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
The effects of elevated CO2 on feeding guild responses of biological control agents of Pontederia crassipes Mart. (Pontederiaceae)
- Authors: Paper, Matthew Keenan
- Date: 2022-04-06
- Subjects: Carbon dioxide , Pontederia crassipes , Biological pest control agents , Invasive plants Biological control , Pontederiaceae Climatic factors
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/455338 , vital:75422
- Description: Elevated CO2 (eCO2) and rising global temperatures have the potential to alter plant-insect interactions with important implications for plant community structure and function. Previous studies on plant-insect interactions have shown that eCO2 will affect insect feeding guilds differently, impacting negatively, positively or having very little effect. The implications of this on the global invasive plant biological control programme is largely unknown. This study investigates the response of one of the world’s most invasive aquatic plants, Pontederia ( = Eichhornia) crassipes Mart. (Pontederiaceae), to predicted eCO2 conditions of 800 ppm and how this may affect the feeding response of two biological control agents representing different feeding guilds; the leaf chewing Cornops aquaticum Brüner (Orthoptera: Acrididae) and the phloem-feeding Megamelus scutellaris Berg (Hemiptera: Delphacidae). A factorial eCO2 x feeding impact study was conducted at the Rhodes University Elevated CO2 Facility in the Eastern Cape Province of South Africa over 13 weeks in the growing season of 2019. The effect of insect herbivory by C. aquaticum and M. scutellaris at two atmospheric CO2 concentrations, representing current and future predicted concentrations (400 ppm and 800 ppm) on P. crassipes was examined through both biomass and ecophysiological measures. Assimilation rates, C:N ratio, total dry weight and relative growth rate of P. crassipes were unaffected by eCO2 conditions, and plants experienced no CO2 fertilization in eutrophic water conditions representative of South African waterways. Effects of eCO2 on insect herbivory varied depending on the feeding guild. Pontederia crassipes showed compensatory growth responses when exposed to C. aquaticum herbivory regardless of CO2 treatment, but chewing herbivory damage remained constant, and the agent maintained efficacy. Pontederia crassipes showed down-regulation of photosynthesis when exposed to M. scutellaris due to eCO2-related feeding responses by M. scutellaris increasing substantially through a significant (30%) increase in adult population density under eCO2 conditions. These results indicate that the plant-insect interactions that underpin biological control programmes for P. crassipes should remain successful under future CO2 conditions. Phloem-feeding insect damage (M. scutellaris) was significantly greater than chewing damage in this study, suggesting that invasive plant biological control programmes will need to shift focus away from the charismatic chewing insect herbivores and onto the often-neglected phloem-feeding biological control agents due to their overwhelmingly positive response to eCO2. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Date Issued: 2022-04-06
An investigation into yeast-baculovirus synergism for the improved control of Thaumatotibia leucotreta, an economically important pest of citrus
- Authors: Van der Merwe, Marcél
- Date: 2021-10-29
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Yeast , Natural pesticides , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control , Thaumatotibia leucotreta
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191236 , vital:45073
- Description: A mutualistic association between Cydia pomonella and yeasts belonging to the genus Metschnikowia has previously been demonstrated. Larval feeding galleries inoculated with M. andauensis, reduced larval mortality and enhanced larval development. Additionally, adult C. pomonella female oviposition preference was also shown to be influenced by the volatiles produced by M. andauensis. This mutualistic relationship was manipulated for biological control purposes, by combining M. pulcherrima with the baculovirus Cydia pomonella granulovirus. The combination of M. pulcherrima with brown cane sugar and CpGV in laboratory assays and field trials resulted in a significant increase in larval mortality. A similar observation was made when M. pulcherrima was substituted for Saccharomyces cerevisiae. This indicates that yeasts harbour the potential for use in biological control, especially when combined with other well-established biocontrol methods. Thaumatotibia leucotreta is a phytophagous insect endemic to southern Africa. It is highly significant to the South African citrus industry due to its classification as a phytosanitary pest by most international markets. An integrated pest management programme has been implemented to control T. leucotreta. The baculovirus Cryptophlebia leucotreta granulovirus forms one component of this programme and is highly effective. In this study, we proposed to determine which yeast species occur naturally in the gut of T. leucotreta larvae and to examine whether any of the isolated yeast species, when combined with the CrleGV-SA, enhance its effectiveness. Firstly, Navel oranges infested with T. leucotreta larvae were collected from geographically distinct citrus-producing regions across South Africa. This led to the isolation and identification of six yeast species from the gut of T. leucotreta larvae via PCR amplification and sequencing of the internal transcribed spacer region and D1/D2 domain of the large subunit. Six yeast species were identified, viz. Meyerozyma guilliermondii, Hanseniaspora uvarum, Clavispora lusitaniae, Kluyveromyces marxianus, Pichia kudriavzevii and Pichia kluyveri. Additionally, Saccharomyces cerevisiae was included as a control in all trials due to its commercial availability and use in the artificial diet used to rear T. leucotreta. Secondly, larval development and attraction assays were conducted with the isolated yeast species. Thaumatotibia leucotreta larvae that fed on Navel oranges inoculated with M. guilliermondii, P. kluyveri, H. uvarum, and S. cerevisiae had accelerated developmental periods and reduced mortality rates. Additionally, it was demonstrated that T. leucotreta neonates were attracted to YPD broth cultures inoculated with P. kluyveri, H. uvarum, P. kudriavzevii and K. marxianus for feeding. Thirdly, oviposition preference assays were conducted with adult T. leucotreta females to determine whether the isolated yeast species influence their egg-laying in two-choice and multiple-choice tests. Navel oranges were inoculated with a specific yeast isolate, and mated adult females were left to oviposit. Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to influence adult T. leucotreta female oviposition preference in two-choice tests. However, multiple-choice tests using the aforementioned yeast species did not mimic these results. Lastly, a series of detached fruit bioassays were performed to determine the optimal yeast:virus ratio, test all isolated yeast species in combination with CrleGV-SA and to further enhance yeast/virus formulation through the addition of an adjuvant and surfactant. CrleGV-SA was applied at a lethal concentration that would kill 50 % of T. leucotreta larvae. The optimal yeast concentration to use alongside CrleGV-SA was determined. Pichia kluyveri, P. kudriavzevii, K. marxianus and S. cerevisiae in combination with CrleGV-SA increased larval mortality compared to CrleGV-SA alone. The inclusion of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae plus CrleGV-SA formulations greatly enhanced their efficacy. Additionally, semi-field trials were initiated using P. kudriavzevii and S. cerevisiae, with promising preliminary results being obtained, although more replicates need to be performed. The experiments performed in this study provide a platform for further research into the application of a yeast/virus combination as a novel control and monitoring option for T. leucotreta in the field. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Date Issued: 2021-10-29
Potential Synergism between Entomopathogenic Fungi and Entomopathogenic Nematodes for the control of false codling moth (Thaumatotibia leucotreta)
- Authors: Prinsloo, Samantha Lee
- Date: 2021-10
- Subjects: Cryptophlebia leucotreta , Entomopathogenic fungi , Insect nematodes , Citrus Diseases and pests , Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents
- Language: English
- Type: Masters theses , text
- Identifier: http://hdl.handle.net/10962/188832 , vital:44790
- Description: False codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) (FCM), is a major phytosanitary pest of citrus in South Africa. Sufficient control measures for the soil-dwelling life stages of FCM have yet to be identified and owing to restrictions on the use of insecticides, non-chemical control options have been investigated including the use of entomopathogenic fungi (EPF) and entomopathogenic nematodes (EPN). Laboratory and field trials on an indigenous EPF, Metarhizium anisopliae FCM Ar 23 B3, have shown that this isolate is capable of inducing mortality in FCM soil-dwelling life stages. Other agents that have been highlighted as potential controls for soil-dwelling FCM life stages are the EPN species Steinernema yirgalemense 157-C, S. jeffreyense J194 and H. noenieputensis 158-C. This study conducted laboratory bioassays to assess the virulence of these four control agents on fifth instar FCM, in 24-well plates. These results reaffirmed the virulence of the four microbial control agents at their recommended doses of 50 IJs (EPN) and 1×107 conidia/ml (EPF) against fifth instar FCM with 80 to 96% larval mortality recorded. The EPF isolate exhibited the lowest mortality whilst S. yirgalemense induced the greatest mortality. In addition, the lethal concentration (LC) values for each isolate were determined using dose response bioassays. These values were previously unknown for all EPN species and for the EPF isolate based on the methodology used in this study. The LC50 results in order from lowest to highest EPN IJ concentration requirements were 4.38 IJs (S. yirgalemense), 4.47 IJs (S. jeffreyense) and 7.11 IJs (H. noenieputensis). The EPF isolate exhibited an LC50 of 3.42×105 conidia/ml. Lastly, research has shown that the combination of two control agents may increase control of late instar lepidopteran and coleopteran larvae, through synergistic interactions. Thus, the interactions that occurred between the combination of these EPN species with the EPF isolate were determined. This study found that when all three EPN species were combined simultaneously and sequentially with the EPF isolate M. anisopliae FCM AR 23 B3, additive interactions took place with exception of the simultaneous application of S. yirgalemense and H. noenieputensis, with the EPF and S. jeffreyense applied 24 h post EPF application. For the former, a synergistic interaction was found, whilst for the latter two, an antagonistic interaction. Although no strongly synergistic interactions were observed, additive interactions have been shown to reach a synergistic level when certain parameters are changed. Moving forward, a uniform methodology for conducting EPF/EPN interaction experiments has been suggested. It has also been recommended that due to the additive interactions observed in this study, laboratory soil-bioassays and field trials should be carried out for all three EPN species in combination with the EPF isolate. This research will inevitably facilitate the constant knowledge into management strategies for the phytosanitary pest, FCM in South African citrus. , Thesis (MSc) -- Science, Zoology and Entomology, 2021
- Full Text:
- Date Issued: 2021-10
Baculovirus synergism for improved management of false codling moth Thaumatotibia leucotreta Meyr. (Lepidoptera: Tortricidae)
- Authors: Taylor, David Graham
- Date: 2021-04
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Codling moth , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/176942 , vital:42774
- Description: Baculoviruses are an environmentally friendly and effective agent for managing lepidopteran pests. This includes the management of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a serious pest of citrus in Southern Africa and a major threat to the South African citrus export industry. For more than 15 years, CrleGV-SA- based biopesticides have been used as part of an integrated pest management strategy for the control of T. leucotreta in citrus orchards in South Africa, under the names Cryptogran™ and Cryptex®. While these biopesticides have been effective during this period, there are some areas in which baculovirus use could potentially be improved. Baculoviruses are notoriously slow to kill in comparison to chemical-based pesticides, and lately, pest resistance to baculoviruses has become a major concern with the development of resistance by Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) to its granulovirus occurring in the field in Europe. The consistent use of CrleGV-SA for more than 15 years in the field has raised concern that T. leucotreta could develop resistance to this virus, and has made it necessary to alter baculovirus-based management strategies to prevent this from occurring. A second baculovirus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), has recently been isolated and was shown to be effective against T. leucotreta. However, the interactions between CrleGV-SA and CrpeNPV are not yet understood and so it is important to test these interactions before both viruses are applied on the same orchards. Not only is it important to know whether these viruses could negatively impact each other, but it is also important to test whether they could interact synergistically. A synergistic interaction could not only provide a potential tool for the management of resistance, but it could also be exploited to improve baculovirus-based management of T. leucotreta. In this study, a stock of CrleGV-SA was purified by glycerol gradient centrifugation from T. leucotreta cadavers, while a stock of CrpeNPV purified from Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) cadavers was provided by River Bioscience (Pty) Ltd. These stocks were screened for purity by a multiplex polymerase chain reaction (mPCR) protocol designed to detect CrleGV-SA and CrpeNPV. The occlusion body (OB) density was then calculated using darkfield microscopy and a counting chamber. Both stocks were shown to be pure within the limits of the mPCR protocol, and the CrleGV-SA and CrpeNPV stocks were calculated to contain 3.08 × 1011 OBs/mL and 1.92 × 1011 OBs/mL respectively The first aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the dose mortality, in terms of lethal concentration. This was calculated using 7-day surface-dose biological assays for each virus and a 1:1 mixture of OBs of the two against T. leucotreta neonates. The lethal concentrations of each treatment required to kill 50 % of larvae (LC50) and 90 % of larvae (LC90) for each treatment were then calculated and compared using a probit regression. The mixed infection performed significantly better than either virus by itself, while each virus by itself did not differ significantly from the other. The LC50 for CrleGV-SA, CrpeNPV and the mixed infection were 1.53 × 104 OBs/mL, 1.15 × 104 OBs/mL and 4.38 × 103 OBs/mL respectively. The LC90 of CrleGV-SA, CrpeNPV and the mixed infection were calculated to be 4.10 × 105 OBs/mL, 1.05 × 105 OBs/mL, and 4.09 × 104 OBs/mL respectively. The second aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the speed of kill. A time-response biological assay protocol was created that allowed for effective observation of the larvae. This was then used to generate time-mortality data that were analysed by a logit regression function to calculate and compare the treatments at the time of 50 % larval mortality (LT50) and the time of 90 % mortality (LT90). Each virus by itself did not differ significantly from the other, while the mixed infection took significantly longer to kill 50 % and 90 % of the larvae, suggesting that there is competition for resources between viruses during the secondary, systemic phase of infection. The LT50 for CrleGV-SA, CrpeNPV and the mixed infection were 117.5 hours, 113.5 hours and 139.0 hours respectively. The LT90 for CrleGV-SA, CrpeNPV and the mixed infection were 153.2 hours, 159.3, and 193.4 hours respectively. Finally, the composition of OBs recovered from the cadavers produced by the time-response biological assays were investigated by mPCR. A method for extracting gDNA from OBs in neonate-sized T. leucotreta larvae is described. The presence of CrpeNPV along with CrleGV-SA was noted in 4 out of 9 larvae inoculated with only CrleGV-SA. The presence of CrleGV-SA as well as CrpeNPV was noted in all but one larva inoculated with only CrpeNPV, and both CrleGV-SA and CrpeNPV were noted in all but one larva inoculated with a 1:1 mixture of the two, with one larva only being positive for CrleGV-SA. This suggests either stock contamination or the presence of covert infections of CrleGV-SA and CrpeNPV in the T. leucotreta population used in this study. This is the second study to report an improved lethal concentration of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates, and the first study to report the slower speed of kill of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates. While the improved lethal concentration of the mixed infection is a promising step in the future improvement of baculovirus-based biopesticides, it is at the cost of a slower speed of kill. , Thesis (MSc) -- Faculty of Science, Department of Zoology and Entomology, 2021
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- Date Issued: 2021-04
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
Post-release evaluation of Megamelus scutellaris Berg. (hemiptera: delphacidae): a biological control agent of water hyacinth Eichhornia crassipes (Mart.) Solms-Laub (Pontederiaceae) in South Africa
- Authors: Miller, Benjamin Erich
- Date: 2019
- Subjects: Megamelus scutellaris Berg. , Delphacidae , Noxious weeds -- Biological control -- South Africa , Aquatic weeds -- Biological control -- South Africa , Water hyacinth -- Biological control -- South Africa , Biological pest control agents
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/92330 , vital:30710
- Description: Water hyacinth, Eichhornia crassipes (Mart.) Solms-Laub. (Pontederiaceae) is a free-floating aquatic macrophyte from South America that was introduced to South Africa in the 1900s for its attractive ornamental flowers. The plant was classified as a serious invader in the country in the 1970s, eventually becoming the worst invasive aquatic plant in South Africa. Biological control is widely regarded as the most effective method of managing water hyacinth, as it is ecologically safe, cost-effective, and self-sustaining. To date, nine biological control agents have been released in South Africa against water hyacinth, including eight arthropods and a pathogen. Due to the cumulative effects of highly eutrophic waterbodies, which mitigate the damage caused by biological control, and the cold winters which inhibit the rate of biological control agent population build up, South Africa currently has more biological control agents released on water hyacinth than anywhere else in the world. The need for a cold-tolerant agent that can reproduce and develop quickly, while still being damaging to water hyacinth in eutrophic systems, led to the introduction of the most recently released water hyacinth biological control agent, the planthopper Megamelus scutellaris Berg (Hemiptera: Delphacidae), which was initially collected from Argentina. This thesis formed the first post-release evaluation of M. scutellaris since its release in South Africa in 2013. It included a greenhouse experiment to measure the agent’s feeding damage in relation to different nutrient levels and stocking rates, as well as a field component to evaluate both the post-winter recovery of M. scutellaris, and a nationwide survey to measure the establishment of the agent around the country in relation to climate, water quality, and plant health. In the greenhouse experiment, the feeding damage was quantified using measurements of plant growth parameters and chlorophyll fluorometry. It was found that, like other biological control agents of water hyacinth, M. scutellaris was most damaging when released in high numbers on plants grown at medium nutrient levels, and less effective on plants grown at elevated nutrient levels. A water hyacinth infestation on the Kubusi River was selected for the evaluation of the post-winter recovery of M. scutellaris. The Kubusi River is both the first site where M. scutellaris was released, and the coldest site where water hyacinth biological control agents have established successfully in South Africa. Monthly visits tracking seasonal plant health characteristics and agent population densities indicated that the populations of M. scutellaris were impacted most significantly by the season. Low temperatures led to the water hyacinth plants being of poor quality during the winter, which had a subsequent negative effect on the agent populations. The agents could only fully recover by late summer, which meant that the plants were without any significant biological control through the initial phases of the growing season, when they were most vulnerable, and a significant lag-phase occurred between the recovery of the plants and the recovery of the agent population after the winter bottleneck. A survey of all sites where M. scutellaris had been released in South Africa yielded 16 sites where the agents had successfully established, having survived at least one full winter. Among these sites were four sites where the agents were found without them having been released, indicating that they can disperse unaided to new sites. The temperature was a major factor responsible for the success or failure of establishment, with very few agents surviving in the hot areas of South Africa or in areas with a high frost incidence. The density of M. scutellaris was higher in nutrient-rich water, and on plants with more leaves, suggesting that the quality of the plants also contributed to establishment. The results of this thesis showed that M. scutellaris is able to establish successfully in South Africa, and that the agents are capable of causing significant damage to water hyacinth, making it a promising addition to the biological control programme. Novel methods of measuring subtle insect feeding damage in plants and quantifying agent populations are also discussed, along with suggestions for the future implementation of M. scutellaris in South Africa.
- Full Text:
- Date Issued: 2019
Reproductive isolation mechanisms of two cryptic species of Eccritotarsus (Hemiptera: Miridae), biological control agents of water hyacinth, Eichhornia crassipes (Martius) Solms-Laubach (Pontederiaceae)
- Authors: Mnguni, Sandiso
- Date: 2019
- Subjects: Eccritotarsus , Meridae , Noxious weeds -- Biological control -- South Africa , Aquatic weeds -- Biological control -- South Africa , Water hyacinth -- Biological control -- South Africa , Biological pest control agents
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/68133 , vital:29202
- Description: Water hyacinth, Eichhornia crassipes (Martius) Solms-Laubach (Pontederiaceae), is one of the world’s worst alien invasive plants. It is indigenous to the Amazon basin in South America but has become a problematic alien invasive in other parts of the world. As such, several host-specific biological control agents have been sourced from the native distributions in South America and have been released to control this plant where it has become problematic. Two of these agents include the geographically and reproductively isolated cryptic species of Eccritotarsus (Hemiptera: Miridae). One of these species was collected in the upper reaches of the Amazon River in Peru, while the other was collected over 3500km away from that site, in Florianopolis, southern Brazil. These cryptic species were thought to be a single species until recently, when DNA barcoding indicated that they were likely to be two species, and the species status has now been confirmed by interbreeding experiments and detailed morphological studies. The Brazilian population remains Eccritotarsus catarinensis (Carvalho), while the Peruvian population is now known as Eccritotarsus eichhorniae (Henry). The aim of this project was to investigate the mating behaviour and other behavioural traits of the two species that have resulted in reproductive isolation, and which could have led to speciation. In addition, investigations involving analysis of chemical compound compositions of the two species aimed to determine the extent to which the compounds played a role in the development and maintenance of reproductive isolation. To achieve the aims, behavioural-observation experiments were conducted in the form of no-choice, bi-choice and multi-choice tests in 1:1, 2:1 and 3:1 sex ratio assessments, both within and between species. Chemical compound compositions of E. catarinensis and E. eichhorniae were also assessed using Nuclear Magnetic Resonance (NMR), Solid-phase micro-extraction (SPME) and Gas-Chromatography Mass-Spectrometry (GC-MS) techniques. In no-choice experiments, the highest number of single and multiple copula incidences, and average total copula duration was found within species while copulation between species was much rarer. In bi-choice experiments, E. eichhorniae females and E. catarinensis males only chose to mate with their respective conspecifics, and within species copulations continued to have higher average total copula duration. In multi-choice experiments, the highest number of single and multiple copula incidences and average total copula duration was also found within species. GC-MS analysis suggested that E. catarinensis females and E. eichhorniae males have unique chemical compounds missing in their conspecifics and same sex of the other species. Further analysis suggested that E. catarinensis females and E. eichhorniae males have similar chemical compound compositions, whereas as E. eichhorniae females and E. catarinensis males have similar chemical compound compositions. These results suggest that there are behavioural differences that led to the development and maintenance of prezygotic reproductive isolation mechanisms, and that this is probably driven by pheromones in chemical compound compositions. These two species were geographically isolated in the native range and the populations have diverged to the point that they are now reproductively incompatible and therefore, distinct species. The main driver of the speciation is most likely mate recognition and attraction, as only reproductively important traits such as pheromones, genitalia, the scent glands and antennae have changed, while other traits, including host range and morphology, have remained remarkably stable. This provides evidence that differences in sexual selection in isolated populations may be important drivers of speciation and reproductive isolation in cryptic species.
- Full Text:
- Date Issued: 2019
Potential impact and host range of Pereskiophaga brasiliensis Anderson (Curculionidae): a new candidate biological control agent for the control of Pereskia aculeata Miller (Cactaceae) in South Africa
- Authors: Mdodana, Lumka Anita
- Date: 2018
- Subjects: Curculionidae -- South Africa , Cactus -- South Africa , Biological pest control agents , Alien plants-- South Africa , Pereskiophaga brasiliensis Anderson (Curculionidae) , Pereskia aculeata Miller (Cactaceae)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/62350 , vital:28157
- Description: Pereskia aculeata Miller (Cactaceae) is a damaging invasive alien plant in South Africa that has negative impacts to indigenous biodiversity and ecosystem functioning. Mechanical and chemical control are not effective against P. aculeata so biological control is considered the only viable option. Two biological control agents, the leaf-feeding beetle Phenrica guerini Bechyne (Chrysomelidae) and the stem-wilting bug Catorhintha schaffneri (Coreidae), have been released in South Africa thus far. Post-release evaluations have indicated that P. guerini will not reduce P. aculeata densities to acceptable levels alone, while C. schaffneri was released very recently, so it is too soon to determine how effective that agent will be. Even if C. schaffneri is extremely damaging, it is likely that further agents will be required to reduce the densities of P. aculeata to acceptable levels within a reasonable time-scale. Additional agents should target the woody stems of P. aculeata which are not impacted by the damage of either of the released agents. Pereskiophaga brasiliensis Anderson (Curculionidae) is a promising potential candidate agent that feeds on the thick woody stems of the plant in the larval stage. Climatic matching, genetic matching and field based host specificity observations all indicated that P. brasiliensis was a promising candidate. In this study, the impact of P. brasiliensis to the target weed, P. aculeata, was quantified under quarantine conditions to determine whether it was sufficiently damaging to warrant release. This was followed by host specificity testing to determine whether P. brasiliensis was suitably host specific for release in South Africa. Impact studies indicated that P. brasiliensis was damaging to P. aculeata at insect densities that would be expected in the field. Pereskiophaga brasiliensis reduced the number of leaves of P. aculeata to a greater extent than it reduced shoot lengths, but both plant parameters were significantly reduced due to the feeding damage from the insect. This suggests that the damage from P. brasiliensis may be compatible with that of C. schaffneri which reduces shoot length to a greater degree than the number of leaves. Pereskiophaga brasiliensis is therefore sufficiently damaging to warrant release, and although interaction studies with the other agents would be required, it is expected that it should complement other existing agents. Although P. brasiliensis is sufficiently damaging, at present the host specificity data indicates that it is not suitably specific for release in South Africa because oviposition and larval development to the adult stage was recorded on both indigenous and alien plant species within the families Cactaceae and Basellaceae. This non-target feeding was recorded during no-choice tests, which are very conservative, but significant non-target damage and development to the adult stage was recorded on an indigenous plant from a different family to the target weed. Further host specificity testing, including paired and multiple choice tests, are required to confirm the broad host range of P. brasiliensis. Other biological control agents that damage the woody stems of P. aculeata should be considered. The stem-borer, Acanthodoxus machacalis (Cerambycidae) is considered the most promising of the other candidate agents as it can be sourced from a climatically matched region where genetically suitable P. aculeata plants are found, it is sufficiently damaging to the woody stems of P. aculeata and there is no evidence that the species has a broad host range. Acanthodoxus machacalis should be sourced from Rio de Janeiro, Brazil, and imported into quarantine in South Africa for host specificity testing.
- Full Text:
- Date Issued: 2018
Studies in leaf domatia-mite mutualism in South Africa
- Authors: Situngu, Sivuyisiwe
- Date: 2018
- Subjects: Insect-plant relationships , Mites , Mutualism (Biology) , Biological pest control agents
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/63334 , vital:28394
- Description: Plants have various traits which allow them to cope and resist their enemies including both insects and fungi . In some cases such traits allow plants to build mutualistic relationships with natural enemies of plant pests. This is the case in many dicotyledonous plants which produce leaf domatia. Leaf domatia are plant cavities usually found in the axils of major veins in the abaxial side of leaves. They are usually associated with mites and often mediate mutualistic relationships with predacious mites. Mites use leaf domatia primarily for shelter, to reproduce, and to develop. In turn, plants benefit from having predaceous mites on their leaves, because mites act as plant “bodyguards” and offer defence against pathogens and small arthropod herbivores. This phenomenon has been well documented all over the world, but Africa remains disproportionally understudied. The aim of this study was to fill the gap that exists in our knowledge of the extent of the distribution of leaf domatia-mite mutualisms and generate a better understanding of the diversity of mites found within leaf domatia from an African perspective. This was done by surveying plant species that bear leaf domatia from different vegetation types in South Africa. The plants with leaf domatia were examined for the presence of mites in order to determine patterns of mite abundance and diversity and, in so doing, address the following questions: • Does each tree species host have a specific mite or mite assemblage? • Do some mites prefer certain types of leaf domatia? • Do mites prefer a specific place in the tree canopy and does the microclimate in the tree canopy affect the distribution of mites? • Do different vegetation sites and types differ in their mite diversity and species composition? • Does mite abundance and diversity vary with seasons? Do coffee plantations have a different suite of mites than the adjacent forest? The anatomical structures of leaf domatia from six selected plant species(Coffea arabica, Gardenia thunbergia, Rothmannia capensis, Rothmannia globosa (Rubiaceae), Ocotea bullata (Lauraceae) and Tecoma capensis (Bignoniaceae) with different types of leaf domatia were also studied. The results from this study suggested that the key futures which distinguish domatia are the presence of an extra layer of tissue in the lower epidermis, a thick cuticle, cuticular folds, the presence of trichomes and an invagination. This study provides a better understating of the structure of leaf domatia. Leaf domatia bearing plants are widely distributed in South Africa, and species and vegetation-specific associations were assessed. Over 250 plant specimens with leaf domatia were collected and examined and more than 60 different mite species were found in association with the sampled plant species. The majority of mites found within the domatia of these tree species were predaceous and included mites from Stigmatidae, Tydeidae and Phytoseiidae. Furthermore, 15 new species were collected, suggesting that mites are understudied in South Africa. This study showed that the different vegetation types sampled did not differ markedly in terms of their mite biota and that similar mites were found across the region, and the association between leaf domatia and mites was found to be opportunistic and that mites had no preference for any particular domatia types. No host specificity relationship was observed between plants and mites. The assessment of mites associated with Coffea arabica showed that indigenous mites are able to colonise and establish a beneficial mutualism on exotic species. This is important as it ascertains that economically important plants that are cultivated outside their area of natural distribution can still benefit from this mutualism. This study also found that mite abundance and diversity in plants with leaf domatia were influenced by factors such as temperature, relative humidity and rainfall. Mite communities found in association with domatia changed as the year progressed and over the seasons. The seasonal fluctuations varied between the sampled plant species. In addition, this study found that mites were sensitive to extreme environmental conditions, and thus, mites preferred leaves found in the lower parts of the tree canopy and avoided exposed leaves. This study provides a better understanding of the distribution of domatia bearing plants in South Africa and their associated mites and contributes to our knowledge of the biodiversity of mites in the region. Furthermore, this study also adds to our understanding of the leaf domatia - mite mutualism in Africa. The applied example looking at the plant-mite mutualism in Coffea arabica highlights the importance of this mutualism in commercial plants.
- 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
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
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
Screening of entomopathogenic fungi against citrus mealybug (Planococcus citri (Risso)) and citrus thrips (Scirtothrips aurantii (Faure))
- 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
Entomopathogenic fungi for control of soil-borne life stages of false codling moth, Thaumatotibia leucotreta (Meyrick) (1912) (Lepidoptera: Tortricidae)
- Authors: Coombes, Candice Anne
- Date: 2013
- Subjects: Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5607 , http://hdl.handle.net/10962/d1002057 , Tortricidae , Lepidoptera , Cryptophlebia leucotreta , Insect pests -- Biological control -- South Africa -- Eastern Cape , Tortricidae -- Biological control -- South Africa -- Eastern Cape , Citrus -- Diseases and pests -- Biological control -- South Africa -- Eastern Cape , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: False codling moth (FCM), Thaumatotibia leucotreta is an extremely important pest of citrus in South Africa and with the shift away from the use of chemicals, alternate control options are needed. One avenue of control which has only recently been investigated against the soil-borne life stages of FCM is the use of entomopathogenic fungi (EPF). In 2009, 12 entomopathogenic fungal isolates collected from South African citrus orchards showed good control potential during laboratory conducted bioassays. The aim of this study was to further analyse the potential of these isolates through concentration-dose and exposure-time response bioassays. After initial re-screening, concentration-dose response and exposure-time response sandconidial bioassays, three isolates were identified as exhibiting the greatest control potential against FCM in soil, Metarhizium anisopliae var. anisopliae (G 11 3 L6 and FCM Ar 23 B3) and Beauveria bassiana (G Ar 17 B3). Percentage mycosis was found to be directly related to fungal concentration as well as the amount of time FCM 5th instar larvae were exposed to the fungal conidia. LC50 values for the three isolates were not greater than 1.92 x 10⁶ conidia.ml⁻ₑ and at the LC₅₀, FCM 5th instar larvae would need to be exposed to the fungus for a maximum of 13 days to ensure a high mortality level. These isolates along with two commercially available EPF products were subjected to field persistence trials whereby net bags filled with a mixture of autoclaved sand and formulated fungal product were buried in an Eastern Cape citrus orchard. The viability of each isolate was measured on a monthly basis for a period of six months. All isolates were capable of persisting in the soil for six months with the collected isolates persisting far better than the commercially used isolates. Two of the isolates, G 11 3 L6 and G Ar 17 B3, were subjected to small scale laboratory application trials. Two formulations were investigated at two concentrations. For each isolate, each formulation and each concentration, FCM 5th instar larvae were applied and allowed to burrow into the soil to pupate before fungal application or after fungal application. Contact between fungi and FCM host is essential as, in contrast to pre-larval treatments, percentage mortality in post-larval treatments was low for both formulations and both isolates. For isolate G Ar 17 B3, a conidial suspension applied as a spray at a concentration of 1 x 10⁷ conidia.ml⁻ₑ obtained the highest percentage mortality (80 %). For isolate G 11 3 L6 however, both formulations performed equally well at a high, 1 x10⁷ conidia.ml⁻ₑ concentration (conidial suspension: 60 %; granular: 65 %) The results obtained thus far are promising for the control of FCM in citrus, but if these EPFs are to successfully integrate into current FCM control practices more research, some of which is discussed, is essential
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- Date Issued: 2013
Influence of mite predation on the efficacy of the gall midge Dasineura sp. as a biocontrol agent of Australian myrtle Leptospermum laevigatum (Myrtaceae) in South Africa
- Authors: Mdlangu, Thabisa Lynette Honey
- Date: 2010
- Subjects: Dasyneura leguminicola , Myrtaceae -- South Africa , Mites as biological pest control agents , Pests -- Biological control , Biological pest control agents , Predation (Biology)
- Language: English
- Type: Thesis , Masters , MSc (Zoology)
- Identifier: vital:11789 , http://hdl.handle.net/10353/272 , Dasyneura leguminicola , Myrtaceae -- South Africa , Mites as biological pest control agents , Pests -- Biological control , Biological pest control agents , Predation (Biology)
- Description: Dasineura sp. is a gall forming midge that was introduced into South Africa for the biocontrol of the Australian myrtle, Leptospermum laevigatum. It causes galls on both the vegetative and reproductive buds of the plant. Although Dasineura sp. was initially regarded as a potentially successful agent, galling up to 99 percent of the buds of the host plant, it has been preyed on by native opportunistic mites, which caused a decline in the performance of the midge as a biocontrol agent of L. laevigatum. This raised a concern about whether this fly will be able to perform effectively in the presence of its new natural enemies. Therefore, the objectives of this study were to: 1) ascertain whether mite abundance has seasonal variations; 2) determine if plant density and plant size have an effect on midge predation by the mites; and 3) determine if midge predation varies in different locations. The study was conducted at three sites in the Hermanus area, Western Cape Province. Every three weeks for thirteen months, galls were collected and dissected so as to count and record the numbers of midge larvae, pupae, adults and mites that were found. Data collected showed that predation varied with season, and the mites were scarce during the flowering season. Predation also varied among the study sites and plant density had an effect on midge predation. Midges in smaller plants (saplings) were more vulnerable to predation than those in the bigger plants (plants from isolates and thickets). It was concluded that although mites have an effect on midge populations, they do not prevent their establishment on the plant. Therefore, a survey should be done in two to three years time to check if the midges are still persisting on the plant, vi and recommendations are that a new agent should be released to supplement the midges.
- Full Text:
- Date Issued: 2010
Investigation of entomopathogenic fungi for control of false codling moth, Thaumatotibia leucotrata, Mediterranean fruit fly, Ceratitis capitata and Natal fruit fly, C. rosa in South African citrus
- Authors: Goble, Tarryn Anne
- Date: 2010
- Subjects: Insect pests -- Biological control , Tortricidae -- Biological control -- South Africa , Tephritidae -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5723 , http://hdl.handle.net/10962/d1005409 , Insect pests -- Biological control , Tortricidae -- Biological control -- South Africa , Tephritidae -- Biological control -- South Africa , Citrus -- Diseases and pests -- Biological control -- South Africa , Entomopathogenic fungi , Fungi as biological pest control agents , Biological pest control agents
- Description: The biology of key citrus pests Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Ceratitis capitata Wiedemann (Diptera: Tephritidae) and Ceratitis rosa Karsch (Diptera: Tephritidae) includes their dropping from host plants to pupate in the soil below citrus trees. Since most EP fungi are soil-borne microorganisms, the development and formulation of alternative control strategies using these fungi as subterranean control agents, targeted at larvae and pupae in the soil, can potentially benefit existing IPM management of citrus in South Africa. Thus, a survey of occurrence of entomopathogenic fungi was undertaken on soils from citrus orchards and natural vegetation (refugia) on conventionally and organically managed farms in the Eastern Cape Province in South Africa. A method for baiting soil samples with citrus pest T. leucotreta and C. capitata larvae, as well as with the standard bait insect, Galleria mellonella Linnaeus (Lepidoptera: Pyralidae), was implemented. Sixty-two potentially useful entomopathogenic fungal isolates belonging to four genera were collected from 288 soil samples, an occurrence frequency of 21.53%. The most frequently isolated entomopathogenic fungal species was Beauveria bassiana (Balsamo) Vuillemin (15.63%), followed by Metarhizium anisopliae var. anisopliae (Metschnikoff) Sorokin (3.82%). Galleria mellonella was the most effective insect used to isolate fungal species (χ2=40.13, df=2, P≤ 0.005), with a total of 45 isolates obtained, followed by C. capitata with 11 isolates, and T. leucotreta with six isolates recovered. There was a significantly (χ2=11.65, df=1, P≤ 0.005) higher occurrence of entomopathogenic fungi in soil samples taken from refugia compared to cultivated orchards of both organically and conventionally managed farms. No significant differences were observed in the recovery of fungal isolates when soil samples from both farming systems were compared. The physiological effects and host range of 21 indigenous fungal isolates obtained in the Eastern Cape were investigated in the laboratory to establish whether these isolates could be effectively used as biological control agents against the subterranean life stages of C. rosa, C. capitata and T. leucotreta. When these pests were treated with a fungal concentration of 1 x 10⁷ conidia ml⁻¹, the percentage of T. leucotreta adults which emerged in fungal treated sand ranged from 5 to 60% (F=33.295; df=21; P=0.0001) depending on fungal isolate and the percentage of pupae with visible signs of mycosis ranged from 21 to 93% (F= 96.436; df=21; P=0.0001). Based on fungal isolates, the percentage adult survival in C. rosa and C. capitata ranged from 30 to 90% and 55 to 86% respectively. The percentage of C. rosa and C. capitata puparia with visible signs of mycosis ranged from 1 to 14% and 1 to 11% respectively. Deferred mortality due to mycosis in C. rosa and C. capitata adult flies ranged from 1 to 58% and 1 to 33% respectively, depending on fungal isolate. Entomopathogenic fungal isolates had a significantly greater effect on the adults of C. rosa and C. capitata than they did on the puparia of these two fruit fly species. Further, C. rosa and C. capitata did not differ significantly in their response to entomopathogenic fungi when adult survival or adult and pupal mycosis were considered. The relative potency of the four most virulent Beauveria isolates as well as the commercially available Beauveria bassiana product, Bb Plus® (Biological Control Products, South Africa), were compared against one another as log-probit regressions of mortality against C. rosa, C. capitata and T. leucotreta which all exhibited a dose-dependent response. Against fruit flies the estimated LC50 values of all five Beauveria isolates ranged from 5.5 x 10¹¹ to 2.8 x 10¹² conidia/ml⁻¹. There were no significant differences between the relative potencies of these five fungal isolates. When T. leucotreta was considered, isolates: G Moss R10 and G 14 2 B5 and Bb Plus® were significantly more pathogenic than G B Ar 23 B3 and FCM 10 13 L1. The estimated LC₅₀ values of the three most pathogenic isolates ranged from 6.8 x 10⁵ to 2.1 x 10⁶ conidia/ml⁻¹, while those of the least pathogenic ranged from 1.6 x 10⁷ to 3.7 x 10⁷ conidia/ml⁻¹. Thaumatotibia leucotreta final instar larvae were exposed to two conidial concentrations, at four different exposure times (12, 48, 72 and 96 hrs) and showed an exposure time-dependant relationship (F=5.43; df=3; P=0.001). At 1 x 10⁷conidia/ml⁻¹ two Beauveria isolates: G Moss R10 and G 14 2 B5 were able to elicit a response in 50% of test insects at 72 hrs (3 days) exposure. Although a limited amount of mycosis was observed in the puparia of both fruit fly species, deferred adult mortality due to mycosis was high. The increased incidence of adult mortality suggests that post emergence mycosis in adult fruit flies may play a more significant role in field suppression than the control of fruit flies at the pupal stage. The increased incidence of pupal mortality, as well as the relatively low concentrations of conidia required to elicit meaningful responses in T. leucotreta pupae may suggest that pre-emergent control of false codling moth will play a more significant role in field suppression than the control of adult life stages using indigenous isolates of entomopathogenic fungi. Various entomopathogenic fungal application techniques targeted at key insect pests within integrated pest management (IPM) systems of citrus are discussed.
- Full Text:
- Date Issued: 2010
The development of a putative microbial product for use in crop production
- Authors: Gumede, Halalisani
- Date: 2008
- Subjects: Agricultural productivity , Agriculture -- Economic aspects , Microbial products , Bacterial diseases of plants , Biological pest control agents , Lettuce -- Diseases and pests , Crops -- Nutrition , Bacillus (Bacteria) , Phytopathogenic microorganisms -- Control
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3945 , http://hdl.handle.net/10962/d1004004 , Agricultural productivity , Agriculture -- Economic aspects , Microbial products , Bacterial diseases of plants , Biological pest control agents , Lettuce -- Diseases and pests , Crops -- Nutrition , Bacillus (Bacteria) , Phytopathogenic microorganisms -- Control
- Description: The challenges faced by the agricultural sector especially around improving production yields using environmentally friendly solutions have received market attention. Biological intervention can range from application of biological products to enhance the nutritional value of crops or to control plant pathogens. Biostart, a biological product that demonstrated growth enhancement when applied in lettuce crops is currently in the market. The product is comprised of a consortium of bacterial isolates (Bacillus licheniformis, Brevibacillus laterosporus and Bacillus laterosporus) but the contribution of the individual isolates to growth enhancement had not been elucidated. Green house experiments on lettuce seedlings with individual and mixed treatments were commissioned to determine such contribution. There was either no or marginal growth enhancement observed in the experiments. The results showed that the product was effective as a consortium and not as individual isolates. Further isolation and screening for potential Bacilli with antifungal properties was undertaken. An isolate identified as Bacillus subtilis that demonstrated inhibition against a wide spectrum of fungi, and especially the phytopathogenic Verticillium dahliae and Fusarium oxysporum, was successfully identified. The isolate was cryo-preserved and cultivated to significant levels at bench scale. A characterized comparison of different putative products with known systematic fungicide showed potential application even of heat treated products. The product showed control V. dahliae when tested in green houses with potatoes and tomatoes as test crops. This isolate has been targeted for further development as a biological control product.
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- Date Issued: 2008