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ethanol vials and identified to the lowest possible taxonomic level (i.e. genus or species) using both published and unpublished dichotomous identification keys.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
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- Date Issued: 2024-10-11
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
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- Date Issued: 2024-10-11
Augmentation of Aphytis melinus DeBach (Hymenoptera: Aphelinidae) for the control of California red scale Aonidiella aurantii Maskell (Hemiptera: Diaspididae) on citrus
- 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
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- Date Issued: 2024-04-05
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
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- Date Issued: 2024-04-04
Composition and physiological roles of gut microbiota in the False Coding Moth (Thaumatotibia leucotreta)
- Authors: Richardson, Perryn Heather
- Date: 2023-10-13
- Subjects: False codling moth , Microbiomes , Insect physiology , Citrus Diseases and pests South Africa , Biological pest control , Cryptophlebia leucotreta
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424457 , vital:72155
- Description: Gut microbiota can have a profound influence on host performance, behaviour and fitness. For False Codling Moth (FCM), Thaumatotibia leucotreta (Lepidoptera: Tortricidae), a major pest of citrus in South Africa, little work has been undertaken to date on gut microbe diversity or its influence on the host. This thesis aimed to i) characterise the gut microbiome of FCM under laboratory conditions and in FCM from the field, ii) and produce moths with reduced gut microbiota through egg dechorionation, which was followed by iii) the measurement of a suite of physiological traits, namely mass, survival and thermal stress in FCM from normal laboratory, dechorionated laboratory and field collected larvae that may be indicative of overall field performance. We aimed to directly test the hypothesis that gut microbial diversity partly determines insect performance and fitness by measuring its effects on growth, development, and tolerance to cold temperatures in FCM. FCM eggs that underwent dechorionation with sodium hypochlorite had an overall effect on larval survival, egg morphology and both larval and adult moth physiological measures. Increasing concentrations of sodium hypochlorite significantly decreased insect survival, (𝜒2(1, n = 10 850) = 21.724, p-value < 0.0001), with a concentration of ≈3.69% as the concentration limit (p-value < 0.001). Successful dechorionation of FCM was achieved with a wash of sodium hypochlorite at around 3.69% concentration and was visually confirmed by reduction of FCM egg surface area, (𝜒2(25, n = 260) p-value < 0.0001) and Scanning Electron Micrographs of the egg morphology. The gut microbiome of FCM from the different focus treatments was successfully characterized. Identification of the dominant bacterial families in these microbiomes revealed Xanthobacteraceae, Beijerinckiaceae and Burkholderiaceae in both the laboratory reared and field collected larvae, which suggests their systematic association with T. leucotreta. The most abundant genera were revealed as Bradyrhizobium, Methylobacterium and Burkholderia-Caballeronia-Paraburkholderia. Comparison of larval mass showed that treatment (dechorionated or not) had a significant effect on larval mass (𝜒2(2, n = 230) = 22.703, p-value < 0.001), field larvae were heavier than both control larvae and larvae with a disrupted gut microbiome. However, adult insects with a disrupted gut microbiome had more mass than individuals from the control and field-collected larvae with intact gut microbiomes (𝜒2(2, n = 230) = 39.074, p-value < 0.001). Despite the difference in mass between larval treatments, there was no significant difference in relative protein (𝜒2(2, n = 24) = 5.680, p-value = 0.06), carbohydrate (𝜒2(2, n = 24) = 3.940, p-value = 0.14) or lipid (𝜒2(2, n = 24) = 6.032, p-value = 0.05) content between individuals from the control and dechorionated treatments and field-collected individuals. Turning to thermal physiology, insects collected from the field took significantly longer to recover from chill coma than both laboratory treatments with intact and disrupted gut microbiomes (𝜒2(2, n = 129 = 39.659, p-value < 0.001). In addition, exposure to cold stress showed that treatment had a significant effect on insect mortality (𝜒2(2, n = 272) = 9.176, p-value = 0.01), with individuals from the control and dechorionated treatment being less likely to die after experiencing cold stress compared to field-collected individuals. Differences in the mass and thermal tolerance of insects with intact and disrupted gut microbiota suggest that gut microbiota may play an important role in the cold performance of T. leucotreta, and these findings constitute the basis for future molecular work on the functions of these bacterial taxa. This research highlights the need for consideration of the effects of T. leucotreta microbiome in current pest control programs. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
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- Date Issued: 2023-10-13