The time response was determined using GraphPad Prism software (version 6.0). The LC₅₀ and LC₉₀ values for the neonate larvae were 3.56 × 10⁵ and 1.14 × 10⁷ OBs/ml respectively. The LT₅₀ was determined to be 104 hours. The neonate larvae were found to be more susceptible to infection than the fourth instar larvae with the same virus concentration. The concentrations used for the neonate larvae assay did not have a significant effect on the fourth instar as no mortality was recorded. This is the first study to describe a novel South African PlxyGV isolate and the results suggest that PlxyGV-SA has significant potential for development as an effective biopesticide for the control of P. xylostella in the field.
- Full Text:
- Date Issued: 2014
Screening of entomopathogenic fungi against citrus mealybug (Planococcus citri (Risso)) and citrus thrips (Scirtothrips aurantii (Faure))
- FitzGerald, Véronique Chartier
- Authors: FitzGerald, Véronique Chartier
- Date: 2014
- Subjects: Entomopathogenic fungi , Citrus mealybug -- South Africa -- Eastern Cape , Citrus thrips -- South Africa -- Eastern Cape , Citrus -- Diseases and pests , Citrus mealybug -- Biological control , Citrus thrips -- Biological control , Biological pest control agents , Scanning electron microscopy , Mycoses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4166 , http://hdl.handle.net/10962/d1020887
- Description: Mealybugs (Planococcus citri) and thrips (Scirtothrips aurantii) are common and extremely damaging citrus crop pests which have proven difficult to control via conventional methods, such as chemical pesticides and insect growth regulators. The objective of this study was to determine the efficacy of entomopathogenic fungi against these pests in laboratory bioassays. Isolates of Metarhizium anisopliae and Beauveria bassiana from citrus orchards in the Eastern Cape, South Africa were maintained on Sabouraud Dextrose 4% Agar supplemented with Dodine, chloramphenicol and rifampicin at 25°C. Infectivity of the fungal isolates was initially assessed using 5th instar false codling moth, Thaumatotibia leucotreta, larvae. Mealybug bioassays were performed in 24 well plates using 1 x 107 ml-1 conidial suspensions and kept at 26°C for 5 days with a photoperiod of 12 L:12 D. A Beauveria commercial product and an un-inoculated control were also screened for comparison. Isolates GAR 17 B3 (B. bassiana) and FCM AR 23 B3 (M. anisopliae) both resulted in 67.5% mealybug crawler mortality and GB AR 23 13 3 (B. bassiana) resulted in 64% crawler mortality. These 3 isolates were further tested in dose-dependent assays. Probit analyses were conducted on the dose-dependent assays data using PROBAN to determine LC₅₀ values. For both the mealybug adult and crawlers FCM AR 23 B3 required the lowest concentration to achieve LC₅₀ at 4.96 x 10⁶ conidia ml-1 and 5.29 x 10⁵ conidia ml-1, respectively. Bioassays on adult thrips were conducted in munger cells with leaf buds inoculated with the conidial suspensions. Isolate GAR 17 B3 had the highest mortality rate at 70% on thrips while FCM AR 23 B3 resulted in 60% mortality. Identification of the isolates, FCM AR 23 B3, GAR 17 B3 and GB AR 23 13 3, were confirmed to be correct using both microscopic and molecularly techniques. ITS sequences were compared to other sequences from GenBank and confirmed phylogenetically using MEGA6. Mealybug infection was investigated using scanning electron microscopy, mycosis was confirmed but the infection process could not be followed due to the extensive waxy cuticle. These results indicate that there is potential for the isolates FCM AR 23 B3 and GAR 17 B3 to be developed as biological control agents for the control of citrus mealybug and thrips. Further research would be required to determine their ability to perform under field conditions.
- Full Text:
- Date Issued: 2014
- Authors: FitzGerald, Véronique Chartier
- Date: 2014
- Subjects: Entomopathogenic fungi , Citrus mealybug -- South Africa -- Eastern Cape , Citrus thrips -- South Africa -- Eastern Cape , Citrus -- Diseases and pests , Citrus mealybug -- Biological control , Citrus thrips -- Biological control , Biological pest control agents , Scanning electron microscopy , Mycoses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4166 , http://hdl.handle.net/10962/d1020887
- Description: Mealybugs (Planococcus citri) and thrips (Scirtothrips aurantii) are common and extremely damaging citrus crop pests which have proven difficult to control via conventional methods, such as chemical pesticides and insect growth regulators. The objective of this study was to determine the efficacy of entomopathogenic fungi against these pests in laboratory bioassays. Isolates of Metarhizium anisopliae and Beauveria bassiana from citrus orchards in the Eastern Cape, South Africa were maintained on Sabouraud Dextrose 4% Agar supplemented with Dodine, chloramphenicol and rifampicin at 25°C. Infectivity of the fungal isolates was initially assessed using 5th instar false codling moth, Thaumatotibia leucotreta, larvae. Mealybug bioassays were performed in 24 well plates using 1 x 107 ml-1 conidial suspensions and kept at 26°C for 5 days with a photoperiod of 12 L:12 D. A Beauveria commercial product and an un-inoculated control were also screened for comparison. Isolates GAR 17 B3 (B. bassiana) and FCM AR 23 B3 (M. anisopliae) both resulted in 67.5% mealybug crawler mortality and GB AR 23 13 3 (B. bassiana) resulted in 64% crawler mortality. These 3 isolates were further tested in dose-dependent assays. Probit analyses were conducted on the dose-dependent assays data using PROBAN to determine LC₅₀ values. For both the mealybug adult and crawlers FCM AR 23 B3 required the lowest concentration to achieve LC₅₀ at 4.96 x 10⁶ conidia ml-1 and 5.29 x 10⁵ conidia ml-1, respectively. Bioassays on adult thrips were conducted in munger cells with leaf buds inoculated with the conidial suspensions. Isolate GAR 17 B3 had the highest mortality rate at 70% on thrips while FCM AR 23 B3 resulted in 60% mortality. Identification of the isolates, FCM AR 23 B3, GAR 17 B3 and GB AR 23 13 3, were confirmed to be correct using both microscopic and molecularly techniques. ITS sequences were compared to other sequences from GenBank and confirmed phylogenetically using MEGA6. Mealybug infection was investigated using scanning electron microscopy, mycosis was confirmed but the infection process could not be followed due to the extensive waxy cuticle. These results indicate that there is potential for the isolates FCM AR 23 B3 and GAR 17 B3 to be developed as biological control agents for the control of citrus mealybug and thrips. Further research would be required to determine their ability to perform under field conditions.
- Full Text:
- Date Issued: 2014