Thesis (MScAgric (Soil Science))--University of Stellenbosch, 2008. / In order to produce table grapes of export quality economically, irrigation must be practised conservatively without adversely affecting the crop. To use water as conservatively as possible effective irrigation scheduling practices must be applied. The highest water use efficiency (WUE) is only possible if irrigation scheduling practices lower the amount of water applied, while at the same time they increase the yield. The first aim of this project is to investigate whether current irrigation practices make efficient use of water by comparing irrigation requirements determined using theoretical models with actual irrigation applied for two seasons (2005/6 and 2006/7). Secondly, the effect of cumulative irrigation on the chemical status of soil in 16 blocks was investigated to establish whether nutrient leaching as a result of differential water use may have had an influence on yield. Six blocks (three dripper and three microsprinkler blocks) were selected and irrigation requirements were determined using evaporation pan calculations, SAPWAT and Vinet and compared with actual irrigation applications. Furthermore, a yield-irrigation index (kg/m3) and an income-irrigation index (R/m3) were determined for each of the six blocks and compared. To investigate the effect of cumulative water use on the chemical status of the soils of 16 blocks, soil samples were taken and analysed for pH (1M KCl), EC (1:5); soluble cations and anions (Ca, Mg, Na, K, SO4, NO3, and Cl), ammonium acetate extractable cations (Ca, Mg, Na and K) and micro elements (Zn, Fe, Mn, Cu and B). The irrigation requirements predicted by the different irrigation scheduling methods are variable. For Vinet, the irrigation requirement determined for microsprinkler irrigation is much higher than that determined using the evaporation pan or SAPWAT approaches. Comparison of the irrigation applied to each of these blocks does not clarify whether any irrigation scheduling takes place. Results showed a relationship between the yield-irrigation index and income-irrigation index. It has not however been verified whether this relationship is statistically significant.

Thesis (MBA)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: After the deregulation of the South African deciduous fruit industry in 1997 an unregulated grape marketing environment replaced the single channel marketing system. Many new South African grape exporters were now involved in an uncoordinated and uncooperative marketing environment. Each marketing organisation implemented their own marketing plans resulting in an increase in competition among South African suppliers. The South African grape supplies originate from various production areas, each with their unique characteristics and qualities, making it a very competitive environment. South African grapes also share the various markets with other competing Southern Hemisphere grape suppliers. This level of competition makes it very important for the South African grape industry to have a uniform marketing strategy. To achieve a uniform industry marketing strategy the industry has to develop a common vision - to be the preferred table grape supplier in our target markets - that forms the basis of developing a generic South African table grape marketing strategy. A single vision gives the competitive export companies a common marketing objective. This study includes recommendations to industry stakeholders that will enable them to formulate their own marketing strategy, all within the guidelines of the common vision that is adjusted to the specific needs of their respective target markets. / AFRIKAANSE OPSOMMING: Na die deregulering van die Suid-Afrikaanse sagtevrugtebedryf in 1997 het 'n ongereguleerde bemarkingsomgewing die enkelkanaalbemarking vervang. Verskeie nuwe Suid-Afrikaanse tafeldruifuitvoerders was nou betrokke by 'n ongekoërdineerde bemarkingsomgewing waar daar geen samewerking tussen uitvoerders was nie. Elke bemarkingsagent het sy eie bemarkingsplan gevolg wat gelei het tot 'n toename in kompetisie tussen Suid-Afrikaanse produsente. Die Suid-Afrikaanse tafeldruiwe word in verskillende produksieareas geoes, elk met hul unieke eienskappe en kwaliteite. Dit maak dit 'n baie kompeterende omgewing. Die Suid-Afrikaanse druiwe kompeteer ook in die mark met ander Suidelike Halfrond druiwe produsente. Met so 'n hoë vlak van kompetisie, is dit belangrik dat die Suid- Afrikaanse industrie 'n eenvormige bemarkingstrategie het. Om 'n eenvormige bemarkingstrategie vir die industrie te formuleer, moet die industrie visie - om die voorkeurverskaffer van tafeldruiwe in ons teikenmarkte te wees - die basis vorm vir die formulering van 'n generiese bemarkingstrategie vir Suid-Afrikaanse tafeldruiwe. 'n Eenvormige visie bied vir die kompeterende bemarkingsagente 'n gemene doelwit. Hierdie werkstuk sluit aanbevelings in wat vir rolspelers leiding sal gee om hul eie bemarkingstrategie te formuleer wat die doelwitte van 'n eenvormige visie onderskryf, maar nog steeds die behoeftes van die onderskeie teikenmarkte aanspreek.

Thesis (MBA)--Stellenbosch University, 2012. / The South African table grape industry has a very fruitful history. The landscape of the South African table grape industry has changed at a rapid rate since deregulation in 1997. The sustainability of these changes was questioned and created the opportunity for this research. The orientation of this research report outlines the structure to this research. It indicates that the South African table grape industry is experiencing a strong trend of consolidation of the production and exporter base. Economically sustainable growers and marketing companies produce or attract more volumes of table grapes to export to global destinations. The environment provides the background to the South African table grape industry, since deregulation. Five major trends in this industry are important to mention and give perspective to the research question, namely: - The number of producers has declined at a rapid rate (about 52%) since deregulation. - Traditional production regions like the Berg and Hex River have become less important due to the timing of product supplied to the market. New geographic production regions have grown in importance, like the Orange River and Northern Province. - Production volumes of table grapes have slowed down over the last ten years and in certain regions have become stagnant. - The cultivar spectrum of table grapes has changed from seeded grape that is marginal to the market to a seedless product that earns premiums in the market. - There has been a shift in marketing of table grapes from Western countries like the UK and EU to Eastern countries. There are also certain factors that have a profound impact on the South African table grape industry, namely economic, environmental, political, social and global factors, which required further investigation. The evaluation of trends in the industry and the factors that affect the industry revealed the problem statement for this research and set the foundation for the research question. The research question of this study is: What are the distinguishable marketing models currently used by South African exporters and how sustainable is each model? The research methodology demonstrates that the South African table grapes industry is split into two dominant marketing models, which are the marketing agent and the grower-exporter model. A marketing model metrics was designed, based on literature and consultations with industry experts via a questionnaire, to evaluate the economic, social and environmental sustainability of these two marketing models. The method of data analysis was a qualitative investigation into the sustainability of marketing models that exist in the South African table grape industry. The structure of the holistic marketing dimension model which was used has the following four dimensions (Kotler & Keller, 2009:61): - Relationship marketing - Performance marketing - Integrated marketing - Internal marketing. Semi-structured interviews were held with six different marketing entities in the South African table grape industry, which represented 40 percent of the total population. The interviews were held by utilising a discussion guide that comprised of standard questions to all the interviewees. The information gathered from the interviews was used to design a conceptual marketing model, with the main objective of being sustainable on an economic, social and environmental level. This relevance and practicality of this conceptual marketing model was tested against a grower-exporter model in the South African table grape industry, called Angon Fruit. The findings of this research report focused on the triple bottom-line approach of building economic, social and environmental capital. The economic sustainability revealed the following factors to consider: - Grower-exporter model is a more sustainable marketing model, due to low cost operations. - Marketing agent model should have a definite competitive advantage, to justify relevance in the cost-chain. - Sustainable market share in volume of table grapes to export by a marketing company is five percent. - Prices paid to growers by a marketing company should keep track of inflation on inputs and a ten percent premium earned on top of the production cost is regarded as sustainable. - A sustainable commission percentage charged by marketing companies should not exceed five percent of free on board (FOB) value at Cape Town port. - Growers need to replace marginal seeded cultivars with premium seedless cultivars that are high in demand in global markets. - Marketing companies should supply table grapes at a split of 50 percent to Western countries and 50 percent to Eastern countries. - The sustainability of the Berg and Hex River production regions is at risk, due to global competitions from South American countries. The South African table grape industry revealed the following social sustainability findings: - A marketing company should have a social policy, which governs social responsibility projects (CSI). - The marketing company should be involved one major project instead of a few smaller projects. - Corporate social investment (CSI) projects should aim to provide the opportunity to improve education and training, improve health status and to develop sport among the youth. - The marketing company should not invest in CSI projects if the economic sustainability is at risk. The objective is to invest five percent of net profit in CSI projects, annually. Environmental sustainability revealed the following factors to consider: - A marketing company should have an environmental policy. - The company should measure its carbon and water footprint at least every two years, with the objective to reduce or optimise the level of use. - The company should embrace and implement green technology that utilises renewable energy to reduce the dependency on fossil fuels and coal-generated electricity. - The use of water should be optimised, through implementation of advanced technology. - The marketing company should adopt the practice of recycling. The conclusion to this research report is that the table grape industry is still economically sustainable, however more work can be done on the social and environmental sustainability. Finally, ten recommendations are made to the South African table grape industry to consider from a sustainability perspective. Companies can consider implementing these best practices into their marketing of table grapes.

Thesis (MscAgric (Viticulture and Oenology))--University of Stellenbosch, 2007. / Regal Seedless is a white, seedless grape which has the potential to become a profitable cultivar for the table grape producer since it has the advantages of early season harvesting and inherently large berries. There is, however, a downside to this cultivar, namely the seasonal occurrence of an unacceptable, astringent taste. This negative taste affects the demand by local and international markets. The astringency perception is due to the presence of phenolic compounds. It is well known that the phenolic composition and concentration change during the ripening of the grape. Different postharvest treatments are applied to fresh fruit like persimmons to remove astringency. These treatments include the use of carbon dioxide, nitrogen and ethanol. The aim of this study was to determine the optimum maturity level for Regal Seedless where the phenolic concentration is the lowest and the astringent taste acceptable. The use of postharvest treatments to manipulate the taste and the phenolic content, were also investigated. The effect on other quality parameters like total soluble solids (TSS), pH and total titratable acidity (TTA) were also evaluated.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Previous research has shown that carotenoids are precursors of C13-norisoprenoid aroma compounds in wine. C13-norisoprenoids have low threshold values in wine with the most prominent C13-norisoprenoids being β-damascanone and β-ionone which contribute honey and floral like aroma to wine. Chlorophyll and its derivates have also been detected in wine with potential to be precursors to aroma compounds. Apart from the contribution of these pigments to wine aroma and quality they are vital role players in photosynthesis and are widely found in plants and plant products. The main functions of these pigments in plants are light collection and light-protection. Research has shown that environmental conditions, climate, light exposure of bunches and soil water deficit influence the carotenoid content of grape berries. Furthermore the concentration of carotenoids and chlorophylls has also been shown to differ between cultivars. No research in this regard has been done on Merlot grape berries. With this in mind, the aim of this study was to evaluate the effect of vigour and soil water content on the evolution of carotenoids and chlorophylls through ripening of grape berries from the cv. Merlot. However, when looking at methods to analyse carotenoids and chlorophylls in berry tissue, especially lyophilised tissue, there were no readily available methods. Thus, an extraction method to identify and quantify the carotenoid and chlorophyll profile of lyophilised tissue from unripe (green) to ripe (red) Merlot grape berries was needed. In this study the RPHPLC method of Taylor et al. (2006) for carotenoids and the extraction method of Mendes-Pinto et al. (2004) were adapted to analyse both carotenoids and chlorophylls in lyophilised grape tissue. The RP-HPLC method baseline separated all the carotenoids and chlorophylls and their derivatives. Recovery of standards from mock extractions was high, indicating that the extraction procedure was acceptable. However, extraction recovery tested in the matrix of the grape tissue showed less promising results due to the high acid content of grape tissue. Violaxanthin, neoxanthin and the chlorophylls were especially sensitive to low pH conditions which facilitated their degradation. The degradation products of these compounds under acidic conditions were identified as pheophytin a, b, chlorophillide a, pyropheophytin b, cisviolaxanthin, cis-neoxanthin, neochrome, mutatoxanthin and luteoxanthin. There is a possibility that some degradation products were already present in the tissue due to lyophilisation (since the water in the berry was then removed and the acid concentrated). More work is needed to investigate the effect of lyophilisation and storage on the composition of grape tissue of different maturity. The extraction method for grape berry tissue at different ripening stages should also be optimised further to effectively neutralise tissue acidity, without compromising the extraction of carotenoids significantly, in especially green berry tissue. The question as to whether cisisomers and chlorophyll degradation products are naturally present in grape berries or are formed during sampling and processing remains unanswered in the current study. This study confirmed that in general carotenoids and chlorophylls decrease on a per berry (μg/berry) and concentration (μg/g) basis from veraison to harvest. Furthermore, this study was inconclusive in showing that vigour differences have an effect on the rate of synthesis/degradation of carotenoids, chlorophyll and some other ripening parameters, namely malic acid, total glucose and fructose, total tannin and total anthocyanin, from pre-veraison (pea size) to harvest. Additionally, no significant effect of soil water content on carotenoids, chlorophylls and ripeness parameters was found in this study, most likely due the fact that high soil water capacity was found in lower soil layers which may have prevented significant differences in grapevine water status. Experimental plots selected for vigour differences based on normalised difference vegetation index (NDVI) images, pruning mass and soil water measurements by means of a neutron probe, showed significant differences in soil water content in only the first 30 cm of the soil for the ripening seasons studied. Predawn plant water potential measurements, however, indicated that none of the experimental vines experienced severe water stress which was previously shown to effect carotenoid content of grapes. The carotenoid 5,8-epoxy--carotene was quantified for the first time in grapes and represents a significant amount of the total carotenoids present at harvest. All the carotenoids and chlorophylls except -carotene appeared to be sensitive to seasonal variation in climatic conditions. Lutein and β-carotene were found to be the most abundant carotenoids present in Merlot grape berries together with chlorophyll a for both seasons studied. The values of these carotenoids also correlated well with previous research. However, chlorophyll a was found in much larger quantities in Merlot berries compared to reported data. This is possibly because in this study the chlorophyll degradation products were included in the calculation of chlorophyll a. Multivariate analysis showed promising preliminary prediction models (with correlation values of above 0.8 for both seasons analysed) for the prediction of the concentration of ripeness parameters (glucose, fructose, malic acid, total tannins and anthocyanins) with carotenoid and chlorophyll content. This result highlights the opportunity for the development of a rapid non-destructive method to measure carotenoids and chlorophylls in berries which in turn can predict optimal ripeness. Furthermore, since carotenoids are the precursors to C13- norisoprenoid aroma compounds in wine a preview of the potential contribution of these aromas to wine might be evaluated. Further research is necessary to investigate the possibility of building and validating such models. / AFRIKAANSE OPSOMMING: Vorige navorsing het getoon dat karotenoïede die voorlopers is van C13-norisoprenoïed aromaverbindings in wyn. C13-norisoprenoïede het lae drempelwaardes in wyn, met β- damassenoon en β-jonoon as die prominentste C13-norisoprenoïede wat ‘n bydrae tot die heuning en blomagtige aroma van die wyn maak. Chlorofil en sy derivate is ook reeds in wyn bespeur, met die potensiaal om voorlopers van aromaverbindings te wees. Buiten die bydrae van hierdie pigmente tot wynaroma en -kwaliteit is hulle ook belangrike rolspelers in fotosintese en kom hulle wydverspreid in plante en plantprodukte voor. Die vernaamste funksies van hierdie pigmente in plante is om lig te versamel en om as beskerming teen lig op te tree. Navorsing het getoon dat omgewingstoestande, klimaat, ligblootstelling van die trosse en grondwatertekorte die karotenoïedinhoud van druiwekorrels beïnvloed. Verder is ook getoon dat die konsentrasie van karotenoïede en chlorofille tussen kultivars verskil. Geen navorsing is al in hierdie opsig op Merlot-druiwekorrels gedoen nie. Met hierdie aspek in gedagte was die doelwit van hierdie studie om die effek van groeikrag en grondwaterinhoud op die evolusie van karotenoïede en chlorofille tydens die rypwording van druiwekorrels van die cv. Merlot te evalueer. Wanneer mens egter kyk na die metodes waarvolgens die karotenoïede en chlorofille in korrelweefsel geanaliseer word, is daar geen geredelik beskikbare metodes nie. ‘n Ekstraksiemetode om die karotenoïed- en chlorofilprofiel van geliofiliseerde weefsel van onryp (groen) tot ryp (rooi) Merlot-bessies te identifiseer en kwantifiseer was dus nodig. In hierdie studie is die RP-HPLC metode van Taylor et al. (2006) vir karotenoïede en die ekstraksiemetode van Mendes-Pinto et al. (2004) aangepas om beide karotenoïede en chlorofille in geliofiliseerde druiweweefsel te analiseer. Die basislyn van die RP-HPLC metode het all karotenoïede en chlorofille en hul derivate geskei. Herwinning van die standaarde vanaf skynekstraksies was hoog, wat aandui dat die ekstraksieprosedure aanvaarbaar was. Ekstraksieherwinning wat in die matriks van die druiweweefsel getoets is, het egter minder belowende resultate getoon as gevolg van die hoë suurinhoud van die druifweefsel. Violaxantien, neoxantien en die chlorofille was veral sensitief vir toestande van lae pH, wat hulle afbreking gefasiliteer het. Die afbrekingsprodukte van hierdie verbindings onder suurtoestande is geïdentifiseer as feofitien a en b, chlorofillied a, pirofeofitien b, cis-violaxantien, cis-neoxantien, neochroom, mutatoxantien en luteoxantien. Daar is ‘n moontlikheid dat sommige afbreekprodukte reeds in die weefsel teenwoordig was as gevolg van liofilisering (aangesien die water in die korrel reeds verwyder was en die suur gekonsentreerd was). Meer werk is nodig om die effek van liofilisering en berging op die samestelling van druifweefsel van verskillende rypheid te bepaal. Die ekstraksiemetode vir druifkorrelweefsel op verskillende stadia van rypwording moet ook verder geoptimaliseer word om weefselsuurheid doeltreffend te neutraliseer, sonder om die ekstraksie van karotenoïede noemenswaardig te kompromitteer, veral in groen korrelweefsel. Die vraag of cis-isomere en chlorofil afbreekprodukte natuurlik in die druifkorrels teenwoordig is en of hulle tydens monsterneming en prosessering gevorm word, kon nie in hierdie studie beantwoord word nie. Hierdie studie het bevestig dat karotenoïede en chlorofille oor die algemeen op ‘n korrel (μg/korrel) en konsentrasie (μg/g) basis afneem vanaf deurslaan tot oes. Hierdie studie het nie daarin geslaag om te toon dat groeikragverskille vanaf voor-deurslaan (ertjiekorrelgrootte) tot oes ‘n effek het op die tempo van sintese/afbreking van karotenoïede, chlorofil en ander rypwordingsparameters nie, naamlik op appelsuur, totale glukose en fruktose, totale tannien en totale antosianien. Daar is ook in hierdie studie geen noemenswaardige effek van grondwaterinhoud op karotenoïede, chlorofille en rypheidsparameters gevind nie, heel moontlik as gevolg van die feit dat hoë grondwaterkapasiteit in die laer grondlae gevind is, wat betekenisvolle verskille in wingerdwaterstatus kon verhoed het. Eksperimentele persele wat gekies is vir groeikragverskille op grond van genormaliseerde verskil plantegroei indeks (NDVI) beelde, snoeimassa en grondwatermetings met ‘n neutronvogmeter het net in die eerste 30 cm van die grond noemenswaardige verskille in grondwaterinhoud getoon vir die rypwordingseisoene wat bestudeer is. Voor-sonopkoms plantwaterpotensiaalmetings het egter aangedui dat geen van die eksperimentele wingerdstokke ernstige waterstres ervaar het nie. Sulke stres is voorheen aangedui om ‘n effek op die karotenoïedinhoud van druiwe te hê. Die karotenoïed 5,8-epoksi--karoteen is vir die eerste keer in druiwe gekwantifiseer en verteenwoordig ‘n noemenswaardige hoeveelheid van die totale karotenoïede wat met oes teenwoordig is. Al die karotenoïede en chlorofille behalwe -karoteen blyk sensitief vir seisoenale verskille in klimaatstoestande te wees. Luteïen en β-karoteen was die volopste karotenoïede in die Merlot-druifkorrels, tesame met chlorofil a, vir beide seisoene wat bestudeer is. Die waardes van hierdie karotenoïede was ook goed gekorreleer met vorige navorsing. Chlorofil a is egter in baie groter hoeveelhede in Merlot-korrels gevind in vergelyking met dít wat in die data gerapporteer is. Die rede hiervoor is moontlik dat die chlorofil-afbreekprodukte in hierdie studie in die berekening van chlorofil a ingesluit is. Meerveranderlikeontleding het belowende voorlopige voorspellingsmodelle getoon (met korrelasiewaardes van meer as 0.8 vir beide die seisoene wat geanaliseer is) vir die voorspelling van die konsentrasie van rypheidsparameters (glukose, fruktose, appelsuur, totale tanniene en antosianiene) met karotenoïed- en chlorofilinhoud. Hierdie resultaat beklemtoon die geleentheid vir die ontwikkeling van ‘n vinnige, nie-destruktiewe metode om karotenoïede en chlorofille in korrels te meet, wat op sy beurt optimate rypheid kan voorspel. Aangesien karotenoïede die voorlopers van C13-norisoprenoïed aromaverbindings in wyn is, kan ‘n voorskou van die potensiële bydrae van hierdie aromas tot wyn moontlik verder evalueer word. Verdere navorsing is nodig om die moontlikheid van die bou en geldigheidsbepaling van sulke modelle te ondersoek.

Thesis (MScAgric)--Stellenbosch University, 1946. / No Abstract Available

Thesis (MScAgric)--Stellenbosch University, 1977. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The importance of small antimicrobial peptides in the innate immune system of plants became increasingly apparent over the past decade. Antimicrobial peptides are unique and diverse molecules that are found in many tissue types in a variety of invertebrate, plant and animal species. Many of these peptides, such as plant defensins, have been found to be ubiquitous throughout the plant kingdom and have been isolated from flowers, leaves, roots, seeds, seedlings, pods, tubers and bark. The growing relevance of antimicrobial peptides (including plant defensins) in research can be largely attributed to their broad-spectrum antifungal activity. This makes them promising potential targets, both as therapeutic agents and for their use in crop protection and disease resistance. The continuing discovery of novel antimicrobial peptides has advanced the development of strategies to overexpress these genes in plants to attempt to enhance the plant’s natural ability to resist pathogenic attack. The first grapevine antifungal peptide, Vv-AMP1, was isolated and characterized and was shown to be tissue specific and developmentally regulated, being expressed only in berries at the onset of berry ripening. The peptide showed strong antifungal activity against a number of plant pathogenic fungi in vitro. In this study, the biological role of the Vv-AMP1 peptide was further investigated, both within its native host (Vitis vinifera) and under in vitro conditions against a panel of grapevine-specific pathogens. As a first step, recombinant production of Vv-AMP1 using an existing bacterial expression system was evaluated and the heterologous production of the Vv-AMP1 peptide improved. Specific optimizations targeting both production and purification of the peptide showed to improve the yield of Vv-AMP1. Steps in the production process targeted for improvement included induction conditions of peptide production by the bacterial culture as well as a number of purification steps, such as lysate preparation, binding conditions, column washing, elution conditions and thrombin protease cleavage. The optimized purification method produced up to 3 mg of pure Vv-AMP1 peptide from 1.6 L of overnight culture. While production was markedly improved, the resultant purified Vv-AMP1 proved biologically inactive and structurally unstable. This is uncharacteristic of the peptide, suggesting that an important aspect necessary for peptide activity, such as folding or the presence of specific co-factors might not be supported in this non-host prokaryotic production system. The study also entailed the characterization and evaluation of the Vv-AMP1 peptide against a panel of grapevine-specific pathogens that are culturable to sporulating cultures using in vitro antifungal assays and microscopy analysis. Vv-AMP1 showed strong inhibitory activity against all pathogens tested, inhibiting the growth of Diplodia seriata and Cylindrocarpon liriodendri by 50% at concentrations between 4.8 μg/ml and 9.6 μg/ml. Phaemoniella chlamydospora and Phomopsis viticola proved particularly sensitive, with IC50 values of 5.5 μg/ml and 4.0 μg/ml respectively. Microscopy analysis of the effect of the Vv-AMP1 peptide on P. viticola showed a severe inhibition on fungal germination and growth. The peptide did not induce morphological changes in fungal hyphae but compromises the fungal membranes, supporting the theory that the peptide induces membrane permeabilization. Functional analysis of a transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was included in this study to provide the opportunity to study the in planta role of the peptide in its native host. The genetic characterization of the putative population included confirming gene integration and copy number through PCR and Southern blot analysis as well as gene expression through northern blot analysis. A confirmed transgenic population was evaluated for improved disease resistance against Botrytis cinerea as a first test organism in an attempt to link the overexpression of the Vv-AMP1 gene to a disease resistance phenotype. Observations of lesion type, average lesion size and further statistical analysis concluded that the transgenic population showed a definite, albeit slight, improved resistance when compared to the untransformed control lines. In conclusion, the study determined that Vv-AMP1 had a strong antifungal action against grapevine-specific pathogenic fungi when tested in vitro. A definite link could be established between the overexpression of Vv-AMP1 and a mild resistance phenotype within its native host plant. The characterized transgenic population is important for further work to evaluate the in planta activity of the peptide against more grapevine pathogens such as the stem pathogens that were proven sensitive and specifically those that cannot be cultured and are obligate pathogens, such as the downy and powdery mildews. / AFRIKAANSE OPSOMMING: Die belang van klein antimikrobiese peptiede in die ingebore immuunstelsel van plante het tydens die afgelope dekade toenemend duidelik geraak. Antimikrobiese peptide is unieke en diverse molekules wat in verskeie weefseltipes in ‘n verskeidenheid van invertebraat-, plant- en dierspesies gevind word. Baie van hierdie peptiede, soos bv. “plant defensins”, word bevind om alomteenwoordig in die plantryk te wees en is reeds geïsoleer vanuit blomme, blare, wortels, sade, saailinge, peule, knolle en bas. Die toenemende belang van antimikrobiese peptiede (insluitend “plant defensins”) in navorsing kan grootliks toegeskryf word aan hul breë-spektrum antifungiese aktiwiteit. Hierdie eienskap maak hul belowende potensiële teikens, beide as terapeutiese middels asook vir gebruik in gewasbeskerming en siekteweerstand. Die voortdurende ontdekking van nuwe antimikrobiese peptiede bevorder tans die ontwikkeling van strategieë om hierdie gene in plante uit te druk in ‘n poging om die plant se natuurlike vermoeë om patogeniese aanval teen te staan te verbeter. Die eerste wingerd antifungale peptied, Vv-AMP1, is geïsoleer en gekarakteriseer as ‘n ontwikkelings-gereguleerde peptied wat slegs uitgedruk word in korrels, tydens die aanvang van bessie rypwording. Die peptied het tydens in vitro toetse sterk antifungale aktiwiteit getoon teen ‘n verskeidenheid plant-patogeniese swamme. In hierdie studie word die biologiese rol van die Vv-AMP1 peptied verder ondersoek, beide binne sy natuurlike gasheerplant, (Vitis vinifera) asook onder in vitro kondisies teen ‘n paneel van wingerd-spesifieke patogene. As ‘n beginpunt is rekombinante produksie van Vv-AMP1 met behulp van ‘n bakteriële ekspressie sisteem evalueer en die hetereloë produksie van die Vv-AMP1 peptied stelselmatig verbeter. Spesifieke optimerings het gefokus op beide die produksie en suiwering van die peptied en het die algehele opbrengs van Vv-AMP1 verhoog. Spesifieke stappe wat in die produksieproses vir verbetering geteiken is sluit beide induksietoestande van peptiedproduksie deur die bakteriële kultuur in sowel as ‘n aantal suiweringsstappe, soos lisaatvoorbereiding, bindingskondisies, kolom wasstappe, eluasie kondisies en “thrombin” protease snyding in. Die optimale suiweringsmetode het tot 3 mg suiwer Vv-AMP1 peptied opgelewer vanaf ‘n 1.6 L oornag bakteriële kultuur. Hoewel die produksie van die peptide noemenswaardig verbeter is, was die gesuiwerde Vv-AMP1 beide onaktief en struktureel onstabiel. Dit is buitengewoon vir hierdie peptied, wat daarop dui dat belangrike aspekte benodig vir antifungiese aktiwiteit, soos korrekte vou of die teenwoordigheid van spesifieke kofaktore, moontlik ontbreek in hierdie nie-gasheer prokariotiese produksiesisteem. Die studie het ook die karakterisering en evaluering van die Vv-AMP1 peptied teen 'n paneel van wingerd-spesifieke patogene wat kultureerbaar is en sporuleer, insluitend in vitro antifungale toetse en mikroskopiese analise, behels. Vv-AMP1 toon sterk inhiberende aktiwiteit teen alle patogene getoets. Dit inhibeer die groei van Diplodia seriata en Cylindrocarpon liriodendri met 50% teen konsentrasies tussen 4.8 μg/ml en 9.6 μg/ml. Phaemoniella chlamydospora en Phomopsis viticola was besonders sensitief, met IC50 waardes van 5.5 μg/ml en 4.0 μg/ml, onderskeidelik. Mikroskopiese analise van die effek van die Vv-AMP1 peptied op P. viticola het 'n ernstige inhibisie op swam ontkieming en groei aangedui. Die peptied het geen morfologiese veranderinge in swam hifes veroorsaak nie maar het wel die swam membraan beskadig. Hierdie bevinding ondersteun die teorie dat die peptied membraan permeabilisasie induseer. Funksionele analise van ‘n transgeniese V. vinifera (cv. Sultana) populasie wat die Vv-AMP1 geen ooruitdruk is by die studie ingesluit om ‘n geleentheid te bied om die in planta rol van die peptide binne sy natuurlike gasheerplant te bestudeer. Die genetiese karakterisering van die vermeende transgeniese bevolking het die bevestiging van beide geenintegrasie en kopiegetal deur PKR en Southern-klad analise ingesluit, sowel as geenuitdrukking d.m.v. noordelike-klad analise. ‘n Bevestigde transgeniese bevolking is evalueer vir potensiële verbeterde weerstand (in vergelyking met die wilde tipe) deur infeksie met Botrytis cinerea as ‘n eerste toetsorganisme in ‘n poging om ‘n weerstandbiedende fenotipe met die ooruitdrukking van Vv-AMP1 te assosieer. Waarnemings van letsel tipe, letsel grootte en verdere statistiese analise het tot die gevolgtrekking gelei dat die transgeniese bevolking ‘n definitiewe (dog geringe) verbeterde weerstand toon in vergelyking met die ongetransformeerde lyne. Ten slotte bepaal die studie dat Vv-AMP1 ‘n sterk antifungale effek teen wingerdspesifieke patogene toon tydens in vitro toetse. ‘n Definitiewe korrelasie is vasgestel tussen die ooruitdrukking van Vv-AMP1 in wingerd en ‘n weerstandsfenotipe in die transgeniese bevolking. Die gekarakteriseerde transgeniese bevolking is uiteraard belangrik vir toekomstige werk om die in planta aktiwiteit van die peptied te evalueer teen verdere wingerdpatogene soos bv. die stampatogene wat sensitief getoets het teen die peptide, asook patogene wat nie kultureerbaar is nie, insluitend verpligte patogene soos dons- en poeierskimmel.

Thesis (MScAgric)--Stellenbosch University, 1975. / ENGLISH ABSTRACT: no abstract available / AFRIKAANSE OPSOMMING: geen opsomming

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Plants are constantly exposed to biotic and abiotic stress inducing factors that threaten their existence. Biotic factors such as pathogens are the cause of huge yield losses to crop plants worldwide with fungal pathogens debatably constituting the worst damage. Fungal pathogens such as Botrytis cinerea, which has a wide host range, release cell wall degrading enzymes called endopolygalacturonases (ePGs) during plant infection. These ePGs break down the pectin component of the cell wall, thus providing an entry route, as well as nutrients for the fungus. Plants have evolved mechanisms to counteract and suppress the action of the ePGs. This is achieved through the action of cell wall associated proteins called polygalacturonaseinhibiting proteins, PGIPs. PGIPs directly inhibit ePGs and their inhibitory action also prolongs the existence of longer chain oligogalacturonide residues which are believed to elicit a cascade of defence responses. In grapevine, a PGIP encoding gene, VvPGIP1, was previously isolated and characterised. VvPGIP1, as well as nine non-vinifera grapevine PGIPs have been expressed in tobacco and shown to be potent antifungal proteins that caused the transgenic tobacco to have strong resistance phenotypes against Botrytis in whole plant infection assays. Following on the tobacco study, two of the non-vinifera PGIPs were expressed in cultivars of the susceptible Vitis vinifera. Characterisation of the putative transgenic population showed that transgene integration was successful, the transgenes were being expressed and there were at least 29 transgenic lines with independent integration events. The transgenic lines were confirmed to have active PGIPs (transgene-derived) in their leaves. Crude protein extracts from 22 lines exhibited 100% inhibition against crude B. cinerea PGs (BcPGs). The plant lines with positive transgene integration, expression, independent integration events and exhibiting 100% transgene-derived PGIP activity were further selected for whole plant and detached leaf antifungal assays where they were challenged with B. cinerea. The whole plant infection assay showed that expression of the non-vinifera PGIPs in V. vinifera promotes susceptibility to B. cinerea, not resistance. This surprising result could perhaps be explained by a quicker and stronger recognition between the pathogen and the host and the stronger activation of defence responses in the host. A more active hypersensitive response in the host would benefit Botrytis being a necrotroph. The type of lesions and the onset and speed of lesion development observed on the transgenics lines versus the wild type support this possibility. Knowledge gaps with regards to the efficiency of the ePG inhibition by the nonvinifera PGIPs during infection of grapevine tissue; the potential changes that might be caused by expressing PGIPs in a grapevine host with a native PGIP with high homology to the transgenes (including potential gene silencing) and the potential impact on defence signalling and defence responses all provides further avenues of study to elucidate this very interesting phenotype further. Overall, this study provides a comprehensively characterised population of transgenic plants that provides useful resources for in vivo analysis of PGIP function in defence, where the host plant harbours a native copy of the PGIP encoding gene. / AFRIKAANSE OPSOMMING: Plante word voortdurend blootgestel aan biotiese en abiotiese faktore, wat stres veroorsaak en hul bestaan bedreig. Biotiese faktore, soos patogene, veroorsaak groot verliese in wêreldwye gewasopbrengste, met swampatogene wat moontlik die grootste skade veroorsaak. Swampatogene, soos Botrytis cinerea, wat ‘n wye reeks gasheerplante kan infekteer, stel selwand-afbrekende ensieme tydens plantinfeksie vry, wat as endo-poligalakturonases (ePG’s). bekend staan. Hierdie ePG’s breek die pektienkomponent van die selwand af, wat gevolglik as ‘n ingangspunt dien,asook voedingstowwe vir die swam verskaf. Plante het meganismes ontwikkel om die aktiwiteit van hierdie ePG’s te bekamp en te onderdruk. Die aktiwiteit van die selwand-geassosieërde proteïene, genaamd poligalakturonase-inhiberende proteïene (PGIP’s), speel hier ‘n rol. PGIP’s inhibeer ePG’s direk en hul inhiberende aktiwiteit verleng ook die bestaan van langketting oligogalakturoniedresidu’s, wat blykbaar ‘n kaskade van weerstandsreaksies kan inisieer. ‘n PGIP-koderende geen, VvPGIP1, is voorheen uit wingerd geïsoleer en gekarakteriseer. VvPGIP1, asook nege nie-vinifera wingerd-PGIP’s is voorheen in tabak uitgedruk en bevestig as proteïene met sterk anti-swamaktiwiteit, soos bevestig deur die bevinding dat die transgeniese tabak ‘n weerstandsfenotipe teen Botrytis in heelplant-infeksietoetse het. Ná die tabakstudie is twee van die nie-vinifera PGIP’s uitgedruk in vatbare V. vinifera-kultivars. Karakterisering van die vermeende transgeniese bevolking het getoon dat die transgeen-integrasie suksesvol was, dat die transgeen uitgedruk word en dat daar ten minste 29 transgeniese lyne met onafhanklike integrasie gebeurtenisse geskep is. Daar is verder bevestig dat die transgeniese lyne aktiewe PGIP’s (transgeen-afkomstig) in hul blare het. Ongesuiwerde proteïenekstrakte van 22 lyne het 100% inhibisie teen ‘n mengsel van ongesuiwerde B. cinerea PGs (BcPGs) getoon. Die plantlyne met positiewe transgeenintegrasie en -uitdrukking, asook onafhanklike integrasiegebeure en wat 100% transgeen-afkomstige PGIP-aktiwiteit getoon het, is verder aan heel-plant en verwyderde blaarswaminfeksies met B cinerea onderwerp. Die heelplantinfeksietoetse het getoon dat uitdrukking van nie-vinifera PGIP’s in V. vinifera ‘n toename, in plaas van ‘n afname, in vatbaarheid teen B. cinerea veroorsaak. Hierdie verbasende resultaat kan moontlik toegeskryf word aan ‘n vinniger en sterker herkenningsreaksie tussen patogeen en gasheer en die moontlike sterker stimulering van weerstandsreaksies in die gasheer. ‘n Meer aktiewe hipersensitiewe reaksie in die gasheer sal tot die voordeel van Botrytis, wat ‘n nektrotroof is, wees. Die tipe letsel, asook die aanvang en spoed van letselontwikkeling wat waargeneem is in transgeniese lyne teenoor die wilde-tipe ondersteun hierdie moontlikheid. Gapings in kennis ten opsigte van die doeltreffendheid van die ePG-inhibisie deur die nievinifera PGIP’s tydens infeksie van wingerdweefsel, die moontlike veranderinge (insluitend ‘n moontlike geenuitdowingseffek) wat veroorsaak kan word deur die uitdrukking van PGIP-gene in ‘n kultivar met ‘n inheemse en baie homoloë PGIP-geen, kon ‘n invloed op weerstandseine en weerstandsreaksies gehad het. Hierdie aspekte lewer verdere studiemoontlikhede om hierdie interessante fenotipe verder te verklaar.Algeheel lewer hierdie studie ‘n breedvoeriggekarakteriseerde bevolking trangeniese plante, wat dien as nuttige hulpbronne vir in vivoanalise van PGIP se funksie in siekteweerstandbiedendheid, veral waar die gasheerplant ‘n inheemse kopie van die PGIP-koderende geen huisves.