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
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- Date Issued: 2022-10-14
Effect of Helicosporidium sp. (Chlorophyta; Trebouxiophyceae) infection on Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), a biological control agent for the invasive Salvinia molesta D.S. Mitchell (Salviniaceae) in South
- Authors: Mphephu, Tshililo Emmanuel
- Date: 2022-10-14
- Subjects: Salvinia molesta South Africa , Weeds Biological control , Cyrtobagous salviniae , Ketoconazole
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365815 , vital:65792 , DOI https://doi.org/10.21504/10962/365815
- Description: The effectiveness of established biological control agents depends on biotic and abiotic interactions in the introduced range. The weevil, Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), was released as a biological control against Salvinia molesta D.S. Mitchell (Salviniaceae) in South Africa in 1985. This agent has been highly successful against S. molesta and has significantly reduced the weed’s populations around the country. However, in 2007, the parasitic alga, Helicosporidium sp. (an undescribed species), was detected in field-collected C. salviniae adults in South Africa. The distribution and impacts of this disease on the weevil and its efficacy as a control agent were not known. In this thesis, the prevalence, infection load, and impact of Helicosporidium sp. on C. salviniae was determined. In 2019, adult weevils were collected from 10 sites across the Eastern Cape, KwaZulu-Natal, Limpopo, and Western Cape provinces and screened to determine the occurrence, infection load, and geographic distribution of Helicosporidium sp. Transmission mechanisms of this disease in C. salviniae were then evaluated. The possible impact of Helicosporidium sp. was assessed by comparing the feeding rates and the reproductive output of the diseased and healthy adults of C. salviniae. An attempt was then made to eliminate the disease in C. salviniae through the application of the antibiotic, ketoconazole. Further, the role of temperature on infection load in C. salviniae was also assessed. Finally, recommendations for the long-term biological control programme against S. molesta in South Africa were made. The disease covers the entire distribution range of C. salviniae in South Africa, with the disease occurrence rate ranging from 92.15% to 100% insects infected per site. Helicosporidium sp. was found to transmit vertically within the populations of C. salviniae. Infection by the Helicosporidium sp. disease reduced the reproductive output of C. salviniae as well its impact on biomass reduction of S. molesta when a diseased culture was compared to a healthy culture from the USA. 98.44 to 98.55% of Helicosporidium sp. loads were reduced through multiple applications of ketoconazole concentrations under in vitro trials. In vivo treatments resulted in 70% control of Helicosporidium sp. in the adults of C. salviniae that were fed ketoconazole three times over a 21 day period. Adult C. salviniae feeding and survival performances were similar when fed fronds of S. molesta inoculated with ketoconazole and water. The lowest and highest disease loads of Helicosporidium sp. were recorded when the weevils were reared at 30°C and 14°C, respectively. As expected, the highest impact and reproductive output of C. salviniae were at 30°C. The evaluations discussed in this thesis highlight the role of diseases in biological control agents, and gaps in both the pre-release and post-release monitoring that should integrate screening of diseases in these studies. Although the combined application of the antibiotic and temperature will reduce Helicosporidium sp. loads and impact, this technology is most likely only applicable where the weevils are reared in small numbers in a rearing facility and not really applicable to the field situation. It is important to release healthy agents that will cause efficient control of the target weed plant species, therefore, when introducing new biological control agents, the health status of such agents needs to be understood. Therefore, long-term field monitoring and assessment of the impact of C. salviniae on S. molesta should be conducted to track all the changes that may result due to the presence of Helicosporidium sp. This long-term monitoring and assessment will give a more informative role of Helicosporidium sp. in field populations of C. salviniae. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2022
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- 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
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- Date Issued: 2022-10-14
The effect of orchard sanitation and predatory ants on the eclosion of the internal feeding pests and Oriental fruit fly, in South Africa
- Authors: Makitla, Tshepang
- Date: 2022-10-14
- Subjects: Orchards South Africa , Phytosanitation , Citrus Diseases and pests Biological control , Ants , Insects as biological pest control agents
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362927 , vital:65375
- Description: There are several pests of phytosanitary concern in the citrus industry in South Africa. Orchard sanitation can play an important role in suppressing the populations of these pests, however there are little data on the efficacy of sanitation techniques. Therefore, the current study investigated the effect of fruit disposal techniques and burying depths on the eclosion of the most important pests of citrus in South Africa, false codling moth Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Mediterranean fruit fly or Medfly Ceratitis capitata Wiedemann (Diptera: Tephritidae), Natal fruit fly Ceratitis rosa Karsh (Diptera: Tephritidae), and Oriental fruit fly Bactrocera dorsalis Hendel (Diptera: Tephritidae). Abscised C. sinensis fruits were inoculated with larvae of T. leucotreta, and eggs of C. capitata, C. rosa, and B. dorsalis, before being disposed as pulped, or whole, and buried at different depths (0 cm, 5 cm, 25 cm, and 50 cm). Abundance and richness of predatory ants were monitored using pitfall traps to ascertain their effect on the mortality of the immature stages of these pests. Ceratitis capitata and C. rosa failed to eclose from the inoculated fruits disposed at different depths, however, T. leucotreta and B. dorsalis adults did eclosed. Significantly fewer B. dorsalis eclosed from fruits that were pulped in comparison to eclosion where the fruit were left whole (F (3, 16) = 11.45, P < 0.01). Furthermore, depth of burial had a significant effect on the number of eclosed adults of Drosophila sp (F (3, 112) = 3.43, P < 0.01). Burying fruits at 50 cm suppressed the eclosion of all the internal feeding pests tested. Twenty-seven thousand seventy-three individual ants (Hymenoptera: Formicidae) were sampled from the same plots as used above, with at least 47% and 53% sampled from plots where pulped and whole C. sinensis fruits were disposed of, respectively. The ants were identified to morphospecies which included Pheidole1, Pheidole2, Formicinae1, Formicinae2, Formicinae3, and Myrmicinae1. The disposal of the inoculated C. sinensis fruits either as pulped or whole and burying at different depths significantly suppressed and/or delayed the eclosion of either of the tested internal feeding pests of citrus. Although, predacious ants were sampled from the same treatment plots they did not affect the survival or eclosion of the tested pests, and this could be attributed to the application of the slow toxic ant bait. Therefore, based on the observed results B. dorsalis adults showed the ability to eclose from 50 cm depth where fruit was either disposed as pulped or whole, thus, citrus farmers are advised to use hammer mill that will finely crush sanitised fruit, and/or bury fruit beyond 50 cm depth to prevent the adult od this pest from eclosing. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
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- Date Issued: 2022-10-14