Thesis (MscAgric (Viticulture and Oenology))--University of Stellenbosch, 2007. / A common phenomenon in most South African vineyards, especially in the Western Cape region, is that of within vineyard variation. This variation phenomenon is caused by an array of controllable and non-controllable factors that interact with each other to affect vine vigour. Controllable factors can be managed by the grape grower, while the non-controllable factors have to be managed in the planning process in order not to negatively affect productivity or product quality. The main goal of any grape grower is to optimise vine performance in an attempt to achieve the best possible yield while at the same time allowing vines to optimally ripen grapes towards optimal wine quality. A grape grower has to use every possible means and technique available to him in order to manage his vineyards in such a manner as to achieve this goal. In the past, it was difficult to visualize the extent and distribution of vigour variation in vineyards, but with modern technological improvements in the field of remote sensing, grape growers are able to identify and specify different vigour levels within a vineyard. When remote sensing is applied in a vineyard, the grape grower can identify certain areas that may need more specific attention than others. Consequently, managerial decisions based on detailed information can be made in an attempt to improve the general condition and performance of a vine. Not only can the acquired information be used to plan managerial actions throughout the season, but it can also be used to plan and devise harvest strategies. Some areas in a vineyard may be at a certain point in the ripening process and need to be harvested, while grapes from other areas still need to develop the wanted flavours. One managerial action applied at véraison by some grape growers, is that of crop thinning. Different vigour areas can now be subjected to various crop thinning actions in an attempt to determine the best crop load for a vigour level. With this in mind, two studies were launched to firstly investigate the interaction between vine vigour and harvest dates; and secondly to investigate the interaction between vine vigour and crop load and how their combined interaction might influence a vine’s characteristics, grape composition and wine quality. Vigour variation was firstly identified through multispectral aerial imagery, and then visually verified by visits to the experimental vineyards. The multispectral aerial image was then “orthorectified” in order to produce a classified multispectral image. The image was classified through different colour codes that were assigned to the different vigour levels to clearly distinguish between them. A series of vegetative and reproductive measurements were conducted to try and establish if any correlations could be obtained of the interaction between vine vigour, different harvest dates and crop loads. In order to verify differences in vine vigour, underlying causes were also determined through soil analyses of which chemical analysis, bulk density, porosity, as well as root penetration and distribution were determined. Vegetative measurements that were conducted for both studies indicated good correlations between the different vigour levels and the image classifications. The results also identified the effect that topping (mechanical or manual) had on the main and lateral leaf areas. Reproductive measurements throughout the season, in the form of berry sampling, showed changes in berry composition and accentuated the effects of the different treatments, which could also be confirmed through sensorial analysis of the wines. The results also emphasized the need to not only make use of one of two chemical parameters to identify grape ripeness, but to incorporate a number of parameters, such as sugar, pH and acid levels. From the varying grape chemical characteristics, a wine style can be identified that might carry the approval of the winemaker for the production of a specific type of wine. Soil studies of both vineyards also gave important evidence for the causes of vigour variation. The data collected will hopefully provide grape growers with information that will enable them to make educated decisions concerning grape production and how vigour, in conjunction with different harvest dates and crop loads, will enable them to produce fruit of good quality and, so doing, improve their financial position.

Thesis (MscAgric (Viticulture and Oenology))--Stellenbosch University, 2008. / The effect of various irrigation strategies on grapevine water relations during the berry ripening period was investigated in a Shiraz/Richter 99 vineyard. Comparisons between different irrigation strategies (full/seasonal, véraison+post véraison, post véraison and no irrigation) were made. During the day, the seasonally irrigated vines experienced less water stress than the deficit treatments. Non-irrigated vines seemed to maintain higher diurnal leaf water potentials. Lower leaf water potentials indicated lower water contents in the vegetative and reproductive tissue. Full irrigation seemed to stimulate primary shoot length. Longer water deficit induced earlier and more complete shoot maturation (reserve accumulation). Re-distribution of leaf area on the shoot may occur when vines are subjected to water deficit. Extended water deficit seemed to induce earlier and restricted water loss from vegetative tissue. The water relations were reflected in the berry size. Irrigation during ripening seemed to induce a continuation of berry water loss. Transpiration losses were apparently much higher in fully irrigated vines whereas stomatal control efficiently maintained water relations in non-irrigated vines. Water deficit seemed to have enhanced the soluble solid accumulation. Irrigation treatments did not seem to affect the titratable acid and pH. The post véraison irrigation in particular seemed to favour a wide window for harvesting. Irrigation at post véraison and especially véraison+post veraison seemed to have a greater effect on the synthesis and extraction of phenolics, anthocyanins and tannins in the berry skins. Different irrigation strategies may affect grapes in such a way that different wine styles are obtained.

Thesis (MscAgric (Viticulture and Oenology))--Stellenbosch University, 2008. / Over the years, different techniques have been used to legally reduce the ethanol content of wines. Several physical processes are available for producing wines with less alcohol. Despite their efficacy, these treatments have a capital and operational cost influence. They can also affect the concentration of other wine components. On the other hand, vast amount of research has been conducted through genetic modification of wine yeast strains in order to reduce the ethanol yield of Saccharomyces cerevisiae by diverting sugar metabolism towards various byproducts. However, genetically modified yeasts are not currently accepted in most wine industries worldwide, including South Africa. Therefore, other approaches need to be envisaged. Commercial enzymes are commonly added during winemaking. Most enzymes essential for vinification naturally occur in grapes, but are inefficient under pH and sulphur levels associated with winemaking. Enzymes of fungal origin are resistant to such conditions. The most widely used commercial enzymes include pectinases, hemicellulases, glucanases and glycosidases. With the exception of glucanases, produced by Trichoderma harzianium, all the other enzymes are produced by Aspergillus niger. In this study, the possibility of using Gluzyme Mono® 10.000 BG (Gluzyme) (Novozymes, South Africa) to reduce the glucose content of synthetic grape must and grape must before fermentation in order to produce wine with a reduced alcohol content was investigated. Gluzyme is a glucose oxidase preparation from Aspergillus oryzae, currently being used in the baking industry. Glucose oxidase catalyses the oxidation of glucose to gluconic acid and hydrogen peroxide in the presence of molecular oxygen. Gluzyme was initially used in synthetic grape must where different enzyme concentrations and factors influencing its activity were investigated for its use in winemaking. The results showed that up to 0.5% v/v less alcohol were obtained using an enzyme concentration of 20 kU compared to the control. This reduction in alcohol was increased to 1 and 1.3% v/v alcohol at pH 3.5 and pH 5.5 respectively in aerated synthetic grape must using 30 kU enzyme. Secondly, Gluzyme trials were carried out using Pinotage grape must. Gluzyme treated wines after fermentation contained 0.68% v/v less alcohol than the control samples at 30 kU enzyme. Colour and volatile flavour compounds of treated wine did not differ significantly from the untreated samples. Lower free anthocyanin and total phenol concentrations in treated than control samples were observed, possibly due to the hydrogen peroxide oxidation which could have led to polymerisation. The present study has clearly demonstrated that Gluzyme may be used in winemaking to produce reduced-alcohol wine without affecting its colour and aroma compounds. The enzyme in its current form is however, not ideal for winemaking; other forms such as liquid or powder form should be considered if the enzyme is to be used under winemaking conditions. Future work should focus on evaluating the potential new form of the enzyme and studying the effects of Gluzyme in various grape must in semi-industrial scale. A tasting panel should also evaluate its impact on the organoleptic properties and the overall quality of the resulting wines.

MScAgric / ENGLISH ABSTRACT: The Little Karoo region of South Africa stretches from Montagu in the west, through Barrydale on the Langeberg Mountain, towards Ladismith, Calitzdorp, Oudtshoorn and De Rust in the east, with the Swartberg mountain range in the north. The Wine of Origin district of Calitzdorp is a small, demarcated area around Calitzdorp in the Little Karoo, surrounded by the Rooiberg, Swartberg and Kleinberg mountains. With a mean February temperature (MFT) of 23.7ºC and a low annual rainfall of 233 mm, the district of Calitzdorp has a similar climate to that of the Douro Demarcated Region (DDR). The MFT is comparable to the DDR mean July temperature, and it seems that the Douro Superior sub-region to the east of the DDR has a mean July temperature of higher than 25°C. In the Cima Corgo sub-region (in the centre of the DDR), and the Baixo Cargo sub-region, the mean July temperatures are ±25°C and ±22°C respectively. Annual rainfall in the DDR is much higher, with Baixo Cargo recording 1 018 mm, Cima Corgo recording 658 mm and Douro Superior in the east recording only 437 mm. Touriga Naçional is one of the highest quality Portuguese red grape varieties. It produces high-quality port-style wine as well as table wines. Excellent quality Touriga Naçional wines have a dark black/purple colour, good extract, high, elegant tannin content and intense aromas, with typical plum, raisin, wild fruit, mulberry, “fynbos” and cherry aromas. The most suitable terroir for Touriga Naçional in the DDR has been found to be on sites that restrain the natural vigour of the grapevine. Soils with moderate to low water-holding capacity, in association with low rainfall, result in water deficits during the growing season and are considered optimal to restrict growth vigour. A steep, northern middle slope is ideal in the southern hemisphere for high temperatures and sunlight interception. Warm temperatures (25 to 30 °C) during the day and cooler temperatures during the night are optimal for photosynthesis and colour development. In order to study factors affecting the quality of Touriga Naçional in Calitzdorp, two Vitis vinifera L. cv. Touriga Naçional commercial vineyards in the Calitzdorp district were selected. Each vineyard was divided into two separate management blocks based on their empirically determined quality of production. Two crop-reduction treatments, the standard 50% crop reduction (which was considered to be the control) and a further less drastic treatment of 25% crop reduction, were applied. Significant differences were found in viticultural performance between the two adjacent Touriga Naçional management blocks in each vineyard, especially with respect to vigour. The upper management blocks, which provided grapes for reserve-quality port-style wines, experienced a higher water deficit due to the moderate soil water-holding capacity and higher temperatures in comparison to the lower sites. The higher water deficits had a restraining effect on the Touriga Naçional vines, and therefore the upper sites had lower vigour, which contributed to better quality of both the wine and port-style wine, and this could be recognised sensorially. However, it was not reflected in the chemical analytical results. Crop load also appeared to have an effect on the Touriga Naçional grapevines, but this appeared to be dependent on the management block. The 50% crop reduction had a significant positive effect on the sensory analyses, but did not significantly affect the chemical analyses. Calitzdorp terroir has a similar effect on Touriga Naçional compared to the DDR terroir, and that is why Calitzdorp can produce good table and port-style wines from Touriga Naçional. / AFRIKAANSE OPSOMMING: Die Klein Karoo-streek in Suid Afrika strek vanaf Montagu in die weste, deur Barrydale teen die Langeberg, na Ladismith, Calitzdorp, Oudtshoorn en De Rust in die ooste, met die Swartberg in die noorde. Die distrik van Calitzdorp is ‘n klein area rondom Calitzdorp, in die Klein Karoo, wat deur die Rooiberg, Swartberg en Kleinberg omring is. Calitzdorp het ‘n gemiddelde Februarie-temperatuur (GFT) van 23.7°C en ‘n lae jaarlikse reënval van 223 mm, wat soortgelyk is aan die klimaat van die Douro Vallei in Portugal. Die Douro Vallei se gemiddelde Julie-temperatuur (GJT) in vergelyking met die GFT van Calitzdorp is hoër, met temperature van meer as 25°C in die substreek Douro Superior. Vir die substreke Cima Cargo en Baixo Cargo is die GJT ±25°C en ±22°C onderskeidelik. Die jaarlikse reënval is ook hoër by Baixo Cargo, met 1 018 mm, Cima Cargo met 658 mm en Douro Superior met slegs 437 mm. Touriga Naçional is een van die beste Portugese rooi kultivars wat hoëkwaliteit tafel- en portwyne produseer. ‘n Tipiese hoëkwaliteit Touriga Naçional-wyn het ‘n swartpers kleur, hoë ekstrak, hoë elegante tanniene en intense aromatiese geure wat tipiese pruim, rosyne, wilde vrugte, moerbei, fynbos en kersie aromas insluit. Die geskikste terroir vir Touriga Naçional is op swak gronde wat die natuurlike groeikrag van die wingerdstok strem. Gronde met matige tot lae grondwaterhouvermoë tesame met lae reënval veroorsaak ‘n waterstremming in die wingerdstok gedurende die groeiseisoen en word as optimaal beskou omdat dit beheersde groei veroorsaak. In die suidelike halfrond word relatief steil, noordelike middelhange as ideaal beskou vir hoë temperature en maksimale sonligonderskepping. Gepaardgaande hiermee is die interne dreinasie verantwoordelik vir vinniger uitdroging van die grond. Hoë temperature (25 tot 30°C) gedurende die dag en koue nagte is optimaal vir fotosintese en kleurontwikkeling. Twee Vitis vinifera L. cv. Touriga Naçional kommersiële wingerde in die Calitzdorp-distrik is geselekteer en in twee afsonderlike bewerkingsblokke verdeel, gebaseer op kwaliteitsverskille. In elke blok was die verdeling van so ‘n aard dat daar ‘n hoërliggende helfte en ‘n laerliggende helfte was. Twee trosverminderingsbehandelings, nl. 50% (kontrole) en 25%, gebaseer op trosgetalle, is toegepas. By elkeen van die wingerde was daar betekenisvolle groeiverskille tussen die twee aangrensende helftes. Die boonste helftes (of gedeeltes) het minder gegroei a.g.v. ‘n hoër waterstremming sowel as hoër temperatuur as die laer helftes. Dit het geblyk om ‘n positiewe invloed op die kwaliteit van beide die tafel- en portwyne uit te oefen. Troslading het ook ‘n effek op die Touriga Naçional-wingerde gehad, maar dit blyk of dit blok-afhanklik is. Die 50% trosverminderingsbehandeling het ‘n beduidende positiewe verskil in die sensoriese analises gemaak, maar nie ‘n beduidende verskil in die chemiese analises van die wyne nie. Calitzdorp se terroir het ‘n soortgelyke effek op Touriga Naçional as dié van die DDR terroir en daarom kan Calitzdorp soortgelyke goeie tafel- en portwyne van Touriga Naçional produseer.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Grapevine water status is considered to be the most important factor limiting plant growth and production in the Mediterranean zones. In these regions with limited summer rainfall and limited water resources for irrigation grapevines may experience water deficits for an extended period of time. The demand of water for agriculture is constantly increasing, and will continue to do so due to the rise in the world population and to the effects of climate change on rainfall and evaporative demand in these regions. The Western Cape wine region is also classified as Mediterranean and grapevines grown in this region are often exposed to water “stress” conditions due to high evaporative demand and low water availability in the soil. Plant water status of grapevines may dependent on, amongst other factors, the water potential of soil layers close to the root system, canopy size and evaporative demand. The canopy size of a grapevine can inherently be seen as a measure of grapevine vigour, and vigour variation among grapevines within a vineyard is a common phenomenon in the Western Cape. The importance of the contributions from several factors causing vigour variation within vineyards is still a subject of debate. This may be largely ascribed to the significant amount of variability in vineyards that researchers have to deal with during viticultural studies. However, the recent advances in remote sensing technology have established new methods to assess grapevine vigour variability. In the face of the recognized variation within vineyards and the importance of a sustained grapevine water status, for wine grape productivity and -quality, it is alarming to think that a vineyard block is generally managed as a homogeneous entity when it comes to irrigation scheduling. What is more alarming is the assumption that grape, juice and wine quality will be homogeneous throughout a vineyard block – even without irrigation. With this in mind, a study was conducted to study the interaction between grapevine vigour and grapevine water status within a commercial vineyard with variable vigour by implementing various irrigation regimes. Vigour variation was identified through multispectral aerial imagery and plant-based water status determinants were used to assess grapevine water status in plots of differing vigour within the vineyard. Soil water status was also assessed, and vegetative growth quantified to ultimately determine the variability in vigour and its possible contribution to the variability through the water status of the plant. Reproductive growth was monitored continually before evaluating the effect of water status and grapevine vigour on grape composition and subsequent wine quality. The various methods used to evaluate grapevine vigour showed good correspondence. Pruning mass measured at the end of the season confirmed leaf area measurement (main leaves and lateral leaves) during vegetative growth, and corresponded well, in terms of main vigour classifications with the NDVI images collected. Berry weight and volume responded to the various classifications, with a decrease in water deficits from one classification to the next accompanying an increase in berry weight and volume. Analyses of the berry composition and wines showed statistically significant differences between the classifications. This was found for sugar content per berry, total phenols, total red pigment, malic acid, nitrogen and pH for the grape juice analyses. Wine pH and total acidity also differed significantly. / AFRIKAANSE OPSOMMING: In die Mediterreense sones word plantwaterstatus beskou as `n hooffaktor wat groei en produksie van `n wingerdstok negatief beinvloed. In hierdie sones kan wingerdstokke vir lang periodes `n tekort aan water ervaar a.g.v `n tekort aan reënwater gedurende die somer en lae beskikbaarheid van besproeingswater. Die vraag na water vir landbou is ook konstant besig om toe te neem in dié sones en die tendens sal voorduur a.g.v die groei in die wêreldbevolking, die effek van klimaatsveranderig op reënvalpatrone en die hoë verdampingsfaktor. Die wingerd- en wynstreek van die Wes-Kaap word ook geklassifiseer as Mediterreens en wingerdstokke in hierdie streek ervaar dikwels waterspanning wat deur hoë evapotranspirasie en min beskikbare grondwater veroorsaak word. Van die faktore wat die waterstatus van `n wingerdstok bepaal is onder andere die waterpotensiaal van die grondlae rondom die wortelstelsel, die grootte van die wingerdlowerraamwerk en die evapotranspirasiebehoefte. Die omvang van `n wingerdstok se lower binne die prieel word beskou as `n aanduiding van wingerdstokgroeikrag en variasie in groeikrag tussen wingerdstokke is `n algemene verskynsel in die Wes-Kaap. Die rangorde, wat die effek van die verskeie faktore wat groeikragvariasie tussen wingerdstokke bepaal, word steeds gedebatteer. Die debat kan groottendeels toegeskryf word aan die beduidende hoeveelheid variasie tussen wingerde waarmee navorsers te doen kry in wingerdkundige studies. Hoewel, met onlangse vordering aangaande afstandswaarnemingstegnologie is daar nou nuwe metodes beskikbaar om wingerdgroeikrag te evalueer. Dit is kommerwekend om te dink dat `n wyndruifwingerd normaalweg as `n homogene eenheid bestuur word as dit kom by besproeiing. Veral met die wete dat groeikragvariasie tussen wingerde algemeen erken en aangeteken word, en dat volhoubare waterstatus van `n wingerdstok van kardinale belang is vir produksie en kwaliteit van wyndruiwe. Die aanname dat wyndruiwe, die sap- en ook wynkwaliteit homogeen sal wees regdeur `n wingerdblok is egter meer kommerwekkend. Na aanvang van dié denke is daar `n studie geloods om die interaksie tussen wingerdstokgroeikrag en wingerdstokwaterstatus te evalueer. Met die studie is verskeie besproeiingsregimes aangebring binne `n kommersiële wingerd wat interne groeikragvariasie tentoonstel. Groeikragvariasie was geïdentifiseer deur middel van multispektrale lugfotos terwyl die wingerdstok se waterstatus geëvalueer is met behulp van plantgebaseerde metings in die verskillende groeikragareas. Die waterstatus van die grond is geëvalueer tesame met die vegetatiewe groei van die wingerd sodat die groeikragvariasie en die invloed van die plantwaterstatus op die groeikrag bepaal kon word. Die reproduktiewe groei is deurlopend gemonitor voor die effek van wingerdstokwaterstatus en wingerdstokgroeikrag op druifsamestelling en wynkwaliteit bepaal is. Daar was `n goeie ooreenkoms tussen die verskeie metodes wat gebruik is om wingerdgroeikrag te bepaal. Snoeimassa aan die einde van die seisoen was ooreenkomstig met die blaaroppervakte (hooflootblare en sylootblare) wat tydens vegetatiewe groei gemeet is, en het ook goed korreleer, met die multispektrale lugfotos se hoof groeikragklassifikasie. Korrelgewig en -volume het reageer op die verskeie besproeiingsregimes, en daar was `n toename in korrelgewig en -volume saam met die afname in watertekort van een regime tot `n ander. Daar was beduidende verskille tussen die verskeie klassifikasies t.o.v. korrelsamestelling analise en wynevaluasie. Die suikerinhoud per korrel, totale fenole, totale rooi pigment, appelsuur, stikstof en pH het verskil in druiwesap analises. Die pH en suur van die wyne het ook beduidend verskil.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Because of long-term climate changes, apparently associated with higher temperatures and fewer rainfall events, factors such as water-use efficiency and site selection for new cultivars are a matter of increasing importance for viticulture. Within this context, the root system is expected to play a key role. Its relevance to grapevine functioning is due to the numerous functions in which it is involved. In the light of this, the development of the root system is highly relevant to the viticulturist because of the fact that grapevine growth and functioning are dependent on the development of the root system. Differences can, therefore, be expected in terms of berry ripening on single grapevines of the same scion for situations with differing development of root systems, despite being grafted on the same rootstock. Root growth is influenced by several factors, among the ecological aspects. Soil parameters have a predominant influence on root growth and distribution but also annual root production can be altered by canopy manipulation. Due to the importance of root growth to the aboveground development of the vine, it is critical to gain understanding of the relationship between soil factors and root growth and distribution, and the central role that the subterranean environment plays in the concept of terroir. This study aimed to investigate the effect of selected soil physical and chemical parameters on root growth and distribution and to investigate whether having very different canopies influences root growth. In order to achieve these goals, two experiments were conducted; the first was performed in two commercial Sauvignon blanc vineyards each grafted onto Richter 110, non-irrigated, with two treatments: undisturbed lateral growth and complete lateral removal. The second study included the analysis of eight commercial Sauvignon blanc vineyards grafted onto Richter 99 and Richter 110 located in the Stellenbosch Wine of Origin District. Measurements of physical and chemical soil parameters, root growth and distribution, canopy growth and functioning, vine water status and berry composition were performed. The edaphic factors appeared to be one of the most important parameters that affected root development by changing soil water availability and possibly causing physical or chemical limitations on root growth. From the results of this study, it is clear that severe water stress and a pH (KCl) lower than 4.5 play a key role in the limitation of root growth. Due to the fact that most of the soils from the Stellenbosch Wine of Origin District, especially the subsoils, are acidic, this is a factor to consider before planting. On the other hand, the combination of favourable edaphic conditions, such as a subsoil pH of higher than 5.0, light- to mediumtextured subsoil and moderate water stress, allow increased growth of thin roots. However, the effect of canopy management on root growth cannot be discounted due to its importance in the variation of carbohydrate demand by competing sinks. This study showed that lateral removal done from when the berries are at pea size results in an increase in the number of thin roots (0.5-2.0 mm). The secondary leaf area represents at least the same leaf area as the primary leaf area in all the vineyards evaluated, which reveals the relative importance of the laterals in the total leaf area of the vine and the potential importance in terms of microclimate and leaf area available for photosynthesis. Studies of root growth should take the vineyard canopy architecture into account. / AFRIKAANSE OPSOMMING: As gevolg van langtermyn klimaatsveranderinge wat toegeskryf kan word aan die voorkoms van hoër temperature en laer reënval, is faktore soos effektiwiteit van waterverbruik en liggingseleksie vir nuwe kultivars van kardinale belang vir wingerdkunde. Binne hierdie konteks, speel die wortelsisteem ‘n belangrike rol. Die belangrikheid hiervan vir wingerdfunksionering kan toegeskryf word aan die talle funksies waarby dit betrokke is. Die ontwikkeling van die wortelsisteem is dus hoogs relevant vir die wingerdkundige, omdat wingerdgroei en funksionering afhanklik is van die ontwikkeling van die wortelsisteem. Verskille kan daarom dus verwag word in terme van korrelrypwording op ‘n enkele wingerdstok van dieselfde onderstok vir gevalle met verskillende ontwikkeling van die wortelsisteem, ten spyte daarvan dat dit op dieselfde onderstok geënt is. Wortelgroei word, onder ekologiese aspekte, deur verskillende faktore beïnvloed. Grondfaktore het meerendeels ‘n predominante invloed op wortelgroei en -verspreiding, terwyl jaarlikse wortelproduksie deur lowermanipulasie beïnvloed kan word. Weens die belangrikheid van wortelgroei vir die bogrondse ontwikkeling van die wingerd, is dit krities om kennis op te doen oor die verhouding tussen grondfaktore en wortelgroei en –verspreiding, asook die sentrale rol wat die subterreinomgewing op die terroir-konsep speel. Die studie was daarop gemik om die invloed van geselekteerde fisiese en chemiese parameters van grond op wortelgroei en -verspreiding vas te stel, en ook te ondersoek of verskillende lowers wortelgroei sal beïnvloed. Om laasgenoemde doelwitte te bereik, is twee eksperimente uitgevoer. Die eerste is uitgevoer in ‘n kommersïele Sauvignon blanc-wingerd wat geënt is op Richter 110, sonder besproeïng en met twee behandelings, naamlik onversteurde sêkondere lootgroei en volledige sêkondere lootverwydering. Die tweede studie het die analise van agt kommersïele Sauvignon blancwingerde geënt op Richter 99 en Richter 110 in die Stellenbosch Wyn van Oorsprong Distrik. Metings van fisiese en chemiese grondfaktore, wortelgroei en -verspreiding, lowergroei en -funksionering, plantwaterstatus en korrelsamestelling is uitgevoer. Dit blyk dat edafiese faktore een van die belangrikste parameters is wat wortelontwikkeling beïnvloed deur beskikbaarheid van grondwater te verander, en wat moontlik fisiese en chemiese beperkings op wortelgroei kan veroorsaak. Uit die resultate van die studie is dit duidelik dat intense waterspanning en ‘n pH (KCl) laer as 4.5 ‘n belangrike rol in die beperking van wortelgroei speel. Aangesien die meeste van die grondsoorte in die Stellenbosch Wyn van Oorsprong Distrik, veral al die subgronde, suur is, is dit ‘n faktor wat in oorweging geneem moet word voor aanplantings. Die kombinasie van gunstige edafiese toestande, soos ‘n subgrond met ‘n pH hoër as 5.0, ‘n lig tot medium tekstuur en matige waterspanning, sal dus aanleiding gee tot ‘n toename in die groei van dun wortels. Die effek van lowerbestuur op wortelgroei kan egter nie buite rekening gelaat word nie weens die belangrikheid daarvan in die variasie van koolhidraataanvraag deur kompeterende vraagpunte. Hierdie studie toon dat, indien sêkondere lootverwydering tydens ertjiekorrelgrootte toegepas is, dit aanleiding gee tot ‘n toename in die dun wortels (0.5 tot 2.0 mm). Die sêkondere blaaroppervlakte verteenwoordig minstens dieselfde blaaroppervlakte as die primêre blaaroppervlakte in al die wingerde wat ondersoek is, wat dui op die belangrikheid van sêkondere lote in die totale blaaroppervlakte van die wingerd en die potensiële belangrikheid daarvan in terme van mikroklimaat en blaaroppervlakte wat vir fotosintese beskikbaar is. Studies van wortelgroei moet lowerargitektuur in ag neem.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: One of the most striking qualities of wine is its tart, sour taste. The sensory perception of sourness is mainly attributed to the presence of hydrogen ions (protons) at high concentrations. Large amounts of weak carboxylic acids (organic acids) are the main sources of these ions within wine. Once wine enters a person's mouth, the dissociable protons of the weak organic acids within wine are partially neutralized or, in other words, titrated by the saliva secreted inside one's mouth. This explains why the duration and intensity of a wine's sourness is related to its titratable acidity content. The sour taste of wine is usually considered refreshing and it helps balance wine flavour. In fact, wines become watery when its titratable acidity content is too low. After alcoholic fermentation, the titratable acidity of wine will usually be less than that of the grape juice from which was made due to ethanol-induced precipitation of potassium bitartrate crystals and partial consumption of malic acid by fermenting wine yeasts. Occasionally however, increases in titratable acidity are observed during alcoholic fermentation. If wine is produced from grape juice with optimum levels of titratable acidity, unforeseen increases in titratable acidity during alcoholic fermentation can be detrimental to the quality of the final product. Although the net production of malic acid by wine yeasts contributes to increases in titratable acidity seen during grape juice fermentations, the production of succinic acid is regarded as the primary contributor. In fact, succinic acid accounts for approximately 90% of the non-volatile acids produced during fermentation of grape juice. Between 0.5 and 1.5 g/L succinic acid is normally found in wine, but higher concentrations thereof (up to 3.0 g/L) have been detected within certain red wines. Acidity adjustments should preferably be carried out before the onset of alcoholic fermentation to allow better integration of the added compound(s) and to ensure that conditions during fermentation favour the quality and microbial stability of the final product. In doing so unfortunately, winemakers run the risk of ending up with wines that may taste too sour if they are unable to accurately predict and take into consideration the amount of succinic acid produced during alcoholic fermentation. Knowledge with regard to the factors involved in succinic acid's production by fermenting wine yeasts is therefore required in order to manage the titratable acidity of wines more accurately. Ever since Louis Pasteur first noticed succinic acid amongst the by-products of alcoholic fermentation, attempts have been made to determine the metabolic pathways and factors involved in its production by fermenting wine yeasts. Up until now however, it remains unclear why wines sometimes end up with exceptionally high levels of succinic acid. For these reasons it was decided to investigate the possible causes of very high succinic acid concentrations within wine. Due to complexity of grape juice's chemical composition and the problems associated with sterilizing grape juice, fermentation experiments were conducted within a chemically defined grape juice-like medium. Succinic acid production by nine different industrial wine yeast strains was studied under various conditions with regard to the nutrient status of the synthetic grape juice, temperature and availability of molecular oxygen during alcoholic fermentation. The amount of succinic acid produced during alcoholic fermentation was found to depend on the yeast strain, fermentation temperature and chemical composition of the synthetic grape juice. Out of the nine commercial yeast strains selected for this study, strain WE372 produced the largest amount of succinic acid in synthetic grape juice at 28°C. Strain WE372 produced significantly smaller amounts of acetic acid than the other yeast strains of this study and very little acetic acid at 28°C, which indicated that strain WE372 may have less acetaldehyde dehydroganase activity than the other yeast strains of this study under the conditions tested. The effect this has on NAD: NADH balance is the probable cause for its ability to form more glycerol, succinic and malic acid than the other strains. Results from our study show that succinic acid production is influenced primarily by the metabolizable fraction of YAN, which we termed metabolically available nitrogen (MAN). Succinic acid production by fermenting yeasts will be favoured by moderate to high fermentation temperatures (20°C to 28°C) in grape juice with a nicotinic acid and/ or nicotinamide deficiency, high sugar content (200 g/L to 240 g/L), moderate amounts of metabolically available nitrogen (300 ± 50 mg/L MAN), the presence of flavonoids and large supplies of unsaturated long-chain fatty acids. Even higher concentrations of succinic acid were produced when oxygen was made available to fermenting yeasts by aerating the fermenting grape juice. Fermentation temperatures below 18°C, too much metabolizable nitrogen (> 450 mg/L MAN), very high concentrations of fermentable sugar (> 240 g/L), lipid deficiencies and a lack of pantothenic acid, thiamine, biotin or pyridoxine will decrease the amount of succinic acid produced fermenting yeasts. / No Afrikaans summary available.

Thesis (MScAgric) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Fungi are an extremely diverse group of organisms and, by acting as pathogens, they can colonise various other organisms, including humans, plants and animals. The effect of this is usually detrimental, not only to agricultural crops and livestock, but also to human well-being. The extensive farming of crops and livestock requires persistent control of fungal populations, commonly through the use of chemical fungicides. However, the exclusive use of fungicides is no longer a sustainable practice, as a result of serious problems, such as increasing fungicide resistance in pathogen strains, the high costs of fungicides, as well as concern about the environment. The search by producers and scientists for alternative control measures is an ongoing process. The fungal cell wall consists of polysaccharides that not only playa role in protection of the fungi, but also in relaying signals for the invasion and infection of susceptible hosts. Chitin, a polysaccharide composed of N-acteylglucosamine (GleNAc) residues linked by P-1,4 glucosidic linkages, is one of the major components of the fungal cell wall, where it plays an important role in the apical growth of the vegetative hyphae. Chitinases (EC 3.2.1.14) are abundant proteins produced by a variety of microorganisms and plants and are necessary for the hydrolysis of the chitin polymer. During the invasion of many plant species by a pathogen, the production of a specific group of proteins, designated pathogenesis-related (PR) proteins that include chitinases, is induced as part of their defence response. Due to the facts that pathogenic fungi contain chitin in their cell walls and that plant chitinases are induced upon pathogen attack, chitinases have been confirmed as an integral and crucial part of the plant's natural defence response. Chitinases have increasingly been targeted to upregulate plants' endogenous disease resistance mechanisms through transgenic overexpression in a variety of hosts. Several species of fungi, including various Trichoderma spp., are potent biocontrol agents of plant pathogenic fungi and insects. The antagonistic activities of these biological control agents towards phytopathogens are based on the secretion of extracellular hydrolytic enzymes, such as cell wall-degrading chitinase enzymes. However, biological control is not restricted to naturally occurring biocontrol agents. Through the process of genetic transformation, other fungal or yeast species can be enhanced to produce their own chitinases or other antimicrobial substances more effectively in order to yield potent biocontrol agents. Various types of chitinases have been applied in the production of fungal resistant plants and some research has been done on the application of chitinases, from a variety of microorganisms, as biological control agents. In contrast, very little is known about the antifungal activity of the Saccharomyces cerevisiae chitinase enzyme, encoded by the CTS1-2 gene. The CTS1-2 gene was utilised in this study as a candidate for overexpression in both yeast and plant expression systems to analyse the ability of the encoding chitinase to inhibit fungal growth. The first objective of this study involved the high level expression and optimisation of the secretion of the CTS1-2 gene in S. cerevisiae to render recombinant yeast with enhanced antifungal abilities and with possible applications as a biocontrol agent to control plant pathogenic fungi. It was hypothesised that high-level expression and efficient secretion would be prerequisites in a biocontrol yeast strain. To this end, two strong promoters and terminators were included in the study and the secretion of the chitinase gene was evaluated by testing three different secretion signals. The secretion signals included: the native CTS1-2 secretion signal, the S. cerevisiae mating pheromone a-factor (MFa1) secretion signal, as well as the Trichoderma reesei f3-xylanase 2 (XYN2) secretion signal. The phosphoglycerate kinase 1 (PGK1) and alcohol dehydrogenase 2 (ADH2) promoters and terminators were employed to achieve high-level expression. The results obtained from the analysis of the recombinant yeasts showed that the PGK1 promoter-terminator constructs yielded high level CTS1-2-expressing and chitinase-producing strains of S. cerevisiae PRY488. The ability of the different secretion signals to efficiently secrete the overexpressed chitinase was analysed and it was found that the non-native secretion signals delivered significantly more protein to the extracellular environment. It was thus evident that the performance of the MFa1 and XYN2 secretion signals was superior to that of the native secretion signal. The antifungal activities of the recombinant chitinases produced by these constructs were tested in in vitro assays against Botrytis cinerea. The enzymes led to a significant reduction in hyphal development, caused by extreme structural damage to the hyphal tips, the hyphal cell walls as well as the ability of the fungus to form reproductive and survival structures, thereby confirming the antifungal abilities of this enzyme. The ADH2 promoter-terminator constructs yielded CTS1-2 transcripts, but no chitinase activity could be detected with any of these strains. The reasons for this still remain unclear. The second objective of this study was to assess the potential of the yeast chitinase gene to upregulate defence against fungal infection in planta. In order to elucidate this, the CTS1-2 gene was constitutively overexpressed in tobacco plants, targeting the chitinase both to the intra- and the extracellular environment. The results obtained showed that the transgenic tobacco lines regenerated in this study stably integrated the transgene, exhibiting transgene expression as well as the production of a biologically active yeast chitinase enzyme. The F, progeny were rigorously tested for resistance to B. cinerea, and both in vitro and in planta assays confirmed that the yeast chitinase increased the plant's tolerance to fungal infection; some of the lines showed disease resistance of 65 and 70%. The plants expressing an extracellularly targeted chitinase gene are still under evaluation. Interesting results are expected relating to the effect of the chitinase on the plant surface with regards to disease resistance to fungal pathogens. In conclusion, the combined set of results from both the yeast and plant overexpression studies has confirmed the strong antifungal effect of yeast chitinases. The yeast CTS1-2 chitinase could be instrumental in the development of a new generation of yeast strains with improved antifungal capabilities. This enzyme could also play an important role in genetic transformation technologies aimed at enhanced disease resistance. / AFRIKAANSE OPSOMMING: Swamme omsluit 'n uiterste diverse groep organismes wat mense, plante en diere deur patogeniese aksie kan koloniseer. Die uitkoms hiervan op landbougewasse, die veebedryf en menslike gesondheid is gewoonlik skadelik. Uitgebreide gewas- en veeboerderye benodig voortdurende beheer van fungiese populasies, tipies deur van chemiese swamdoders gebruik te maak. Die uitsluitlike gebruik van swamdoders is egter nie meer 'n lewensvatbare praktyk nie, hoofsaaklik as gevolg van probleme soos die opbou van weerstand van patogeniese rasse teen swamdoders, die hoë kostes van die middels, asook besorgheid oor die omgewing. Die soektog na alternatiewe beheermaatreëls deur produsente en wetenskaplikes bly 'n aaneenlopende proses. Die swamselwand bestaan uit polisakkariede wat nie net In rol in die beskerming van die swam speel nie, maar ook betrokke is in die oordrag van aanvals- en infeksieverwante seine in 'n vatbare gasheer. Chitien, 'n polisakkaried bestaande uit N-asetielglukosamien (GlcNAc) residu's gekoppel deur 13-1,4glukosidiese bindings, is een van die hoofkomponente van die swamselwand, waar dit 'n belangrike rol in die apikale groei van vegetatiewe hifes speel. Chitinases (EC 3.2.1.14) is proteïene wat oorvloedig deur 'n verskeidenheid van mikroërganismes en plante geproduseer word, waar hulle vir die hidrolise van die chitien polimeer noodsaaklik is. Tydens die infeksie van verskeie plantspesies deur In patogeen, word die produksie van 'n spesifieke groep proteïene, die sogenaamde patogeen-verwante (PR) proteïene wat chitinases insluit, as deel van die plant se verdedigingsreaksie geïnduseer. Die feit dat patogeniese swamselwande chitien bevat en dat plantchitinases tydens infeksie geïnduseer word, het daartoe gelei dat dit bevestig is dat chitinases In integrale en kritiese deel van die plant se natuurlike verdedigingsreaksie uitmaak. Chitinases word toenemend geteiken in pogings om die plant se intrinsieke siekteweerstandsmeganismes te verbeter deur transgeniese ooruitdrukking daarvan in 'n verskeidenheid van gashere. Verskeie swamspesies, insluitend verskillende Trichodenna-spesies, is kragtige bio-antagoniste van plantpatogeniese swamme. Die antagonistiese aksies van hierdie biologiese beheeragente teenoor fitopatogene is gebaseer op die uitskeiding van ekstrasellulêre hidrolitiese ensieme, soos die selwandverterende chitinase ensieme. Nietemin is biologiese beheer nie net tot bio-antagoniste wat natuurlik voorkom beperk nie. Deur die proses van genetiese transformasie kan ander swam- of gisspesies verbeter word om hul eie chitinases of ander antimikrobiese substanse meer effektief te produseer, wat aanleiding sal gee tot kragtige bio-antagoniste. Verskeie tipes chitinases is al in die produksie van swambestande plante ingespan en uitgebreide navorsing is gedoen op die toepassing van 'n reeks chitinases, afkomstig van 'n verskeidenheid van mikroërganismes, as biologiese beheeragente. In teenstelling is baie min bekend oor die antifungiese aktiwiteite van die Saccharomyces cerevisiae chitinase ensiem, wat deur die CTS1-2 geen ge-enkodeer word. Die CTS1-2-geen is in hierdie studie gebruik vir ooruitdrukking in beide gis- en plantuitdrukkingsisteme om die chitinase se vermoë om swamgroei te inhibeer, te ondersoek. Die eerste oorkoepelende oogmerk van hierdie studie het hoë-vlak uitdrukking en optimalisering van sekresie van die CTS1-2-geen in S. cerevisiae behels, met die toekomstige doelwit om 'n rekombinante gis met verbeterde antifungiese eienskappe en met moontlike toepassings as 'n bio-antagonis teen plantpatogeniese swamme te ontwikkel. Die hipotese was dat hoë-vlak uitdrukking en voldoende sekresie voorvereistes vir 'n bio-antagonisras is. Omdié rede is twee sterk promotors en termineerders by hierdie studie ingesluit en is die sekresie van die chitinase-geen geëvalueer deur drie verskillende sekresieseine te toets. Die sekresieseine sluit in: die wilde-tipe CTS1-2 sekresiesein, die S. cerevisiae paringsferomoon a-faktor (MFa1) sekresiesein, en die Trichoderma reesei p-xilanase (XYN2) sekresiesein. Die fosfogliseraat kinase 1 (PGK1) en alkohol dehidrogenase 2 (ADH2) promotors en termineerders is gebruik om hoë-vlak uitdrukking te dryf. Die resultate wat vanaf die analises van die rekombinante giste verkry is, het getoon dat die PGK1 promotor-termineerder konstrukte hoë-vlak CTS1-2-uitdrukkende en chitinase-produserende S. cerevisiae PRY488 rasse opgelewer het. Die vermoë van die verskillende sekresieseine om die ooruitgedrukte chitinase voldoende uit te skei, is geanaliseer, en daar is gevind dat die heteroloë sekresieseine aansienlik meer proteïene na die ekstrasellulêre omgewing geloods het. Dit was dus duidelik dat die MFa1 en XYN2 sekresieseine beter as die wilde-tipe sekresiesein presteer het. Die antifungiese aktiwiteit van die rekombinante chitinases wat deur hierdie konstrukte geproduseer is, is ook in in vitrotoetse teen Botryits cinerea getoets. Die teenwoordigheid van die ensieme het gelei tot 'n aansienlike afname in hife-ontwikkeling, veroorsaak deur ekstreme strukturele skade aan die hifepunte, die hifeselwande, asook die vermoë van die swam om voortplanting- en oorlewingstrukture te vorm. Die ADH2 promotor-termineerderkonstrukte het CTS1-2 transkripte vertoon, maar geen chitinase-aktiwiteite kon in hierdie konstrukte waargeneem word nie. Die redes hiervoor is tot op hede onbekend. Die tweede oogmerk van hierdie studie was om die potensiaal van die gischitinase om swaminfeksie in planta teë te werk, te ondersoek. Die CTS1-2-geen is konstitutief ooruitgedruk in tabakplante, waarin die chitinase na beide die intra- en ekstrasellulêre omgewing geteiken is. Resultate het getoon dat die geregenereerde transgeniese tabaklyne die transgeen stabiel geïntegreer het, transgeenuitdrukking vertoon en dat 'n biologies aktiewe chitinase-ensiem geproduseer is. 'n F1-generasie is aan strawwe toetse onderwerp om weerstand teen B. cinerea te ondersoek. Beide die in vitro en in planta toetse het bevestig dat die gischitinase die plant se verdraagsaamheid teenoor swaminfeksie verhoog het; sommige lyne het siekteweerstand van tussen 65 en 70% getoon. Die plante wat 'n ekstrasellulêre chitinase produseer, word steeds geëvalueer. Interessante resultate word verwag aangaande die effek van die chitinase op die plant se oppervlak met betrekking tot siekteweerstand teen swampatogene. Ten slotte, die gekombineerde stel resultate wat vanaf beide die gis- en plantuitdrukkingstudies verkry is, het die sterk antifungiese effek van gischitinases bevestig. Die gis CTS1-2 kan instrumenteel wees in die ontwikkeling van 'n nuwe generasie gisrasse met verbeterde antifungiese eienskappe. Die ensiem kan ook 'n belangrike rol in genetiese transformasietegnologieë, wat op verbeterde siekteweerstand gemik is, speel.

Thesis (MScAgric (Viticulture and Oenology)--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: ‘Crimson Seedless’ is currently one of the most important and popular table grape cultivars produced in South Africa, and as such it is of great economic value for table grape producers. Major concerns with ‘Crimson Seedless’ is that it is prone to inadequate colouring, and with increased yields the berry size decreases. An additional difficulty is that methods used to increase berry size, further impede berry colouring. A plant growth regulator (PGR) commonly used in table grape production, to enhance colour formation, is ethephon (2-chloro-ethyl-phosphonic acid, 2-CEPA). In recent years significant research has been done on the effect of sunlight on anthocyanin production in grapes, although this has primarily been on wine grape cultivars. Currently, there is limited knowledge on the effect of sunlight on table grapes, and how this might influence their anthocyanin composition and content. The effect of ethephon on colour of grapes and other fruit have been extensively researched and well documented. However, the effect of ethephon on the anthocyanin composition of ‘Crimson Seedless’ is not well known. The current study aimed to explore the effect of sunlight (by matter of exclusion) and management practices, namely defoliation and ethephon application, on the anthocyanin profile and content of ‘Crimson Seedless’. Four different treatments were applied to two ‘Crimson Seedless’ vineyards, the first site located in Paarl, and the second in De Doorns. The treatments were: 1. Naturally exposed bunches, 2. Exposed bunches treated with ethephon, 3. Bunches kept in shade boxes, 4. Shaded bunches treated with ethephon. At the De Doorns site an additional defoliation treatment was superimposed over the above treatments. An HPLC technique was modified for the separation and detailed profiling of ‘Crimson Seedless’ anthocyanins and was used to analyse the effect of the reported treatments on the anthocyanin profile of berry skins. The predominant anthocyanin in ‘Crimson Seedless’ is peonidin-3-glucoside (Pn-gluc), and this was found to be significantly increased only by ethephon application, and was not altered by sunlight or leaf removal. The responses of the other anthocyanin types varied according to the respective treatments applied. However, a general observation was that ethephon application more consistently increased the concentration of anthocyanins in berry skins than did sunlight. Leaf removal had the least significant effect on anthocyanin concentration. / AFRIKAANSE OPSOMMIMG: ‘Crimson Seedless’ is tans een van die belangrikste en gewildste tafeldruif cultivars wat in Suid-Afrika verbou word en daarom is dit van groot ekonomiese waarde vir tafeldruifprodusente. ‘Crimson Seedless’ is egter daarvoor bekend dat dit te swak kleur (volgens uitvoer spesifikasies) en tweedens is die cultivar geneig om kleiner korrels te ontwikkel wanneer die oeslading vermeerder word. ‘n Addisionele probleem is dat die praktyke wat in die industrie gebruik word om korrels te vergroot ‘n verdere negatiewe impak op ‘Crimson Seedless’ se kleur ontwikkeling kan veroorsaak. Die plant-groei-reguleerder wat algemeen in tafeldruif verbouing gebruik word, ten einde beter gekleurde druiwe te produseer, is ethephon (2-chloro-ethyl-phosphonic acid, 2-CEPA). In die laaste paar jaar was daar baie navorsing gedoen oor die effek wat sonlig het op die antosianien produksie van druiwe, maar navorsing was gefokus op wyndruif cultivars. Huidiglik is daar beperkte tegniese kennis oor die effek wat sonlig op tafeldruiwe het, en hoe dit moontlik die antosianien samestelling en inhoud kan beïnvloed. Daar is ook reeds verskeie studies gedoen en data gepubliseer oor die invloed wat ethephon op die kleur het van druiwe en ander vrugte, maar die invloed wat ethephon op die antosianien samestelling van ‘Crimson Seedless’ het, is nie wel bekend nie. Die doel van hierdie studie was om die effek van sonlig (deur uitsluiting) en bestuurspraktyke (blaarverwydering en ethephon toediening) te bestudeer en hoe dit die antosianien samestelling van ‘Crimson Seedless’ beïnvloed. Vier verskillende behandelings is toegedien in twee ‘Crimson Seedless’ wingerde, die eerste proefperseel in die Paarl en die tweede proefperseel in De Doorns. Die behandelings was: 1. Natuurlik blootgestelde trosse, 2. blootgestelde trosse met ethephon, 3. Trosse met skadubokse omhul, 4. Skaduboks trosse met ethephon. By De Doorns is ‘n addisionele blaarverwydering proef bygebring. ‘n HPLC tegniek was aangepas om die antosianien samestelling en inhoud van ‘Crimson Seedless’ te bepaal, en om die effek van die behandelings te ondersoek. Die HPLC data het getoon dat peonodien-3-glukosied (Pn-gluc) die primêre antosianien in ‘Crimson Seedless’ is met die hoogste inhoud van al die antosianiene. Pn-gluc was betekenisvol beïnvloed deur ethephon toediening, terwyl die ander behandelings geen betekenisvolle effekte daarop gehad het nie. Die effekte wat die ander antosianiene gehad het, het gevarieer volgens die behandelings wat toegedien was. ‘n Algemene observasie was dat ethephon toediening die antosianien konsentrasie in ‘Crimson Seedless’ druiwe skille meer konsekwent vermeerder het as die sonlig blootstelling. Die blaarverwydering het die minste betekenisvolle effek op die antosianien inhoud van ‘Crimson Seedless’ gehad.

Thesis (PhD (Viticulture and Oenlogy))--University of Stellenbosch, 2008. / Includes bibliography and list of tables and figures. / ENGLISH ABSTRACT: Over the last decade research has shown the importance of small antimicrobial peptides in the innate immunity of plants. These peptides do not only play a critical role in the multilayered defense systems of plants, but have proven valuable in the engineering of disease resistant food crops towards the ultimate aim of reducing the dependency on chemical fungicides. As the lists of isolated and characterized peptides grew, it became clear that other biological activities, in addition to the antimicrobial capacity, could be linked to some of these peptides; these alternative activities could have important applications in the field of medicine. This has made the defensin encoding genes prime targets for the agricultural and medical biotechnology sectors. To this end we set out to evaluate South African flora for the presence of plant defensin sequences and to isolate plant defensin genes that might be useful in biotechnology applications. Moreover, by isolating and characterizing these novel peptides, also in an in planta environment and in interaction with fungal pathogens, important knowledge will be gained of the biological role and importance of the peptides in the plant body. The plant host targets were South Africa Brassicaceae species including indigenous species, as well as Vitis vinifera, as the most important fruit crop in the world and since no defensins have been isolated from this economically important crop plant. The Brassicaceae family has been shown to be abundant in defensin peptides and several of the best characterized peptides with potent activity have been isolated from this family. Based on initial activity screens conducted on selected South African Brassicaceae spp. we concluded that these spp. contain promising antifungal peptide activities, warranting further efforts to isolate the genes and encoding peptides and to characterize them further. The preliminary activity screens used a peptide-enrichment isolation strategy that favored the isolation of basic, heat-stable peptides; these properties are characteristic features of plant antimicrobial peptides. These peptide fractions showed strong antifungal activities against the test organisms. A PCR-amplification strategy was subsequently designed and implemented, leading to the isolation of 14 novel defensin peptide encoding genes from four South African Brassicaceae spp., including the indigenous South African species Heliophila coronopifolia. Amino acid sequence analysis of these peptides revealed that they are diverse in amino acid composition and share only 42% homology at amino acid level. This divergence in amino acid composition is important for the identification of new biological activities within closely related plant defensins. Single amino acid changes have been contributed with the divergent biological activities observed in closely related plant defensin peptides. Phylogenetic analysis conducted on the deduced amino acid sequences revealed that all the new defensins share a close relationship to other Brassicaceae members of the plant defensin superfamily and was furthest removed from the defensins isolated from the families Solanaceae and Poaceae. Classification analysis of these peptides showed that they belong to subgroup A3 of the defensin superfamily. A putative defensin sequence was also isolated from V. vinifera cultivar, Pinotage, and termed Vv-AMP1. Genetic characterization showed that only a single gene copy of this peptide is present within the V. vinifera genome, situated on chromosome 1. Genetic characterization of this peptide encoding gene within the Vitis genus showed that this gene has stayed conserved throughout the divergent evolution of the Vitis genus. Expression studies of Vv-AMP1 revealed that this gene is expressed in a tissue specific and developmentally regulated manner, being only expressed in grape berries and only at the onset of vèraison. Induction of Vv-AMP1 in grapevine leaf material could never be achieved through the external application of hormones, osmotic stress, wounding, or pathogen infection by Botrytis cinerea. Deduced amino acid analysis showed that Vv-AMP1 encoded for a 77 amino acid peptide consisting of a 30 amino acid signal peptide and a 47 amino acid mature peptide, with putative antifungal activity. The Vv-AMP1 peptide grouped with the subclass B type defensins, which have been documented to have both antifungal and antibacterial activities. The Vv-AMP1 signal peptide directed the green fluorescent protein (GFP) reporter gene to the apoplastic regions in cells with high levels of accumulation in the vascular tissue and the guard cells of the stomata. Recombinant Vv-AMP1 peptide was successfully purified from a bacterial host and shown to have a size of 5.495 kDa. Recombinant Vv-AMP1 showed strong antifungal activity at low concentrations against a broad spectrum of fungal pathogens, which included Verticillium dahliae (IC50 of 1.8 μg mL-1) and the necrotrophic pathogen Botrytis cinerea (IC50 of 12-13 μg mL-1). Antifungal activity of Vv-AMP1 did not induce morphological changes in fungal hyphae, but its activity was associated with induced membrane permeabilization in treated hyphae. Vv-AMP1 was successfully introduced into Nicotiana tabacum as confirmed by Southern blot analysis and 20 individual lines were generated. Genetic characterization confirmed the integration and expression of the gene in the heterologous tobacco environment. The peptide was under control of its native signal sequence which has been shown to direct its product to the apoplastic regions of cells. The transgenic lines were analyzed to determine the presence and activity of the grapevine defensin peptide. Western blot analyses of partially purified plant extracts detected a signal of the expected size in both the untransformed control and the transgenic lines. Comprehensive analysis of EST databases identified three highly homologous sequences from tobacco that probably caused the background signal in the control. These crude protein extracts were able to inhibit the growth of V. dahliae in vitro when tested in a microtiter plate assay, but the inhibition could not be conclusively linked to the presence of the transgenic peptide, since non-expressing transgenic lines, included as controls, also showed inhibition. Similar results were obtained with infection studies, clearly showing that despite successful integration and expression of the transgene, the peptides was either not functional in the heterologous environment, or perhaps unstable under the particular regulatory conditions. This peptide belongs to a subclass of peptides known for associated activities that might activate tight control by plant hosts if threshold levels are reached. These aspects need further investigation, specifically since it is in stark contrast to previous results obtained with defensins from a different subclass. This study has also yielded significant other related resources that would be instrumental for further possible biotechnology exploitation of some of the novel peptides, but also to provide genetic constructs and plant material that would be invaluable to address fundamentally important questions such as the regulation and mode of action of defensin peptides, specifically in interaction with pathogen hosts. The novel peptides have been transformed to various hosts, including grapevine and these transgenic populations are available to facilitate the next rounds of research into this extremely promising group of antifungal peptides. / AFRIKAANSE OPSOMMING: In die laaste dekade het navorsing die belangrike rol van klein antimikrobiese peptiede in plantweerstandsmeganismes beklemtoon. Hierdie peptiede speel nie alleenlik 'n belangrike rol in die komplekse lae van plantweerstandstelsels nie, maar het ook hulle ekonomiese potensiaal getoon in die manipulering van siekteweerstandbiedendheid in voedselgewasse met die oorkoepelende doel om landbougewasse minder afhanklik van chemiese spuitstowwe te maak. Soos wat die hoeveelheid geïsoleerde en gekarakteriseerde peptiede toeneem, het dit duidelik geword dat ander biologiese aktiwiteite, bykomend tot die antimikrobiese kapasiteit, met sommige van dié peptiede verbind kan word; hierdie alternatiewe aktiwiteite het belangrike toepassing in veral die mediese veld. Dit het die defensin-koderende gene kernteikens vir die landbou- en mediese biotegnologiesektore gemaak. In die studie is daar begin om die Suid-Afrikaanse blommeryk te evalueer vir die teenwoordigheid van plantdefensingene en om dié gene te isoleer wat van ekonomiese belang vir die biotegnologiebedryf kan wees. Deur die in vitro- én in planta karakterisering van die unieke plantdefensinpeptiede word daar gemik daarna om belangrike inligting in te win oor die biologiese rol van die peptiede binne die plantligggaam. Die plantgashere wat geteiken is sluit in die Suid-Afrikaanse Brassicaceae-spesies, insluitende inheemse spesies, asook Vitis vinifera, wat as die belangrikste vrugtegewas ter wêreld beskou word. Die Brassicaceae-familie is welbekend daarvoor dat dit 'n ryk bron van plantdefensinpeptiede is en verskeie van die bes gekarakteriseerde antifungiese defensinpeptiede is van dié familie afkomstig. Aanvanklike aktiwiteitstoetse het getoon dat die Suid-Afrikaanse Brassicaceae-spesies belowende antifungiese aktiwiteit toon, wat die verdere isolering en karakterisering van dié gene en hul peptiedprodukte regverdig. Die aanvanklike aktiwiteitstoetse het 'n selektiewe peptiedverrykingstrategie gevolg wat die isolering van basiese, hittestabiele peptiede bevoordeel het; hierdie eienskappe is baie kenmerkend van plant-antimikrobiese peptiede. Die peptiedfraksies wat met hierdie metode geïsoleer is, het sterk antifungiese aktiwiteit teen die toetsorganismes getoon. Die resultate het gelei tot die ontwikkeling en toepassing van 'n polimerasekettingreaksie-strategie, wat daartoe gelei het dat 14 nuwe defensingene van vier Suid-Afrikaanse Brassicaceae-genera, insluitend die inheemse spesie Heliophila coronopifolia, geïsoleer kon word. Afgeleide aminosuurvolgorde-analises van die nuwe defensinpeptiede het gewys dat hulle slegs 42% homologie het. Hierdie diversiteit in aminosuurvolgorde is belangrik vir die identifisering van nuwe biologiese aktiwitiete binne die groep van verwante peptiede. Navorsing het verder getoon dat enkel-aminosuurverskille bydra tot die diverse spektrum van biologiese aktiwiteite binne 'n groep van verwante defensinpeptiede. Filogenetiese analise van die aminosuurvolgordes het getoon dat al die nuwe defensinpeptiede 'n sterk verwantskap met plantdefensinpeptiede, wat van ander Brassicaceae-spesies geïsoleer is, toon. Daarteenoor het dit die kleinste verwantskap getoon met plantdefensinpeptiede wat van die Solanaceae- en Poaceae-families geïsoleer is. Klassifikasiestudies het bewys dat die nuwe peptiede saam met subgroep A3 van die plantdefensin-superfamilie groepeer. 'n Moontlike plantdefensingeen, genaamd Vv-AMP1, is ook van die V vinifera-kultivar, Pinotage, geïsoleer. Genetiese karakterisering het aangedui dat slegs 'n enkele kopie van die geen in die V. vinifera-genoom teenwoordig en op chromosoom 1 geleë is. Genetiese karakterisering van Vv-AMP1 binne die Vitus-genus het gewys dat die geen binne die genus evolusionêr gekonserveerd is. Uitdrukkingstudies van Vv-AMP1 het verder bewys dat die geen uitgedruk word op 'n weefselspesifieke, ontwikkelingsgekoppelde wyse, naamlik slegs in druiwekorrels en slegs tydens rypwording. Vv-AMP1-uitdrukking kon nooit geïnduseer word in wingerdblare deur die uitwendige toediening van hormone, osmotiese stres, wonding of patogeeninfeksie deur Botrtys cinerea nie. Ontleding van die afgeleide aminosuurvolgorde het gewys dat Vv-AMP1 kodeer vir 'n 77-aminosuurpeptied, wat uit 'n 30-aminosuurseinpeptied en 'n 47-aminosuur-aktiewe peptied met voorspelde antifungiese aktiwiteit bestaan. Die Vv-AMP1-peptied is gegroepeer met subgroep B van die plantdefensin-superfamilie, 'n subgroep wat vir beide antifungiese en antibakteriese aktiwiteit gedokumenteer is. Die Vv-AMP1-seinpeptied het die groen fluoressensie-indikatorproteïen (GFP) na die apoplastiese areas van die plantselle gelei, met hoë vlakke van lokalisering in die vaatbundelweefsel en sluitselle van die huidmondjies. Die rekombinante Vv-AMP1-peptied is suksesvol geproduseer en uit 'n bakteriese produksieras gesuiwer, en het 'n molekulêre massa van 5.495 kDa gehad. Die gesuiwerde peptide het by lae konsentrasies 'n sterk aktiwiteit getoon teen 'n breë spektrum van fungiese patogene, wat Verticllium dahliae (IC50 van 1.8 μg mL-1) en die nekrotrofiese patogeen, B. cinerea (IC50 van 12-13 μg mL-1), ingesluit het. Vv-AMP1-aktiwiteit het geen ooglopende morfologiese veranderinge in die fungi-hifes veroorsaak nie, maar hulle aktiwiteit is verbind met 'n verhoogde membraandeurdringbaarheid in behandelde fungi-hifes. Suksesvolle intergrasie van Vv-AMP1 in die Nicotiana tabacum-genoom is deur Southern-kladontledings bevestig en 20 individuele transgeniese lyne is ontwikkel. Genetiese karakterisering van die transgeniese lyne het gewys dat Vv-AMP1 suksesvol geïntegreer is en ook in die transgeniese tabakomgewing uitgedruk word. Die peptied is uitgedruk onder beheer van sy eie seinpeptied, wat die aktiewe produk na die apoplastiese areas van die plantselle teiken. Die transgeniese tabaklyne is ook ontleed om te bepaal of die wingerdpeptied suksesvol geproduseer word en sy aktiwiteit in die transgeniese omgewing behou. Western-kladanalise van semi-gesuiwerde plantproteïenekstrakte het 'n positiewe sein gelewer in beide die kontroleplante en die transgeniese plantlyne. Bestudering van tabakgeenuitdrukkings-databasisse het drie nukleotiedvolgordes opgelewer wat homologie met Vv-AMP1 toon en moontlik verantwoordelik kan wees vir die positiewe sein in die ongetransformeerde kontroleplante. Kru proteïenekstrakte van die transgeniese tabaklyne het in vitro-aktiwiteit teen V. dahliae getoon. Geen oortuigende ooreenkoms kon egter gevind word tussen V. dahliae-inhibisie en die teenwoordigheid van die transgeniese Vv-AMP1-peptied nie, aangesien kontroleplante wat Southern-klad-positief is, maar nie geenuitdrukking toon nie, ook inhibisie van V. dahliae veroorsaak het. Soortgelyke resultate is met infeksiestudies verkry. Alle resultate dui daarop dat, al is daar suksesvolle integrasie en uitdrukking van die geen in tabak verkry, dat die Vv-AMP1 peptied óf onaktief óf onstabiel in die transgeniese tabakomgewing is. Die peptied behoort aan 'n subgroep peptiede met aktiwiteite wat, sodra sekere vlakke van peptied oorskry word, die moontlik streng kontrole op proteïenvlak in die gasheerplant kan uitlok. Sekere aspekte van die studie sal verder bestudeer moet word, aangesien die data teenstrydig is met data wat verkry is met soortgelyke plantdefensinpeptiede wat aan 'n ander subgroep behoort. Die studie het baie hulpbronne gegenereer wat vir die biotegnologiesektor belangrik kan wees, veral op ekonomiese gebied. Verder is die geenkonstrukte en plantlyne wat ontwikkel is waardevol om fundamentele vrae rondom die regulering en meganisme van aksie van defensinpeptiede, spesifiek plantpatogeeninteraksie, te beantwoord. Die nuwe plantdefensingene is na verskeie gasheerplante, insluitende wingerd, getransformeer waar die transgeniese lyne die volgende rondte van navorsing oor die bestudering oor die belangrike groep van antifungiese peptiede, sal aanvul.