Bioprospecting for entomopathogenic fungi against a foliar citrus pest
- Authors: Boon, Erin Ashley
- Date: 2025-04-02
- Subjects: Citrus Diseases and pests South Africa , Entomopathogenic fungi , Pests Integrated control , Biological assay , Cryptophlebia leucotreta , Ultraviolet radiation Physiological effect
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/478991 , vital:78247
- Description: Historically, pest management was highly dependent on the use of chemical insecticides for the control of agriculturally important pests. However, more recently, key export markets have imposed stringent chemical residue restrictions for citrus export. This deterring factor for chemical use has been coupled with the fact that these economically important pests are experiencing insecticidal resistance. As a result, the use of entomopathogenic fungi (EPF) has been explored as a complementary control option in integrated pest management (IPM) regimes. Previous research in South Africa identified several strains of Beauveria bassiana and Metarhizium spp. (including isolate M. pinghaense FCM Ar 23 B3). Laboratory bioassays evaluating the virulence of these isolates against major pests such as the false codling moth (FCM) (Thaumatotibia leucotreta, Lepidoptera: Tortricidae), citrus thrips (Scirtothrips aurantii, Thysanoptera: Thripidae), and citrus mealybugs (Planococcus citri, Hemiptera: Pseudococcidae) highlighted the potential of these EPF. While field trials targeting FCM with soil-applied treatments yielded encouraging results, foliar applications aimed at controlling citrus thrips and mealybugs showed limited success. These findings highlighted the need to assess the biological traits of the recovered isolates. Varying temperature ranges and humidity levels were found to not hinder the isolates' efficacy in the field. Conidial inactivation induced by ultraviolet (UV) radiation however, was. As these strains were recovered from the soil environment, it stood to reason that EPF isolates recovered from the foliar environment may be more suited for foliar application. Thus, bioprospecting for isolates from the aboveground environment was initiated and was the focal point of this thesis. Following the isolation and identification, the pathogenic ability and virulence, as well as the UV tolerance of these novel strains were established. Of the isolates recovered from the aboveground environment and identified using morphological and molecular techniques, four were B. bassiana (Px LM 4, Ha LM 11, Ha LM 12, Coe 18), one M. anisopliae (Hu LM 14), one Fusarium oxysporum (Pc HV 9), and one Geotrichum candidum yeast (Ha LM 2). The majority were isolated from insect cadavers, but one (Coe 18) was isolated as a foliar endophyte from an organically managed citrus farm in the Eastern Cape. Using standard protocols and conidial doses, the virulence of the recovered isolates was established against a common foliar pest of citrus, citrus mealybug. Isolate FCM Ar 23 B3 was included as a comparative control in this study as the virulence against citrus mealybug has previously been established. The initial screening of the isolates ranged between 15 and 90 % mortality. Isolates Px LM 4 and FCM Ar 23 B3 both induced an average mortality of 90 %. Isolates Ha LM 11, Ha LM 12, Hu LM 14, and Coe 18 caused mortalities greater than 60 % and were further investigated under dose-response assays. Of the six isolates measured for LC50, FCM Ar 23 B3 was the most virulent (5.25 × 105 conidia/ml), followed by Px LM 4 (1.09 × 106 conidia/ml) and Hu LM 14 (1.32 × 106 conidia/ml). The UV susceptibility to simulated sunlight of the six most virulent isolates was investigated. Whilst UV radiation certainly delayed the conidial germination of all the isolates, all the strains isolated from the aboveground environment demonstrated significant initial tolerance to UV radiation compared to the most virulent M. pinghaense FCM Ar 23 B3, which was recovered from the soil environment. Even though the B. bassiana Coe 18, which was recovered as an endophytic EPF, was not the most virulent, it stood out with strong initial UV tolerance and sustained a relatively high germination rate over time, establishing it as the most UV-tolerant isolate. Although formulation for development as a microbial biocontrol programme should not be overlooked for these isolates, the initial UV and sustained tolerance demonstrated by these aboveground isolates warrants further investigation under field conditions. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2025
- Full Text:
- Authors: Boon, Erin Ashley
- Date: 2025-04-02
- Subjects: Citrus Diseases and pests South Africa , Entomopathogenic fungi , Pests Integrated control , Biological assay , Cryptophlebia leucotreta , Ultraviolet radiation Physiological effect
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/478991 , vital:78247
- Description: Historically, pest management was highly dependent on the use of chemical insecticides for the control of agriculturally important pests. However, more recently, key export markets have imposed stringent chemical residue restrictions for citrus export. This deterring factor for chemical use has been coupled with the fact that these economically important pests are experiencing insecticidal resistance. As a result, the use of entomopathogenic fungi (EPF) has been explored as a complementary control option in integrated pest management (IPM) regimes. Previous research in South Africa identified several strains of Beauveria bassiana and Metarhizium spp. (including isolate M. pinghaense FCM Ar 23 B3). Laboratory bioassays evaluating the virulence of these isolates against major pests such as the false codling moth (FCM) (Thaumatotibia leucotreta, Lepidoptera: Tortricidae), citrus thrips (Scirtothrips aurantii, Thysanoptera: Thripidae), and citrus mealybugs (Planococcus citri, Hemiptera: Pseudococcidae) highlighted the potential of these EPF. While field trials targeting FCM with soil-applied treatments yielded encouraging results, foliar applications aimed at controlling citrus thrips and mealybugs showed limited success. These findings highlighted the need to assess the biological traits of the recovered isolates. Varying temperature ranges and humidity levels were found to not hinder the isolates' efficacy in the field. Conidial inactivation induced by ultraviolet (UV) radiation however, was. As these strains were recovered from the soil environment, it stood to reason that EPF isolates recovered from the foliar environment may be more suited for foliar application. Thus, bioprospecting for isolates from the aboveground environment was initiated and was the focal point of this thesis. Following the isolation and identification, the pathogenic ability and virulence, as well as the UV tolerance of these novel strains were established. Of the isolates recovered from the aboveground environment and identified using morphological and molecular techniques, four were B. bassiana (Px LM 4, Ha LM 11, Ha LM 12, Coe 18), one M. anisopliae (Hu LM 14), one Fusarium oxysporum (Pc HV 9), and one Geotrichum candidum yeast (Ha LM 2). The majority were isolated from insect cadavers, but one (Coe 18) was isolated as a foliar endophyte from an organically managed citrus farm in the Eastern Cape. Using standard protocols and conidial doses, the virulence of the recovered isolates was established against a common foliar pest of citrus, citrus mealybug. Isolate FCM Ar 23 B3 was included as a comparative control in this study as the virulence against citrus mealybug has previously been established. The initial screening of the isolates ranged between 15 and 90 % mortality. Isolates Px LM 4 and FCM Ar 23 B3 both induced an average mortality of 90 %. Isolates Ha LM 11, Ha LM 12, Hu LM 14, and Coe 18 caused mortalities greater than 60 % and were further investigated under dose-response assays. Of the six isolates measured for LC50, FCM Ar 23 B3 was the most virulent (5.25 × 105 conidia/ml), followed by Px LM 4 (1.09 × 106 conidia/ml) and Hu LM 14 (1.32 × 106 conidia/ml). The UV susceptibility to simulated sunlight of the six most virulent isolates was investigated. Whilst UV radiation certainly delayed the conidial germination of all the isolates, all the strains isolated from the aboveground environment demonstrated significant initial tolerance to UV radiation compared to the most virulent M. pinghaense FCM Ar 23 B3, which was recovered from the soil environment. Even though the B. bassiana Coe 18, which was recovered as an endophytic EPF, was not the most virulent, it stood out with strong initial UV tolerance and sustained a relatively high germination rate over time, establishing it as the most UV-tolerant isolate. Although formulation for development as a microbial biocontrol programme should not be overlooked for these isolates, the initial UV and sustained tolerance demonstrated by these aboveground isolates warrants further investigation under field conditions. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2025
- Full Text:
An assessment of the status of psylloid species (Hemiptera: Psylloidea) as potential pests of commercial citrus in southern Africa: implications for pest management
- Authors: Moagi, Raynold
- Date: 2024-10-11
- Subjects: Citrus Diseases and pests South Africa , Candidatus Liberibacter , Psylloidea , Polymerase chain reaction , Insect trapping Equipment and supplies , Pests Control
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/464417 , vital:76509
- Description: Psylloids (Hemiptera: Psylloidea), constitute a group of plant sap-sucking insects, some of which are economically significant pests in different ecosystems due to their potential to transmit Gram-negative bacteria, such as the Candidatus Liberibacter species. The African citrus triozid (ACT), Trioza erytreae (Del Guercio), which transmits African citrus greening and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, which transmits Asian citrus greening are significant threats to citrus. Asian citrus psyllid poses a global economic threat due to its ability to vector “Candidatus Liberibacter asiaticus” (CLas), which can rapidly kill citrus trees. However, both ACP and CLas are currently not present in southern Africa but are present in East and West Africa. In the Afrotropical region, 71 triozid species are known to occur and approximately 41 described Diaphorina species in southern Africa. Currently, two indigenous Diaphorina species, Diaphorina punctulata and Diaphorina zebrana have been documented to feed on citrus. There is a significant knowledge gap regarding the ecological roles of other indigenous psylloid species occurring within the citrus environments. Therefore, this study aimed to: (i) determine the diversity and community structure of psylloid species in citrus environments, and (ii) their host ranges through DNA analysis of gut contents to determine if they fed on citrus. Field surveys were carried out across 12 distinct commercial citrus environments across Limpopo and Mpumalanga provinces between 2022 and 2023. Psylloids were collected using yellow sticky traps and an insect sweep-net. Collected psylloid specimens were preserved in 70% ethanol vials and identified to the lowest possible taxonomic level (i.e. genus or species) using both published and unpublished dichotomous identification keys. Furthermore, citrus leaf samples were collected from the same plants on which psylloids were found in the orchards. Genomic DNA (gDNA) was extracted from both leaf and psylloid samples using two different DNA extraction methods. To confirm if citrus DNA could be detected in the psylloid guts, all leaf gDNA samples were initially amplified using the rbcLaF/R primer pair, targeting a 530-bp region of the chloroplast rbcL gene through the polymerase chain reaction (PCR). Lastly, gut content analysis was performed on 11 psylloid species using the same primer pair through PCR to detect citrus DNA. A total of 4,900 psylloids belonging to five families (i.e. Aphalaridae, Carsidaridae, Liviidae, Psyllidae and Triozidae), 19 genera and 47 species, were collected in citrus environments. More psylloids were recorded in Limpopo (3,754) than in Mpumalanga (1,146). The most abundant species were Pauropsylla trichaeta (1,680), followed by Diaphorina punctulata (466), Trioza erytreae (426), Diaphorina virgata (371), Euryconus sp. (358), Cacopsylla sp. (311), Retroacizzia mopanei (263), Acizzia russellae-group (240), Acizzia sp.3 (216) and Acizzia sp.2 (140). Yellow sticky traps captured 3,265 psylloids in citrus orchards, while an insect sweep-net collected 1,635 psylloids (477 from citrus orchards and 1,158 from adjacent natural vegetation). Data from the insect sweep-net revealed that 22 psylloid species were recorded on citrus. In comparison, nine psylloid species were found on Vachellia spp. and unidentified plant species separately, whereas six, three and two psylloid species were recorded on marula, Ficus sp. and mopane, respectively. The abundance, richness and community structure of psylloids differed significantly between the collection methods, provinces and among plant species. The rbcLaF/R primer pair amplified all citrus leaf gDNA samples, producing amplicons of the targeted 530-bp size. The PCR analysis of 11 psylloid species showed that the rbcLaF/R primer pair amplified plant DNA, with PCR-amplified plant DNA samples producing amplicons between 500-bp and 750-bp in the gut contents of five psyllid species: Diaphorina punctulata, Diaphorina virgata, Diaphorina zebrana, Euryconus sp. and Trioza erytreae. However, the targeted 530-bp plant DNA region was only amplified from the gut contents of Euryconus sp. and Diaphorina punctulata. This study documented psylloid diversity and community structure within commercial citrus environments. The findings indicate that the community of psylloids was diverse in citrus environments, with yellow sticky traps being more effective in monitoring different psyllid species within these environments. Furthermore, the PCR analysis detected citrus DNA in the gut contents of Euryconus sp. and Diaphorina punctulata, suggesting that they could be nibbling on citrus when their specific or main host-plants adjacent to citrus orchards are depleted. However, these insects do not lay their eggs or complete their life cycle on citrus, further confirming that citrus is not their host-plant. Thus, further studies, including Sanger sequencing of PCR-amplified plant DNA, are recommended to confirm the ingested plant species, and host-specific testing including infection trials needs to be conducted. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Authors: Moagi, Raynold
- Date: 2024-10-11
- Subjects: Citrus Diseases and pests South Africa , Candidatus Liberibacter , Psylloidea , Polymerase chain reaction , Insect trapping Equipment and supplies , Pests Control
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/464417 , vital:76509
- Description: Psylloids (Hemiptera: Psylloidea), constitute a group of plant sap-sucking insects, some of which are economically significant pests in different ecosystems due to their potential to transmit Gram-negative bacteria, such as the Candidatus Liberibacter species. The African citrus triozid (ACT), Trioza erytreae (Del Guercio), which transmits African citrus greening and the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, which transmits Asian citrus greening are significant threats to citrus. Asian citrus psyllid poses a global economic threat due to its ability to vector “Candidatus Liberibacter asiaticus” (CLas), which can rapidly kill citrus trees. However, both ACP and CLas are currently not present in southern Africa but are present in East and West Africa. In the Afrotropical region, 71 triozid species are known to occur and approximately 41 described Diaphorina species in southern Africa. Currently, two indigenous Diaphorina species, Diaphorina punctulata and Diaphorina zebrana have been documented to feed on citrus. There is a significant knowledge gap regarding the ecological roles of other indigenous psylloid species occurring within the citrus environments. Therefore, this study aimed to: (i) determine the diversity and community structure of psylloid species in citrus environments, and (ii) their host ranges through DNA analysis of gut contents to determine if they fed on citrus. Field surveys were carried out across 12 distinct commercial citrus environments across Limpopo and Mpumalanga provinces between 2022 and 2023. Psylloids were collected using yellow sticky traps and an insect sweep-net. Collected psylloid specimens were preserved in 70% ethanol vials and identified to the lowest possible taxonomic level (i.e. genus or species) using both published and unpublished dichotomous identification keys. Furthermore, citrus leaf samples were collected from the same plants on which psylloids were found in the orchards. Genomic DNA (gDNA) was extracted from both leaf and psylloid samples using two different DNA extraction methods. To confirm if citrus DNA could be detected in the psylloid guts, all leaf gDNA samples were initially amplified using the rbcLaF/R primer pair, targeting a 530-bp region of the chloroplast rbcL gene through the polymerase chain reaction (PCR). Lastly, gut content analysis was performed on 11 psylloid species using the same primer pair through PCR to detect citrus DNA. A total of 4,900 psylloids belonging to five families (i.e. Aphalaridae, Carsidaridae, Liviidae, Psyllidae and Triozidae), 19 genera and 47 species, were collected in citrus environments. More psylloids were recorded in Limpopo (3,754) than in Mpumalanga (1,146). The most abundant species were Pauropsylla trichaeta (1,680), followed by Diaphorina punctulata (466), Trioza erytreae (426), Diaphorina virgata (371), Euryconus sp. (358), Cacopsylla sp. (311), Retroacizzia mopanei (263), Acizzia russellae-group (240), Acizzia sp.3 (216) and Acizzia sp.2 (140). Yellow sticky traps captured 3,265 psylloids in citrus orchards, while an insect sweep-net collected 1,635 psylloids (477 from citrus orchards and 1,158 from adjacent natural vegetation). Data from the insect sweep-net revealed that 22 psylloid species were recorded on citrus. In comparison, nine psylloid species were found on Vachellia spp. and unidentified plant species separately, whereas six, three and two psylloid species were recorded on marula, Ficus sp. and mopane, respectively. The abundance, richness and community structure of psylloids differed significantly between the collection methods, provinces and among plant species. The rbcLaF/R primer pair amplified all citrus leaf gDNA samples, producing amplicons of the targeted 530-bp size. The PCR analysis of 11 psylloid species showed that the rbcLaF/R primer pair amplified plant DNA, with PCR-amplified plant DNA samples producing amplicons between 500-bp and 750-bp in the gut contents of five psyllid species: Diaphorina punctulata, Diaphorina virgata, Diaphorina zebrana, Euryconus sp. and Trioza erytreae. However, the targeted 530-bp plant DNA region was only amplified from the gut contents of Euryconus sp. and Diaphorina punctulata. This study documented psylloid diversity and community structure within commercial citrus environments. The findings indicate that the community of psylloids was diverse in citrus environments, with yellow sticky traps being more effective in monitoring different psyllid species within these environments. Furthermore, the PCR analysis detected citrus DNA in the gut contents of Euryconus sp. and Diaphorina punctulata, suggesting that they could be nibbling on citrus when their specific or main host-plants adjacent to citrus orchards are depleted. However, these insects do not lay their eggs or complete their life cycle on citrus, further confirming that citrus is not their host-plant. Thus, further studies, including Sanger sequencing of PCR-amplified plant DNA, are recommended to confirm the ingested plant species, and host-specific testing including infection trials needs to be conducted. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
Biology and management of the fruit piercing moth Serrodes partita in citrus orchards
- Authors: Mushore, Tapiwa Gift
- Date: 2024-10-11
- Subjects: Baculoviruses , Moths Monitoring , Pests Control , Insect traps , Citrus Diseases and pests South Africa Kat River Valley , Catapult moth
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/466792 , vital:76779 , DOI https://doi.org/10.21504/10962/466792
- Description: The fruit-piercing moth, Serrodes partita (Fabricius) (Lepidoptera: Erebidae), is a polyphagous, multivoltine pest of citrus. This insect has a distinct geographical separation between its larval and adult stages, each with different feeding patterns. During the larval stage, it primarily acts as a forest defoliator, feeding mostly on Jacket plum, Pappea capensis Eckl. & Zeyh. (Sapindaceae). In contrast, the adult stage of this moth feeds on both tropical and subtropical fruit, including citrus and can cause serious economic losses. The adult moth uses its sclerotised proboscis to pierce the skin of ripening or ripe fruit, from which it extracts the juice. This piercing action initiates a fermentation process within the fruit, attracting other secondary-feeding moths, commonly referred to as fruit-sucking moths. As a result of the feeding activity, the affected fruit eventually rot, drop to the ground, and become unsuitable for the market. Serrodes partita exhibits an outbreak life strategy, reoccurring every 5 to 10 years. In South Africa's Eastern Cape Province, specifically in the Upper Kat River Valley, citrus growers have expressed concerns about the impact of this moth on soft citrus (Satsumas and Clementines). This raises the possibility of a shift in the population dynamics of S. partita, where these occurrences become more frequent and less sporadic. Such a trend poses a significant threat to fruit arboriculture in the Eastern Cape region. Currently, there are limited management strategies available for managing fruit-piecing moths. The use of pesticides is not a feasible option for ripe or nearly ripe fruit, and is ineffective against the adult moth. Alternative control methods, such as orchard netting and light barriers, either come with high costs or are impractical for large-scale citrus production. Given the limited range of management options, combined with the moth's tendency for sudden outbreaks, citrus growers find themselves without effective means to manage this pest. The objective of this study was, therefore, to investigate the biology of S. partita and explore various control options to effectively manage this pest. Research focused on the biology and laboratory rearing of larval stages of S. partita. The flight behaviour, feeding patterns, and preferences of adult S. partita within citrus orchards were also explored. The aim was to elucidate key fundamental aspects, including whether the same population frequents a particular orchard, and if infestations within orchards exhibit a specific direction. Lure type and lure presentation method trials were conducted to determine the most effective lure and trap design. Seasonal monitoring of S. partita in soft citrus orchards was conducted over three years to determine its outbreak status in the Committee’s Drift area and the role of weather variables in the activity of the moth. Damage assessments were also conducted alongside monitoring to determine the level of damage inflicted by S. partita. Natural enemies associated with S. partita were explored to determine the prevalence and causes of mortality in late instars during laboratory rearing. Rearing S. partita on an artificial diet was unsuccessful despite several modifications. The moth, however, completed its entire life cycle on its natural host, P. capensis in the laboratory. The total life cycle from egg to adult took 80.7 ± 3.6 days, the larval stage lasted 52.3 ± 2.8 days, and the pupal stage lasted 25.8 ± 3.6 days at 21°C. The investigation into the biology of S. partita also brought attention to the most susceptible stages of its growth, with high mortality rates recorded among neonates and late instars. The findings of the study revealed directional patterns of moth infestations, with higher numbers observed at the orchard's periphery leading towards natural vegetation. This raises the prospect of using sacrificial rows on the edge of a citrus orchard to concentrate moth feeding damage during outbreak years. Using a mark and recapture technique, the study showed that a relatively small proportion (4.5 %) of moths tended to revisit the same orchard. The moths strongly preferred damaged fruit (85 %) over undamaged fruit. Visible damage (rotting symptoms) typically became apparent within 3 to 5 days. Satsumas had a higher number of feeding scars (2.1) than Clementines (1.08), highlighting their susceptibility. The study also established that, on average, pierced soft citrus fruit takes about four days to display symptoms of decay. Synthetic proprietary Australian lures were ineffective at attracting the moth, whereas fresh bananas proved to be a successful lure. Furthermore, the addition of both Agar and Super absorbent polymer showed promise as thickening agents to enhance the longevity of fresh bananas in traps. The effectiveness of various trap designs was compared, including the funnel trap, delta trap, bucket trap, and circular trap, in capturing fruit-feeding moths. The funnel trap performed best as it captured the most moths, followed by the delta trap, Lynfield trap and disc trap, respectively. Additionally, an electronic enhancement to the funnel trap, incorporating a zapper element, improved efficiency. However, efforts to exploit both visual and olfactory cues through the inclusion of an Ultraviolet (UV) light component did not improve its effectiveness. No extensive outbreaks were recorded during the study; however, population variations of S. partita populations were recorded. Annual trends showed two population peaks, with the first peak recorded from December to March, while the second peak was recorded from April to July. The activity of the moths also differed across different months, with the highest peaks recorded in May, while no moths were recorded from August to November. Both cultivar type and farm location did not influence the occurrence of the moth. Meanwhile cumulative weather parameters (rainfall, temperature and humidity) from the four months prior to occurrence influenced the activity of S. partita. Temperature determined the timing of the outbreak, while rainfall determined the magnitude of the outbreak. Damage assessment showed very low fruit damage by S. partita throughout the monitoring period. Varying levels of infestation by a tachinid fly, 4 % and 35 %, were recorded for 2021 and 2022, respectively. The tachinid parasitoid could not be identified at the species level. A novel baculovirus, tentatively classified as S. partita NPV (SepaNPV), was identified as the larval mortality causative agent. This study enhanced our understanding of S. partita's biology and population dynamics, providing valuable insights for developing effective management strategies against this economically impactful citrus pest. Future research should focus on refining control measures and addressing the challenges of the adult moth's elusive nature. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Authors: Mushore, Tapiwa Gift
- Date: 2024-10-11
- Subjects: Baculoviruses , Moths Monitoring , Pests Control , Insect traps , Citrus Diseases and pests South Africa Kat River Valley , Catapult moth
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/466792 , vital:76779 , DOI https://doi.org/10.21504/10962/466792
- Description: The fruit-piercing moth, Serrodes partita (Fabricius) (Lepidoptera: Erebidae), is a polyphagous, multivoltine pest of citrus. This insect has a distinct geographical separation between its larval and adult stages, each with different feeding patterns. During the larval stage, it primarily acts as a forest defoliator, feeding mostly on Jacket plum, Pappea capensis Eckl. & Zeyh. (Sapindaceae). In contrast, the adult stage of this moth feeds on both tropical and subtropical fruit, including citrus and can cause serious economic losses. The adult moth uses its sclerotised proboscis to pierce the skin of ripening or ripe fruit, from which it extracts the juice. This piercing action initiates a fermentation process within the fruit, attracting other secondary-feeding moths, commonly referred to as fruit-sucking moths. As a result of the feeding activity, the affected fruit eventually rot, drop to the ground, and become unsuitable for the market. Serrodes partita exhibits an outbreak life strategy, reoccurring every 5 to 10 years. In South Africa's Eastern Cape Province, specifically in the Upper Kat River Valley, citrus growers have expressed concerns about the impact of this moth on soft citrus (Satsumas and Clementines). This raises the possibility of a shift in the population dynamics of S. partita, where these occurrences become more frequent and less sporadic. Such a trend poses a significant threat to fruit arboriculture in the Eastern Cape region. Currently, there are limited management strategies available for managing fruit-piecing moths. The use of pesticides is not a feasible option for ripe or nearly ripe fruit, and is ineffective against the adult moth. Alternative control methods, such as orchard netting and light barriers, either come with high costs or are impractical for large-scale citrus production. Given the limited range of management options, combined with the moth's tendency for sudden outbreaks, citrus growers find themselves without effective means to manage this pest. The objective of this study was, therefore, to investigate the biology of S. partita and explore various control options to effectively manage this pest. Research focused on the biology and laboratory rearing of larval stages of S. partita. The flight behaviour, feeding patterns, and preferences of adult S. partita within citrus orchards were also explored. The aim was to elucidate key fundamental aspects, including whether the same population frequents a particular orchard, and if infestations within orchards exhibit a specific direction. Lure type and lure presentation method trials were conducted to determine the most effective lure and trap design. Seasonal monitoring of S. partita in soft citrus orchards was conducted over three years to determine its outbreak status in the Committee’s Drift area and the role of weather variables in the activity of the moth. Damage assessments were also conducted alongside monitoring to determine the level of damage inflicted by S. partita. Natural enemies associated with S. partita were explored to determine the prevalence and causes of mortality in late instars during laboratory rearing. Rearing S. partita on an artificial diet was unsuccessful despite several modifications. The moth, however, completed its entire life cycle on its natural host, P. capensis in the laboratory. The total life cycle from egg to adult took 80.7 ± 3.6 days, the larval stage lasted 52.3 ± 2.8 days, and the pupal stage lasted 25.8 ± 3.6 days at 21°C. The investigation into the biology of S. partita also brought attention to the most susceptible stages of its growth, with high mortality rates recorded among neonates and late instars. The findings of the study revealed directional patterns of moth infestations, with higher numbers observed at the orchard's periphery leading towards natural vegetation. This raises the prospect of using sacrificial rows on the edge of a citrus orchard to concentrate moth feeding damage during outbreak years. Using a mark and recapture technique, the study showed that a relatively small proportion (4.5 %) of moths tended to revisit the same orchard. The moths strongly preferred damaged fruit (85 %) over undamaged fruit. Visible damage (rotting symptoms) typically became apparent within 3 to 5 days. Satsumas had a higher number of feeding scars (2.1) than Clementines (1.08), highlighting their susceptibility. The study also established that, on average, pierced soft citrus fruit takes about four days to display symptoms of decay. Synthetic proprietary Australian lures were ineffective at attracting the moth, whereas fresh bananas proved to be a successful lure. Furthermore, the addition of both Agar and Super absorbent polymer showed promise as thickening agents to enhance the longevity of fresh bananas in traps. The effectiveness of various trap designs was compared, including the funnel trap, delta trap, bucket trap, and circular trap, in capturing fruit-feeding moths. The funnel trap performed best as it captured the most moths, followed by the delta trap, Lynfield trap and disc trap, respectively. Additionally, an electronic enhancement to the funnel trap, incorporating a zapper element, improved efficiency. However, efforts to exploit both visual and olfactory cues through the inclusion of an Ultraviolet (UV) light component did not improve its effectiveness. No extensive outbreaks were recorded during the study; however, population variations of S. partita populations were recorded. Annual trends showed two population peaks, with the first peak recorded from December to March, while the second peak was recorded from April to July. The activity of the moths also differed across different months, with the highest peaks recorded in May, while no moths were recorded from August to November. Both cultivar type and farm location did not influence the occurrence of the moth. Meanwhile cumulative weather parameters (rainfall, temperature and humidity) from the four months prior to occurrence influenced the activity of S. partita. Temperature determined the timing of the outbreak, while rainfall determined the magnitude of the outbreak. Damage assessment showed very low fruit damage by S. partita throughout the monitoring period. Varying levels of infestation by a tachinid fly, 4 % and 35 %, were recorded for 2021 and 2022, respectively. The tachinid parasitoid could not be identified at the species level. A novel baculovirus, tentatively classified as S. partita NPV (SepaNPV), was identified as the larval mortality causative agent. This study enhanced our understanding of S. partita's biology and population dynamics, providing valuable insights for developing effective management strategies against this economically impactful citrus pest. Future research should focus on refining control measures and addressing the challenges of the adult moth's elusive nature. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
Evaluating baculovirus mixtures against false codling moth Thaumatotibia leucotreta Meyrick. (Lepidoptera: Tortricidae)
- Authors: Tole, Siviwe
- Date: 2024-10-11
- Subjects: False codling moth Biological control , Baculoviruses , Integrated pest management , Natural pesticides , Granulovirus
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/463996 , vital:76464
- Description: False codling moth (FCM), Thaumatotibia leucotreta, is an important pest of citrus, stone fruit, avocados, peppers, and other important agricultural crops in southern Africa. Baculovirus-based biopesticides are components in an integrated pest management (IPM) programme to manage the pest in the field. Cryptogran™ and Cryptex™ which are CrleGV-SA based-biopesticides have been effective in the control of T. leucotreta for the past 15 years. Recently, CrpeNPV-based Multimax™ and Codlmax™ have been commercialised to control T. leucotreta and other important agricultural pests. Despite these viruses being relatively host-specific and safe to humans and animals in comparison to chemical insecticides, their application is hindered by their slow speed of kill, sensitivity to UV light, and the potential for insect resistance. Research investigating the effects of mixed baculoviral interactions against target pests has been a growing field of interest due to their potential to overcome such shortcomings. Previous studies using a combination of CrleGV-SA and CrpeNPV against T. leucotreta observed a reduction in lethal concentration in laboratory bioassays, indicating that such mixtures may have the potential for application in the field. This has led to the motivation to investigate further interactions between CrleGV-SA in combination with CrpeNPV, CpGV-M, and HearNPV-Au to understand better how these viruses interact and to determine whether synergistic, additive, or antagonistic interactions can occur against T. leucotreta. The outcome of these interactions will inform researchers and farmers about best practices concerning these viruses should they be combined against T. leucotreta in the future. Prior to performing mixed baculovirus infections in laboratory bioassays, oligonucleotides targeting unique regions in the viral genomes of CrleGV-SA, CrpeNPV, CpGV-M, and HearNPV-Au were designed using Primer-BLAST. The specificity of these oligonucleotides was further tested in silico using Geneious R11 software (11.1.5). The stocks of CrpeNPV, CpGV-M, and HearNPV-Au were purified using crude OB extraction from diseased C. peltastica, C. pomonella, and H. armigera larval cadavers provided by River Bioscience (Pty) Ltd (Gqeberha, South Africa). The stock of CrleGV-SA was purified using crude OB extraction from infected T. leucotreta cadavers. Subsequently, the unique oligonucleotides were used in PCR assays to detect if the samples contained the baculoviruses of interest. Amplicons of the expected sizes were generated indicating the presence of CrleGV-SA, CrpeNPV, CpGV-M, and HearNPV-Au in each of the samples. The OBs were counted using darkfield microscopy and a counting chamber before the single and mixed infections were initiated against T. leucotreta neonate larvae. Surface-dose biological assays were used to evaluate the relative virulence in terms of lethal concentration of CrleGV-SA, CrpeNPV, and CpGV-M, alone against T. leucotreta. After 7 days, the dose mortality data was analysed using “drc” in R studio and the LC50 and LC90 were compared amongst each virus. The CrleGV-SA treatment was estimated to be the most virulent in comparison to CrpeNPV and CpGV-M. A dose discriminate assay confirmed that HearNPV does not cause mortality in T. leucotreta. Similarly, the relative virulence in terms of lethal concentration of CrleGV-SA in various ratios in combination with CrpeNPV, CpGV-M, and HearNPV-Au was determined using 7-day surface dose biological assays. The CrleGV/CrpeNPV was the most virulent mixture with lower LC50 and LC90 values measured in comparison to CrleGV/CpGV and CrleGV/HearNPV, respectively. The Tammes Bakuniak graphic method confirmed the CrleGV/CrpeNPV, CrleGV/CpGV, and CrleGV/HearNPV mixtures to be antagonistic against T. leucotreta neonate larvae in terms of lethal concentration. The last aspect of the study was to determine the probable cause of larval death. A modified CTAB protocol was used to extract genomic DNA from neonate-sized T. leucotreta cadavers collected in single and mixed infection assays. The gDNA served as templates in PCR assays using the unique oligonucleotides. In single infections, the presence of CrleGV-SA in CrpeNPV and HearNPV inoculated larvae was observed. The results suggest possible covert infections of CrleGV-SA in the T. leucotreta colony which may be caused by virus infection or an unknown stress factor. The results from the mixed infections showed the presence of each virus in all replicates except for the CrleGV/CpGV and CrleGV/HearNPV mixtures. In the CrleGV/CpGV mixture, only CrleGV-SA was present in the last replicate, suggesting a possible competition for host resources. In the CrleGV/HearNPV mixture, only CrleGV-SA was detected in all 3 replicates, suggesting that HearNPV did not have any effect and the larvae died of the CrleGV-SA infection. This is the first study to report mixtures of CrleGV-SA in combination with CpGV-M and HearNPV-Au against T. leucotreta neonate larvae. Despite the antagonistic interactions observed in the evaluated mixtures, this study has laid a foundation to further investigate how these viruses interact in dual infections for the improved control of T. leucotreta. This may be done by evaluating different ratios and combinations of baculoviruses to those used in this study. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
- Full Text:
- Authors: Tole, Siviwe
- Date: 2024-10-11
- Subjects: False codling moth Biological control , Baculoviruses , Integrated pest management , Natural pesticides , Granulovirus
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/463996 , vital:76464
- Description: False codling moth (FCM), Thaumatotibia leucotreta, is an important pest of citrus, stone fruit, avocados, peppers, and other important agricultural crops in southern Africa. Baculovirus-based biopesticides are components in an integrated pest management (IPM) programme to manage the pest in the field. Cryptogran™ and Cryptex™ which are CrleGV-SA based-biopesticides have been effective in the control of T. leucotreta for the past 15 years. Recently, CrpeNPV-based Multimax™ and Codlmax™ have been commercialised to control T. leucotreta and other important agricultural pests. Despite these viruses being relatively host-specific and safe to humans and animals in comparison to chemical insecticides, their application is hindered by their slow speed of kill, sensitivity to UV light, and the potential for insect resistance. Research investigating the effects of mixed baculoviral interactions against target pests has been a growing field of interest due to their potential to overcome such shortcomings. Previous studies using a combination of CrleGV-SA and CrpeNPV against T. leucotreta observed a reduction in lethal concentration in laboratory bioassays, indicating that such mixtures may have the potential for application in the field. This has led to the motivation to investigate further interactions between CrleGV-SA in combination with CrpeNPV, CpGV-M, and HearNPV-Au to understand better how these viruses interact and to determine whether synergistic, additive, or antagonistic interactions can occur against T. leucotreta. The outcome of these interactions will inform researchers and farmers about best practices concerning these viruses should they be combined against T. leucotreta in the future. Prior to performing mixed baculovirus infections in laboratory bioassays, oligonucleotides targeting unique regions in the viral genomes of CrleGV-SA, CrpeNPV, CpGV-M, and HearNPV-Au were designed using Primer-BLAST. The specificity of these oligonucleotides was further tested in silico using Geneious R11 software (11.1.5). The stocks of CrpeNPV, CpGV-M, and HearNPV-Au were purified using crude OB extraction from diseased C. peltastica, C. pomonella, and H. armigera larval cadavers provided by River Bioscience (Pty) Ltd (Gqeberha, South Africa). The stock of CrleGV-SA was purified using crude OB extraction from infected T. leucotreta cadavers. Subsequently, the unique oligonucleotides were used in PCR assays to detect if the samples contained the baculoviruses of interest. Amplicons of the expected sizes were generated indicating the presence of CrleGV-SA, CrpeNPV, CpGV-M, and HearNPV-Au in each of the samples. The OBs were counted using darkfield microscopy and a counting chamber before the single and mixed infections were initiated against T. leucotreta neonate larvae. Surface-dose biological assays were used to evaluate the relative virulence in terms of lethal concentration of CrleGV-SA, CrpeNPV, and CpGV-M, alone against T. leucotreta. After 7 days, the dose mortality data was analysed using “drc” in R studio and the LC50 and LC90 were compared amongst each virus. The CrleGV-SA treatment was estimated to be the most virulent in comparison to CrpeNPV and CpGV-M. A dose discriminate assay confirmed that HearNPV does not cause mortality in T. leucotreta. Similarly, the relative virulence in terms of lethal concentration of CrleGV-SA in various ratios in combination with CrpeNPV, CpGV-M, and HearNPV-Au was determined using 7-day surface dose biological assays. The CrleGV/CrpeNPV was the most virulent mixture with lower LC50 and LC90 values measured in comparison to CrleGV/CpGV and CrleGV/HearNPV, respectively. The Tammes Bakuniak graphic method confirmed the CrleGV/CrpeNPV, CrleGV/CpGV, and CrleGV/HearNPV mixtures to be antagonistic against T. leucotreta neonate larvae in terms of lethal concentration. The last aspect of the study was to determine the probable cause of larval death. A modified CTAB protocol was used to extract genomic DNA from neonate-sized T. leucotreta cadavers collected in single and mixed infection assays. The gDNA served as templates in PCR assays using the unique oligonucleotides. In single infections, the presence of CrleGV-SA in CrpeNPV and HearNPV inoculated larvae was observed. The results suggest possible covert infections of CrleGV-SA in the T. leucotreta colony which may be caused by virus infection or an unknown stress factor. The results from the mixed infections showed the presence of each virus in all replicates except for the CrleGV/CpGV and CrleGV/HearNPV mixtures. In the CrleGV/CpGV mixture, only CrleGV-SA was present in the last replicate, suggesting a possible competition for host resources. In the CrleGV/HearNPV mixture, only CrleGV-SA was detected in all 3 replicates, suggesting that HearNPV did not have any effect and the larvae died of the CrleGV-SA infection. This is the first study to report mixtures of CrleGV-SA in combination with CpGV-M and HearNPV-Au against T. leucotreta neonate larvae. Despite the antagonistic interactions observed in the evaluated mixtures, this study has laid a foundation to further investigate how these viruses interact in dual infections for the improved control of T. leucotreta. This may be done by evaluating different ratios and combinations of baculoviruses to those used in this study. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
- Full Text:
The effect of biological control on the population dynamics of Pontederia crassipes Mart. (Pontederiaceae) and Salvinia minima Baker (Salviniales: Salviniaceae)
- Authors: Chikodza, Tressia
- Date: 2024-10-11
- Subjects: Pontederia crassipes , Salvinia minima , Pontederiaceae Biological control South Africa , Salviniaceae Biological control South Africa , Population dynamics , Secondary invasion
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/464462 , vital:76513
- Description: Pontederia crassipes is widely regarded as the most damaging floating aquatic weed in terms of its invasive traits, and its impact on aquatic ecosystems. Biological control using host-specific natural enemies is widely used for its control, with the most recent agent released being a planthopper, Megamelus scutellaris, in South Africa and the USA. In South Africa, inundative releases of M. scutellaris have been shown to control the weed even at eutrophic, and high elevation cold sites, such as Hartbeespoort Dam, arguably Africa’s most hypertrophic impoundment. However, subsequent to the control of P. crassipes on Hartbeespoort Dam, in 2021, a secondary invader, Salvinia minima, dominated the water system. Salvinia minima is only known from a handful sites near Hartbeespoort Dam and there is currently no approved biocontrol agent for it. The population dynamics of P. crassipes and S. minima at the Dam were overall interchanging due to insect feeding on M. scutellaris reducing P. crassipes populations thus allowing S. minima to become dominant. Intense feeding from M. scutellaris resulted in the reduction of P. crassipes populations. During late autumn and winter, remaining P. crassipes plants experienced frost damage from cold temperatures. The decrease in P. crassipes allowed S. minima to successfully invade, potentially as the result of less competition from P. crassipes, and therefore available resources, such as space, nutrients, and light. However, as spring approached, P. crassipes regenerated from its seed bank, and S. minima populations diminished. The changes in dominance were observed in 2021 and 2022 but in 2023, this trend was not evident as S. minima did not dominate as in previous years. This thesis investigated the combination of competition and herbivory by M. scutellaris on the vigour of P. crassipes and S. minima to understand the dynamics of these two highly invasive species. Competitive abilities of P. crassipes and S. minima were determined using an inverse linear model with plant weight as the yield variable. In the absence of herbivory, P. crassipes, was 4 times more competitive than S. minima, but as competitive when exposed to M. scutellaris feeding. Salvinia minima was 1.2 times as aggressive as P. crassipes in the absence of herbivory, but 2.6 times as competitive when M. scutellaris was established on P. crassipes. In the presence of herbivory on P. crassipes, interspecific competition coefficients from P. crassipes on S. minima were no longer statistically significant. These results indicate that the competitive ability of P. crassipes was reduced through herbivory when grown with S. minima, explaining the temporal dominance between the two species at Hartbeespoort Dam. Some studies have reported that P. crassipes releases allelochemicals as a competitive strategy to algae and phytoplankton. Previously identified allelochemicals include N-phenyl-1-naphthylamine, N-phenyl-2-naphthylamine and linoleic acid. This thesis investigated the presence or absence of key chemical compounds released by P. crassipes during allelopathy with microbes and phytoplankton to determine its potential to inhibit S. minima growth. Methanol extracts from P. crassipes roots, leaves, and field samples underwent LC-ESI-MS/MS analysis, creating a molecular network to match chemical profiles. Of the investigated compounds, N-phenyl-1-naphthylamine and N-phenyl-2-naphthylamine were absent in P. crassipes and field samples, while linoleic acid was consistently found. Its presence suggests its potential defensive role against S. minima. Future research should explore allelochemical production in the presence of S. minima and algae to confirm if S. minima elicits an allelochemical response by P. crassipes, or whether the methods used here were insufficient to detect allelochemical production. Understanding whether P. crassipes employs allelochemicals, especially in the presence of S. minima, could shed light on its competitive advantage beyond its invasive nature at the Dam. , Thesis (MSc) -- Faculty of Science, Botany, 2024
- Full Text:
- Authors: Chikodza, Tressia
- Date: 2024-10-11
- Subjects: Pontederia crassipes , Salvinia minima , Pontederiaceae Biological control South Africa , Salviniaceae Biological control South Africa , Population dynamics , Secondary invasion
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/464462 , vital:76513
- Description: Pontederia crassipes is widely regarded as the most damaging floating aquatic weed in terms of its invasive traits, and its impact on aquatic ecosystems. Biological control using host-specific natural enemies is widely used for its control, with the most recent agent released being a planthopper, Megamelus scutellaris, in South Africa and the USA. In South Africa, inundative releases of M. scutellaris have been shown to control the weed even at eutrophic, and high elevation cold sites, such as Hartbeespoort Dam, arguably Africa’s most hypertrophic impoundment. However, subsequent to the control of P. crassipes on Hartbeespoort Dam, in 2021, a secondary invader, Salvinia minima, dominated the water system. Salvinia minima is only known from a handful sites near Hartbeespoort Dam and there is currently no approved biocontrol agent for it. The population dynamics of P. crassipes and S. minima at the Dam were overall interchanging due to insect feeding on M. scutellaris reducing P. crassipes populations thus allowing S. minima to become dominant. Intense feeding from M. scutellaris resulted in the reduction of P. crassipes populations. During late autumn and winter, remaining P. crassipes plants experienced frost damage from cold temperatures. The decrease in P. crassipes allowed S. minima to successfully invade, potentially as the result of less competition from P. crassipes, and therefore available resources, such as space, nutrients, and light. However, as spring approached, P. crassipes regenerated from its seed bank, and S. minima populations diminished. The changes in dominance were observed in 2021 and 2022 but in 2023, this trend was not evident as S. minima did not dominate as in previous years. This thesis investigated the combination of competition and herbivory by M. scutellaris on the vigour of P. crassipes and S. minima to understand the dynamics of these two highly invasive species. Competitive abilities of P. crassipes and S. minima were determined using an inverse linear model with plant weight as the yield variable. In the absence of herbivory, P. crassipes, was 4 times more competitive than S. minima, but as competitive when exposed to M. scutellaris feeding. Salvinia minima was 1.2 times as aggressive as P. crassipes in the absence of herbivory, but 2.6 times as competitive when M. scutellaris was established on P. crassipes. In the presence of herbivory on P. crassipes, interspecific competition coefficients from P. crassipes on S. minima were no longer statistically significant. These results indicate that the competitive ability of P. crassipes was reduced through herbivory when grown with S. minima, explaining the temporal dominance between the two species at Hartbeespoort Dam. Some studies have reported that P. crassipes releases allelochemicals as a competitive strategy to algae and phytoplankton. Previously identified allelochemicals include N-phenyl-1-naphthylamine, N-phenyl-2-naphthylamine and linoleic acid. This thesis investigated the presence or absence of key chemical compounds released by P. crassipes during allelopathy with microbes and phytoplankton to determine its potential to inhibit S. minima growth. Methanol extracts from P. crassipes roots, leaves, and field samples underwent LC-ESI-MS/MS analysis, creating a molecular network to match chemical profiles. Of the investigated compounds, N-phenyl-1-naphthylamine and N-phenyl-2-naphthylamine were absent in P. crassipes and field samples, while linoleic acid was consistently found. Its presence suggests its potential defensive role against S. minima. Future research should explore allelochemical production in the presence of S. minima and algae to confirm if S. minima elicits an allelochemical response by P. crassipes, or whether the methods used here were insufficient to detect allelochemical production. Understanding whether P. crassipes employs allelochemicals, especially in the presence of S. minima, could shed light on its competitive advantage beyond its invasive nature at the Dam. , Thesis (MSc) -- Faculty of Science, Botany, 2024
- Full Text:
The expression and evaluation of CrpeNPV gp37 as a formulation additive for enhanced infectivity with CrleGV-SA and improved Thaumatotibia leucotreta control
- Authors: Muleya, Naho
- Date: 2024-10-11
- Subjects: Cryptophlebia leucotreta Biological control , False Codling Moth , Cryptophlebia leucotreta granulovirus , Cryptophlebia peltastica nucleopolyhedrovirus , Citrus Diseases and pests South Africa , Baculoviruses
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/463919 , vital:76457
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a significant pest native to Africa, causing damage to citrus and posing a threat to the export of fresh citrus in South Africa. Classified as a phytosanitary risk by several South African export markets, this pest necessitates effective control measures. Baculoviruses emerge as promising biological control agents against T. leucotreta due to their inherent safety and eco-friendly characteristics. Among these, Cryptophlebia leucotreta Granulovirus (CrleGV-SA) and Cryptophlebia peltastica Nucleopolyhedrovirus (CrpeNPV) stand out, both causing larval mortality upon infecting T. leucotreta. CrleGV-SA has been formulated into the products Cryptogran™, CryptoMax™ and Cryptex®, while CrpeNPV has been formulated into the product Multimax™. Both viruses are used in integrated pest management programmes to reduce fruit damage in agricultural fields, with CrleGV-SA having been employed against T. leucotreta for nearly 20 years in South Africa. However, these control options are limited by factors such as virulence and the slow speed of kill. This limitation can be addressed by exploiting potential synergistic relationships between baculoviruses infecting the same host. Previous studies have demonstrated that the truncated CpGV gp37 can enhance the infectivity of NPVs on other lepidopteran pests, such as Spodoptera exigua (Hübner). Although the mechanism behind this phenomenon remains unclear, it presents an opportunity to enhance the effectiveness of baculovirus-based management strategies. Notably, the genome of CrpeNPV encodes gp37, while CrleGV-SA lacks this gene. The potential interaction between CrleGV-SA and CrpeNPV gp37 remains unexplored. Therefore, investigating whether they exhibit synergistic or antagonistic effects is essential for optimising baculovirus-based management of T. leucotreta. This study aims to express CrpeNPV gp37 in a bacterial system and then evaluate its effect on larval mortality when combined with CrleGV-SA in laboratory bioassays. The initial step involved extracting genomic DNA (gDNA) from occlusion bodies (OBs) of CrpeNPV. A modified Quick DNA Miniprep plus kit was utilised, which entailed pre-treatment with Na2CO3 followed by neutralisation with Tris-HCI before gDNA extraction using the kit. Subsequently, the concentration of the gDNA was estimated using a Nanodrop spectrophotometer. Oligonucleotides targeting the CrpeNPV gp37 gene were designed for PCR amplification, with the gDNA serving as a template. The gp37 amplicon was identified through agarose gel electrophoresis and then gel purified in preparation for cloning. Secondly, the purified PCR product was cloned into the intermediate vector pJET1.2/blunt and then subcloned into the bacterial expression vector pCA528 through DNA ligation. The construction of recombinant plasmids (pJET-gp37 and pCA-gp37) was conducted and verified using Colony PCR, plasmid extraction, restriction enzyme analysis, and Sanger sequencing. Thirdly, the recombinant protein (6×His-SUMO-gp37) was expressed and purified using Nickel affinity chromatography and analysed through SDS-PAGE and Western blot techniques. The expression of 6×His-SUMO-gp37 was carried out at both 25 °C and 18 °C. A time course induction study was conducted, inducing transformed cells for 0-, 3-, 5-, and 24-hours post induction (hpi). SDS-PAGE and Western blotting of samples collected at various time points revealed that 6×His-SUMO-gp37, approximately 42 kDa in size, was visible from 3 hpi, with maximal expression at 24 hpi. Solubility analysis of 6×His-SUMO-gp37 was performed at both temperatures, showing solubility at 18 °C but predominantly present in the insoluble fraction. The soluble protein was purified under native conditions, while the insoluble protein was purified under denaturing conditions. Despite being unable to elute 6×His-SUMO-gp37 under native conditions, successful elution was achieved under denaturing conditions, confirmed via Western blot analysis. No further experiments were conducted on the eluted 6×His-SUMO-gp37 under denaturing conditions. Lastly, a preliminary surface dose bioassay was conducted to evaluate the efficacy of pelleted bacteria expressing 6×His-SUMO-gp37 in combination with CrleGV-SA against T. leucotreta neonates. Two lethal concentration doses of CrleGV-SA were prepared: a low concentration (2.96×104 OBs/mL) capable of killing 40 % of the T. leucotreta population, and a high concentration (2.96×105 OBs/mL) capable of killing 90 % of the population. The target protein, 6×His-SUMO-gp37, and the control, pCA528, were obtained by lysing the cells, centrifuging the samples, and collecting the insoluble fractions in pellet form. These fractions were then resuspended in PBS and used as treatments in combination with the prepared CrleGV-SA concentration doses. The concentration of the pellets was estimated using a Nanodrop spectrophotometer by measuring the absorbance at 280 nm. The bioassay results revealed that the combination of 100 μg/mL of pelleted bacteria expressing 6×His-SUMO-gp37 with CrleGV-SA had no effect on T. leucotreta larval mortality compared to CrleGV-SA alone. A one-way ANOVA was performed to assess differences among the virus treatment groups, concluding that no statistically significant differences were observed among the groups. The experiments in this study provided valuable insights for future research, particularly in exploring the use of a protein-virus combination as a novel method for pest control. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
- Full Text:
- Authors: Muleya, Naho
- Date: 2024-10-11
- Subjects: Cryptophlebia leucotreta Biological control , False Codling Moth , Cryptophlebia leucotreta granulovirus , Cryptophlebia peltastica nucleopolyhedrovirus , Citrus Diseases and pests South Africa , Baculoviruses
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/463919 , vital:76457
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a significant pest native to Africa, causing damage to citrus and posing a threat to the export of fresh citrus in South Africa. Classified as a phytosanitary risk by several South African export markets, this pest necessitates effective control measures. Baculoviruses emerge as promising biological control agents against T. leucotreta due to their inherent safety and eco-friendly characteristics. Among these, Cryptophlebia leucotreta Granulovirus (CrleGV-SA) and Cryptophlebia peltastica Nucleopolyhedrovirus (CrpeNPV) stand out, both causing larval mortality upon infecting T. leucotreta. CrleGV-SA has been formulated into the products Cryptogran™, CryptoMax™ and Cryptex®, while CrpeNPV has been formulated into the product Multimax™. Both viruses are used in integrated pest management programmes to reduce fruit damage in agricultural fields, with CrleGV-SA having been employed against T. leucotreta for nearly 20 years in South Africa. However, these control options are limited by factors such as virulence and the slow speed of kill. This limitation can be addressed by exploiting potential synergistic relationships between baculoviruses infecting the same host. Previous studies have demonstrated that the truncated CpGV gp37 can enhance the infectivity of NPVs on other lepidopteran pests, such as Spodoptera exigua (Hübner). Although the mechanism behind this phenomenon remains unclear, it presents an opportunity to enhance the effectiveness of baculovirus-based management strategies. Notably, the genome of CrpeNPV encodes gp37, while CrleGV-SA lacks this gene. The potential interaction between CrleGV-SA and CrpeNPV gp37 remains unexplored. Therefore, investigating whether they exhibit synergistic or antagonistic effects is essential for optimising baculovirus-based management of T. leucotreta. This study aims to express CrpeNPV gp37 in a bacterial system and then evaluate its effect on larval mortality when combined with CrleGV-SA in laboratory bioassays. The initial step involved extracting genomic DNA (gDNA) from occlusion bodies (OBs) of CrpeNPV. A modified Quick DNA Miniprep plus kit was utilised, which entailed pre-treatment with Na2CO3 followed by neutralisation with Tris-HCI before gDNA extraction using the kit. Subsequently, the concentration of the gDNA was estimated using a Nanodrop spectrophotometer. Oligonucleotides targeting the CrpeNPV gp37 gene were designed for PCR amplification, with the gDNA serving as a template. The gp37 amplicon was identified through agarose gel electrophoresis and then gel purified in preparation for cloning. Secondly, the purified PCR product was cloned into the intermediate vector pJET1.2/blunt and then subcloned into the bacterial expression vector pCA528 through DNA ligation. The construction of recombinant plasmids (pJET-gp37 and pCA-gp37) was conducted and verified using Colony PCR, plasmid extraction, restriction enzyme analysis, and Sanger sequencing. Thirdly, the recombinant protein (6×His-SUMO-gp37) was expressed and purified using Nickel affinity chromatography and analysed through SDS-PAGE and Western blot techniques. The expression of 6×His-SUMO-gp37 was carried out at both 25 °C and 18 °C. A time course induction study was conducted, inducing transformed cells for 0-, 3-, 5-, and 24-hours post induction (hpi). SDS-PAGE and Western blotting of samples collected at various time points revealed that 6×His-SUMO-gp37, approximately 42 kDa in size, was visible from 3 hpi, with maximal expression at 24 hpi. Solubility analysis of 6×His-SUMO-gp37 was performed at both temperatures, showing solubility at 18 °C but predominantly present in the insoluble fraction. The soluble protein was purified under native conditions, while the insoluble protein was purified under denaturing conditions. Despite being unable to elute 6×His-SUMO-gp37 under native conditions, successful elution was achieved under denaturing conditions, confirmed via Western blot analysis. No further experiments were conducted on the eluted 6×His-SUMO-gp37 under denaturing conditions. Lastly, a preliminary surface dose bioassay was conducted to evaluate the efficacy of pelleted bacteria expressing 6×His-SUMO-gp37 in combination with CrleGV-SA against T. leucotreta neonates. Two lethal concentration doses of CrleGV-SA were prepared: a low concentration (2.96×104 OBs/mL) capable of killing 40 % of the T. leucotreta population, and a high concentration (2.96×105 OBs/mL) capable of killing 90 % of the population. The target protein, 6×His-SUMO-gp37, and the control, pCA528, were obtained by lysing the cells, centrifuging the samples, and collecting the insoluble fractions in pellet form. These fractions were then resuspended in PBS and used as treatments in combination with the prepared CrleGV-SA concentration doses. The concentration of the pellets was estimated using a Nanodrop spectrophotometer by measuring the absorbance at 280 nm. The bioassay results revealed that the combination of 100 μg/mL of pelleted bacteria expressing 6×His-SUMO-gp37 with CrleGV-SA had no effect on T. leucotreta larval mortality compared to CrleGV-SA alone. A one-way ANOVA was performed to assess differences among the virus treatment groups, concluding that no statistically significant differences were observed among the groups. The experiments in this study provided valuable insights for future research, particularly in exploring the use of a protein-virus combination as a novel method for pest control. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
- Full Text:
Augmentation of Aphytis melinus DeBach (Hymenoptera: Aphelinidae) for the control of California red scale Aonidiella aurantii Maskell (Hemiptera: Diaspididae) on citrus
- De Beer, Ernst Friedrich Ludwig
- Authors: De Beer, Ernst Friedrich Ludwig
- Date: 2024-04-05
- Subjects: Aphytis melinus , Aphytis , Aonidiella aurantii , Citrus Diseases and pests South Africa , Pests Biological control
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/435638 , vital:73175 , DOI 10.21504/10962/435638
- Description: Aphytis lingnanensis was reared and tested in South Africa in the early 2000s for augmentation against red scale on citrus and was found to be ineffective. Aphytis melinus is now commercially available and it is important that the efficacy of augmentation thereof on red scale is determined locally. Field trials, fitness assessments and molecular identification on A. melinus from two insectaries were done. Field trials was done in seven, five and six pairs of comparable release and control orchards across the Eastern and Western Cape during the seasons of 2019/2020, 2020/2021 and 2021/2022 respectively. Red scale infestation was monitored and a sample of 20 infested fruit from each orchard was randomly collected every four weeks. Aphytis spp. responsible for parasitism were identified and the percentage parasitism recorded. Results of this study of field trials suggest that the augmentation of A. melinus did not significantly increase the level of parasitism above that of the untreated control. Five repetitions with six replicates of flight and longevity tests were performed with wasps from each insectary. Wasps in the longevity test from two insectaries were kept at 23 °C and 65% RH with honey. Flight tests were performed in tubes of 16 by 30 cm, with a light above a clear, sticky ceiling at 23 °C and 65% RH. On average in five replicates, 65%, 33% and 17% A. melinus wasps were alive on day one, five and 10 respectively. The overall sex ratio was 1.58 for females to males, but 1.05, 2.19 and 2.66 for non-flyers, non-crawlers, crawlers, and flyers respectively. In flight tests for both insectaries combined, only 36.97% of wasps could initiate flight in 24 h while 56.96% remained on the tube floor, and 6.05% attempted to crawl upwards. No significant differences in flight performance were recorded between the two insectaries. Wasps from the local insectary lived significantly longer during the longevity tests but were shorter in transit than wasps from the overseas insectary. COI genes were sequenced and compared against Genbank sequences using BLAST. Molecular identifications did not confirm morphological identifications for all species, indicating unexpected genetic complexity. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Authors: De Beer, Ernst Friedrich Ludwig
- Date: 2024-04-05
- Subjects: Aphytis melinus , Aphytis , Aonidiella aurantii , Citrus Diseases and pests South Africa , Pests Biological control
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/435638 , vital:73175 , DOI 10.21504/10962/435638
- Description: Aphytis lingnanensis was reared and tested in South Africa in the early 2000s for augmentation against red scale on citrus and was found to be ineffective. Aphytis melinus is now commercially available and it is important that the efficacy of augmentation thereof on red scale is determined locally. Field trials, fitness assessments and molecular identification on A. melinus from two insectaries were done. Field trials was done in seven, five and six pairs of comparable release and control orchards across the Eastern and Western Cape during the seasons of 2019/2020, 2020/2021 and 2021/2022 respectively. Red scale infestation was monitored and a sample of 20 infested fruit from each orchard was randomly collected every four weeks. Aphytis spp. responsible for parasitism were identified and the percentage parasitism recorded. Results of this study of field trials suggest that the augmentation of A. melinus did not significantly increase the level of parasitism above that of the untreated control. Five repetitions with six replicates of flight and longevity tests were performed with wasps from each insectary. Wasps in the longevity test from two insectaries were kept at 23 °C and 65% RH with honey. Flight tests were performed in tubes of 16 by 30 cm, with a light above a clear, sticky ceiling at 23 °C and 65% RH. On average in five replicates, 65%, 33% and 17% A. melinus wasps were alive on day one, five and 10 respectively. The overall sex ratio was 1.58 for females to males, but 1.05, 2.19 and 2.66 for non-flyers, non-crawlers, crawlers, and flyers respectively. In flight tests for both insectaries combined, only 36.97% of wasps could initiate flight in 24 h while 56.96% remained on the tube floor, and 6.05% attempted to crawl upwards. No significant differences in flight performance were recorded between the two insectaries. Wasps from the local insectary lived significantly longer during the longevity tests but were shorter in transit than wasps from the overseas insectary. COI genes were sequenced and compared against Genbank sequences using BLAST. Molecular identifications did not confirm morphological identifications for all species, indicating unexpected genetic complexity. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
The enemy release hypothesis and beyond: Lagarosiphon major invasion dynamics and management options for New Zealand using native natural enemies from South Africa
- Authors: Baso, Nompumelelo Catherine
- Date: 2024-04-05
- Subjects: Enemy release hypothesis , Lagarosiphon major Biological control New Zealand , Hydrellia , Submerged aquatic vegetation , Invasion ecology
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/435627 , vital:73174 , DOI 10.21504/10962/435627
- Description: Numerous scientific investigations have demonstrated the destructive impact that exotic species can have on ecosystem services beyond a specific threshold. There are many explanations for why introduced plants are likely to be more successful outside their native range. One such explanation is offered by the Enemy Release Hypothesis (ERH), which states that plants automatically become superior competitors outside of their natural range due to release from top-down stressors (herbivory, parasites, and diseases) that is evident in the absence of their natural enemies. The underlying assumption of the ERH is that natural enemies are important regulators of plant species populations, and that the pressures from these natural enemies are felt more readily by native species compared to alien plants. Consequently, in the absence of such pressures, the ERH assumes that exotic plants can allocate more resources towards growth and reproduction, while effectively maintaining accumulated biomass. Classical biological control has previously been cited as evidence for the enemy release hypothesis. Therefore, the overarching aim and theme of this thesis was to investigate the role of ERH on the invasiveness of Lagarosiphon major (Ridl.) Moss ex Wager (Hydrocharitaceae) in New Zealand. Firstly, a literature search and a meta-analysis was used to synthesize existing studies in order to test for general applicability of this hypothesis to aquatic plant invasions. Furthermore, an empirical investigation was conducted in order to directly quantify enemy release in L. major populations invaded areas of New Zealand. To achieve this, various plant parameters of this plant, overall macrophyte and invertebrate diversity were measured and compared between sites in the native range in South Africa and the invaded areas in New Zealand. Although the meta-analysis showed variable evidence for this hypothesis depending on various modulating factors such as study type, plant growth form and measured parameters, for L. major, there was strong evidence of enemy release. The biogeographical comparisons showed that L. major exhibited increased fitness in most of the invaded sites, marked by elevated biomass accumulation, significantly higher shoot production, and the displacement of native plant species. The observed fitness advantages were directly correlated to a decrease in herbivory diversity and pressure upon the plant's introduction to New Zealand. Unlike the native populations, which contend with the presence of at least four co-occurring herbivores, including specialist herbivores, the invaded range had a substantially lower herbivore diversity, with only Hygraula nitens Butler (Lepidoptera: Crambidae) syn. Nymphula nitens, significantly damaging L. major. These findings emphasize the importance of understanding invasion ecology and theories such as ERH in order to advance aquatic plant management and also present valuable insights for developing effective strategies to mitigate the impact of invasive alien species on aquatic ecosystems. Specifically, results from the empirical investigation provide evidence in support of the ERH and highlight the suitability of implementing biological control strategies to manage the L. major invasion in New Zealand. Previous studies have shown the suitability of two specialist herbivores, Hydrellia lagarosiphon Deeming (Diptera: Ephydridae), and Polypedilum tuburcinatum Andersen (Diptera: Chironomidae), as potential biological control agents. This control strategy presents a sustainable and ecologically responsible approach, promoting coexistence between exotic plants and native species rather than displacement through competitive exclusion. With the apparent dominance of L. major at various New Zealand localities, the subsequent objective of this thesis was to investigate the competitive interactions between L. major and another invasive Hydrocharitaceae, Egeria densa Planchon, as driven by herbivory. Combinations of two host specific Ephydrid flies, H. lagarosiphon and H. egeriae, were used at eight different factorial combination of planting densities. The analysis of plant parameters and the application of inverse linear models revealed that L. major often exhibits relatively higher fitness, especially in low monoculture treatments when the two insects were isolated. However, multiple inverse linear models revealed that actual competitive outcomes are dependent on factors such as initial plant density and herbivory regime, with competitive interactions generally being mild. Nevertheless, the presence of H. lagarosiphon resulted in facilitation of E. densa growth. Thus, even at lower densities, these insects still had an impact on the observed interactions, further emphasizing suitability as damaging biological control agents. Lastly, focusing on the abiotic component of L. major invasion, Species Distribution Models (SDMs) were employed to map potential suitable habitat for this species, as well as predict the consequences of climate change on this. Correlative and mechanistic modelling was also used to simulate suitable habitat for potential biological control agents, thus addressing the potential for mismatches between host plant distribution and insect suitable range. The Maximum Entropy Species Distribution Modelling (MaxEnt) algorithm revealed that more than 90% of all freshwater ecosystems in New Zealand are susceptible to L. major invasion, with suitability projected to expand further under future climate scenarios. Moreover, correlative modelling using this method suggests limited suitable habitat for both herbivores. However, degree-day modelling, which also takes into account the physiological requirements, showed that H. lagarosiphon has the potential to produce viable populations in several parts of New Zealand. Overall, this thesis explored the intricate web of biotic and abiotic factors influencing the success of L. major outside its native range. The results emphasize the potential impacts of climate change on the invasion potential and management strategies for L. major. The findings also advocate for the implementation of sustainable and ecologically sound management solutions, such as biological control, to manage this species. , Thesis (PhD) -- Faculty of Science, Botany, 2024
- Full Text:
- Authors: Baso, Nompumelelo Catherine
- Date: 2024-04-05
- Subjects: Enemy release hypothesis , Lagarosiphon major Biological control New Zealand , Hydrellia , Submerged aquatic vegetation , Invasion ecology
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/435627 , vital:73174 , DOI 10.21504/10962/435627
- Description: Numerous scientific investigations have demonstrated the destructive impact that exotic species can have on ecosystem services beyond a specific threshold. There are many explanations for why introduced plants are likely to be more successful outside their native range. One such explanation is offered by the Enemy Release Hypothesis (ERH), which states that plants automatically become superior competitors outside of their natural range due to release from top-down stressors (herbivory, parasites, and diseases) that is evident in the absence of their natural enemies. The underlying assumption of the ERH is that natural enemies are important regulators of plant species populations, and that the pressures from these natural enemies are felt more readily by native species compared to alien plants. Consequently, in the absence of such pressures, the ERH assumes that exotic plants can allocate more resources towards growth and reproduction, while effectively maintaining accumulated biomass. Classical biological control has previously been cited as evidence for the enemy release hypothesis. Therefore, the overarching aim and theme of this thesis was to investigate the role of ERH on the invasiveness of Lagarosiphon major (Ridl.) Moss ex Wager (Hydrocharitaceae) in New Zealand. Firstly, a literature search and a meta-analysis was used to synthesize existing studies in order to test for general applicability of this hypothesis to aquatic plant invasions. Furthermore, an empirical investigation was conducted in order to directly quantify enemy release in L. major populations invaded areas of New Zealand. To achieve this, various plant parameters of this plant, overall macrophyte and invertebrate diversity were measured and compared between sites in the native range in South Africa and the invaded areas in New Zealand. Although the meta-analysis showed variable evidence for this hypothesis depending on various modulating factors such as study type, plant growth form and measured parameters, for L. major, there was strong evidence of enemy release. The biogeographical comparisons showed that L. major exhibited increased fitness in most of the invaded sites, marked by elevated biomass accumulation, significantly higher shoot production, and the displacement of native plant species. The observed fitness advantages were directly correlated to a decrease in herbivory diversity and pressure upon the plant's introduction to New Zealand. Unlike the native populations, which contend with the presence of at least four co-occurring herbivores, including specialist herbivores, the invaded range had a substantially lower herbivore diversity, with only Hygraula nitens Butler (Lepidoptera: Crambidae) syn. Nymphula nitens, significantly damaging L. major. These findings emphasize the importance of understanding invasion ecology and theories such as ERH in order to advance aquatic plant management and also present valuable insights for developing effective strategies to mitigate the impact of invasive alien species on aquatic ecosystems. Specifically, results from the empirical investigation provide evidence in support of the ERH and highlight the suitability of implementing biological control strategies to manage the L. major invasion in New Zealand. Previous studies have shown the suitability of two specialist herbivores, Hydrellia lagarosiphon Deeming (Diptera: Ephydridae), and Polypedilum tuburcinatum Andersen (Diptera: Chironomidae), as potential biological control agents. This control strategy presents a sustainable and ecologically responsible approach, promoting coexistence between exotic plants and native species rather than displacement through competitive exclusion. With the apparent dominance of L. major at various New Zealand localities, the subsequent objective of this thesis was to investigate the competitive interactions between L. major and another invasive Hydrocharitaceae, Egeria densa Planchon, as driven by herbivory. Combinations of two host specific Ephydrid flies, H. lagarosiphon and H. egeriae, were used at eight different factorial combination of planting densities. The analysis of plant parameters and the application of inverse linear models revealed that L. major often exhibits relatively higher fitness, especially in low monoculture treatments when the two insects were isolated. However, multiple inverse linear models revealed that actual competitive outcomes are dependent on factors such as initial plant density and herbivory regime, with competitive interactions generally being mild. Nevertheless, the presence of H. lagarosiphon resulted in facilitation of E. densa growth. Thus, even at lower densities, these insects still had an impact on the observed interactions, further emphasizing suitability as damaging biological control agents. Lastly, focusing on the abiotic component of L. major invasion, Species Distribution Models (SDMs) were employed to map potential suitable habitat for this species, as well as predict the consequences of climate change on this. Correlative and mechanistic modelling was also used to simulate suitable habitat for potential biological control agents, thus addressing the potential for mismatches between host plant distribution and insect suitable range. The Maximum Entropy Species Distribution Modelling (MaxEnt) algorithm revealed that more than 90% of all freshwater ecosystems in New Zealand are susceptible to L. major invasion, with suitability projected to expand further under future climate scenarios. Moreover, correlative modelling using this method suggests limited suitable habitat for both herbivores. However, degree-day modelling, which also takes into account the physiological requirements, showed that H. lagarosiphon has the potential to produce viable populations in several parts of New Zealand. Overall, this thesis explored the intricate web of biotic and abiotic factors influencing the success of L. major outside its native range. The results emphasize the potential impacts of climate change on the invasion potential and management strategies for L. major. The findings also advocate for the implementation of sustainable and ecologically sound management solutions, such as biological control, to manage this species. , Thesis (PhD) -- Faculty of Science, Botany, 2024
- Full Text:
Developing a community of practice to promote the use of biological control in the integrated management of Prosopis in South Africa
- Authors: Van Staden, Gretha
- Date: 2024-04-04
- Subjects: Mesquite South Africa Northern Cape , Community of practice , Stakeholder participation , Mesquite Control , Invasive plants Biological control , Prosopis
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/434998 , vital:73122
- Description: Prosopis spp. are non-native species present in the arid parts of southern Africa. These trees originated in the Americas and were first introduced as a source of fodder, shade, and wood, but have become invasive, negatively impacting local biodiversity, and disrupting the delivery of ecosystem services. Some species of Prosopis hybridise freely, complicating identification and subsequent control. The control of Prosopis in the Northern Cape Province of South Africa is still contentious, because of the perceived benefits to some landowners. The effectiveness of biological control agents to control the spread of Prosopis has been quantified, more damaging agents have been considered in recent years because of the continued increase in Prosopis density. Research into the establishment of a Community of Practice (CoP) to address the differences in perceptions regarding the control of Prosopis in the Northern Cape Province is considered an appropriate starting point to conceptualise the challenges to the successful integrated management of Prosopis. As farmers are the main stakeholders involved with Prosopis control, understanding the role of farmers and the functioning of farming enterprises in the Northern Cape is imperative for the development of both a CoP as well as the appropriate management of Prosopis. To foster engagement with as many stakeholders as possible, workshops dealing with biological control as part on an integrated approach to the control of Prosopis were held in towns in the Northern Cape Province: Groblershoop, Brandvlei, Kenhardt, Upington, Prieska and Williston. Some of the main concerns of the landusers include the host specificity of the released biocontrol agents, as well as increased transparency and communication. This is especially in regards to the research on present and future biological control agents that will possibly improve the perceptions of stakeholders. The mechanical harvesting and use of biomass as a control method was supported, especially where farmers were removing Prosopis from their properties, leaving large amounts of biomass in the veld. Continued research into Prosopis in the Northern Cape needs to consider the possibility of secondary invasions, especially in areas where native bush encroachment is already a concern. The impact of clearing in terms of natural grazing and animal improvement needs to be quantified, to conceptualise the importance of maintaining better veld quality versus using Prosopis for fodder. Biological control targeting not only the reproductive output of the trees, but also the standing biomass has support from the majority of the land users in the Northern Cape Province following these workshops and this study shows the importance of a socio-ecological approach to the control of landscape scale invasion. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Authors: Van Staden, Gretha
- Date: 2024-04-04
- Subjects: Mesquite South Africa Northern Cape , Community of practice , Stakeholder participation , Mesquite Control , Invasive plants Biological control , Prosopis
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/434998 , vital:73122
- Description: Prosopis spp. are non-native species present in the arid parts of southern Africa. These trees originated in the Americas and were first introduced as a source of fodder, shade, and wood, but have become invasive, negatively impacting local biodiversity, and disrupting the delivery of ecosystem services. Some species of Prosopis hybridise freely, complicating identification and subsequent control. The control of Prosopis in the Northern Cape Province of South Africa is still contentious, because of the perceived benefits to some landowners. The effectiveness of biological control agents to control the spread of Prosopis has been quantified, more damaging agents have been considered in recent years because of the continued increase in Prosopis density. Research into the establishment of a Community of Practice (CoP) to address the differences in perceptions regarding the control of Prosopis in the Northern Cape Province is considered an appropriate starting point to conceptualise the challenges to the successful integrated management of Prosopis. As farmers are the main stakeholders involved with Prosopis control, understanding the role of farmers and the functioning of farming enterprises in the Northern Cape is imperative for the development of both a CoP as well as the appropriate management of Prosopis. To foster engagement with as many stakeholders as possible, workshops dealing with biological control as part on an integrated approach to the control of Prosopis were held in towns in the Northern Cape Province: Groblershoop, Brandvlei, Kenhardt, Upington, Prieska and Williston. Some of the main concerns of the landusers include the host specificity of the released biocontrol agents, as well as increased transparency and communication. This is especially in regards to the research on present and future biological control agents that will possibly improve the perceptions of stakeholders. The mechanical harvesting and use of biomass as a control method was supported, especially where farmers were removing Prosopis from their properties, leaving large amounts of biomass in the veld. Continued research into Prosopis in the Northern Cape needs to consider the possibility of secondary invasions, especially in areas where native bush encroachment is already a concern. The impact of clearing in terms of natural grazing and animal improvement needs to be quantified, to conceptualise the importance of maintaining better veld quality versus using Prosopis for fodder. Biological control targeting not only the reproductive output of the trees, but also the standing biomass has support from the majority of the land users in the Northern Cape Province following these workshops and this study shows the importance of a socio-ecological approach to the control of landscape scale invasion. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
Managing releases of Anagyrus vladimiri (Triapitsyn) to augment biocontrol of the citrus mealybug Planococcus citri (Risso) in South African citrus orchards
- Authors: Mommsen, Wayne Trevor
- Date: 2024-04-04
- Subjects: Citrus Diseases and pests South Africa , Citrus mealybug , Anagyrus vladimiri , Parasitism , Hyperparasite , Pests Biological control
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/434952 , vital:73118
- Description: In May 2019, South Korean inspectors rejected numerous grapefruit consignments from Letsitele, Hoedspruit and Onderberg in South Arica, because of live mealybug found on fruit. Growers expressed deep concern as mealybug management to a phytosanitary level was almost unattainable. Regular spray interventions for control of citrus black spot fungus, Phyllosticta citricarpa, and citrus thrips, Scirtothrips aurantii, cause repercussions in mealybug populations because they undermine the naturally occurring biocontrol complex. As part of an Integrated Pest Management (IPM) strategy, release of commercially produced parasitoids is common practice, to augment the naturally occurring beneficial insect populations. Prior knowledge of the harmful effects of insecticides on parasitoids is essential to IPM planning and the success of the biocontrol component in such a programme. Timing of augmentative releases to coincide with the phenology of citrus and the mealybug pest is also considered important for the successful establishment and control. Consequently, field trials were conducted to compare efficacy of early vs. late releases of Anagyrus vladimiri (Triapitsyn), an effective parasitoid of the citrus mealybug, Planococcus citri (Risso). Semi-field bioassays were conducted concurrently to determine the impact of various thripicides on A. vladimiri. The impact of sulfoxaflor, spinetoram, spirotetramat and prothiofos were rated harmless, as A. vladimiri mortality was lower than 25% after coming into contact with aged residues between 7 and 14 days old. October and November releases of A. vladimiri resulted in early parasitism and lowered peak-infestation of mealybug. January releases are possibly too late in grapefruit and lemon, open field, orchards, considering parasitism by A. vladimiri peaked in February. In mandarin orchards under net, percentage parasitism of 3rd instar mealybug increased a month later. Notably, at harvest, the difference in efficacy between treatments was not clear. This could be explained by high levels of natural parasitism observed in the treated and untreated orchards, which emphasises the importance of conservation biocontrol. In a second season, the proportion of hyperparasitoids captured (61%) from samples of mealybug-infested fruit was larger than the proportion of primary parasitoids, Anagyrus vladimiri, Coccidoxenoides perminutus (Girault) and Leptomastix dactylopii (Howard) (39%), which was far lower than the captures of eclosing primary parasitoids the previous season, which was 60%. The new discovery of Pseudaphycus sp. in citrus orchards in South Africa could be a key in explaining the uncontrollable levels of mealybug experienced and has drawn attention to a need for further understanding of ecological factors that influence biological control in citrus. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Authors: Mommsen, Wayne Trevor
- Date: 2024-04-04
- Subjects: Citrus Diseases and pests South Africa , Citrus mealybug , Anagyrus vladimiri , Parasitism , Hyperparasite , Pests Biological control
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/434952 , vital:73118
- Description: In May 2019, South Korean inspectors rejected numerous grapefruit consignments from Letsitele, Hoedspruit and Onderberg in South Arica, because of live mealybug found on fruit. Growers expressed deep concern as mealybug management to a phytosanitary level was almost unattainable. Regular spray interventions for control of citrus black spot fungus, Phyllosticta citricarpa, and citrus thrips, Scirtothrips aurantii, cause repercussions in mealybug populations because they undermine the naturally occurring biocontrol complex. As part of an Integrated Pest Management (IPM) strategy, release of commercially produced parasitoids is common practice, to augment the naturally occurring beneficial insect populations. Prior knowledge of the harmful effects of insecticides on parasitoids is essential to IPM planning and the success of the biocontrol component in such a programme. Timing of augmentative releases to coincide with the phenology of citrus and the mealybug pest is also considered important for the successful establishment and control. Consequently, field trials were conducted to compare efficacy of early vs. late releases of Anagyrus vladimiri (Triapitsyn), an effective parasitoid of the citrus mealybug, Planococcus citri (Risso). Semi-field bioassays were conducted concurrently to determine the impact of various thripicides on A. vladimiri. The impact of sulfoxaflor, spinetoram, spirotetramat and prothiofos were rated harmless, as A. vladimiri mortality was lower than 25% after coming into contact with aged residues between 7 and 14 days old. October and November releases of A. vladimiri resulted in early parasitism and lowered peak-infestation of mealybug. January releases are possibly too late in grapefruit and lemon, open field, orchards, considering parasitism by A. vladimiri peaked in February. In mandarin orchards under net, percentage parasitism of 3rd instar mealybug increased a month later. Notably, at harvest, the difference in efficacy between treatments was not clear. This could be explained by high levels of natural parasitism observed in the treated and untreated orchards, which emphasises the importance of conservation biocontrol. In a second season, the proportion of hyperparasitoids captured (61%) from samples of mealybug-infested fruit was larger than the proportion of primary parasitoids, Anagyrus vladimiri, Coccidoxenoides perminutus (Girault) and Leptomastix dactylopii (Howard) (39%), which was far lower than the captures of eclosing primary parasitoids the previous season, which was 60%. The new discovery of Pseudaphycus sp. in citrus orchards in South Africa could be a key in explaining the uncontrollable levels of mealybug experienced and has drawn attention to a need for further understanding of ecological factors that influence biological control in citrus. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
Evaluation of potential oviposition deterrents for false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae)
- Authors: Dambuza, Khalipha
- Date: 2023-10-13
- Subjects: Cryptophlebia leucotreta , Pests Integrated control , Semiochemicals , Agricultural pests Control , Oviposition , Essences and essential oils
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424479 , vital:72157
- Description: There has been extensive research on the use of semiochemicals as deterrents or true repellents in insect pest management, particularly in push-pull strategies. Much of this research has focused on pests of medical and veterinary importance and has been limited for agricultural pests. This means there is an opportunity to study use of deterrents to manage pests of agricultural importance. No study has been conducted on deterrents for false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a key phytosanitary pest in citrus orchards across South Africa. This study assessed FCM oviposition deterrence in botanicals (plants (n = 11) and essential oils (n = 15)), and some commercial pesticides (n = 7) used for FCM control in South Africa. All tested botanicals were selected based on an extensive literature review of plant compounds that have been reported to deter or repel lepidopteran pests. Choice and no-choice oviposition bioassays were conducted in complete darkness in a controlled environment room. Oranges treated with solutions/suspensions of potential oviposition deterrents were placed into a cage with gravid FCM females for four hours, with oviposition being recorded every hour. Of the 33 tested compounds, only eight significantly reduced FCM oviposition (P < 0.05) compared to the control in oviposition bioassays i.e. two essential oils (lavender and peppermint), two plant crude extracts (garlic and marigold), one fruit (Mango), and three commercial FCM insecticides (Delegate, Coragen, and Warlock). All identified oviposition deterrents, except for Mango, were further investigated for their ovicidal properties in concentration response bioassays, where all botanicals were identified to have dual action (both deterrent and ovicidal properties), as they significantly (P < 0.05) reduced FCM oviposition and egg hatch. Garlic was the most efficacious botanical whilst Warlock was the only commercial insecticide that did not show ovicidal activity (F = 41.17, P = 0.0622). Larval penetration of the host fruit was less than egg hatch for all tested compounds in concentration response bioassays. Oviposition, egg hatch, and larval penetration were all affected by concentration, with the higher concentrations being the most effective. The efficacy of these deterrent compounds should be further tested in semi-field and/or field trials, and they may have potential in FCM management as allomone dispensers or sprays. They can also be implemented in push-pull strategies where they can be used in conjunction with FCM attractants. Lastly, repellence studies should be conducted in absentia of the host fruit to determine whether oviposition deterrence was a result of true repellence or odour masking. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Authors: Dambuza, Khalipha
- Date: 2023-10-13
- Subjects: Cryptophlebia leucotreta , Pests Integrated control , Semiochemicals , Agricultural pests Control , Oviposition , Essences and essential oils
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424479 , vital:72157
- Description: There has been extensive research on the use of semiochemicals as deterrents or true repellents in insect pest management, particularly in push-pull strategies. Much of this research has focused on pests of medical and veterinary importance and has been limited for agricultural pests. This means there is an opportunity to study use of deterrents to manage pests of agricultural importance. No study has been conducted on deterrents for false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a key phytosanitary pest in citrus orchards across South Africa. This study assessed FCM oviposition deterrence in botanicals (plants (n = 11) and essential oils (n = 15)), and some commercial pesticides (n = 7) used for FCM control in South Africa. All tested botanicals were selected based on an extensive literature review of plant compounds that have been reported to deter or repel lepidopteran pests. Choice and no-choice oviposition bioassays were conducted in complete darkness in a controlled environment room. Oranges treated with solutions/suspensions of potential oviposition deterrents were placed into a cage with gravid FCM females for four hours, with oviposition being recorded every hour. Of the 33 tested compounds, only eight significantly reduced FCM oviposition (P < 0.05) compared to the control in oviposition bioassays i.e. two essential oils (lavender and peppermint), two plant crude extracts (garlic and marigold), one fruit (Mango), and three commercial FCM insecticides (Delegate, Coragen, and Warlock). All identified oviposition deterrents, except for Mango, were further investigated for their ovicidal properties in concentration response bioassays, where all botanicals were identified to have dual action (both deterrent and ovicidal properties), as they significantly (P < 0.05) reduced FCM oviposition and egg hatch. Garlic was the most efficacious botanical whilst Warlock was the only commercial insecticide that did not show ovicidal activity (F = 41.17, P = 0.0622). Larval penetration of the host fruit was less than egg hatch for all tested compounds in concentration response bioassays. Oviposition, egg hatch, and larval penetration were all affected by concentration, with the higher concentrations being the most effective. The efficacy of these deterrent compounds should be further tested in semi-field and/or field trials, and they may have potential in FCM management as allomone dispensers or sprays. They can also be implemented in push-pull strategies where they can be used in conjunction with FCM attractants. Lastly, repellence studies should be conducted in absentia of the host fruit to determine whether oviposition deterrence was a result of true repellence or odour masking. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
The possible effect of insecticide drift from citrus orchards, and acute toxicity of insecticides on the biocontrol agents of Pontederia crassipes (Mart.) Solms-Laub (Pontederiaceae) established along citrus orchards in the Lowveld region of Mpumalanga Province, South Africa
- Authors: Mabuza, Mefika Michael
- Date: 2023-10-13
- Subjects: Acute toxicity , Pontederia crassipes , Biocontrol , Water hyacinth Biological control , Insecticides Toxicology , Nutrient , Citrus Diseases and pests
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424468 , vital:72156
- Description: This study investigated the possible effect of insecticide drift on naturalized biological control agents of Pontederia crassipes (Mart.) Solms-Laub (Pontederiaceae), in the Lowveld region of Mpumalanga Province of South Africa. Occurrence and abundance of biocontrol agents were recorded at three sites on the Crocodile River and at three dams adjacent to citrus orchards. Leaves of P. crassipes and water samples were collected for insecticide residues and also nutrient status of the water and plants. Eccritotarsus catarinensis Carvalho (Hemiptera: Miridae), Neochetina spp. (combined) (Coleoptera: Curculionidae), and Orthogalumna terebrantis Wallwork (Sarcoptiformes: Galumnidae) were recorded with notable variation in abundance between the river and dams across regions. Insecticide residues were not detected on all leaves sampled across study regions, however, nutrients were detected with nitrate ranging between oligotrophic and mesotrophic. Phosphorus was also detected, but, neither of the nutrients correlated with the occurrence and abundance of naturalized biological control agents of P. crassipes. Bioassays were conducted to measure the effect of commonly used insecticides (viz. Methomyl and Chlorpyrifos) on the survival and feeding damage of biological control agents of P. crassipes. Survival of individual insects was recorded between 0.5 and 120 hours for Megamelus scutellaris and Neochetina eichhorniae Warner (Coleoptera: Curculionidae) adults for treatments where insecticides were topically applied onto the insects or leaves were dipped into the pesticides. Concentrations below field rates, recommended and above field rates of Methomyl and Chlorpyrifos on either exposure techniques significantly reduced survival and feeding of biocontrol agents. Methomyl was more toxic compared to Chlorpyrifos and it significantly reduced the survival of M. scutellaris and N. eichhorniae. In conclusion, in this study, population abundance of biocontrol agents of P. crassipes at the Lowveld region of Mpumalanga was not influenced by pesticide drift, but, insecticides commonly used in the citrus orchards has the potential to negatively impact naturalized biological control of P. crassipes as demonstrated by the bioassays. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Authors: Mabuza, Mefika Michael
- Date: 2023-10-13
- Subjects: Acute toxicity , Pontederia crassipes , Biocontrol , Water hyacinth Biological control , Insecticides Toxicology , Nutrient , Citrus Diseases and pests
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424468 , vital:72156
- Description: This study investigated the possible effect of insecticide drift on naturalized biological control agents of Pontederia crassipes (Mart.) Solms-Laub (Pontederiaceae), in the Lowveld region of Mpumalanga Province of South Africa. Occurrence and abundance of biocontrol agents were recorded at three sites on the Crocodile River and at three dams adjacent to citrus orchards. Leaves of P. crassipes and water samples were collected for insecticide residues and also nutrient status of the water and plants. Eccritotarsus catarinensis Carvalho (Hemiptera: Miridae), Neochetina spp. (combined) (Coleoptera: Curculionidae), and Orthogalumna terebrantis Wallwork (Sarcoptiformes: Galumnidae) were recorded with notable variation in abundance between the river and dams across regions. Insecticide residues were not detected on all leaves sampled across study regions, however, nutrients were detected with nitrate ranging between oligotrophic and mesotrophic. Phosphorus was also detected, but, neither of the nutrients correlated with the occurrence and abundance of naturalized biological control agents of P. crassipes. Bioassays were conducted to measure the effect of commonly used insecticides (viz. Methomyl and Chlorpyrifos) on the survival and feeding damage of biological control agents of P. crassipes. Survival of individual insects was recorded between 0.5 and 120 hours for Megamelus scutellaris and Neochetina eichhorniae Warner (Coleoptera: Curculionidae) adults for treatments where insecticides were topically applied onto the insects or leaves were dipped into the pesticides. Concentrations below field rates, recommended and above field rates of Methomyl and Chlorpyrifos on either exposure techniques significantly reduced survival and feeding of biocontrol agents. Methomyl was more toxic compared to Chlorpyrifos and it significantly reduced the survival of M. scutellaris and N. eichhorniae. In conclusion, in this study, population abundance of biocontrol agents of P. crassipes at the Lowveld region of Mpumalanga was not influenced by pesticide drift, but, insecticides commonly used in the citrus orchards has the potential to negatively impact naturalized biological control of P. crassipes as demonstrated by the bioassays. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
A native weevil and an exotic planthopper: investigating potential biological control agents for nymphaea mexicana zuccarini (nymphaeaceae) and its hybrids in South Africa
- Authors: Reid, Megan Kim
- Date: 2023-03-31
- Subjects: Nymphaeaceae South Africa , Water lilies Biological control South Africa , Host specificity , Genetic variation , Bagous longulus
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422554 , vital:71957 , DOI 10.21504/10962/422554
- Description: Nymphaea mexicana Zuccarini (Nymphaeaceae) is an invasive plant originating from southern USA and Mexico that has become problematic in South Africa, invading several water bodies around the country. Manual removal of this plant is very labour intensive and is not cost efficient or effective for long term control, while the use of herbicides is damaging to the environment and expensive. Consequently, this plant is a desirable candidate for biological control, which takes advantage of enemy release of the target weed and aims to re-establish population suppression induced by host specific natural enemies. Initiating biological control requires that several steps are followed to maximise the success of the programme, and the first few of these, including overseas surveys in the native range of the plant, have already been completed. This thesis aimed to continue biological control research for this species to take further steps at effectively managing the plant. Firstly, pre-release surveys in the invaded range are necessary to: determine what factors (including enemy release) contribute to the invasiveness of the target weed; establish a baseline of information to allow for comparison after biological control agents have been released; and identify any insect herbivores that may already be present in the country. The pre-release surveys conducted in this study revealed useful information about N. mexicana invasions in South Africa and provided evidence that enemy release is applicable to this case. However, these studies determined that a native weevil, Bagous longulus Gyllenhal (Coleoptera: Curculionidae), has expanded its host range to include the exotic N. mexicana at three sites, and may thus have potential for management of the species through augmentative releases. The invasion of N. mexicana in South Africa is further complicated by the presence of several Nymphaea hybrids originating from a complex history of horticultural trade. Although previous research has shown that several hybrid groups are present in South Africa, their parentage is not known. As biological control requires the use of host specific insects adapted to overcome the unique chemical and morphological defences utilised by plant species, hybrids are notoriously difficult to manage because they possess intermediate characters inherited from parent species to which natural enemies may not have adapted. Although biological control of hybrids is challenging, other case studies have demonstrated that it is possible to find suitable agents, but the chances of success are increased if putative parents of the hybrids are known so that they can be surveyed for natural enemies. Further molecular studies including possible parents of the Nymphaea hybrids in South Africa were thus carried out in this thesis to focus future surveying efforts. Two main hybrid groups were identified with genetic similarity to two tested putative Nymphaea parents, and this will allow further investigations of these species to improve the chances of successfully managing these hybrid groups. Some of the tested hybrids showed genetic contributions from multiple groups, some of which were unidentified, so it is necessary to prioritise the most problematic hybrids for biological control. With more insight into the genetic makeup of the Nymphaea hybrids in South Africa, investigations into the host specificity of potential biological control agents can be conducted. The ideal biological control agent should have a broad enough host range to impact and survive on both N. mexicana and its hybrids, but without a host range so broad that it would pose risk to native South African species. Host specificity trials are thus necessary to determine the suitability of potential agents. The identification of B. longulus feeding on N. mexicana during pre-release surveys motivated further investigations to determine the natural distribution, field host range, and host specificity of B. longulus in experimentally controlled conditions. Further surveys were therefore conducted at native Nymphaea sites around South Africa in addition to host specificity trials using the native Nymphaea nouchali Burm. f. (Nymphaeaceae), two populations of N. mexicana, and a cultivated hybrid. Results from the surveys and host specificity tests suggest that B. longulus is widely distributed across South Africa, is specific to Nymphaea with no observed preference between N. mexicana and the native N. nouchali, and does not perform well on Nymphaea hybrids. Hence, B. longulus is promising for use in new association biological control through augmentative releases but is not suitable for management of hybrids. In addition to the potential use of the South African B. longulus, it is necessary to conduct host specificity trials for natural enemies from the native range of N. mexicana that were prioritised in previous studies. Megamelus toddi Beamer (Hemiptera: Delphacidae) is one such species that was imported into quarantined laboratory conditions from Florida, USA. Host specificity trials were conducted using the same test plants as described for the studies on B. longulus, in addition to multigeneration trials to determine how long M. toddi could survive on non-target host plants. As with the B. longulus studies, no statistically significant differences in preference were observed between N. mexicana and N. nouchali, but M. toddi could not complete development on the test hybrid, indicating that this species is also unsuitable for the management of Nymphaea hybrids. Despite suboptimal plant health, M. toddi completed development for three generations on the native N. nouchali. This lack of host specificity deems M. toddi unsafe for release in South Africa but highlights the importance of following predefined steps to develop a biological control programme. The concluding chapter of this thesis discusses the aforementioned findings in a broader context by considering the driving forces of plant invasions in general and specifically for N. mexicana in South Africa. Case studies are also consulted to provide insight into how to proceed with managing Nymphaea hybrids in South Africa, while the factors governing host specificity and host range expansion are also discussed and considered in the context of B. longulus and M. toddi. Finally, after a consideration of the limitations of these studies, recommendations are made to continue the development of biological control for N. mexicana in South Africa. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Authors: Reid, Megan Kim
- Date: 2023-03-31
- Subjects: Nymphaeaceae South Africa , Water lilies Biological control South Africa , Host specificity , Genetic variation , Bagous longulus
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/422554 , vital:71957 , DOI 10.21504/10962/422554
- Description: Nymphaea mexicana Zuccarini (Nymphaeaceae) is an invasive plant originating from southern USA and Mexico that has become problematic in South Africa, invading several water bodies around the country. Manual removal of this plant is very labour intensive and is not cost efficient or effective for long term control, while the use of herbicides is damaging to the environment and expensive. Consequently, this plant is a desirable candidate for biological control, which takes advantage of enemy release of the target weed and aims to re-establish population suppression induced by host specific natural enemies. Initiating biological control requires that several steps are followed to maximise the success of the programme, and the first few of these, including overseas surveys in the native range of the plant, have already been completed. This thesis aimed to continue biological control research for this species to take further steps at effectively managing the plant. Firstly, pre-release surveys in the invaded range are necessary to: determine what factors (including enemy release) contribute to the invasiveness of the target weed; establish a baseline of information to allow for comparison after biological control agents have been released; and identify any insect herbivores that may already be present in the country. The pre-release surveys conducted in this study revealed useful information about N. mexicana invasions in South Africa and provided evidence that enemy release is applicable to this case. However, these studies determined that a native weevil, Bagous longulus Gyllenhal (Coleoptera: Curculionidae), has expanded its host range to include the exotic N. mexicana at three sites, and may thus have potential for management of the species through augmentative releases. The invasion of N. mexicana in South Africa is further complicated by the presence of several Nymphaea hybrids originating from a complex history of horticultural trade. Although previous research has shown that several hybrid groups are present in South Africa, their parentage is not known. As biological control requires the use of host specific insects adapted to overcome the unique chemical and morphological defences utilised by plant species, hybrids are notoriously difficult to manage because they possess intermediate characters inherited from parent species to which natural enemies may not have adapted. Although biological control of hybrids is challenging, other case studies have demonstrated that it is possible to find suitable agents, but the chances of success are increased if putative parents of the hybrids are known so that they can be surveyed for natural enemies. Further molecular studies including possible parents of the Nymphaea hybrids in South Africa were thus carried out in this thesis to focus future surveying efforts. Two main hybrid groups were identified with genetic similarity to two tested putative Nymphaea parents, and this will allow further investigations of these species to improve the chances of successfully managing these hybrid groups. Some of the tested hybrids showed genetic contributions from multiple groups, some of which were unidentified, so it is necessary to prioritise the most problematic hybrids for biological control. With more insight into the genetic makeup of the Nymphaea hybrids in South Africa, investigations into the host specificity of potential biological control agents can be conducted. The ideal biological control agent should have a broad enough host range to impact and survive on both N. mexicana and its hybrids, but without a host range so broad that it would pose risk to native South African species. Host specificity trials are thus necessary to determine the suitability of potential agents. The identification of B. longulus feeding on N. mexicana during pre-release surveys motivated further investigations to determine the natural distribution, field host range, and host specificity of B. longulus in experimentally controlled conditions. Further surveys were therefore conducted at native Nymphaea sites around South Africa in addition to host specificity trials using the native Nymphaea nouchali Burm. f. (Nymphaeaceae), two populations of N. mexicana, and a cultivated hybrid. Results from the surveys and host specificity tests suggest that B. longulus is widely distributed across South Africa, is specific to Nymphaea with no observed preference between N. mexicana and the native N. nouchali, and does not perform well on Nymphaea hybrids. Hence, B. longulus is promising for use in new association biological control through augmentative releases but is not suitable for management of hybrids. In addition to the potential use of the South African B. longulus, it is necessary to conduct host specificity trials for natural enemies from the native range of N. mexicana that were prioritised in previous studies. Megamelus toddi Beamer (Hemiptera: Delphacidae) is one such species that was imported into quarantined laboratory conditions from Florida, USA. Host specificity trials were conducted using the same test plants as described for the studies on B. longulus, in addition to multigeneration trials to determine how long M. toddi could survive on non-target host plants. As with the B. longulus studies, no statistically significant differences in preference were observed between N. mexicana and N. nouchali, but M. toddi could not complete development on the test hybrid, indicating that this species is also unsuitable for the management of Nymphaea hybrids. Despite suboptimal plant health, M. toddi completed development for three generations on the native N. nouchali. This lack of host specificity deems M. toddi unsafe for release in South Africa but highlights the importance of following predefined steps to develop a biological control programme. The concluding chapter of this thesis discusses the aforementioned findings in a broader context by considering the driving forces of plant invasions in general and specifically for N. mexicana in South Africa. Case studies are also consulted to provide insight into how to proceed with managing Nymphaea hybrids in South Africa, while the factors governing host specificity and host range expansion are also discussed and considered in the context of B. longulus and M. toddi. Finally, after a consideration of the limitations of these studies, recommendations are made to continue the development of biological control for N. mexicana in South Africa. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
Development and optimisation of a qPCR assay for the enumeration of Cryptophlebia leucotreta granulovirus (CrleGV) used for commercial applications
- Authors: Mela, Thuthula
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362949 , vital:65377
- Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Authors: Mela, Thuthula
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362949 , vital:65377
- Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
Effect of Helicosporidium sp. (Chlorophyta; Trebouxiophyceae) infection on Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), a biological control agent for the invasive Salvinia molesta D.S. Mitchell (Salviniaceae) in South
- Authors: Mphephu, Tshililo Emmanuel
- Date: 2022-10-14
- Subjects: Salvinia molesta South Africa , Weeds Biological control , Cyrtobagous salviniae , Ketoconazole
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365815 , vital:65792 , DOI https://doi.org/10.21504/10962/365815
- Description: The effectiveness of established biological control agents depends on biotic and abiotic interactions in the introduced range. The weevil, Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), was released as a biological control against Salvinia molesta D.S. Mitchell (Salviniaceae) in South Africa in 1985. This agent has been highly successful against S. molesta and has significantly reduced the weed’s populations around the country. However, in 2007, the parasitic alga, Helicosporidium sp. (an undescribed species), was detected in field-collected C. salviniae adults in South Africa. The distribution and impacts of this disease on the weevil and its efficacy as a control agent were not known. In this thesis, the prevalence, infection load, and impact of Helicosporidium sp. on C. salviniae was determined. In 2019, adult weevils were collected from 10 sites across the Eastern Cape, KwaZulu-Natal, Limpopo, and Western Cape provinces and screened to determine the occurrence, infection load, and geographic distribution of Helicosporidium sp. Transmission mechanisms of this disease in C. salviniae were then evaluated. The possible impact of Helicosporidium sp. was assessed by comparing the feeding rates and the reproductive output of the diseased and healthy adults of C. salviniae. An attempt was then made to eliminate the disease in C. salviniae through the application of the antibiotic, ketoconazole. Further, the role of temperature on infection load in C. salviniae was also assessed. Finally, recommendations for the long-term biological control programme against S. molesta in South Africa were made. The disease covers the entire distribution range of C. salviniae in South Africa, with the disease occurrence rate ranging from 92.15% to 100% insects infected per site. Helicosporidium sp. was found to transmit vertically within the populations of C. salviniae. Infection by the Helicosporidium sp. disease reduced the reproductive output of C. salviniae as well its impact on biomass reduction of S. molesta when a diseased culture was compared to a healthy culture from the USA. 98.44 to 98.55% of Helicosporidium sp. loads were reduced through multiple applications of ketoconazole concentrations under in vitro trials. In vivo treatments resulted in 70% control of Helicosporidium sp. in the adults of C. salviniae that were fed ketoconazole three times over a 21 day period. Adult C. salviniae feeding and survival performances were similar when fed fronds of S. molesta inoculated with ketoconazole and water. The lowest and highest disease loads of Helicosporidium sp. were recorded when the weevils were reared at 30°C and 14°C, respectively. As expected, the highest impact and reproductive output of C. salviniae were at 30°C. The evaluations discussed in this thesis highlight the role of diseases in biological control agents, and gaps in both the pre-release and post-release monitoring that should integrate screening of diseases in these studies. Although the combined application of the antibiotic and temperature will reduce Helicosporidium sp. loads and impact, this technology is most likely only applicable where the weevils are reared in small numbers in a rearing facility and not really applicable to the field situation. It is important to release healthy agents that will cause efficient control of the target weed plant species, therefore, when introducing new biological control agents, the health status of such agents needs to be understood. Therefore, long-term field monitoring and assessment of the impact of C. salviniae on S. molesta should be conducted to track all the changes that may result due to the presence of Helicosporidium sp. This long-term monitoring and assessment will give a more informative role of Helicosporidium sp. in field populations of C. salviniae. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Authors: Mphephu, Tshililo Emmanuel
- Date: 2022-10-14
- Subjects: Salvinia molesta South Africa , Weeds Biological control , Cyrtobagous salviniae , Ketoconazole
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/365815 , vital:65792 , DOI https://doi.org/10.21504/10962/365815
- Description: The effectiveness of established biological control agents depends on biotic and abiotic interactions in the introduced range. The weevil, Cyrtobagous salviniae Calder and Sands (Coleoptera: Curculionidae), was released as a biological control against Salvinia molesta D.S. Mitchell (Salviniaceae) in South Africa in 1985. This agent has been highly successful against S. molesta and has significantly reduced the weed’s populations around the country. However, in 2007, the parasitic alga, Helicosporidium sp. (an undescribed species), was detected in field-collected C. salviniae adults in South Africa. The distribution and impacts of this disease on the weevil and its efficacy as a control agent were not known. In this thesis, the prevalence, infection load, and impact of Helicosporidium sp. on C. salviniae was determined. In 2019, adult weevils were collected from 10 sites across the Eastern Cape, KwaZulu-Natal, Limpopo, and Western Cape provinces and screened to determine the occurrence, infection load, and geographic distribution of Helicosporidium sp. Transmission mechanisms of this disease in C. salviniae were then evaluated. The possible impact of Helicosporidium sp. was assessed by comparing the feeding rates and the reproductive output of the diseased and healthy adults of C. salviniae. An attempt was then made to eliminate the disease in C. salviniae through the application of the antibiotic, ketoconazole. Further, the role of temperature on infection load in C. salviniae was also assessed. Finally, recommendations for the long-term biological control programme against S. molesta in South Africa were made. The disease covers the entire distribution range of C. salviniae in South Africa, with the disease occurrence rate ranging from 92.15% to 100% insects infected per site. Helicosporidium sp. was found to transmit vertically within the populations of C. salviniae. Infection by the Helicosporidium sp. disease reduced the reproductive output of C. salviniae as well its impact on biomass reduction of S. molesta when a diseased culture was compared to a healthy culture from the USA. 98.44 to 98.55% of Helicosporidium sp. loads were reduced through multiple applications of ketoconazole concentrations under in vitro trials. In vivo treatments resulted in 70% control of Helicosporidium sp. in the adults of C. salviniae that were fed ketoconazole three times over a 21 day period. Adult C. salviniae feeding and survival performances were similar when fed fronds of S. molesta inoculated with ketoconazole and water. The lowest and highest disease loads of Helicosporidium sp. were recorded when the weevils were reared at 30°C and 14°C, respectively. As expected, the highest impact and reproductive output of C. salviniae were at 30°C. The evaluations discussed in this thesis highlight the role of diseases in biological control agents, and gaps in both the pre-release and post-release monitoring that should integrate screening of diseases in these studies. Although the combined application of the antibiotic and temperature will reduce Helicosporidium sp. loads and impact, this technology is most likely only applicable where the weevils are reared in small numbers in a rearing facility and not really applicable to the field situation. It is important to release healthy agents that will cause efficient control of the target weed plant species, therefore, when introducing new biological control agents, the health status of such agents needs to be understood. Therefore, long-term field monitoring and assessment of the impact of C. salviniae on S. molesta should be conducted to track all the changes that may result due to the presence of Helicosporidium sp. This long-term monitoring and assessment will give a more informative role of Helicosporidium sp. in field populations of C. salviniae. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
Genetic analysis and field application of a UV-tolerant strain of CrleGV for improved control of Thaumatotibia leucotreta
- Authors: Bennett, Tahnee Tashia
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents , Ultraviolet radiation , Oligonucleotides
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362741 , vital:65358
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), also known as false codling moth (FCM), is indigenous to sub-Saharan Africa. Thaumatotibia leucotreta has been controlled through an integrated pest management (IPM) programme, which includes chemical control, sterile insect technique (SIT), cultural and biological control. As part of the biological control, a key component is the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA). Currently, CryptogranTM, a commercial formulation of CrleGV, is the preferred product to use in South Africa for the control of T. leucotreta. The registration of the biopesticide Cryptogran (River bioscience, South Africa) was established after conducting extensive field trials with CrleGV-SA. One of the major factors affecting the baculovirus efficacy in the field is UV irradiation. A UV-tolerant Cryptophlebia leucotreta granulovirus (CrleGV-SA-C5) isolate was isolated after consecutive cycles of UV exposure. This UV-tolerant isolate is genetically distinct from the CrleGV-SA isolate. The CrleGV-SA-C5 isolate has the potential as a biological control agent. The control of T. leucotreta in South Africa could be improved by the development of novel isolates into new biopesticide formulations. To date, there has not been any field trials conducted on the CrleGV-SA-C5 isolate. Therefore, it is important to determine the biological and genetic stability of this isolate and to conduct field trials with CrleGV-SA- C5 to test the efficacy of the isolate before possible production into a biopesticide. A de novo assembly was conducted to reassemble the genome of CrleGV-SA-C5 which was followed by a sequence comparison with the CrleGV-SA genome. The identification of SNPs, led to the design of oligonucleotides flanking the regions where the SNPs were detected. Polymerase chain reaction amplification of the target regions was conducted using the oligonucleotides. After sequence comparison, seven SNPs were detected and PCR amplification was successful using the three oligonucleotides, Pif-2, HypoP and Lef-8/HP. To differentiate between CrleGV-SA-C5 and CrleGV-SA genomes and confirm the presence of the SNPs, two methods of screening were conducted. The first was the construction of six plasmids, the plasmids contained the targeted pif-2, HypoP, and the Lef-8/HP insert regions from both the CrleGV-SA-C5 and CrleGV-SA genome region where the SNPs were identified, followed by sequencing. The Five recombinant plasmids, pC5_Pif-2, pSA_Pif-2, pC5_HypoP, pSA_HypoP, and pC5_Lef-8/HP were successfully sequenced. No amplicon was obtained for one of the plasmids used as template (pSA_Lef-8/HP) and therefore the PCR product used for cloning was sequenced instead. Sequence alignment confirmed the presence of four of the five targeted SNPs in the genome of the CrleGV-SA-C5 isolate. However, of these only one SNP (UV_7) rendered a suitable marker for the differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates as the SNPs, UV_2, UV_3 and UV_5, were also present in the CrleGV- SA sequences. The second screening method was a quantitative polymerase chain reaction (qPCR) melt curve analysis to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. qPCR melt curve analysis was done using the CrleGV-SA-C5 and CrleGV-SA HypoP PCR products. This technique was unable to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. However, this may be as a result of sequence data confirming that SNP UV_5 originally identified in the CrleGV-SA-C5 HypoP region was identical to the SNP at the same position in the CrleGV-SA HypoP region. Following the differentiation of the CrleGV-SA-C5 and CrleGV-SA isolates through two screening methods, the genetic integrity of the CrleGV-SA-C5 isolate after two virus bulk-ups was determined by PCR amplification of the target regions in the bulk-up virus followed by sequencing. Prior to virus bulk-up, surface dose bioassays were conducted on 4th instar larvae and LC50 and LC90 values of 4.01 x 106 OBs/ml and 8.75 x 109 OBs/ml respectively were obtained. The CrleGV-SA-C5 isolate was then bulked up in fourth instar T. leucotreta larvae using the LC90 value that was determined. Sequencing of the target regions from the CrleGV- SA-C5_BU2 (bulk-up 2) was conducted. Sequencing results confirmed the presence of the target SNPs in the CrleGV-SA-C5_BU2 genome. The UV-tolerance of the CrleGV-SA-C5 isolate in comparison to the CrleGV-SA isolate was evaluated by detached fruit bioassays under natural UV irradiation. Two detached fruit bioassays were set-up, a UV exposure and a non-UV exposure bioassay set-up. Three treatments were used for each bioassay set-up which were the viruses CrleGV-SA-C5 and CrleGV-SA and a ddH2O control. Statistical analysis indicated that there was no significant difference between the virus treatments in both the UV exposed detached fruit bioassay and the non-UV exposed detached fruit bioassay. This study is the second study to report on the de novo assembly of the CrleGV-SA-C5 and sequence comparison with the CrleGV-SA genome, and the first to report on the UV-tolerance of the CrleGV-SA-C5 isolate by detached fruit bioassays. Future work could involve further evaluation of intraspecific genetic variability in the CrleGV-SA-C5 isolate and to identify any additional SNPs present within the genome that can be used as suitable markers for differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates. It was recognised that it is required to conduct further detached fruit bioassays and field trials, but with improved protocols, for the efficacy and UV-tolerance of the CrleGV-SA-C5 isolate to be conclusively determined. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Authors: Bennett, Tahnee Tashia
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta Biological control , Pests Integrated control , Biological pest control agents , Ultraviolet radiation , Oligonucleotides
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362741 , vital:65358
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), also known as false codling moth (FCM), is indigenous to sub-Saharan Africa. Thaumatotibia leucotreta has been controlled through an integrated pest management (IPM) programme, which includes chemical control, sterile insect technique (SIT), cultural and biological control. As part of the biological control, a key component is the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA). Currently, CryptogranTM, a commercial formulation of CrleGV, is the preferred product to use in South Africa for the control of T. leucotreta. The registration of the biopesticide Cryptogran (River bioscience, South Africa) was established after conducting extensive field trials with CrleGV-SA. One of the major factors affecting the baculovirus efficacy in the field is UV irradiation. A UV-tolerant Cryptophlebia leucotreta granulovirus (CrleGV-SA-C5) isolate was isolated after consecutive cycles of UV exposure. This UV-tolerant isolate is genetically distinct from the CrleGV-SA isolate. The CrleGV-SA-C5 isolate has the potential as a biological control agent. The control of T. leucotreta in South Africa could be improved by the development of novel isolates into new biopesticide formulations. To date, there has not been any field trials conducted on the CrleGV-SA-C5 isolate. Therefore, it is important to determine the biological and genetic stability of this isolate and to conduct field trials with CrleGV-SA- C5 to test the efficacy of the isolate before possible production into a biopesticide. A de novo assembly was conducted to reassemble the genome of CrleGV-SA-C5 which was followed by a sequence comparison with the CrleGV-SA genome. The identification of SNPs, led to the design of oligonucleotides flanking the regions where the SNPs were detected. Polymerase chain reaction amplification of the target regions was conducted using the oligonucleotides. After sequence comparison, seven SNPs were detected and PCR amplification was successful using the three oligonucleotides, Pif-2, HypoP and Lef-8/HP. To differentiate between CrleGV-SA-C5 and CrleGV-SA genomes and confirm the presence of the SNPs, two methods of screening were conducted. The first was the construction of six plasmids, the plasmids contained the targeted pif-2, HypoP, and the Lef-8/HP insert regions from both the CrleGV-SA-C5 and CrleGV-SA genome region where the SNPs were identified, followed by sequencing. The Five recombinant plasmids, pC5_Pif-2, pSA_Pif-2, pC5_HypoP, pSA_HypoP, and pC5_Lef-8/HP were successfully sequenced. No amplicon was obtained for one of the plasmids used as template (pSA_Lef-8/HP) and therefore the PCR product used for cloning was sequenced instead. Sequence alignment confirmed the presence of four of the five targeted SNPs in the genome of the CrleGV-SA-C5 isolate. However, of these only one SNP (UV_7) rendered a suitable marker for the differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates as the SNPs, UV_2, UV_3 and UV_5, were also present in the CrleGV- SA sequences. The second screening method was a quantitative polymerase chain reaction (qPCR) melt curve analysis to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. qPCR melt curve analysis was done using the CrleGV-SA-C5 and CrleGV-SA HypoP PCR products. This technique was unable to differentiate between the CrleGV-SA-C5 and CrleGV-SA isolates. However, this may be as a result of sequence data confirming that SNP UV_5 originally identified in the CrleGV-SA-C5 HypoP region was identical to the SNP at the same position in the CrleGV-SA HypoP region. Following the differentiation of the CrleGV-SA-C5 and CrleGV-SA isolates through two screening methods, the genetic integrity of the CrleGV-SA-C5 isolate after two virus bulk-ups was determined by PCR amplification of the target regions in the bulk-up virus followed by sequencing. Prior to virus bulk-up, surface dose bioassays were conducted on 4th instar larvae and LC50 and LC90 values of 4.01 x 106 OBs/ml and 8.75 x 109 OBs/ml respectively were obtained. The CrleGV-SA-C5 isolate was then bulked up in fourth instar T. leucotreta larvae using the LC90 value that was determined. Sequencing of the target regions from the CrleGV- SA-C5_BU2 (bulk-up 2) was conducted. Sequencing results confirmed the presence of the target SNPs in the CrleGV-SA-C5_BU2 genome. The UV-tolerance of the CrleGV-SA-C5 isolate in comparison to the CrleGV-SA isolate was evaluated by detached fruit bioassays under natural UV irradiation. Two detached fruit bioassays were set-up, a UV exposure and a non-UV exposure bioassay set-up. Three treatments were used for each bioassay set-up which were the viruses CrleGV-SA-C5 and CrleGV-SA and a ddH2O control. Statistical analysis indicated that there was no significant difference between the virus treatments in both the UV exposed detached fruit bioassay and the non-UV exposed detached fruit bioassay. This study is the second study to report on the de novo assembly of the CrleGV-SA-C5 and sequence comparison with the CrleGV-SA genome, and the first to report on the UV-tolerance of the CrleGV-SA-C5 isolate by detached fruit bioassays. Future work could involve further evaluation of intraspecific genetic variability in the CrleGV-SA-C5 isolate and to identify any additional SNPs present within the genome that can be used as suitable markers for differentiation between the CrleGV-SA-C5 and CrleGV-SA isolates. It was recognised that it is required to conduct further detached fruit bioassays and field trials, but with improved protocols, for the efficacy and UV-tolerance of the CrleGV-SA-C5 isolate to be conclusively determined. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
The effect of orchard sanitation and predatory ants on the eclosion of the internal feeding pests and Oriental fruit fly, in South Africa
- Authors: Makitla, Tshepang
- Date: 2022-10-14
- Subjects: Orchards South Africa , Phytosanitation , Citrus Diseases and pests Biological control , Ants , Insects as biological pest control agents
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362927 , vital:65375
- Description: There are several pests of phytosanitary concern in the citrus industry in South Africa. Orchard sanitation can play an important role in suppressing the populations of these pests, however there are little data on the efficacy of sanitation techniques. Therefore, the current study investigated the effect of fruit disposal techniques and burying depths on the eclosion of the most important pests of citrus in South Africa, false codling moth Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Mediterranean fruit fly or Medfly Ceratitis capitata Wiedemann (Diptera: Tephritidae), Natal fruit fly Ceratitis rosa Karsh (Diptera: Tephritidae), and Oriental fruit fly Bactrocera dorsalis Hendel (Diptera: Tephritidae). Abscised C. sinensis fruits were inoculated with larvae of T. leucotreta, and eggs of C. capitata, C. rosa, and B. dorsalis, before being disposed as pulped, or whole, and buried at different depths (0 cm, 5 cm, 25 cm, and 50 cm). Abundance and richness of predatory ants were monitored using pitfall traps to ascertain their effect on the mortality of the immature stages of these pests. Ceratitis capitata and C. rosa failed to eclose from the inoculated fruits disposed at different depths, however, T. leucotreta and B. dorsalis adults did eclosed. Significantly fewer B. dorsalis eclosed from fruits that were pulped in comparison to eclosion where the fruit were left whole (F (3, 16) = 11.45, P < 0.01). Furthermore, depth of burial had a significant effect on the number of eclosed adults of Drosophila sp (F (3, 112) = 3.43, P < 0.01). Burying fruits at 50 cm suppressed the eclosion of all the internal feeding pests tested. Twenty-seven thousand seventy-three individual ants (Hymenoptera: Formicidae) were sampled from the same plots as used above, with at least 47% and 53% sampled from plots where pulped and whole C. sinensis fruits were disposed of, respectively. The ants were identified to morphospecies which included Pheidole1, Pheidole2, Formicinae1, Formicinae2, Formicinae3, and Myrmicinae1. The disposal of the inoculated C. sinensis fruits either as pulped or whole and burying at different depths significantly suppressed and/or delayed the eclosion of either of the tested internal feeding pests of citrus. Although, predacious ants were sampled from the same treatment plots they did not affect the survival or eclosion of the tested pests, and this could be attributed to the application of the slow toxic ant bait. Therefore, based on the observed results B. dorsalis adults showed the ability to eclose from 50 cm depth where fruit was either disposed as pulped or whole, thus, citrus farmers are advised to use hammer mill that will finely crush sanitised fruit, and/or bury fruit beyond 50 cm depth to prevent the adult od this pest from eclosing. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
- Authors: Makitla, Tshepang
- Date: 2022-10-14
- Subjects: Orchards South Africa , Phytosanitation , Citrus Diseases and pests Biological control , Ants , Insects as biological pest control agents
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362927 , vital:65375
- Description: There are several pests of phytosanitary concern in the citrus industry in South Africa. Orchard sanitation can play an important role in suppressing the populations of these pests, however there are little data on the efficacy of sanitation techniques. Therefore, the current study investigated the effect of fruit disposal techniques and burying depths on the eclosion of the most important pests of citrus in South Africa, false codling moth Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae), Mediterranean fruit fly or Medfly Ceratitis capitata Wiedemann (Diptera: Tephritidae), Natal fruit fly Ceratitis rosa Karsh (Diptera: Tephritidae), and Oriental fruit fly Bactrocera dorsalis Hendel (Diptera: Tephritidae). Abscised C. sinensis fruits were inoculated with larvae of T. leucotreta, and eggs of C. capitata, C. rosa, and B. dorsalis, before being disposed as pulped, or whole, and buried at different depths (0 cm, 5 cm, 25 cm, and 50 cm). Abundance and richness of predatory ants were monitored using pitfall traps to ascertain their effect on the mortality of the immature stages of these pests. Ceratitis capitata and C. rosa failed to eclose from the inoculated fruits disposed at different depths, however, T. leucotreta and B. dorsalis adults did eclosed. Significantly fewer B. dorsalis eclosed from fruits that were pulped in comparison to eclosion where the fruit were left whole (F (3, 16) = 11.45, P < 0.01). Furthermore, depth of burial had a significant effect on the number of eclosed adults of Drosophila sp (F (3, 112) = 3.43, P < 0.01). Burying fruits at 50 cm suppressed the eclosion of all the internal feeding pests tested. Twenty-seven thousand seventy-three individual ants (Hymenoptera: Formicidae) were sampled from the same plots as used above, with at least 47% and 53% sampled from plots where pulped and whole C. sinensis fruits were disposed of, respectively. The ants were identified to morphospecies which included Pheidole1, Pheidole2, Formicinae1, Formicinae2, Formicinae3, and Myrmicinae1. The disposal of the inoculated C. sinensis fruits either as pulped or whole and burying at different depths significantly suppressed and/or delayed the eclosion of either of the tested internal feeding pests of citrus. Although, predacious ants were sampled from the same treatment plots they did not affect the survival or eclosion of the tested pests, and this could be attributed to the application of the slow toxic ant bait. Therefore, based on the observed results B. dorsalis adults showed the ability to eclose from 50 cm depth where fruit was either disposed as pulped or whole, thus, citrus farmers are advised to use hammer mill that will finely crush sanitised fruit, and/or bury fruit beyond 50 cm depth to prevent the adult od this pest from eclosing. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2022
- Full Text:
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:
- 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:
An investigation into yeast-baculovirus synergism for the improved control of Thaumatotibia leucotreta, an economically important pest of citrus
- Authors: Van der Merwe, Marcél
- Date: 2021-10-29
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Yeast , Natural pesticides , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control , Thaumatotibia leucotreta
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191236 , vital:45073
- Description: A mutualistic association between Cydia pomonella and yeasts belonging to the genus Metschnikowia has previously been demonstrated. Larval feeding galleries inoculated with M. andauensis, reduced larval mortality and enhanced larval development. Additionally, adult C. pomonella female oviposition preference was also shown to be influenced by the volatiles produced by M. andauensis. This mutualistic relationship was manipulated for biological control purposes, by combining M. pulcherrima with the baculovirus Cydia pomonella granulovirus. The combination of M. pulcherrima with brown cane sugar and CpGV in laboratory assays and field trials resulted in a significant increase in larval mortality. A similar observation was made when M. pulcherrima was substituted for Saccharomyces cerevisiae. This indicates that yeasts harbour the potential for use in biological control, especially when combined with other well-established biocontrol methods. Thaumatotibia leucotreta is a phytophagous insect endemic to southern Africa. It is highly significant to the South African citrus industry due to its classification as a phytosanitary pest by most international markets. An integrated pest management programme has been implemented to control T. leucotreta. The baculovirus Cryptophlebia leucotreta granulovirus forms one component of this programme and is highly effective. In this study, we proposed to determine which yeast species occur naturally in the gut of T. leucotreta larvae and to examine whether any of the isolated yeast species, when combined with the CrleGV-SA, enhance its effectiveness. Firstly, Navel oranges infested with T. leucotreta larvae were collected from geographically distinct citrus-producing regions across South Africa. This led to the isolation and identification of six yeast species from the gut of T. leucotreta larvae via PCR amplification and sequencing of the internal transcribed spacer region and D1/D2 domain of the large subunit. Six yeast species were identified, viz. Meyerozyma guilliermondii, Hanseniaspora uvarum, Clavispora lusitaniae, Kluyveromyces marxianus, Pichia kudriavzevii and Pichia kluyveri. Additionally, Saccharomyces cerevisiae was included as a control in all trials due to its commercial availability and use in the artificial diet used to rear T. leucotreta. Secondly, larval development and attraction assays were conducted with the isolated yeast species. Thaumatotibia leucotreta larvae that fed on Navel oranges inoculated with M. guilliermondii, P. kluyveri, H. uvarum, and S. cerevisiae had accelerated developmental periods and reduced mortality rates. Additionally, it was demonstrated that T. leucotreta neonates were attracted to YPD broth cultures inoculated with P. kluyveri, H. uvarum, P. kudriavzevii and K. marxianus for feeding. Thirdly, oviposition preference assays were conducted with adult T. leucotreta females to determine whether the isolated yeast species influence their egg-laying in two-choice and multiple-choice tests. Navel oranges were inoculated with a specific yeast isolate, and mated adult females were left to oviposit. Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to influence adult T. leucotreta female oviposition preference in two-choice tests. However, multiple-choice tests using the aforementioned yeast species did not mimic these results. Lastly, a series of detached fruit bioassays were performed to determine the optimal yeast:virus ratio, test all isolated yeast species in combination with CrleGV-SA and to further enhance yeast/virus formulation through the addition of an adjuvant and surfactant. CrleGV-SA was applied at a lethal concentration that would kill 50 % of T. leucotreta larvae. The optimal yeast concentration to use alongside CrleGV-SA was determined. Pichia kluyveri, P. kudriavzevii, K. marxianus and S. cerevisiae in combination with CrleGV-SA increased larval mortality compared to CrleGV-SA alone. The inclusion of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae plus CrleGV-SA formulations greatly enhanced their efficacy. Additionally, semi-field trials were initiated using P. kudriavzevii and S. cerevisiae, with promising preliminary results being obtained, although more replicates need to be performed. The experiments performed in this study provide a platform for further research into the application of a yeast/virus combination as a novel control and monitoring option for T. leucotreta in the field. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
- Authors: Van der Merwe, Marcél
- Date: 2021-10-29
- Subjects: Baculoviruses , Cryptophlebia leucotreta , Yeast , Natural pesticides , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control , Thaumatotibia leucotreta
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191236 , vital:45073
- Description: A mutualistic association between Cydia pomonella and yeasts belonging to the genus Metschnikowia has previously been demonstrated. Larval feeding galleries inoculated with M. andauensis, reduced larval mortality and enhanced larval development. Additionally, adult C. pomonella female oviposition preference was also shown to be influenced by the volatiles produced by M. andauensis. This mutualistic relationship was manipulated for biological control purposes, by combining M. pulcherrima with the baculovirus Cydia pomonella granulovirus. The combination of M. pulcherrima with brown cane sugar and CpGV in laboratory assays and field trials resulted in a significant increase in larval mortality. A similar observation was made when M. pulcherrima was substituted for Saccharomyces cerevisiae. This indicates that yeasts harbour the potential for use in biological control, especially when combined with other well-established biocontrol methods. Thaumatotibia leucotreta is a phytophagous insect endemic to southern Africa. It is highly significant to the South African citrus industry due to its classification as a phytosanitary pest by most international markets. An integrated pest management programme has been implemented to control T. leucotreta. The baculovirus Cryptophlebia leucotreta granulovirus forms one component of this programme and is highly effective. In this study, we proposed to determine which yeast species occur naturally in the gut of T. leucotreta larvae and to examine whether any of the isolated yeast species, when combined with the CrleGV-SA, enhance its effectiveness. Firstly, Navel oranges infested with T. leucotreta larvae were collected from geographically distinct citrus-producing regions across South Africa. This led to the isolation and identification of six yeast species from the gut of T. leucotreta larvae via PCR amplification and sequencing of the internal transcribed spacer region and D1/D2 domain of the large subunit. Six yeast species were identified, viz. Meyerozyma guilliermondii, Hanseniaspora uvarum, Clavispora lusitaniae, Kluyveromyces marxianus, Pichia kudriavzevii and Pichia kluyveri. Additionally, Saccharomyces cerevisiae was included as a control in all trials due to its commercial availability and use in the artificial diet used to rear T. leucotreta. Secondly, larval development and attraction assays were conducted with the isolated yeast species. Thaumatotibia leucotreta larvae that fed on Navel oranges inoculated with M. guilliermondii, P. kluyveri, H. uvarum, and S. cerevisiae had accelerated developmental periods and reduced mortality rates. Additionally, it was demonstrated that T. leucotreta neonates were attracted to YPD broth cultures inoculated with P. kluyveri, H. uvarum, P. kudriavzevii and K. marxianus for feeding. Thirdly, oviposition preference assays were conducted with adult T. leucotreta females to determine whether the isolated yeast species influence their egg-laying in two-choice and multiple-choice tests. Navel oranges were inoculated with a specific yeast isolate, and mated adult females were left to oviposit. Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to influence adult T. leucotreta female oviposition preference in two-choice tests. However, multiple-choice tests using the aforementioned yeast species did not mimic these results. Lastly, a series of detached fruit bioassays were performed to determine the optimal yeast:virus ratio, test all isolated yeast species in combination with CrleGV-SA and to further enhance yeast/virus formulation through the addition of an adjuvant and surfactant. CrleGV-SA was applied at a lethal concentration that would kill 50 % of T. leucotreta larvae. The optimal yeast concentration to use alongside CrleGV-SA was determined. Pichia kluyveri, P. kudriavzevii, K. marxianus and S. cerevisiae in combination with CrleGV-SA increased larval mortality compared to CrleGV-SA alone. The inclusion of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae plus CrleGV-SA formulations greatly enhanced their efficacy. Additionally, semi-field trials were initiated using P. kudriavzevii and S. cerevisiae, with promising preliminary results being obtained, although more replicates need to be performed. The experiments performed in this study provide a platform for further research into the application of a yeast/virus combination as a novel control and monitoring option for T. leucotreta in the field. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
- Full Text:
Distribution, ecological and economic impacts and competition of the invasive alien aquatic weeds (Pontederia crassipes Mart., Pistia stratiotes L., Salvinia molesta D.S. Mitch. and Azolla filiculoides Lam.) in Madagascar
- Authors: Lehavana, Adolphe
- Date: 2021-10-29
- Subjects: Pontederiaceae Madagascar , Water lettuce Madagascar , Salvinia molesta Madagascar , Azolla filiculoides Madagascar , Introduced aquatic organisms , Aquatic weeds Economic aspects , Aquatic weeds Social aspects , Aquatic weeds Geographical distribution
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191225 , vital:45072
- Description: In Madagascar, as in several countries in the world, the invasion by four aquatic weeds (Pontederia crassipes Mart. (Pontederiaceae), Pistia stratiotes L. (Araceae), Salvinia molesta D.S. Mitch Salviniaceae) and Azolla filiculoides Lam. (Azollaceae) are among the drivers of environmental and socio-economic deterioration in aquatic ecosystems. Pistia stratiotes was first recorded on the island in the 19th century, and P. crassipes from the beginning of the 20th century, while S. molesta and A. filiculoides were only documented during in the 21st century. From the 1920s, botanists such as Henri Perrier de la Bathie and Raymond Decary were already aware of the dangers caused, in particular by P. crassipes in other countries, and raised the alarm, but little attention has been paid to these species. The aim of the research conducted for this thesis was to determine the distribution, socio-economic and ecological impacts of these four invasive alien aquatic weeds in Madagascar and to make recommendations for their control. First, the distributions of these four aquatic weeds were mapped. This mapping exercise compiled data from different sources including herbarium records, online data and field visits across Madagascar. The mapping study was undertaken from August 2015 to June 2020. Except for mountainous areas above 1800 m (Tsaratanana Massif, Ankaratra Massif and Andringitra Massif) where no data were available, all of Madagascar's bioclimates were invaded by at least one of the four aquatic weeds. In total, at least one species was recorded in 18 of the 22 Regions. Pontederia crassipes was recorded in 13 Regions, S. molesta in 14 Regions, P. stratiotes in 12 Regions, and A. filiculoides in 13 Regions. Herbarium records revealed the oldest record for P. stratiotes to be 1847, 1931 for P. crassipes, 1995 for S. molesta and there were no herbarium specimens for A. filiculoides prior to the start of the current study in 2015. We now know where these four weeds occur and how abundant they are. An objective of this research was to assess the impacts of the four invasive aquatic plants on the socio-economy of the island, mainly on rice production and fishing. Between 2016 and 2019, 102 households in three regions, Soanierana Ivongo, Foulpointe and Antananarivo, were randomly selected and questioned on the impact of these weeds in their aquatic ecosystems and their livelihoods such as fishing and rice growing. Surveys revealed that the four aquatic weeds significantly threatened household activities. On the east coast of Madagascar, the invasions of these four invasive species decreased fish and freshwater shrimp production by 82%. On the high plateau of Madagascar, they reduced rice yield by 30% despite requiring an additional expense of US$ 1,107/ha for control. Although farmers surveyed only used manual control to manage these weeds, they were receptive to other control methods, including integrated control using herbicides and biological control. Another objective of this research was to determine the ecological impacts of the four weeds and specifically if freshwater ecosystem functioning would return after control. To assess the ecological impact, between February 2017 to August 2019, on Lake Antsokafina, the following abiotic and biotic factors were considered: physico-chemistry of water, succession of macrophyte community and animal diversity. With the exception of turbidity, the values of the physico-chemical parameters of the water (pH, electrical conductivity, water temperature and turbidity), were similar between the infested zone and cleared zone. A study on the invasion process of aquatic weeds showed that the plant community succession of the lake changed over time in the areas that had been cleared. The submerged species Ceratophyllum demersum was the pioneer, followed by creeping species such as Echinochloa colona and Ipomoea aquatica, before the area was recolonized by aquatic weeds. Among the aquatic weeds, S. molesta was the most aggressive, covering 92% of the area one year after the start of the experiment. For animal diversity, bird, shrimp and fish community were assessed. The cleaning of the plots in the lake allowed the resumption of fishing activity providing 50 to 200g/catch for shrimp and from 0.25 to 0.5kg/catch for fish per person per day, while no catch was obtained in the areas infested by aquatic weeds were fishermen still attempting to harvest fish/shrimp from the aquatic weed infested areas. Three species of birds, Humblot’s Heron (Ardea humbloti), the white-faced whistling duck (Dendrocygna viduata) and red-billed teal (Anas erythrorhyncha) returned once the areas had been cleared. A manipulated outdoor as descriptor for laboratory experiment was conducted to determine the level and nature of competition of four aquatic weeds species against the indigenous floating fern, Salvinia hastata Desv. (Salviniaceae), using an additive series density model. It was shown that all four invasive species outcompeted S. hastata, with P. crassipes being 24 times more dominant, followed by P. stratiotes at 12 times, S. molesta at 8 times, and finally A. filiculoides at 1.2 times more dominant. This study provided direct evidence of the biodiversity impact of these four species and thus also provided an environmental argument for their control. Based on the findings of this study, a series of recommendations was formulated to manage the invasions of alien species in Madagascar with particular attention to invasive aquatic weeds. These recommendations mainly concern the establishment of management structures and legal instruments such as the creation of a lead government agency at national level and a cross-sectorial invasive species advisory committee, which should review legislation and regulations related to invasive species. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2021
- Full Text:
- Authors: Lehavana, Adolphe
- Date: 2021-10-29
- Subjects: Pontederiaceae Madagascar , Water lettuce Madagascar , Salvinia molesta Madagascar , Azolla filiculoides Madagascar , Introduced aquatic organisms , Aquatic weeds Economic aspects , Aquatic weeds Social aspects , Aquatic weeds Geographical distribution
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191225 , vital:45072
- Description: In Madagascar, as in several countries in the world, the invasion by four aquatic weeds (Pontederia crassipes Mart. (Pontederiaceae), Pistia stratiotes L. (Araceae), Salvinia molesta D.S. Mitch Salviniaceae) and Azolla filiculoides Lam. (Azollaceae) are among the drivers of environmental and socio-economic deterioration in aquatic ecosystems. Pistia stratiotes was first recorded on the island in the 19th century, and P. crassipes from the beginning of the 20th century, while S. molesta and A. filiculoides were only documented during in the 21st century. From the 1920s, botanists such as Henri Perrier de la Bathie and Raymond Decary were already aware of the dangers caused, in particular by P. crassipes in other countries, and raised the alarm, but little attention has been paid to these species. The aim of the research conducted for this thesis was to determine the distribution, socio-economic and ecological impacts of these four invasive alien aquatic weeds in Madagascar and to make recommendations for their control. First, the distributions of these four aquatic weeds were mapped. This mapping exercise compiled data from different sources including herbarium records, online data and field visits across Madagascar. The mapping study was undertaken from August 2015 to June 2020. Except for mountainous areas above 1800 m (Tsaratanana Massif, Ankaratra Massif and Andringitra Massif) where no data were available, all of Madagascar's bioclimates were invaded by at least one of the four aquatic weeds. In total, at least one species was recorded in 18 of the 22 Regions. Pontederia crassipes was recorded in 13 Regions, S. molesta in 14 Regions, P. stratiotes in 12 Regions, and A. filiculoides in 13 Regions. Herbarium records revealed the oldest record for P. stratiotes to be 1847, 1931 for P. crassipes, 1995 for S. molesta and there were no herbarium specimens for A. filiculoides prior to the start of the current study in 2015. We now know where these four weeds occur and how abundant they are. An objective of this research was to assess the impacts of the four invasive aquatic plants on the socio-economy of the island, mainly on rice production and fishing. Between 2016 and 2019, 102 households in three regions, Soanierana Ivongo, Foulpointe and Antananarivo, were randomly selected and questioned on the impact of these weeds in their aquatic ecosystems and their livelihoods such as fishing and rice growing. Surveys revealed that the four aquatic weeds significantly threatened household activities. On the east coast of Madagascar, the invasions of these four invasive species decreased fish and freshwater shrimp production by 82%. On the high plateau of Madagascar, they reduced rice yield by 30% despite requiring an additional expense of US$ 1,107/ha for control. Although farmers surveyed only used manual control to manage these weeds, they were receptive to other control methods, including integrated control using herbicides and biological control. Another objective of this research was to determine the ecological impacts of the four weeds and specifically if freshwater ecosystem functioning would return after control. To assess the ecological impact, between February 2017 to August 2019, on Lake Antsokafina, the following abiotic and biotic factors were considered: physico-chemistry of water, succession of macrophyte community and animal diversity. With the exception of turbidity, the values of the physico-chemical parameters of the water (pH, electrical conductivity, water temperature and turbidity), were similar between the infested zone and cleared zone. A study on the invasion process of aquatic weeds showed that the plant community succession of the lake changed over time in the areas that had been cleared. The submerged species Ceratophyllum demersum was the pioneer, followed by creeping species such as Echinochloa colona and Ipomoea aquatica, before the area was recolonized by aquatic weeds. Among the aquatic weeds, S. molesta was the most aggressive, covering 92% of the area one year after the start of the experiment. For animal diversity, bird, shrimp and fish community were assessed. The cleaning of the plots in the lake allowed the resumption of fishing activity providing 50 to 200g/catch for shrimp and from 0.25 to 0.5kg/catch for fish per person per day, while no catch was obtained in the areas infested by aquatic weeds were fishermen still attempting to harvest fish/shrimp from the aquatic weed infested areas. Three species of birds, Humblot’s Heron (Ardea humbloti), the white-faced whistling duck (Dendrocygna viduata) and red-billed teal (Anas erythrorhyncha) returned once the areas had been cleared. A manipulated outdoor as descriptor for laboratory experiment was conducted to determine the level and nature of competition of four aquatic weeds species against the indigenous floating fern, Salvinia hastata Desv. (Salviniaceae), using an additive series density model. It was shown that all four invasive species outcompeted S. hastata, with P. crassipes being 24 times more dominant, followed by P. stratiotes at 12 times, S. molesta at 8 times, and finally A. filiculoides at 1.2 times more dominant. This study provided direct evidence of the biodiversity impact of these four species and thus also provided an environmental argument for their control. Based on the findings of this study, a series of recommendations was formulated to manage the invasions of alien species in Madagascar with particular attention to invasive aquatic weeds. These recommendations mainly concern the establishment of management structures and legal instruments such as the creation of a lead government agency at national level and a cross-sectorial invasive species advisory committee, which should review legislation and regulations related to invasive species. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2021
- Full Text: