Doctoral Degrees (Plant Pathology)

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    The diversity and epidemiology of Botryosphaeriaceae species associated with grapevines and woody hosts surrounding vineyards in South Africa
    (Stellenbosch : Stellenbosch University, 2020-12) Du Plessis, Ihan Lambert; Halleen, Francois; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Botryosphaeriaceae species are reported globally as causal agents of grapevine trunk diseases which translate to yield losses as well as a reduction in the productive lifespan of affected vines. Growers rely on management practises to try and prevent vines from becoming infected. However, despite decades of implementation, current disease management strategies do not fully protect grapevines from becoming infected. This highlights a need for an improved understanding of the epidemiology of these pathogens as well as the development of improved disease management strategies. The aim of this study was to investigate the diversity of Botryosphaeriaceae species occurring on both grapevines as well as other woody hosts within the wine growing regions of the Western Cape Province. In addition, the potential threat that these other hosts pose to the grapevine industry by acting as sources of pathogen inoculum was investigated by characterizing and comparing different populations of the pathogen N. stellenboschiana isolated from both grapevines as well as non-grapevine hosts. The species diversity survey reported 20 different Botryosphaeriaceae species from 38 different host species which were located within 50 m of vineyards. These represented 114 different host/ fungi combinations which were not previously known in South Africa. This survey was dominated by three Botryosphaeriaceae species, Diplodia seriata, Neofusicoccum australe and N. stellenboschiana which constituted 85.1% of all the isolates obtained during this survey. These species are also known grapevine pathogens and were reported from 19, 11 and 24 different host species respectively which highlights the broad host range of these economically important pathogens. The species diversity survey also yielded six new Botryosphaeriaceae species which were formally described and their pathogenicity towards grapevines and olive trees, where relevant, were assessed through field pathogenicity trials. All of the new species were shown to form lesions on grapevine or olive shoots which were comparable to those caused by known Botryosphaeriaceae pathogens, demonstrating the capacity of these species to act as pathogens of these economically important hosts. The population genetics study was carried out based on seven microsatellite markers which were demonstrated to be polymorphic in this study. This study reported that N. stellenboschiana populations from grapevines and other hosts at three different locations in the Western Cape Province were genetically homogenous. This indicates that there are no barriers which prevent the movement of N. stellenboschiana between grapevines and other hosts. These results are disconcerting because they imply that woody hosts surrounding grapevines which are infected with Botryosphaeriaceae grapevine pathogens could be acting as disease reservoirs and sources of pathogen inoculum which threaten vineyards. To conclude, this study furthered our understanding of the diversity of Botryosphaeriaceae species occurring in woody hosts that commonly surround vineyards in the Western Cape Province of South Africa and described six new species. Furthermore, this study has contributed to our understanding of the epidemiology of these pathogens by demonstrating that the alternative hosts of Botryosphaeriaceae grapevine trunk disease pathogens represent a threat to grapevines by acting as sources of pathogen inoculum. This helps to lay the groundwork for future studies to address this threat by developing improved pathogen management strategies.
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    Characterisation, epidemiology and management of olive trunk disease pathogens in South Africa
    (Stellenbosch : Stellenbosch University, 2020-04) Van Dyk, Meagan; Halleen, Francois; Mostert, Lizel; Spies, Chris, F. J.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: The Olive Sector Development Plan of the Department of Trade and Industry identified low production and the lack of local research as weaknesses of the olive industry in South Africa. The management of trunk diseases forms an integral part of practices aimed at increasing olive production. A recent olive trunk disease survey performed in the Western Cape Province, South Africa, identified an undescribed Pseudophaeomoniella sp. as the most prevalent fungus associated with the trunk disease symptoms, with other fungal species occurring at much lower frequencies. In the current study, 40 of these isolates were selected for a pathogenicity study. The species forming lesions included several Botryosphaeriaceae, Phaeoacremonium and Phaeomoniellaceae species, as well as Biscogniauxia mediterranea, Coniochaeta velutina, Diaporthe foeniculina, Didymocyrtis banksiae, Eutypa lata, Pleurostoma richardsiae, Symbiotaphrina buchneri, isolates of the Cytospora pruinosa complex, and a Cytospora sp., Fomitiporella sp., Geosmithia sp. and Punctularia sp. The Pseudophaeomoniella sp. formed among the longest lesions, affirming its status as a potentially important trunk pathogen. Long distance dispersal of olive trunk pathogens is expected to occur via infected nursery material, similar to that found in other systems such as in grape and fruit trees. Nurseries as an inoculum source was investigated by making isolations from asymptomatic cuttings from mother blocks (Stage 1), rooted cuttings (Stage 2) and 1–2-year-old trees (Stage 3) of eight cultivars in two nurseries. Known olive trunk pathogens of the Botryosphaeriaceae, Diaporthaceae, Nectriaceae, Phaeomoniellaceae, Pleurostomataceae and Togniniaceae were recovered. Neofusicoccum australe was detected in a single Stage 1 cutting. Stage 3 material showed the highest incidence of fungi from these families, with P. richardsiae having the highest incidence in both nurseries (82.2% and 36.7% of the 1–2-year-old trees). Phaeoacremonium parasiticum was present in 28.9% of the trees from one nursery (Stage 3). The remaining pathogens occurred in 13.3% or less of the material. Pseudophaeomoniella sp. was present in the nurseries but at low frequencies. This suggests that alternative inoculum sources of this pathogen exists. A nested species-specific PCR was developed for the detection of Pseudophaeomoniella sp. from spore washes of pruning debris collected from established olive orchards. Pruning debris identified with a positive PCR was evaluated microscopically. Pycnidia of Pseudophaeomoniella sp. were observed on the pruning debris. Based on previous research, it is expected that the spore release coincides with rainfall and that the spores can be dispersed onto pruning wounds. The susceptibility of wounds from winter and spring pruning to Pseudophaeomoniella sp. was compared. Two-year-old olive branches of 16-year-old olive trees were pruned and inoculated with spore suspensions of Pseudophaeomoniella sp. at different time-points after pruning. The pruning wounds were susceptible for up to 42 days, with no difference between seasons (winter vs. spring). The wounds were the most susceptible within the first week after pruning. Eleven pruning wound protectants were evaluated and applied on pruning wounds made on 16–17-year-old trees directly after pruning. The treated wounds and positive (non-treated) controls were challenged with spore suspensions of Pseudophaeomoniella sp. at 1 or 7 days after pruning. Under low inoculum pressure (first season), Garrison, MT1, Neocil Plus and Tree Seal, reduced Pseudophaeomoniella sp. infections, while the Trichoderma-based protectant, MT1, was considered the most effective water-based protectant. Under higher inoculum pressure (during the second season), Tree Seal and Coprox Super/Bendazid consistently performed the best. In conclusion, several fungal species were identified as olive trunk pathogens, with Pseudophaeomoniella sp. being identified as one of the most important olive trunk pathogens. The propagation process was identified as a source of inoculum for some pathogens, including Pseudophaeomoniella sp. Inoculum sources of Pseudophaeomoniella sp. were also identified in established orchards. Olive pruning wounds are susceptible to Pseudophaeomoniella sp. for prolonged periods. MT1 was highly effective under lower inoculum pressure, while Tree Seal and Coprox/Bendazid were highly effective under high inoculum pressure. This study led to new knowledge with regards to olive trunk diseases, their pathogenicity, detection, epidemiology and control which can be used for the development of improved management strategies of olive trunk diseases in South Africa.
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    Evaluation of adjuvants in fungicide spray application for the control of alternaria brown spot in South African citrus orchards
    (Stellenbosch : Stellenbosch University, 2019-03) Van Zyl, Johannes Gideon; Fourie, Paul H.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Citrus fruit and foliar diseases are mainly controlled through pre-harvest application of fungicides. Fungicides are only as effective as the application process and for effective disease control deposition of a uniformly distributed quantity of active ingredient(s) is required on the intended target(s). Adjuvants have the potential to improve fungicide deposition on a target surface. The influence of adjuvants on the deposition of fungicides, especially at the high spray volumes used in South African citrus production is unknown and was therefore investigated. A previously developed deposition assessment protocol, using a yellow fluorescent pigment as tracer for copper oxychloride (CuOCl) deposition, was improved through photomacrography and digital image analyses which proved accurate in determining the quantity and quality of deposition on citrus leaves. Spray deposition benchmarks indicative of the biologically efficacy of CuOCl against Alternaria alternata [causal agent of Alternaria brown spot (ABS) of mandarins] was developed. The deposition assessment protocol and deposition benchmarks was used to evaluate two organosilicone adjuvants (Break-Thru S240 and Break-Thru Union) at reduced spray volumes in dense and less dense citrus canopies in two separate orchard spray trials. Deposition quantity generally increased with increasing spray volume, but normalised values showed better spray efficiency at lower volumes. In pruned and less dense canopies, a beneficial effect of adjuvants was observed in terms of deposition quantity, efficiency and uniformity, especially at reduced volume applications. Some improvement in deposition quality was generally observed with the use of adjuvants. These benefits were not as evident in very dense canopies, illustrating the importance of canopy management when spraying at reduced volumes. Commercially available adjuvants [Break-Thru, Nu-Film-17, Citrole100, Villa51, Wetcit, Entrée and Exit] were evaluated in three orchard spray trials on different citrus types, cultivars and spray volumes. In trial one, adjuvants improved deposition quantity and canopy penetration. In trial 2 and 3, deposition quantity was generally higher at higher spray volumes, but spray efficiency was significantly better at lower spray volumes. Adjuvants generally improved deposition uniformity and deposition quality, but these benefits were significantly influenced by spray volume and the specific adjuvant treatment. Poor performance by adjuvants was ascribed to high spray volumes and/or too high adjuvant concentration used, which led to increased levels of run-off and poor deposition parameters. The effects of adjuvants on deposition quantity, quality and biological efficacy of CuOCl against ABS on mandarin leaves were determined in laboratory trials. Adjuvant treatments varied significantly in deposition quantity and quality and disease control achieved. Higher deposition quantity, beter quality and higher Cu residues was realized at pre- vs. post-run-off volumes. Adjuvants did not improve deposition parameters compared with the control treatment at both spray volumes. Leaf infection analysis indicated that CuOCl with adjuvant sprays (post-run-off volume) realized similar and in some cases slightly better control (although not significant) than copper oxychloride alone, but that deposition and Cu residue loading in some of these adjuvant treatments were markedly lower. This anomaly could be ascribed to direct or indirect effects of the adjuvant and was investigated further. In vivo and in vitro studies were done to identify possible direct adjuvant effects on pathogen development and potential synergistic effects between the adjuvants and CuOCl. Adjuvants alone did not influence conidial adhesion, appressorium formation, germ tube length and percent viable conidia. Adjuvant sprays together with CuOCl reduced conidial adhesion, germ tube length and percent viable conidia numerically; however, not significantly compared with CuOCl alone. Adjuvants also caused conidium/germ tube stress similar to CuOCl, but did not inhibit germination or growth. In the in vitro microtiter assay, adjuvants together with CuOCl improved germination or growth inhibition compared with the CuOCl treatment alone, although not at significant levels. The findings in Chapter 6 did not fully explain the anomalous findings in Chapter 5, and future studies should focus on developing methodology to support histopathology studies on sensitive leaf surfaces, as well as development of a more sensitive method of measuring deposition quality, especially on a microscopic scale.
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    Genetics of fumonisin biosynthesis and resistance to fusarium verticillioides in maize
    (Stellenbosch : Stellenbosch University, 2018-12) Van Zyl, Karlien; Viljoen, Altus; Rose, Lindy J. ; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Fusarium ear rot (FER) is a serious disease of maize (Zea mays L.) caused by the hemi- biotrophic fungus Fusarium verticillioides. The fungus also produces toxic secondary metabolites, known as fumonisins, in the grain that pose serious animal and human health risks. The role that fumonisins play during F. verticillioides infection is unclear, but they are believed to suppress host basal defence and facilitate necrotrophic proliferation. Disease severity and fumonisin contamination of maize are influenced by host and fungal genetics, and by plant stresses imposed by warm and dry climates. Resistance to F. verticillioides is quantitatively controlled and strongly influenced by the environment. No maize cultivar exists that is immune to FER and fumonisin contamination. The availability of the full genome sequences of both maize and F. verticillioides, however, makes it possible to investigate plant and fungal genetic responses during infection. A cluster of genes responsible for fumonisin biosynthesis in F. verticillioides has been identified, but its regulation is not yet well understood. Random insertional mutagenesis was thus performed to potentially identify genes governing fumonisin production. A mutant was obtained that produced significantly more fumonisins than its wild-type strain. Functional annotation of the single insertion site in the mutant strain showed that it was in a non-protein- coding area of F. verticillioides chromosome 10. The integration potentially causes transcriptional interference of the downstream gene encoding a F. verticillioides 7600 hypothetical protein (FVEG_08564). In future this gene needs to be inactivated by targeted mutagenesis to confirm its function, and the expression of fumonisin in maize grain infected by the mutant determined. Information on the expression of F. verticillioides genes in maize kernels during infection is limited. This is due to the small amount of fungal RNA produced in maize, which makes transcriptome sequencing unreliable. A targeted approach to study the expression of genes involved in fumonisin production in planta was, therefore, employed. A reverse-transcription quantitative PCR (RT-qPCR) assay was first optimized, and then used to study the relative expression of two fumonisin biosynthesis (FUM) genes in maize kernels. The expression of these genes was also correlated with fumonisins levels. A positive but non-significant correlation was obtained between FUM1 and FUM19 gene expression and fumonisin concentration. This finding was in conflict to a significantly positive correlation between FUM1 and FUM19, and fumonisin production, in vitro. The disparity could be attributed to factors affecting FUM gene expression and mycotoxin production in planta, such as host and pathogen genotype, the climate and kernel maturation. The RT-qPCR used can be a valuable tool to further investigate fungal genes expressed in maize kernels. Resistance to F. verticillioides in maize is controlled by many genes that are expressed to protect the plant from early infection, through colonization, to fumonisin production. These defence-related genes are present in both resistant and susceptible genotypes, but their induction is more rapid and stronger in resistant than susceptible plants. When transcript profiles of resistant and susceptible South African maize inbred lines were studied over a 52- day time period, genes associated with pathogen recognition and redox homeostasis were most strongly induced in the resistant than in the susceptible inbred line. During the necrotrophic phase of infection the plant responded by activating jasmonic acid/ethylene signalling and genes that modulate programmed cell death. The study provides novel insights into the upstream host recognition processes over the course of F. verticillioides infection and gene expression during the latter stages of infection.
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    Linking long-term patterns of landscape heterogeneity to changing ecosystem processes in the Kruger National Park, South Africa
    (Stellenbosch : Stellenbosch University, 2018-12) MacFadyen, Sandra; Hui, Cang; Verburg, P. H.; Van Teeffelen, A. J. A.; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Biodiversity loss is a global threat to ecosystem function and human well-being. Environmental heterogeneity is a recognised driver of biodiversity under a niche-based view of available species habitats. As such, an increase in environmental heterogeneity is expected to promote species coexistence, persistence and diversification. Loss of environmental heterogeneity is therefore considered proximal evidence of biodiversity loss. At a landscape scale, this heterogeneity is defined as the degree of difference between landscape elements and is often described as landscape heterogeneity. Patterns of landscape heterogeneity are generated and maintained by the physical landscape template or abiotic environment (e.g. topography, geology and climate), upon which complex adaptive interactions between landscape pattern (structure and composition) and ecological processes (function) occur. Landscape pattern can therefore be described as the self-organising expression of landscape function which varies not only across space but also through time. Accordingly, observable variations in landscape pattern are conjectured to signify divergence in landscape function. This thesis explores this relationship further within the Kruger National Park (Kruger): a large (~ 20,000 km2 ), longestablished (proclaimed 1898) protected area in South Africa’s semi-arid savanna. Results therefore describe landscape heterogeneity, in terms of the abiotic and biotic components (environmental drivers) that generate and maintain landscape pattern in Kruger, to inform strategic biodiversity planning. Chapter 1 introduces the reader to landscape heterogeneity and its relevance to protected area management and biodiversity conservation. Chapter 2 begins by isolating the effects of ‘stationary’ landscape properties on environmental heterogeneity through their relationship with Landsat spectral variance. Results show this relationship is sensitive to season and rainfall with the effects of dynamic ecosystem processes dominating many areas. Thereafter, Chapters 3 and 4 examine in more detail the nature of selected dynamic drivers in Kruger, namely rainfall and elephants. Results demonstrate the existence of longterm spatiotemporal changes in both rainfall and elephant density and distribution patterns in Kruger from 1985-2015. Together these results feed into chapter 5, where a Structural Equation Model (SEM) is used to investigate the causal structure of landscape heterogeneity with stable landscape properties, rainfall, herbivory and fire. Results are presented as path coefficients and long-term driver dominance maps showing the magnitude and direction of the different cause and effect relationships between heterogeneity, the physical landscape template, rainfall, herbivory and fire return interval. Finally the nature of the environmental-heterogeneity theory is operationalised in Chapter 6 using R, Shiny and Leaflet to provide an interactive web interface for protected area managers to explore heterogeneity differences in context with park specific research questions. Chapter 7 concludes the thesis with a brief synthesis of results in context with current literature and highlights future research opportunities and possible directions.