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Doctoral Degrees (Plant Pathology)

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    Screening acacia mearnsii (black wattle) seedlings for frost tolerance using an artificial frost technique, combined with proteomic analysis.
    (2023) Jugmohan, Mayuri.; Laing, Mark Delmege.; Bairu, Michael Wolday.; Chan, Julian Moreno.
    Acacia mearnsii de Wild. (commonly known as black wattle), was first introduced to South Africa in the 1800’s. Today, it is one of the leading commercially grown forestry crops in South Africa due to its usefulness and versatility. Black wattle is a source of high-quality tannin and raw material for wood pulp. It is also excellent for building and as a source of firewood. These uses have contributed to its popularity as a crop for both commercial farmers and small growers. Since its introduction to South Africa, abiotic stresses such as frost damage have affected the silviculture of black wattle, and this has resulted in major financial losses for the forestry industry. Over several decades, breeding research has been conducted with the aim of developing genetically improved seed for frost prone areas. However, this has yet to be achieved. Screening for frost tolerance in black wattle has mainly been conducted using field trials. While this provides the most realistic means of screening, it has several challenges. Field trials are time consuming, expensive and require much effort. Furthermore, frost events are unpredictable in timing and in magnitude, thus affecting frost damage screening trials, with levels of zero to extreme frost occurring in any one year at a selected site. This does not allow for a productive plant breeding program to consistently screen for frost tolerance. The current study focussed on the development of an artificial frost screening method for black wattle. A protocol comprising of eight days of moderately cold temperatures (adaptation) and one day of extremely cold temperature (frost tolerance) was established using a temperature-controlled chamber. The frost damage that resulted from the implementation of this protocol produced similar levels of damage as those experienced in previous field trials. This protocol was thereafter tested in two separate trials involving 100 families of wattle accessions that had previously been ranked in field trials that had been run in frost prone areas. A weak correlation was observed between the results of the artificial frost screening trials and the corresponding field results (r=0.24 to 0.28, rs=0.20 to 0.28). Kruskal-Wallis tests showed a statistical similarity between the medians of one of the artificial frost screening trials and the field frost damage evaluations, and a significant difference between the medians of the two artificial frost screening trials that were conducted. The understanding of the molecular aspects that contribute towards frost tolerance in black wattle is extremely limited. Several studies involving a molecular approach to understanding this trait in other woody species have shown promising results. Frost tolerance is a multigenic trait and therefore a proteomic approach was chosen as the best option to identify biomarkers associated with it. Protein extraction from black wattle has not been previously conducted and therefore a protocol that dealt with the interference of phenolics and that was compatible with downstream proteomic techniques was developed. During the protein extraction protocol development, three different protein extraction methods were compared. These methods differed in terms of their precipitation agent combinations. These combinations included acetone and methanol, phenol and ammonium acetate and ammonium acetate and methanol. The combination of phenol and ammonium acetate produced the highest protein yield, as well as the most distinct protein spots after separation by two-dimensional gel electrophoresis. This method was used to extract proteins from 40 black wattle families with varying levels of frost tolerance, before and after cold stress treatment. These extracted proteins were thereafter separated using two-dimensional gel electrophoresis so that changes in protein expression as a result of cold exposure could be analysed. Multivariate analyses of the proteomic data revealed that six proteins were upregulated in frost-tolerant black wattle families. The identified proteins were: two isoforms of oxygen-evolving enhancer protein 1, probable 1-acyl-sn-glycerol-3-phosphate acyltransferase 4, ribulose bisphosphate carboxylase/oxygenase activate, chaperonin 60 subunit alpha 1 and stromal 70 kDa heat shock-related protein. After identification by mass spectrometry, it was established that these proteins have previously been shown to contribute to the protection of cellular membranes, maintenance of photosynthetic processes and the prevention of protein misfolding and aggregation. These proteomic functions have been observed in previous studies to be associated with the process of cold acclimation in plants and thus seem to play a role in frost tolerance in black wattle. The establishment of an artificial frost screening protocol and the proteomic profiling of black wattle for frost tolerance are important tools for preliminary screening of families prior to field trials. By using these protocols fewer black wattle families will require field testing. This will be beneficial both in terms of cost, effort and time associated with field trials. The development of artificial methods for the induction of stresses and the proteomic changes that result from these are valuable tools for understanding the mechanisms that plants use to cope with abiotic and biotic threats.
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    A study on avocado sunblotch viroid (asbvd) with a focus on symptomless carrier trees.
    Zwane, Zanele Rebecca.; Jooste, Anna Elizabeth Catharina.
    The avocado (Persea americana Mill.) is an important subtropical fruit worldwide (Blakey et al, 2014). South Africa is among the top five avocado exporters and the exports are primarily aimed at the European market (DAFF, 2019). According to the profile of the South African avocado market value chain by DAFF (2019), approximately 54% of total avocados produced in South Africa are exported, 14% are sold through the National Fresh Produce Markets (NFPMs), 12% are sold to the informal markets (bakkies and hawkers), 10% are processed, while the remaining 9% are delivered directly to retailers. Avocado sunblotch disease (ASBD), caused by avocado sunblotch viroid (ASBVd), is one of the smallest pathogens inciting economical losses in the avocado crop worldwide (Palukaitis et al. 1979). The infected trees can either express ASBD symptoms phenotypically or can show no symptoms or signs of infection but still be carriers of ASBVd; these trees are known as symptomless carrier trees (Acheampong et al., 2008). Characteristic ASBD symptoms include the appearance of irregular, sunken areas of white, yellow, or reddish colour in infected fruit; white/yellow sunken streaks on green stems and variegated or bleached symptoms on the leaves (Vallejo-Perez et al., 2014). The ASBVd symptomless carrier trees have been described as the primary sources of infection by spreading the disease through budding and grafting practices thus playing an essential role in the epidemiology of ASBVd (Saucedo Carabez et al., 2019). Therefore, the main aim of the study was to investigate ASBVd in avocado symptomless carrier trees. The specific objectives of the current study were to (1) identify and monitor ASBVd-infected trees from the flowering stage until harvest for any visible physical indications of the disease compared with healthy trees, (2) determine the maturity of ASBVd - infected and healthy fruits over time from early development stages until harvest, (3) determine the productivity of ASBVd - infected fruits of the symptomless carrier trees to healthy ones by counting the number of fruit per tree, (4) investigate direct ripening, storage effects and internal quality of ASBVd - infected fruits, (5) investigate honeybees as possible ASBVd vectors for tree-to-tree transmission during pollination, (6) determine the occurrence of root graft transmission of ASBVd from root systems of infected trees to healthy trees, (7) investigate the impact of cutting tools on the plant to plant transmission, (8) study the movement of ASBVd from initially infected cells to the rest of the tree through the grafting of infected scions on healthy rootstocks and (9) investigate t 85 he genetic differences between ASBVd variants within a clonal symptomless carrier ‘Fuerte’ tree population. Differences were observed in the orchard between infected and healthy trees; medium and highly infected trees excessively produced flowers, lost leaves during flowering and ultimately bore few to no fruit at the end of the season. The dry matter content results showed that ASBVd did not affect the maturity of the fruit; fruit from infected and healthy trees matured at the same time. Yield measurements indicated that medium and highly infected trees produced between 83% and 96% lower yields compared to healthy trees. Postharvest studies showed that medium and highly infected fruit significantly lost firmness and developed colour more rapidly than healthy fruit. Infected fruit also developed external rots and shrivels for non-stored fruit, however, these injuries were reduced for fruit stored at 5°C for 28 days. Therefore, flower overbearing with the shedding of leaves and lower yields can be used as an indication of ASBVd infection in ‘Hass’ orchards; however, confirmation with molecular testing is required. These observations can be incorporated as an ASBD management strategy in 'Hass' orchards. Graft transmission (top-work) was demonstrated by grafting 30 healthy and 30 infected ‘Hass’ scions onto a healthy ‘Bounty’ rootstock. There was a 53% infection success rate for the infected symptomless carrier trees compared to the 43.3% for the healthy trees. The statistical analysis showed that there were no significant differences between grafting the healthy or infected scions to the rootstocks (p≤0.05), implying that ASBVd spread through the infected scions is unavoidable unless viroid-free scions are used. Two separate root graft experiments were conducted, one in the tunnels where a single infected tree was planted in the 10L plastic bag to force root contact with four healthy trees; the healthy trees outcompeted the infected trees in all six experiments and they died. In the field, the healthy young ‘Fuerte’ trees were planted one meter closer to the old infected orchard trees with confirmed ASBVd positive roots to force root contact. Eventually, the trees died from the lack of sunlight and water stress and the experiment was discontinued. Seventy-five ‘Fuerte’ clonal trees were used in the mechanical transmission experiment using pruning shears (secateurs). The trees were infected by cutting the infected branch and then using the same shear to prune the healthy trees. The shears were treated with four different percentages of sodium hypochlorite (3.5% M/V) and the untreated shears were used as controls. ASBVd was not successfully transmitted 118 mechanically using pruning shears, however, when the stability of ASBVd was tested on different surfaces, it showed to survive up to 24 hours. Therefore, if equipment used on an infected tree is used for the next tree, there is a chance that ASBVd will be transmitted to the next tree and lead to ASBVd spread in an orchard. Pollen and bees were sampled from the beehives at four different sites in KwaZulu-Natal. ASBVd was successfully detected in the pollen from all four site sites, however, only detected in the bees from three sites The samples were sequenced and the blast results confirmed the sequences to be ASBVd and the phylogenetic analysis gave a 93% identity between the detected sequences and the existing ASBVd variants retrieved from the GenBank® database. From the current study, it was confirmed that graft transmission is the most prevalent mode of transmission for ASBVd and that honey bees carry pollen from infected trees to healthy trees in the field and can play an important role in pollen transmission. A total of 103 positive symptomless carrier trees were detected with a real-time qRT132 PCR assay from a population of 453 young ‘Fuerte’ trees. Results showed that 22% of this population was infected with ASBVd without showing signs of infection. In a further investigation, complete ASBVd genomes were obtained by using a conventional PCR with primer sequences that yielded a 250 bp product, here only 76 samples tested positive for ASBVd, the remaining 27 samples tested negative. The 76 samples were sequenced and the sequences obtained had lengths that variedbetween 248 and 253 nucleotides. From the original 76 sequences, 42 ASBVd variants were identified and several sequences were repeatedly detected in the population. The variants were deposited to the NCBI GenBank® and were assigned ON135462 to ON135503. The phylogenetic analysis of the variants obtained from this study showed a high sequence identity of 97% with the reference ASBVd variants obtained from GenBank®. The current study is crucial for the development of accurate detection techniques for ASBVd and contributes to the ASBVd action plan goals that promote the removal of all infected material from avocado orchards to prevent the further spread of the viroid in avocado orchards.
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    Banana bunchy top virus in South Africa: the distribution, molecular relationship and transmission studies.
    (2021) Ximba, Sinethemba Patience Fanelwe.; Jooste, Anna Elizabeth Catharina.; Gubba, Augustine.
    The first report of Banana bunchy top virus (BBTV) in KwaZulu-Natal (KZN), South Africa in 2016 has raised the need to study the virus in South Africa. The aim of this research project was to conduct surveys across banana-producing provinces, including KwaZulu-Natal, Mpumalanga and Limpopo in South Africa, to determine the spread of BBTV in banana production regions across the country. To date, the virus has been localized within the province of KZN in the South Coast region. Once positive samples were obtained, the genetic relationships between the South African isolates and those collected globally was investigated. This was done by studying five (DNA-C, -S; -N; -M; -U3) of the six components of the BBTV genome. No major differences between the isolates were observed. As the virus is only transmitted through infected planting material and through the vector, Pentalonia nigronervosa, BBTV transmission studies were conducted. Such studies have been conducted in different countries on this topic with conflicting results and BBTV transmission studies were included here as well. Plant species namely Colocasia esculenta, Alocasia macrorrhizos, Alpinia zerumbet and Strelitzia reginae, that are usually found growing around banana plantations, were investigated to determine if these plants act as reservoirs of the virus vector and also to determine if these potential alternative host plant can be hosts of the virus. Banana plants were included as controls in the experiment. A qPCR was optimised to test for BBTV in the plants and aphids at low concentrations. BBTV was detected in all of the plants except A. macrorrhizos. It was concluded that A. zerumbet was an alternative host of the banana aphid while C.esculenta and S. reginae are assumed to be intermediary hosts of the virus vector while A. macrorrhizos is neither a host of the vector nor of the virus.
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    Scirtothrips aurantii faure (thysanoptera: thripidae) population dynamics, biological control, and the characterisation of bracharoa mixta (snellen) (lepidoptera: erebidae) and wind in scarring avocado, persea americana miller (lauraceae)
    (2020) Bara, Gracian Takudzwa.; Laing, Mark Delmege.
    Avocado (Persea americana miller) production is a growing, multibillion rand, export oriented industry in South Africa employing tens of thousands of people and contributing significantly to the local GDP. To remain competitive in the global market, the South African avocado industry needs to consistently produce high quality fruit. However, poor quality fruit remains one of the biggest challenges to export. The aim of this study was to identify, describe and quantify the role played by biotic and abiotic factors in scarring avocado fruit, as well as to propose mitigatory approaches. The lack of current, updated and scientifically evidenced documentation is a shortcoming addressed in this thesis with a goal to enable policy makers, growers and academics to minimize scarring losses. Using a modified beat cup method, the avocado thrips spectrum during flowering and early fruiting was found to include Frankliniella occidentalis (Pergande), Scirtothrips aurantii Faure, Thrips gowdeyi (Bagnall), Thrips pusillus Bagnall, Thrips tenellus Trybom, Haplothrips gowdeyi (Franklin), Haplothrips bedfordi Jacot-Guillarmod and Megalurothrips sjostedti (Trybom). Tea filter paper (74 μm pore size) and coffee filter paper (53 μm pore size) were determined to be effective thrips screen material for use in thrips exclusion trials. Fruit sampling using naturally infested fruitlets confirmed that avocado is a natural host to S. aurantii (the South African citrus thrips). This is the first study to demonstrate that avocado is a natural host to this pest. The confirmation of S. aurantii, an established and well-studied pest of citrus, in South African avocado fruit, forms the basis for tailor-made IPM programs in avocado fruit and allows for parallels and comparisons to be drawn on its biology, ecology and management from the citrus industry. To monitor the presence and abundance of S. aurantii in avocado orchards during early fruiting, several different coloured sticky traps and placements (border vs interior) were trialled. Yellow sticky traps were found to be effective, with the incidence and distribution of the pest tending to be random, with numbers high during the spring flush, declining thereafter and picking up in summer. This means that when monitoring for S. aurantii, yellow sticky traps can be set-up randomly in an avocado orchard during early fruiting. The spring flush and early fruiting period present a small window of opportunity when control measures can be implemented (if necessary), to prevent economic scarring damage on the young, developing fruit. Several pre-harvest diseases are known to inflict serious economic damage to avocado fruit, among them avocado scab (Sphaceloma perseae Jenkins). Having been confirmed in South Africa previously, S. perseae was initially suspected as the cause of a scarring injury called “wind damage” by growers. However, morphological and DNA fingerprinting did not confirm its presence. Cladosporium spp. were repeatedly isolated from typical scars on avocado fruit but they were neither directly infectious, nor infectious in wounds created by mechanical abrasion (to simulate thrips and wind damage), and pathogenicity was not demonstrated. Thrips and wind abrasion were identified as major quality constraints accounting for 30 % scarring damage, a loss factor of 13.72 % and a combined revenue loss of 5.57 %. Revenue losses in the order of 1.49 % are incurred annually due to S. aurantii downgrading (3.86 % loss factor). Cultivar differences were observed, with ‘Pinkerton’ and ‘Carmen®-Hass’ being the most susceptible cultivars. Proximity to macadamia trees was also found to increase incidence of S. aurantii scarring damage. Avocado growers are therefore advised to take steps to minimise scarring damage by siting avocado orchards away from prevailing and dominant winds, macadamia trees, as well as putting in place suitable windbreaks. Towards the end of the 2018/2019 avocado season, unidentified Lepidoptera larvae were observed scarring avocado fruits and defoliating leaves in Wartburg, KwaZulu-Natal, South Africa. Using morphological and molecular techniques, Bracharoa mixta Snell. (Lepidoptera: Erebidae) (tussock moth) was identified, DNA barcoded (GenBank MN527963) and voucher specimens (voucher I.D AcP 9636) were deposited for future referencing. This is the first report of tussock moths scarring avocado fruit. Potential revenue losses of up to ZAR 1352.90/t (2.26 % revenue loss) through downgrading are possible because this insect is capable of causing economic loss, in sporadic, isolated epizootics. The implications of these findings are that the insect has been identified, characterized and its biology studied, and this will aid in rapid detection and implementation of IPM should there be a further outbreak. Dispersal/Emergence (D/E) traps and fruit infestation indices were used to monitor S. aurantii populations during the critical early fruiting period and soil insecticide applications were applied to control below-ground life stages. Use of fruit infestation indices is recommended ahead of D/E traps for monitoring. Control using soil drenches of biologicals and synthetic insecticides was not demonstrated, probably because only a small percentage of the thrips pupated in the ground (18.40 % under laboratory conditions). The low percentage of S. aurantii pupating on the ground means that soil application of non-systemic insecticides and biocontrol agents is not a viable control option.
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    Study on the use of biopesticides against cotton insect pests under field conditions and their cost benefits.
    (2022) Malinga, Lawrence Nkosikhona.; Laing, Mark Delmege.
    Cotton (Gossypium hirsutum L.) is one of the essential fibre crops. However, production is affected by several factors including low yields, high input costs, pests, and weeds infestations. Biopesticides can play a vital role in the integrated programme to address the challenges that limit production and reduce the profits for cotton farmers. This thesis consists of five chapters covering different aspects of the research on farmers' survey and biological control of cotton pests. Each chapter is presented as an independent study. The focus of this research was to (1) provide the bac-1round on cotton production in South Africa and major pests and their control; (2) survey the current status of pests on cotton and production practices; (3) evaluate the effect of different biological agents on the control of cotton pests under field conditions; (4) evaluate the efficacy of biopesticides in comparison with the insecticides against sucking pests; and (5) perform a cost analysis of cotton production using biological control agents under field conditions. This study would share an insight to build a foundation for management of major cotton insect pests. A survey was done to (1) evaluate farmers' knowledge and perceptions of cotton pests; (2) examine farmers' current practices in managing cotton pests; and (3) identify challenges and intervention opportunities to develop an efficient integrated pest management programme for cotton production. One hundred and forty farmers, mainly smallholder farmers were interviewed, and most of them planted cotton in less than five hectares of land, with 96% planting under dryland. Most farmers neither practiced conservation agriculture (95%) nor conducted soil analyses (87%) and harvested their cotton by handpicking (99%). Their knowledge of insect pests was higher than of diseases, with most of the participants not aware of nematodes (88%), or diseaseresistant cultivars (74%), while 91% were aware of insect-resistant cultivars. Most farmers relied on synthetic pesticides to control cotton pests, and only 7% used biological control. Dryland farmers reported a mean seed cotton yield of 700 kg.ha-1, and 5 000 kg.ha-1 was obtained from irrigated cotton. Most respondents were only mentored and supported by extension officers (82%). Climatic conditions (98%), labour costs (88%), and insect infestations (42%) were identified as the main constraints in cotton production. The study recommends the development of alternative control methods to minimize the use of agrochemicals. Four biopesticides (Eco-Bb®, Bb endophyte, Bolldex®, Delfin®) were compared with a pyrethroid, Karate® against cotton insect pests, particularly the African bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae). The treatments of Karate® and Bolldex® significantly reduced the H. armigera population, while the treatment of Eco- Bb® had the lowest number of damaged bolls. Plots sprayed with Karate® had significantly fewer aphids and leafhoppers. Plots treated with Bolldex® and Bb endophyte exhibited the lowest number of thrips. Plots sprayed with Karate® and Eco- Bb® had a significant effect on the whiteflies, while Delfin® had the least significant number of spider mites. The treatment of Eco-Bb® exhibited a lower cotton stainer population, while the treatment of Karate® had the lowest population of leafhoppers. The highest average seed cotton yield of 6 400 kg.ha-1 was recorded in the plots that were treated with Bolldex®. In summary, the efficacy of different biopesticides against H. armigera varied significantly; however, Karate® and Bolldex® resulted in better control of the pest. Field trials were conducted to evaluate three biopesticides, Eco-Bb®, Bb endophyte, and Eco-Noc in comparison with the insecticides Chlorpyrifos® 480 EC, Karate® EC, and Bandit® 350 SC to determine their efficacy against sucking pests, notably leafhoppers Jacobiella facialis Jacobi (Hemiptera: Cicadellidae), aphids Aphis gossypii Glover (Hemiptera, Aphididae), thrips Thrips tabaci Lind (Thysanoptera: Thripidae), whiteflies Bemisia tabaci Gennadius (Hemiptera, Aleyrodidae), red spider mite Tetranychus urticae Koch (Trombidiformes: Tetranychidae) and cotton stainers Dysdercus spp. (Hemiptera: Pyrrhocoridae). Karate® significantly reduced the leafhopper population while the biopesticides had some control of the aphids. Plots treated with Eco-Bb® and Bandit® 350 SC had the lowest number of thrips, and there were no significant differences in the populations of whiteflies. All the treatments, except for Bandit®, significantly reduced the number of spider mites. The highest average cottonseed yield of 6 395 kg.ha-1 was recorded in plots sprayed with Bandit®. Cost analysis was done by conducting two field trials (bollworm and leafhopper) to evaluate the effect of biopesticides and synthetic pesticides on controlling different cotton insect pests. The cost of biopesticides was higher than synthetic pesticides. Delfin® was the most expensive treatment at R 7 980/ha, while Chlorpyrifos® 480 EC had the lowest price of R 370/ha. The highest input cost of R 7 200/ha was recorded from labour costs incurred during weed control. The highest total costs of R 21 502/ha were incurred where Eco-Bb®, Bb endophyte and Eco-Noc were applied. In the bollworm experiment, the lowest production costs per hectare were observed from the treatment with Karate® EC (R 19 282). The maximum seed cotton yield of 6 818 kg.ha-1 was recorded in Bolldex® treated plots while Karate® EC treated plots had the highest net profit of up to R 19 148 per hectare and mean benefit-cost ratio of 1.8. In the leafhopper trial, the highest seed cotton yield was obtained from the Bandit® 350 SC treated plots (6 394 kg.ha-1). Plots, where Bandit® 350 SC was applied, had the maximum net profit of R 22 686 with a benefit-cost ratio of 2.
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    Biological control of sorghhm and rice stem borers, chilo partellus and sessamia calamistis using endophytic strains of beauveria bassiana.
    (2018) Bancole, Wonroo Bernice Armelle.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
    Sorghum and rice are two of the major cereals grown across the world. Both of these crops are subjected to a range of abiotic and biotic constraints. Insect pests are important biotic stress factors, which affect both of the crops at all of their growth stages. Stem borers from the family of Lepidoptera, e.g. Chilo partellus (Lepidoptera: Pyralidae) and Sesamia calamistis Hampson (Lepidoptera: Noctuidae) are important pests that attack these cereals. Control of C. partellus and S. calamistis has largely been with pesticides. However, chemical pesticides are too expensive for most small-scale farmers in Africa, leaving their crops unprotected. Biological control is one of the measures that have been advocated for the management of stem borers. Various strains of Beauveria bassiana (Vuillemin) have been documented as being endophytes infecting a wide range of plants, as well as being pathogenic on numerous insect pests. Successes in biological control research have led to the development of various B. bassiana products, which are available commercially, but these are largely epiphytic strains. Biological control studies were therefore conducted with several endophytic strains of B. bassiana against sorghum stem borer, C. partellus, and the rice stem borer, S. calamistis. The fungi were tested by endophytic behaviour and the ability to control the 3rd larval instars of both stem borers, in the laboratory and greenhouse. The interactions of B. bassiana strains and a commercially available Trichoderma product, Eco-T®, were tested in sorghum and rice plants. In vivo and in vitro screening were initially undertaken to evaluate the endophytic behaviour of 20 B. bassiana strains, using two inoculation methods. Subsequently, the best endophytic B. bassiana strains and the best inoculation method were tested at 30 and 60 days after inoculation. The strains were screened in vivo using seed treatments and foliar sprays, under greenhouse conditions, for endophytic behaviour in sorghum and rice plants. There were highly significant differences between the B. bassiana strains (P = 0.0001). Depending upon the inoculation method, the B. bassiana strains that successfully colonized the sorghum and rice plants could be selected after 30 and 60 days. Five strains of B. bassiana strains (Bb3, Bb4, Bb10, Bb21 and Bb35) were found to be endophytic in both crops, and to provide biological control against the two borers. The best five B. bassiana strains were tested for their pathogenicity on the 3rd instar larvae of C. partellus and S. calamistis. Out of the five endophytic strains of B. bassiana, two (Bb35 and Bb3) were the most pathogenic on C. partellus, with the greatest mortality of 80 % being achieved within 28 days after treatment. The B. bassiana strains Bb35 and Bb4 were the most effective strains against S. calamistis, killing 93.33 and 76.66% of the 3rd larval instar at 28 days, respectively. The cumulative mortality of the 3rd instar larvae of both stem borers increased over time at 21 days after inoculation for all five B. bassiana strains. A field trial was conducted to evaluate the biocontrol efficacy of the five best endophytic strains of B. bassiana against C. partellus, compared to pyrethroid pesticide, Karate. Three of the endophytic strains of B. bassiana strains were as effective as Karate sprays when they were applied as seed treatments, reducing damage by C. partellus as much as Karate did. In vitro and in vivo screening were carried out under laboratory and greenhouse conditions, using various inoculation methods, to assess the interaction between the five B. bassiana strains and a commercially available Trichoderma harzianum product, Eco-T®. In the in vitro dual culture bioassay, one of the five endophytic B. bassiana strains (Strain Bb35) was not inhibited by T. harzianum Strain Kd (TKD) at 15 days after inoculation at 30 days after in vitro inoculation. None of the five endophytic B. bassiana strains grew in the presence of TKD. Only the TKD grew all over the plates. In greenhouse trials, various interactions occurred between the two fungi, according to the inoculation methods. When a mixture of conidia of the two fungi was used at the same time as a seed treatment, there was a strong inhibitory effect by TKD toward the five B. bassiana strains. However, if sorghum plants were seed treated with the five B. bassiana strains, followed by drenching of the plant roots with a TKD suspension, then the B. bassiana strains appeared to be able to colonize the stems of the plants whilst the TKD colonized the roots. Sorghum roots were rapidly colonized by the TKD when it was used alone for the seed treatment. The endophytic behaviour of some strains of B. bassiana in sorghum and rice plants can be used as powerful tool to enhance their biological control activity against stem borers of these crops. However, the tested TKD and B. bassiana strains were not compatible in the same space, such as the rhizosphere, but could be used sequentially to secure the benefits of insect control by the B. bassiana strains, as well as the biological control and plant growth stimulation activities provided by T. harzianum strains.
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    Ethnopharmacological study on plants used for skincare and beauty by some Xhosa communities.
    (2018) Thibane, Vuyisile Samuel.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
    The paraphrase “beauty lies in the eyes of the beholder” by Plato, a Greek philosopher, has echoed through the fabrics of time and still echoes in our generation. The statement simply ought to refer to that the observer gets to decide what is “beautiful” in their eyes. The decision on what and who is beautiful is heavily influenced by one’s surroundings. In a digital-market environment the concept of beauty enhancement is paraded by bigger forces to drive their own agenda. The desire to enhance one’s facial appearance has significantly contributed to the observed growth of the beauty industry. The notable growth of the industry has in some cases resulted in unpleasant consequences due to the side effects of some of the products in the market. In this global-blooming industry products are traded and exchanged at a rapid rate. Introduction of safer natural beauty enhancement products are required for the South African market if we were to alleviate those with undesirable side effects and could combat some socio-economic challenges through job creation. The use of plants as the source of natural compounds has proven to be a reliable strategy in ethnopharmacological applications. The Eastern Cape Province has a rich plant biodiversity and the communities have immense indigenous knowledge (IK) on the use of these plants. There is a need to explore the pharmacological application of these plants by introducing beauty enhancement product formulations made from local resources. The project was aimed at documenting and conductingethnopharmacological evaluation of plants used for skincare and beauty for their potential in formulation of beauty enhancement products. An ethnobotanical survey was conducted to document plants that are used by communities in the Raymond Mhlaba municipality for skincare and beauty. Knowledge holders were identified by purpose sampling method and the interviews were conducted in isiXhosa using a structured questionnaire. Information on demographics, names of the plant, type of plant, plant part used, method of preparation and administration and frequency of use was collected and captured in the questionnaires. The Asphodelaceae and Asteraceae were the most represented families of the plants used for skincare and beauty. The communities used sustainable harvesting practices as the leaves were the most utilized plant parts. The most reported beauty enhancement uses were for achieving desired skin complexion and for skin smoothness, with both accounting up to 50% of the reported plant usages. Sixteen plants with the highest frequency index (FI) were selected from the ethnobotanical survey for ethnopharmacological studies related to beauty enhancement. This included Acokanthera oblongifolia (Hochst.) Codd, Aloe ferox Mill, Arctotis arctotoides (L.f) O.Hoffm, Bulbine frutescens (L.) Willd, Cassipourea flanaganii (Schinz) Alston, Chenopodium album L, Clausena anisata (Willd.) Hook.f ex Benth, Haemanthus albiflos Jacq, Marrubium vulgare L, Ilex mitis (L.) Radlk, Plantago lanceolata L, Rorippa nasturtium-aquaticum (L.) Hayek, Sonchus asper L, Symphytum officinale L, Ruta graveolens L and Urtica urens L. The antimicrobial activity of plant extracts was assessed using the microdilution bioassay to determine the minimum inhibitory concentrations (MIC). The antimicrobial activity of petroleum ether (PE), dichloromethane (DCM), 70% aqueous ethanol (v/v) and water extracts of the selected plants were assessed against infectious skin microorganisms including Bacillus subtilis ATCC 6051, Brevibacillus agric ATCC 51663, Staphylococcus aureus ATCC 12600, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 11775, Klebsiella pneumoniae ATCC 13883, Candida albicans ATCC 10231 and the dermatophytes Microsporum canis ATCC 36299, Trichophyton mentagrophytes ATCC 9533 and Trichophyton tonsurans ATCC 28942. The majority of the tested plant extracts were effective and inhibited the skin commensal bacteria E. coli with MIC values less than 100 μg/mL. Prolonged infections by commensal bacteria can condition the skin environment and provide favourable conditions for more opportunistic bacteria such as the Staphylococci genus. Ethanol extracts of C. flanaganii and U. urens expressed high antibacterial activity against S. aureus with MIC values less than 100 μg/mL. Ethanol extracts of R. graveolens and dichloromethane extracts of A. arctotoides were effective at inhibiting S. epidermidis and S. aureus, respectively. Inhibition of two opportunistic bacteria has a positive effect on skin tone, due to the scarring and darkening associated with infection by the Staphylococci genus. There was notable activity recorded against C. albicans and dermatophytes M. canis, T. mentagrophytes and T. tonsurans by extracts of A. oblongifolia, A. arctotoides, C. flanaganii, I. mitis and R. graveolens at different polarities with MIC’s less than 1000 μg/mL. The phenolic content and antioxidant activity of the plants were determined by assessing 50% aqueous methanol extracts (v/v) for their total phenolic and flavonoid content, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferric reducing antioxidant power (FRAP) and the coupled oxidation of linoleic acid and bleaching of β-carotene. The antioxidant mechanism of phenolic compounds associated with beauty enhancement has been proposed to be due to their free radical chain breaking capabilities, metal chelation, oxidant quenching and inhibition of enzymatic activity. The total phenolic content of A. ferox, I. mitis and C. flanaganii were significantly high with recorded values ranging from 37.87 to 50.34 mgGAE/g. The flavonoid content of C. flanaganii, A. oblongifolia and P. lanceolata were significantly high. Methanol extracts of R. graveolens and C. flanaganii expressed the highest antioxidant activity, with IC50 values comparable to the standard antioxidant when assessed for their DPPH radical scavenging activity. The presence of antioxidants in the skin structural layers has a positive effect on the health and function of the skin. Extracts of U. urens, A. ferox, C. flanaganii, B. frutescens, P. lanceolata, H. albiflos, M. vulgare, C. anisata, S. officinale and R. nasturtium-aquaticum expressed good metal chelating potential. The highest oxidative protection in the β-carotene linoleic acid model with comparative oxidation rate ratio (ORR) to the positive control was observed for C. flanaganii, S. officinale and U. urens. The results indicate the ability of the plant extracts to provide protection against increased levels of lipid peroxidation in the skin, an important factor in beauty enhancement, due to delaying the age process. The photo-protective effect of the plant extracts was measured by calculating the sun protection factor (SPF). The SPF is the ratio of ultraviolet (UV) radiation required to produce minimal erythema dose (MED) in protected skin to unprotected skin with higher values indicative of increased protection from photo damage. Ethanol extracts of P. lanceolata, C. flanaganii, A. oblongifolia, I. mitis and A. arctotoides exhibited SPF values of more than 15, which translated to photo protection of the skin against UVB radiation by more than 93.3%. The plant extracts demonstrated the highest absorbance of UVB radiation at a wavelength region between 300 – 305 nm. These will protect the skin against UV-induced oxidative damage and enhance the skin’s health and function. The inhibition of enzymes with beauty enhancement potential by the plant extracts was assessed against tyrosinase, secretory phospholipase A2 (sPLA2), lipoxygenase (15-LOX) and cyclooxygenase (COX-1 and COX-2). Ethanol extracts of R. nasturtium-aquaticum, C. anisata, S. officinale and C. flanaganii expressed good anti-tyrosinase activity. The coupled protection against UV-induced damage and modulation of the tyrosinase enzyme activity can be exploited to achieve the desired skin complexion. The anti-inflammatory studies revealed the potential of extracts of C. flanaganii, P. lanceolata and R. nasturtium-aquaticum to serve as dual inhibitors of 15-LOX and COX-2 enzymes. The inhibition of 15-LOX and COX-2 is effective at resolving psoriasis, a skin-inflammatory associated disease which has a negative effect on the health and beauty of the skin. The effectiveness of ethanol extracts of C. flanaganii, C. album, C. anisata and R. nasturtium-aquaticum in maintaining the cells health and function was examined on human epidermal melanocytes (HEM) cell lines. Ethanol extracts of C. flanaganii, C. album, C. anisata and R. nasturtium-aquaticum were able to inhibit cellular tyrosinase activity and therefore reduce melanin production. The effective concentrations of the extracts were further reported as non-toxic to melanocytes. The observed anti-tyrosinase activity of the extracts against HEM cell lines contribute positively in achieving the desired skin complexion while providing photo protection against UV-induced damage. Therefore, plant extracts that are efficient and safe to use can be incorporated into formulations intended for beauty enhancement and further analysed under clinical trials. The study further suggests that the model undertaken be promoted to individuals and corporations interested in formulation of cosmeceuticals to ensure the safety and efficiency of their products.
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    Prevalence, detection and characterization of tomato spotted wilt and iris yellow spot tospoviruses occuring in Zimbabwe.
    (2017) Karavina, Charles.; Gubba, Augustine.
    Abstract available in PDF file.
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    Identification and characterization of viruses infecting cucurbits in the province of KwaZulu-Natal, Republic of South Africa, with the purpose of developing transgenic virus-resistant cucurbits.
    (2016) Ibaba, Jacques Davy.; Gubba, Augustine.; Laing, Mark Delmege.
    The continued presence of cucurbit-infecting viruses across the Province of KwaZulu- Natal (KZN), South Africa requires exploration of alternative methods of controlling these viruses. The aim of this research project was to identify and characterize the viruses infecting cucurbits in KZN with the intention of subsequently developing transgenic cucurbits with broad virus resistance. A systematic virus survey was therefore carried out in all cucurbit growing areas of KZN during the 2011, 2012 and 2013 growing seasons. Symptomatic leaves suspected to be of viral aetiology were sampled and tested using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RTPCR), in order to detect the common viruses causing mosaic and yellowing diseases. Samples were tested for the following viruses: Cucumber mosaic virus (CMV), Squash mosaic virus (SqMV), Moroccan watermelon mosaic virus (MWMV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), Cucurbit yellow stunting disorder virus (CABYV), Beet pseudo-yellows virus (BPYV), Cucumber yellow stunting disorder virus (CYSDV), Lettuce infectious yellows virus (LIYV), and members of the genera Polerovirus and Carlavirus. CMV, BPYV, ZYMV, and MWMV were detected, along with Pepo aphid-borne yellows virus (PABYV), a putative new species in the genus Polerovirus that has been reported in West Africa. MWMV was the most prevalent mosaic-inducing virus and PABYV was the most prevalent yellowing-inducing virus. One common virus symptom consisted of shoe strings leaves and deformed fruits on baby marrows plants (Cucurbita pepo L.). However, the samples tested negative for all the viruses selected. These symptoms were further investigated using Potyvirus universal primers. This led to the detection of a tentative species in the Papaya ringspot virus (PRSV) cluster in the genus Potyvirus. The tentative potyvirus was named Zucchini shoestring virus (ZSSV). Next Generation Sequencing was later used in combination with Sanger Sequencing to elucidate the full genome sequences of MWMV, PABYV, and ZSSV. MWMV isolates from RSA were found to be more closely related to each other than to the isolate from Tunisia. Both PABYV and ZSSV were found to be distinct species in the genera Polerovirus and Potyvirus, respectively, on the basis of their genome organization and the species criteria for each genus. Baby marrow was identified as the most susceptible cucurbit to viral infections in KZN. It was therefore decided to develop baby marrow plants with resistance to the potyviruses identified in the survey, using antisense post-transcriptional gene silencing. A portion of the 5’ coding sequence of the coat protein genes of MWMV, ZYMV and ZSSV were amplified by RT-PCR and inserted into a plant expression vector pEPJ86-m/2N. The expression cassette on Page iii of 136 pEPJ86-m/2N was subsequently sub-cloned into the plant transformation vector pGA482G before being introduced into Rhizobium radiobacter, formerly Agrobacterium tumefaciens, strain LBA4404 (pAL4404)(pBI121) by electroporation. Rhizobium-mediated transformation on baby marrow cotyledon explants was subsequently performed. The resultant putative transgenic regenerated baby marrows plants were subjected to different tests that included PCR to confirm transgene insertion, mechanical inoculation of each virus and DAS-ELISA to evaluate virus resistance. A total of 94 baby marrow plants were successfully regenerated from 250 explants. Out of the 94 plants, 84 were found to have the transgene based on the PCR results. Of these 84 lines, 76 showed resistance to the selected three viruses. Our preliminary results show the potential of using transgenic cucurbits with resistance to three potyviruses as an effective strategy to control virus diseases on cucurbits.
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    Integrating microdosing of fertilizers with biological control agents for maize production in the Eastern Cape, South Africa.
    (2015) Kubheka, Bongani Petros.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
    Maize is an important staple crop in South Africa, and is also used for animal feed. In the Eastern Cape Province in South Africa a larger percentage of the farmers are small scale farmers and lack financial resources to apply the recommended levels of fertilizer inputs for optimal production. The currently promoted system of maize production in the Eastern Cape was designed specifically for commercial production, e.g., it is based on the use of agrochemicals to control plant diseases and pests, combined with the use of synthetic fertilizers to provide nutrients, and the application of large quantities of lime to solve soil acidity issues. The currently available mechanical equipment used to fertilize maize are only for row fertilization, whereas in between rows there may be losses of fertilizer due to the distance to the roots. Small scale farmers of this region do not apply lime. Consequently, maize yields are very low for small scale farmers in the Eastern Cape Province, relative to commercial farmers. Both biotic and abiotic factors combine to reduce maize yields. These include root diseases caused by Rhizoctonia solani Kühn and other root pathogens and poor soils (highly acidic soils with low nutrient content, especially of P and Mo). Given that the farmers do not treat the seed, lime the soil, and apply little or no fertilization, yields are consistently low. One goal of this study was to control root rot on maize caused by R. solani using a biocontrol agent, and a potassium silicate fertilizer as a priming agent of plant disease resistance. A commercial biocontrol agent, Eco-T® (a.i. Trichoderma harzianum Strain kd), is known to control most pathogenic root fungi, including R. solani. This treatment was evaluated alone and in combination with potassium silicate (KSil) in field trials over two seasons. Two KSil formulations were tested, namely a liquid and a slow release formulation. All treatments significantly reduced damage by R. solani, with T. harzianum plus the liquid formulation of KSil resulting in the highest level of control in Season 1, and T. harzianum alone providing the highest level of disease control in Season 2 (p = 0.018). There was no significant difference in the levels of control provided by T. harzianum and KSil applications when they were applied individually. All treatments significantly increased the maize yield relative to inoculated control. The treatment that gave the highest percentage difference relative to the inoculated control was KSil liquid formulation combination with T. harzianum, the combinations gave a significant 45% increased yield over the inoculated control. This means that this combination is an option for the farmers. Small scale farmers in the Eastern Cape produce maize in poor soils that have low pH levels, very high levels of acid saturation and low nutrient levels, especially of P. The second part of this study was to investigate achievable approaches to liming and fertilization for small scale farmers in the Eastern Cape. These included fertilization and liming by micro-dosing of 2:3:2 (34) fertilizer, superphosphate fertilizer and dolomitic lime using a cap of a soda bottle to measure out approximately 5g to each maize plant, applied directly into the planting hole. In order to fix atmospheric nitrogen and to solubilize phosphates in these acidic soils, a nitrogen-fixing isolate of B. megaterium was drenched into the planting holes. Micro-dosing of 2:3:2 (34) fertilizer increased maize yields by 64.6% and 13.6%, over the two seasons of the study. Micro-dosing with superphosphate fertilizer also significantly increased the maize yield (P = 0.001) by 50.5% and 37.4%. The combination of B. megaterium and 2:3:2 (34) fertilizer significantly increased the maize yield (P = 0.001) by 54.7% and 48.1% in season 1 and 2, respectively. The combination of B. megaterium, 2:3:2 (34) fertilizer and lime significantly (P = 0.018) increased maize yield, maize plant height, and stem diameter in both seasons. The increases in both seasons were consistent as a result of this combination compared to the 2:3:2 (34) fertilizer and lime combination. Whenever B. megaterium was included in the treatment combination, yields were increased, although not significantly. It was therefore concluded that micro-dosing of fertilizers can have a significant role in improving the yields for small scale farmers that cannot afford to apply the recommended levels of fertilizer or lime. It was also concluded that the use of B. megaterium is beneficial when combined with NPK and P fertilizers. Field experiments over three seasons were designed to evaluate the integration of the treatments applied in the field experiments mentioned above. The study was conducted in a field with a pH of 4.0 and an acid saturation of 54%. The methods included micro-dosing and spot application of fertilizers and lime. A strain of B. megaterium was used as a nitrogen fixer and phosphate solubilizer. For maize root disease control, the methods employed included the use of a biological control agent, T. harzianum and potassium silicate as a plant defense activator. The aim of the study was to reduce input costs whilst still providing adequate fertilization and root disease management. R. solani significantly reduced maize yields, by up to 34%, but treatment of maize seed with T. harzianum, or B. megaterium reduced losses over the three seasons from 34% to 16% and to 10%, respectively. In Season 1, the integration of all treatments (T. harzianum, B. megaterium and potassium silicate) increased maize yields by 130% relative to the R. solani inoculated control. The plots with the highest yields in the presence of R. solani were treated with T. harzianum (216%), followed by T. harzianum plus potassium silicate (214%), and lastly plots treated with T. harzianum plus B. megaterium (178%). A similar trend was observed over the three seasons. A cost benefit analysis of the integrated management of maize grown under acidic conditions and also in the presence of R. solani was undertaken after the three seasons of field experiments. The first experiment evaluated the control of R. solani using the T. harzianum, priming of plant resistance using potassium silicate, as well as the combination of T. harzianum and potassium silicate. The second experiment evaluated micro-dosing of 2:3:2 (34) fertilizer and lime, and the use of B. megaterium as a nitrogen fixer and phosphate solubilizer. The third experiment evaluated the integration of micro-dosing of 2:3:2 (34) fertilizer, superphosphate, lime and B. megaterium. The current retail prices were used. It was observed that the combination of T. harzianum and the potassium silicate liquid formulation consistently gave the highest returns on investment in controlling R. solani over the three seasons. Full fertilization consistently provided a negative return, with a mean loss of R3, 363 over three seasons, relative to the Untreated Control, which was not fertilized. Micro-dosing with lime plus 2:3:2 fertilizer gave the highest net return on investment. This was significantly different (p = 0.001) to both the Untreated Control and the Full Fertilization in Season 1. However in Season 2, the combination of B. megaterium plus 2:3:2 (34) fertilizer micro-dosed resulted in the highest net return that differed significantly from both the Untreated Control and the Full Fertilization. In the integration experiment all treatments in Season 1 gave a significantly higher yields and increased net returns on investment, relative to the R. solani inoculated control, with the lowest giving a 39% net return and the highest giving a 65% net return. In Season 2 none of the treatments resulted in significantly higher yields. In Season 3 a repeat of Season 1 results was seen where all treatments resulted in a significantly higher yields and net returns relative to the R. solani inoculated control, with the exception of B. megaterium in the presence of the pathogen. Two treatments, namely T. harzianum only and T. harzianum plus B. megaterium were consistently among the top three treatments that significantly controlled R. solani. The combination that gave consistently higher return on investment in the control of R. solani and also in the provision of nutrients was the T. harzianum plus B. megaterium plus micro-dosed 2:3:2 (34) and lime. It was therefore concluded that a cost effective method of fertilization and liming that will suit the Eastern Cape small scale farmers is micro-dosing rather than conventional method. Moreover incorporating B. megaterium improved yield consistently at little cost. For the concurrent control of root pathogens such as R. solani, it is recommended that the small scale farmers use T. harzianum, and possibly potassium silicate.
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    Organogenesis and genetic transformation of dierama erectum hilliard.
    (2016) Koetle, Motselisi Jane.; Van Staden, Johannes.; Finnie, Jeffrey Franklin.
    Abstract available in PDF file.
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    Use of trichoderma and bacillus isolates as seed treatments against the rootknot nematode, meloidogyne javanica (chitwood)
    (2015) Chinheya, Cleopas Chenai.; Laing, Mark Delmege.; Yobo, Kwasi Sackey.
    Abstract available in PDF file.
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    Studies on guava wilt disease.
    Grech, Nigel Mark.; Laing, Mark Delmege.
    No abstract available.
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    Development of a new entomopathogenic nematode species, steinernema innovation : biological characterization and mass production.
    (2014) Ramakuwela, Tshimangadzo.; Laing, Mark Delmege.; Hatting, Justin Louis.; Hazir, Selçuk.
    Entomopathogenic nematodes belonging to the genera Steinernema and Heterorhabditis are employed as environmental friendly biopesticides to control key pests in high value agricultural crops. Their successful application in developed countries has proved their effectiveness. The production of EPN for large scale commercial application has been restricted to developed countries because high capital cost of setting up liquid fermentation, and the high running costs of such units. If EPNs are to be commercialized in developing countries, then this will probably be achieved using in vivo culturing in insect larvae, or by using in vitro solid culture on a small to medium scale. The Republic of South Africa bans the importation of exotic EPN products by Amendment 18 of Act 36 of 1983. Therefore EPN products in South Africa will need to be developed from indigenous species. This study focused on optimizing the solid culture production and biological characterization of an indigenous nematode, Steinernema innovationi, as a biocontrol agent. A good production system must achieve high yield, maintain high virulence and the product must have a reasonable shelf-life. Optimum production temperature was determined using in vivo culturing of S. innovationi inside Galleria mellonella larvae. Performance was measured by quantifying the first day of emergence of infective juveniles (IJs) from cadavers, yield of IJs and IJ length, at five temperatures ranging from 18°C to 25°C. Optimum temperature was selected as 22°C based on high yield and slow emergence. There was a yield of 92,756 ± 28,089 IJs/larva, and 84,056 ± 27,832 IJs/larva, at 22°C and 25°C, respectively, which were not significant different. However, IJ emergence was significantly slower at 22°C, which provided more time for nutrient uptake and therefore these IJs had a greater nutrient reserve. There was no correlation observed between IJ length vs. temperature and IJ length vs. yield. A medium containing a puree of the larvae of the common house fly (Musca domestica) and 3% canola oil produced the highest yield of IJs (781,678 ± 221 IJs/5g), the highest level of live IJs (>84%) and the lowest level of adults (<10%) remaining in the medium at the time of harvest, compared to five other media formulations. All media were subjected to in vitro mass production at 22°C. A liquid inoculum of the EPN gave higher yields than a solid culture inoculum, irrespective of concentration. The length of IJ did not affect virulence against last instar larvae of G. mellonella, which was >90% in all experiments. A study characterizing optimum storage temperature of S. innovationi was carried out at five temperatures ranging from 5°C to 25°C in aqueous suspension over a period of 84 days. Survival was highest and most stable at 15°C, ranging from 84% to 88% after 84 days storage. Storage of the EPN in a sponge at a concentration of 2.5 million IJs in 15ml 0.1% formalin solution was successful, with an improvement of 6% compared to aqueous storage at 15°C. Furthermore, storage of the EPN in a sponge at 25°C, after a period of low temperature (15°C) storage for 84 days, did not have a detrimental impact on IJ survival and infectivity (87% and 95%, respectively). The new EPN (S. innovationi) was characterized by studying traits related to its infectivity (infectivity under a range of temperatures [10°C to 35°C], foraging behaviour & persistence under field conditions). Time till death was shortest at 25 and 30°C (average 1 day). The highest number of established IJs was recovered at 25°C (mean = 27°C). The nematode infected Galleria mellonella larvae at all depths and was capable of covering a distance of up to 15cm in 24 hours. There was no statistical difference between in vitro (40%) and in vivo (27%) cultured IJ persistence 4 weeks post application. Furthermore, there was no statistical difference in nematode recovery after 1 and 4 weeks post application of in vitro (33% & 40%, respectively) and in vivo (60% & 27%, respectively) produced IJs. Thermal activity was optimal at 25°C, the new species was classified as a cruiser and proved to survive under field conditions for at least 4 weeks. Susceptibility of larvae and/or pupae of Eldana saccharina (Walker), Sesamia calamistis (Hampson), Chilo partellus (Swinhoe), Tenebrio molitor (Linnaeus), Galleria mellonella (Linnaeus), Cydia pomonella (Linnaeus), Plutella xylostella (Linnaeus), and Gryllidae acheta (Linnaeus) representing three orders (Coleoptera, Lepidoptera & Orthoptera) was tested at a low and high concentration of 50 and 500 IJs, respectively. The hosts G. acheta, C. partellus and P. xylostella showed least susceptibility with maximum mortalities at the 500 IJs concentration of 28%, 45% and 92%, respectively. All other hosts suffered 100% mortality. Pupal mortality ranged from 47% to 68%. An LC50 and LC70 of 3 IJs/larva and 31 IJs/larva, respectively, was calculated for the black cutworm, Agrotis ipsilon (Hufnagel). These results provided a broad guideline on the relative pathogenicity of this new species against different hosts. A cost analysis was calculated for in vitro solid culture of the EPN, including the cost of rearing an insect-based nutrient component. An estimated retail price was then compared to the costs of market products around the globe. An estimated retail price (R90.61) for S. innovationi was considerably lower than the market price for other Steinernema species, which ranged from R271.50 to R458.55, in South African rands. The production system developed in this study for S. innovationi offers a highly competitive small to medium scale production method to produce EPN products without having to invest in large scale liquid fermentation equipment, by using relatively cheap production media ingredients, and simple solid culture growing conditions.
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    Studies on Puccinia recondita f. sp. tritici with special emphasis on adult plant resistance in wheat.
    (1986) Pretorius, Zacharias Andries.; Rijkenberg, Fredericus Hermanus Johannes.; Wilcoxson, Roy Dell.
    Leaf rust (Puccinia recondita f.sp. tritici) of wheat (Triticum aestivum) was widespread in South Africa during 1983, 1984 and 1985 and often reached epidemic levels, especially on autumn-sown spring wheat in the Cape Province. Nine physiologic races were identified during the study period. The most common race was avirulent to the leaf rust differential genes Lr3a, 3bg, 3ka, 11, 16, 20 and 30 and virulent to Lr1, 2a, 2b, 10, 14a, 15, 17, 24. Resistance genes Lr9, Lr19, Lr21 and Lr26 were effective to all isolates tested. Evaluation of wheat genotypes for components of resistance, viz. infection type, latent period, number of uredinia and uredinium size, revealed three phenotypic reaction classes. The first group exhibited negligible resistance, the second was susceptible or moderately susceptible as seedlings but resistant as adult plants while the third group was resistant at all growth stages tested. Adult plant resistance was expressed by hypersensitive or non-hypersensitive reactions and the combination of components conditioning resistance varied. Adult plant resistance conferred by gene Lr22a was characterized by a long latent period, small uredinia, reduced sporulation and an absence of a differential interaction between components of resistance and different races of Puccinia recondita f.sp. tritici. Numbers of uredinia on flag leaves of RL6044 (Lr22a) were equal to those of a susceptible check, Line E. Lr22a was inherited as a partially recessive gene in crosses with Zaragoza and SST33. Assessment of latent period, number of uredinia and infection type in F4 and FS families homozygous for Lr22a and derived from crosses between RL6044 and Zaragoza or SST33, revealed significantly different levels of resistance between families. Differences were attributed to other genes modifying the expression of Lr22a. Adult plant resistance of Era, Glenlea, RL6044 and sinton was expressed prior to the fifth-leaf stage. Latent period increased and number of uredinia decreased as each wheat matured. While the latent period of the flag, flag-l and flag-2 leaf was similar within Era, Glenlea and RL6044, differences between these genotypes occurred. The latent period of flag leaves of Sinton was shorter than that of the two lower leaves. Significantly fewer uredinia developed on the flag-2 leaf of Glenlea. A reduction in temperature from 21 C to 15 C significantly increased latent period in Era, Glenlea and RL6044, and also restricted uredinium size on flag leaves of RL6044. The adult plant resistance of Glenlea crossed with Line E was conferred by two partially recessive genes. Additionally, F2 to FS progenies of this cross eXhibited high levels of hypersensitive seedling resistance at 29 - 31 C to certain isolates. The latter resistance was not conferred by Lr1 or by the LrT2 gene for mature plant resistance in Glenlea. The high-temperature expression of resistance could be due to a second gene for adult plant resistance or to a previously undetected seedling gene.
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    The use of Agrobacterium for plant improvement.
    (1994) MacRae, Sharmane.; Van Staden, Johannes.; Thomson, Jennifer A.
    Agrobacterium species have potential as tools for plant improvement, in that they can be used as biological control agents against crown gall disease, vectors for gene insertion into plants and plant root-inducing bacteria. For a bacterium to be successful as a biocontrol agent against crown gall disease, it must be able to produce an effective agrocin specific for A. tumefaciens pathogens and be able to colonise host plants effectively. Successful biological control of crown gall disease has been achieved on a limited range of host plants by application of Agrobacterium radiobacter. Strain K84 is non-pathogenic, produces an antibiotic type substance, agrocin 84, which kills a specific spectrum of pathogenic Agrobacterium strains, and is a good plant coloniser. The colonisation abilities of this bacterium and a number of potential biocontrol bacteria, D286, 173 and its derivatives, H8, and H6 and its derivatives, were compared in vitro and in vivo. In addition the ability of these strains to control crown gall pathogens in vitro and in vivo was assessed. Although D286 and H8 were good long term colonisers they were subsequently eliminated from the programme because D286 was unable to control grapevine crown gall disease, and H8 was a pathogen, with a narrow host range for biotype 3 pathogens, which had not been cured. Good long term colonisation ability was shown to be critical to the success of the biological control strain. Both K84 and J73 strains produced fibrils which attached them to tomato root surfaces and have similar colonisation efficiencies up to 14 days after inoculation. However, the ability of J73 to colonise plants for longer periods was significantly less than that of K84. Thus, the presence of fibrils is not sufficient to ensure colonisation. No correlation was found between hydrophobicity and colonisation. Poor in vivo biological control of tomatoes and grapevines by J73 and its derivatives has been shown to be due to poor long term colonisation. The additions of the agrocin 84 plasmid to J73 improved in vitro biological control by J73 but was not effective in vivo. Thus although J73 produced a broad host range agrocin it was ineffective as a biocontrol agent due to its poor long term colonisation ability. H6 and its derivative proved to be good long term colonisers of the grape vine rhizosphere and were able to effectively control crown gall disease on grapevines. Eucalyptus grandis was propagated in vitro from axillary buds. The effect of the gelling agents gelrite, agarose and agar on propagation was determined. Shoot multiplication and elongation on gelrite-containing media was found to be superior to that obtained on agarose- and agar-containing media. Rooting was enhanced with gelrite as the gelling agent. In vitro clone development from seed of selected E. grandis genotypes and the integration of this into vegetative clonal propagation programmes is proposed. Not all clones developed in this manner rooted well under in vitro conditions. Thus the potential use of A. rhizogenes to improve rooting was investigated. A. rhizogenes is a bacterial pathogen which causes 'hairy root' disease on certain plants. Eucalyptus species do not fall into the natural host range of this organism, however, it is able to infect these plant species. In an attempt to improve rooting and root quality of in vitro and in vivo propagated Eucalyptus species and clones, the root-inducing genes on the Ri plasmids of a number of A. rhizogenes strains were inserted into Eucalyptus by inoculating in vitro and in vivo stem cuttings with the selected A. rhizogenes strains. The resultant chimeric plants have transformed roots and normal shoots. Root development was monitored in vitro and after the plantlets had hardened off, and in vivo. Only transformed roots grew as root cultures in hormone-free liquid medium. The potential use of this procedure for improving rooting of clonal material is discussed. Under in vitro conditions for example, one of the broad host range A. rhizogenes strains, LBA9402, was able to induce up to 80 % rooting on E. grandis, E. nitens and E. dunnii explants. While under nursery conditions for example, one of the two E. globulus clones tested, HM15, developed up to ten times as many roots in response to two strains of the A. rhizogenes bacterium (LBA9402 and TR8,3) while the other clone failed to respond. Not only the inherent rooting abilities of the numerous Eucalyptus genotypes and clones tested, but also hormone induced-rooting and Agrobacterium-mediated rooting were found to vary from clone to clone and genotype to genotype. A. rhizogenes strains were however, able to overcome the genetic control on rooting of certain clones but this was found to be dependent on both the genotype of the bacterium and the plant. The novel concept of using bacterial cocktails to improve rooting was investigated, with the aim of overcoming the limited host range of individual A. rhizogenes strains. Both in vitro and nursery trials were established to test the potential use of bacterial cocktails as a means of improving rooting of a range of Eucalyptus genotypes and clones. Agrobacterium cocktails not only improved the ability of certain clones and genotypes to root but also the quality of the roots. The anatomy of transformed roots from chimeric plants was compared to that of non-transformed roots, revealing no difference in root anatomy of these roots. Nutrient uptake studies using a radioisotope nutrient uptake bioassay showed no difference in phosphorus, potassium and nitrogen uptake by transformed and nontransformed roots. The potential of A. rhizogenes strains to improve rooting of two other species, Anacardum occidentale (cashews) and Pinus (a number of pine hybrids) was also shown in preliminary trials.
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    Studies on the expression of resistance to stem rust of wheat caused by Puccinia graminis f.sp. tritici.
    (1991) Lennox, Cheryl Lynne.; Van Staden, Johannes.; Rijkenberg, Fredericus Hermanus Johannes.
    The endogenous cytokinin levels of healthy primary leaves and seeds of a stem-rust susceptible wheat cultivar Little Club were compared with those of Little Club containing the stem rust resistance gene Sr25. Use was made of paper, column and high performance liquid chromatography techniques to separate the endogenous cytokinins in the plant material, and the soybean callus bioassay was used to test for cytokinin-like activity of the chromatography fractions. Leaf material of the resistant Little Club Sr25 had a higher level of total cytokinin activity than Little Club, whereas seed material of Little Club Sr25 did not always have higher levels of cytokinins than Little Club. A number of cultivars would have to be tested before the usefulness of cytokinin levels as an indicator of resistance could be determined. The development of urediospore-derived infection structures of Puccinia graminis f.sp. tritici in wheat, barley, sorghum and maize was examined by scanning electron microscopy (SEM). Infection on and in the four species followed a similar pattern up to, and including, primary infection hyphae formation. In wheat, barley and maize, when a primary infection hypha abutted onto a host epidermal cell, a septum was laid down delimiting a primary haustorial mother cell (HMC); primary HMCs did not form in sorghum. Secondary infection hyphae arose on the substomatal vesicle side of the primary HMC septum; infection did not progress further in maize, but in wheat and barley secondary infection hyphae branched, and proliferated intercellularly forming the fungal thallus. Secondary HMCs were delimited when an intercellular hypha abutted onto host cells. In all four species atypical infection structures were also observed. In an attempt to determine the timing and expression of stem rust resistance gene Sr5, infection structure development of Puccinia graminis f.sp. tritici race 2SA2 in a resistant line (ISr5Ra) and a susceptible line (ISr8Ra) was compared quantitatively using a fluorescence microscopy technique. The results indicated that there were no significant differences in numbers of specific infection structures observed in the two near-isogenic lines up to, and including, 48 hpi, by which time race 2SA2 had successfully formed secondary HMCs in both lines.
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    Studies on Alternaria porri and Stemphylium vesicarium on Allium spp.
    (1993) Aveling, Theresa Ann Sheila.; Rijkenberg, Fredericus Hermanus Johannes.; Wehner, F. C.
    During surveys in South Africa, Alternaria porri (Ellis) cif. and Stemphylium vesicarium (Wallr.) E. Simmons were found to be destructive seed-borne pathogens of onion (Allium cepa L. ). These two pathogens are also reported on garlic (Allium sativum L.) in South Africa for the first time. The development and morphology of conidiophores and conidia of the two pathogens on the onion leaf surface were examined using scanning electron microscopy. In both pathogens, solitary or fasciculate conidiophores emerged through the epidermis. Bud-like conidial initials were produced singly at the apex of conidiophores. As conidia of S. vesicarium matured, they became oblong to ovoid and densely verrucose. Those of A. porri showed slight growth in width but pronounced elongation. Conidial germination, formation of pre-penetration structures, penetration of the onion leaf surface by A. porri and S. vesicarium, and the subsequent infection process by A. porri, were studied using light, scanning electron and transmission electron microscopy. Conidia of both pathogens usually germinated within 24 h of inoculation, forming several germ-tubes which often terminated in bulbous appressoria produced directly on the epidermal cells or on stomata. Following direct penetration of the outer epidermal cell wall or the stoma, bulbous primary hyphae developed below the appressoria. Secondary hyphae of A. porri developed from primary hyphae and grew within the intercellular spaces, penetrating mesophyll cells. The changes in ultrastructure of infected cells, and of cells in close proximity to secondary hyphae, are described. six fungicides, anilazine, benomyl, carbendazim/flusilazol mixture, procymidone, tebuconazole and thiram, as well as a hotwater soak (50 C for 20 min) and sodium hypochlorite treatment, were evaluated for their efficacy in reducing both pathogens on seed and in culture. The effect of the various treatments on seed germination, and seedling emergence and growth, was determined. None of the treatments eradicated A. porri and S. vesicarium from onion seeds. The hot-water soak proved to be the best treatment for reducing these pathogens, although percentage germination and emergence of onion seeds were reduced when compared to the control.
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    A study on the ultrastructure and control of the gladiolus rust pathogen, Uromyces transversalis.
    (1988) Ferreira, Johan Francois.; Rijkenberg, Fredericus Hermanus Johannes.
    The substomatal vesicle of Uromyces transversalis develops in five distinct stages after the formation of a single infection peg in the host plant Gladiolus spp. The primary hyphae of the substomatal vesicle orientate at right angles (rather than at acute angles or parallel) to the stomatal slit and the parallel veins of a gladiolus leaf or to those of the non-host, Zea mays. The transverse orientation of Uromyces transversalis uredia is apparently a genetically controlled phenomenon. The two primary hyphae normally develop asynchronously on opposite sides of the substomatal vesicle, and, after the formation of a haustorium mother cell, secondary hyphae branch off. These subsequently form basal cells each of which give rise to one or more holoblastic protruberances on its distal surface. A septum delimits the protruberance from the basal cell to form a urediospore initial, which in tum elongates and, by septum formation, forms a pedicel and a urediospore. The urediospore is seceded mechanically through the process of schizolysis. The pedicel of a spore thus formed remains on the basal cell and becomes a collar around the next protruberance. The basal layer of the two-layered septum, that delimited the pedicel from the basal cell, grows out to form the wall of the subsequent protruberance, in the process rupturing and laterally displacing the terminal septal layer. A new basipetal septum forms to delimit the subsequent urediospore initial. Therefore, successive urediospores are formed enteroblastically and give rise to a series of basipetal collars. Cultivars, naturally occurring species and breeding lines of gladiolus were evaluated for rust resistance. The cultivars showed no sign of resistance, whereas the populations of wild species and breeding lines had similar resistance profiles. The species G. daleni and G. tristis var. tristis showed promise for breeding. Infection of these species caused an almost immune, fleck reaction. The resistance of the species G. daleni was manifested by the abortion of primary hyphae prior to the formation of the haustorial mother cell. The intercellular hyphae of gladiolus rust only partially adhered to the mesophyll cell of the resistant host. The haustorial apparatus that did form in the mesophyll cells in the resistance reaction was inhibited at various stages of its development. The rust disease could be controlled chemically by either bitertanol or triadimefon. Triadimefon, however, caused a shortening of the internodes of the flower spike. The early development of U. transversalis infection structures in bitertanol-treated leaves was inhibited at, or shortly after, substomatal vesicle development. A number of mature substomatal vesicles with intercellular hyphae, however, did develop in the bitertanol-treated leaf tissue, probably because this fungicide undergoes limited translocation in the gladiolus lamina.