Thesis (MScAgric)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The distinctive varietal flavour of wines is a combination of absolute and relative concentrations of chemical compounds. Volatile compounds are responsible for the odour of wine and non-volatiles cause the sensation of flavour. Accompanying these senses, a third, tactile, sense of ‘mouth-feel’ is recognizable. This forms the complete organoleptic quality of wine. Several hundred different compounds are simultaneously responsible for the odour release in wine, and since there is no real character impact compound, the aroma of wine can be described as a delicate balance of all these compounds. One of the most important groups of volatiles is the monoterpenes, which play a role in both aroma and flavour. This is especially significant for the Muscat varieties, but these flavour compounds are also present in other non-muscat grape varieties, where they supplement the varietal aroma. Monoterpenes occur in wine as free, volatile and odorous molecules, as well as flavourless non-volatile glycosidic complexes. The latter slowly releases monoterpenes by acidic hydrolysis, but the impact on varietal aroma is considered insufficient for wines that are consumed young. It is therefore important to supplement the release mechanism, in order to enhance the varietal aroma of the wine. The enzymatic hydrolysis mechanism functions in two successive steps: firstly, depending on the precursor, the glycosidic linkage is cleaved by α-L-arabinofuranosidase, α-L-rhamnosidase, β-D-xylosidase or β-D-apiosidase. The second step involves the liberation of the monoterpene alcohol by a β-glucosidase. This enzymatic hydrolysis does not influence the intrinsic aromatic characteristics of the wine, as opposed to acid hydrolysis. Pectolytic enzymes play an important role in cell elongation, softening of tissue and decomposition of plant material. These enzymes are used to improve juice yields, release colour and flavour compounds from grape skins, as well as improve clarification and filterability. Pectolytic enzymes work synergistically to break down pectins in wine. Protopectinase produce water-soluble and highly polymerised pectin substances from protopectin, it acts on non-methylated galacturonic acid units. Pectin methylesterase split methyl ester groups from the polygalacturonic chain. Polygalacturonase break down the glycosidic links between galacturonic acid units. Pectin and pectate lyases have a β-eliminative attack on the chain and it results in the formation of a double bond between C4 and C5 in the terminal residues. From the above it can be seen that enzymes play a pivotal role in the winemaking process. Unfortunately, in winemaking a lot of factors can influence the effects of enzymes. One possible factor in the wine medium is the presence of acidprotease, from yeast and/or fungal origin. This type of enzyme utilizes other enzymes as substrates and renders them useless. Pure enzyme preparations were used to study the interactions of a yeast acid-protease and a report activity (β-glucosidase) in vitro. A bottled wine and a buffer were used as in vitro conditions. Enzyme assays were performed to determine the relative activity over a number of days. The results indicated that even though both enzymes showed activity in both the media, the yeast protease did not have any significantly affect on the report activity. Subsequently wine was made from Sauvignon blanc grapes, with varying enzyme preparation additions. Enzyme assays were performed during the fermentation; and chemical, as well as sensory analysis were done on the stabilized wine. The results confirmed that the yeast protease did not have any significant affect on the report activity in these conditions. The protease’s inability to affect the report activity seems unlikely due to the fact that it is active at a low pH range and has been suggested as the only protease to survive the fermentation process. It seems possible that a winerelated factor, possibly ethanol, is responsible. Thus it seems that yeast protease does not threaten the use of commercial enzymes in the winemaking process in any significant way. Future work would entail more detailed enzyme studies of interactions between protease, both from yeast and fungal origin, and other report activities in specified conditions. The degradation capability could be directed towards unwanted enzyme activities that cause oxidation and browning of the must. The characterization of interactions between protease and β-glucosidase activities may hold key to producing wines with enhanced aroma and colour potential, as well as the elimination of unwanted enzyme activities. / AFRIKAANSE OPSOMMING: Die herkenbare kultivar karakter van wyn is ‘n kombinasie van absolute en relatiewe konsentrasies van verskeie chemiese komponente. Vlugtige komponente is verantwoordelik vir die geur, of aroma, van wyn en die nie-vlugtige komponente veroorsaak die sensasie van smaak. ‘n Derde, fisiese sensasie, die ‘mondgevoel’, is ook herkenbaar. Dit vorm die omvattende organoleptiese kwaliteit van die wyn. ‘n Paar honderd verskillende komponente is gelyktydig verantwoordelik vir die aroma vrystelling in wyn en omdat daar geen werklike karakter ‘impak’ komponent is nie, kan die aroma van wyn beskryf word as ‘n delikate balans van al die betrokke komponente. Een van die mees belangrike groepe vlugtige komponente is die monoterpene wat ‘n rol speel in beide aroma en smaak. Dit is veral belangrik by Muskaat kultivars, maar hierdie aroma komponente is ook teenwoordig in niemuskaat druif kultivars, waar hulle bydra tot die kultivar karakter en aroma. Monoterpene kom in wyn voor as vry, vlugtige en aromatiese molekules en in geurlose, nie-vlugtige glikosidies-gebonde komplekse. Die gebonde vorm word stadig vrygestel deur ‘n suurhidrolise, maar dit word as onvoldoende beskou vir wyne wat vroeg gedrink word. Dit is dus belangrik dat die vrystelling van geurstowwe verhoog word om die kultivar karakter van die wyn te versterk. Die ensiematiese hidrolise proses behels twee opeenvolgende stappe: eerstens, afhangende van die aard van die voorloper, word die glikosidiese verbinding deur α-L-arabinofuranosidase, α-Lramnosidase, β-D-xilosidase, of β-D-apiosidase gebreek. In die tweede stap word die monoterpeen-alkohol deur β-glukosidase vrygestel. Hierdie ensiematiese afbraak proses verander nie die intrinsieke aromatiese kenmerke van die wyn, soos met suurhidrolise die geval is nie. Pektolitiese ensieme speel ‘n fundamentele rol in selverlenging, sagwording en afbraak van plant materiaal. Hierdie ensieme word gebruik om sap opbrengs te verhoog, aroma en smaak komponente vry te stel uit die doppe, asook om sapverheldering en filtrasie te verbeter. Die pektolitiese ensieme werk op ‘n sinergistiese wyse om pektien in wyn af te breek. Protopektinase produseer wateroplosbare en hoogs gepolimeriseerde pektien uit protopektien, slegs uit niegemetileerde galakturoonsuur eenhede. Pektien metielesterase verwyder metielester groepe van die poligalakturoonsuurketting. Die glikosidiese bindings tussen galakturoonsuur eenhede word deur poligalakturonase afgebreek. Pektien- en pektaat-liase het ‘n β-eliminasie aanslag op die ketting en as gevolg daarvan word dubbelbindings tussen C4 en C5 in die terminale residue gevorm. Vanuit bogenoemde is dit dus duidelik dat ensieme ‘n kardinale rol speel in die wynbereidingsproses. Ongelukkig is daar ‘n verskeidenhied van faktore wat die werking van ensieme in die wynbereidingsproses kan beïnvloed. Een moontlike faktor is die teenwoordigheid van ‘n suur-protease, van fungisidiese en/of gis oorsprong, in die wynmedium, omdat dit ander ensieme as substraat kan benut en degradeer. Suiwer ensiem preparate is gebruik om die ensiem interaksie tussen ‘n gis suur-protease en ‘n verslag aktiwiteit (β-glukosidase) in vitro te ondersoek. ‘n Gebotteleerde wyn en ‘n buffer is gebruik om die in vitro kondisies na te boots. Relatiewe ensiem aktiwiteit is ontleed oor ‘n aantal dae. Beide die ensieme het aktiwiteit getoon in die media, maar gis protease het geen statisties beduidende invloed gehad op die aktiwiteit van die verslag ensiem nie. Daaropvolgend is wyn berei van Sauvignon blanc druiwe, met verskillende ensiempreparaat toevoegings. Die ensiemaktiwiteit is deurlopend tydens fermentasie gemeet. Na afloop van stabilisasie is chemiese, sowel as sensoriese ontledings op die wyn gedoen. Die resultate het bevestig dat gis protease, onder hierdie kondisies, geen beduidende invloed op die verslag aktiwiteit gehad het nie. Die protease se onvermoë om die verslag aktiwiteit beduidend te beinvloed blyk onwaarskynlik aangesien die suurprotease aktief is by lae pH vlakke en dit as die enigste protease voorgestel is wat die fermentasie proses kan oorleef. Dit blyk asof ‘n wyn-verwante faktor, moontlik etanol, hiervoor verantwoordelik kan wees. Dus hou protease geen gevaar in vir die gebruik van kommersiële ensieme in wynbereiding nie. Navorsing kan in die toekoms fokus op meer gedetailleerde ensiem interaksie studies tussen protease en ander ensiem aktiwiteite, in gespesifiseerde kondisies. Die degradasie kapasiteit kan moontlik aangewend word om ongewenste ensiem aktiwiteite, wat byvoorbeeld oksidasie en verbruining veroorsaak, te verminder. Die karakterisering van die interaksies tussen protease en β-glukosidase kan dus die sleutel wees tot die produksie van wyne met verhoogde aroma potensiaal, asook die eliminasie van ongewenste ensiematiese aktiwiteite.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: In the field of agriculture, plant pathogens are a major concern because of the severe damage these organisms cause to crops yearly. Fundamental studies regarding plant pathogens and their modes of action made it possible for researchers in the field of molecular biology to investigate pathogens further on a molecular level. Botrytis cinerea, has been used to great effect as a model system to investigate various aspects regarding pathogenesis, also on a molecular level. Molecular research done on B. cinerea over the last few years has shown that the endopolygalacturonases (EPGs) of this fungus are key role players in pathogenesis. This hydrolytic enzyme family of six members, encoded by the Bcpg1-6 genes, are important in breaking down the complex cell wall polymers of host plants, enabling the fungus to penetrate its host sufficiently. It has been shown that both BcPG1 and 2 are crucial for virulence of B. cinerea. A leucine-rich repeat inhibitor protein situated in the cell wall of various plant species, the polygalacturonase-inhibiting protein (PGIP), has been proven to interact with and inhibit EPGs, and thus the necrotic actions of B. cinerea. From literature it was clear that specific data regarding individual interactions of fungal EPGs with PGIPs are lacking currently. Furthermore, most experiments regarding the effects of EPG as well as interaction and inhibition studies of EPGs and PGIPs, rely on in vitro methods, without the possibility to contextualize the results on an in vivo or in planta level. The scope of this study was to specifically address the issues of individual EPG:PGIP interactions and the use of possible in vivo methodology by using EPGs from a highly virulent South African strain of B. cinerea and the grapevine VvPGIP1 that has been previously isolated in our laboratory. This PGIP, originally isolated from Vitis vinifera cv Pinotage, has been shown to inhibit a crude EPG extract from this strain with great efficiency. The approach taken relied on heterologous over-expression of the individual Bcpg genes and the isolation of pure and active enzymes to evaluate the inhibition of the EPGs with VvPGIP1. The genes were all successfully over-expressed in Saccharomyces cerevisiae with a strong and inducible promoter, but active enzyme preparations have been obtained only for the encoding Bcpg2 gene, as measured with an agarose diffusion assay. The in vitro PGIP inhibition assay is also based on the agarose diffusion assay and relies on activity of the EPGs to visualize the inhibiting effect of the PGIP being tested. The active EPG2, however, was not inhibited by VvPGIP1 when tested with this assay. The EPG encoding genes from B. cinerea were transiently over-expressed also in Nicotiana benthamiana by using the Agrobacterium-infiltration technique. Transgene expression was confirmed by Northern blot analysis and EPG-related symptoms were observed five to eight days post-infiltration. Differential symptoms appeared with the various EPGs, providing some evidence that the symptoms were not random events due to the infiltration or a hypersensitive response. Moreover, the symptoms observed for EPG2 was similar to those that were reported recently by another group on the same host. In spite of the expression data and the clear symptoms that developed, active preparations, as measured with the agarose diffusion plate asay, could only be obtained for EPG2 again. In our search for a possible in vivo method to detect and quantify EPG activity and inhibition by PGIPs, we tested and evaluated a technique based on chlorophyll fluorescence to detect the effect of EPGs on the rate of photosynthesis. Our results showed that the over-expression of these genes reduced the rate of electrons flowing through photosystem II, indicating metabolic stress occurring in the plant. We used the same technique to evaluate possible interaction between VvPGIP1 respectively with BcPG1 and 2 and found that the co-expressing of the Vvpgip1 gene caused protection of the infiltrated tissue, indicating inhibition of EPG1 and 2 by VvPGIP1. For EPG2, the observed interaction and possible inhibition by VvPGIP1 is the first report to our knowledge of an interaction between this specific EPG2 and a PGIP. Moreover, to further elucidate the in planta interaction between VvPGIP1 and the EPGs from the South African B. cinerea strain, we tested for possible interactions by making use of a plant two-hybrid fusion assay, but the results are inconclusive at this stage. Previous studies in our laboratory have shown that several natural mutations exist between PGIP encoding genes from different V. vinifera cultivars. Based on this finding and the fact that these natural mutations could result in changes with regard to EPG inhibition and ultimately disease susceptibility, we isolated an additional 37 PGIP encoding genes from various grapevine genotypes, some of which are known for their resistance to pathogens. Combined, these results make a valuable contribution to understand plant pathogen interactions, specifically in this case by modeling the interactions of pathogen and plant derived proteins. The possibility to use in vivo methods such as chlorophyll fluorescence to follow these interactions on an in planta level, provides exciting possibilities to strenghten and contextualize in vitro results. / AFRIKAANSE OPSOMMING: Plantpatogene organismes veroorsaak jaarliks erge skade aan landbougewasse en word dus as ’n ernstige probleem in die landbousektor beskou. Diepgaande studies wat handel oor plantpatogene en hul metodes van infeksie het dit vir molekulêre bioloë moontlik gemaak om patogene nou ook op molekulêre vlak verder te bestudeer. Botrytis cinerea is baie effektief as modelsisteem gebruik om verskeie aspekte van patogenese verder te bestudeer, ook op ‘n molekulêre vlak. Molekulêre navorsing op B. cinerea, het getoon dat die endopoligalakturonases (EPGs) van dié swam kernrolbelangrik in patogenese is. Hierdie sesledige hidrolitiese ensiemfamilie word gekodeer deur die Bcpg1-6 gene en is belangrik vir die afbraak van die komplekse selwandpolimere van plantgashere, om suksesvolle gasheerpenetrasie te veroorsaak. Daar is aangetoon dat beide BcPG1 en 2 essensieël vir virulensie van die patogeen is. ’n Leusienryke-herhalings inhibitorproteïen wat in die selwand van verskeie plantspesies voorkom, die poligalakturonase-inhiberende proteïen (PGIP), het interaksie met en inhibeer EPGs en gevolglik ook die nekrotiserende aksies van B. cinerea. Uit die literatuur is dit duidelik dat spesifieke inligting aangaande individuele interaksies van fungiese EPGs met PGIPs tans nog ontbreek. Verder word daar op in vitro metodologie staatgemaak wannneer die effekte van EPGs asook die interaksie en inhibisie met PGIPs bestudeer word, sonder om die konteks van die in vivo- of in planta-omgewing in ag te neem. Die fokus van hierdie studie was om aspekte van individuele EPG:PGIP interaksies, asook die moontlike gebruik van in vivo metodologie te bestudeer deur EPGs, afkomstig van ’n hoogs virulente Suid-Afrikaanse ras van B. cinerea en die wingerd VvPGIP1, wat vroeër in ons laboratorium geïsoleer is, te gebrruik. Hierdie PGIP wat uit Vitis vinifera cv Pinotage geïsoleer is, inhibeer ’n kru EPG-ekstrak van bogenoemde ras baie effektief. Die benadering wat gevolg is het op die ooruitdrukking van die individuele Bcpg-gene in heteroloë sisteme staatgemaak en die gevolglike isolering van suiwer en aktiewe ensieme om EPG-inhibisie deur VvPGIP1 te beoordeel. Al die gene is suksesvol in Saccharomyces cerevisiae ooruitgedruk onder ’n sterk induseerbare promotor, maar volgens ’n agarose-diffundeerbare toets kon aktiewe ensiempreparate slegs vir die enkoderende Bcpg2 verkry word. Die in vitro PGIP-inhibisie toets is ook op die gemelde toets gebasseer en vereis EPG-aktiwiteit om die inhiberende effek van die PGIP, te visualiseer. Die aktiewe EPG2 is egter nie deur VvPGIP1 geïnhibeer met die aanleg van hierdie toets nie. Die EPG-enkoderende gene van B. cinerea is ook tydelik in Nicotiana benthamiana ooruitgedruk deur gebruik te maak van ’n Agrobacterium-infiltrasietegniek. Transgeenuitdrukking kon met die Noordelike kladtegniek bevestig word en EPG-verwante simptome is vyf tot agt dae na infiltrasie waargeneem. Verskillende simptome vir die verskillende EPGs is waargeneem, wat aanduidend is dat die simptome nie lukrake gevolge van die infiltrasies, of ’n hipersensitiewe respons is nie. Verder kon die simptome wat EPG2 vertoon het, gekorreleer word met dié wat onlangs deur ’n ander groep op dieselfde gasheer waargeneem is. Ten spyte van die ekspressiedata en die waargenome simptome, kon aktiewe ensiempreparate op die agarose-diffundeerbare toets, weereens slegs vir EPG2 waargeneem word. ’n Metode wat gebasseer is op chlorofilfluoressensie is getoets en geëvalueer as ’n moontlike in vivo metode om EPG aktiwiteit en inhibisie deur PGIPs waar te neem en te kwantifiseer. Die resultate het bevestig dat die ooruitdrukking van hierdie gene die elektronvloeitempo deur fotosisteem II verminder het wat ’n aanduiding is dat metaboliese stres in die plant heers. Dieselfde tegniek is gebruik om die moontlike interaksies tussen BcPG1 en 2 en VvPGIP1 te bestudeer en het aangetoon dat die mede-uitdrukking van die Vvpgip1-geen aanleiding gee tot ’n beskermende effek van die geinfiltreerde weefsel, wat aanduidend is van inhibisie van EPG1 en 2 deur VvPGIP1. In die geval van EPG2 is hierdie interaksie en moontlike inhibisie met ’n PGIP die eerste waarneming in die verband. In ’n verdere poging om die in planta-interaksie tussen VvPGIP1 en die EPGs van die Suid-Afrikaanse B. cinerea ras uit te klaar, is ’n plantgebasseerde twee-hibriede toets aangelê, maar geen klinkklare resultate kon verkry word nie. Vorige werk het bevestig dat verskeie natuurlike mutasies in PGIP-enkoderende gene, afkomstig van verskillende V. vinifera kultivars, voorkom. Hierdie resultaat en die feit dat hierdie mutasies verskille in EPG inhibisie en uiteindelik vatbaarheid vir siektes kan beïnvloed, het aanleiding gegee tot die isolering van ’n verdere 37 PGIP-enkoderende gene uit ‘n verskeidenheid druifplantgenotipes, sommige waarvan juis bekend vir hul weerstand teen patogene is. Die gekombineerde resultate wat in dié studie verkry is, maak ’n waardevolle bydrae tot die verstaan van plant-patogeeninteraksies, spesifiek met die modelering van interaksies van patogeen- en plantgebasseerde proteïene. Die moontlikheid om in vivo-metodes soos chlorofilfluoressensie te gebruik in in planta-analises, is besonder bemoedigend om in vitro-resultate te versterk en ook in konteks te plaas.

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.