Dissertation (PhD(Agric)) -- Stellenbosch University, 2005. / ENGLISH ABSTRACT: Due to increased consumer demand for products labelled by origin, and the requirement that these labels are a guarantee of both quality and product character, there is an increasing global focus on delimitation of denominations of origin. The integrity of denominations of origin and their defensibility can be ensured through the use of terroirs as a basis for delimitation. The aims of this study were to establish the dominant environmental criteria that affect the viticultural behaviour and wine character of two important cultivars (Cabernet Sauvignon and Sauvignon blanc) in the Stellenbosch Wine of Origin District, to use an appropriate methodology to identify viticultural terroirs in this district based on these criteria and with the use of a geographic information system, and finally to use these viticultural terroirs to identify denominations of origin within the same area. A terroir can be defined as a grouping of homogenous environmental units, or natural terroir units, based on the typicality of the products obtained. Identification and characterisation of terroirs depends on knowledge of environmental parameters, the functioning of the grapevine and the characteristics of the final product. Field studies, resulting in point data, are necessary to investigate the functioning of the grapevine but in order for this information to be of use within zoning studies it must be placed in a spatial context. As a first phase in data acquisition, the Stellenbosch Wine of Origin District was characterised and natural terroir units were identified using existing digital data and a geographic information system. A natural terroir unit (NTU) can be defined as a unit of land that is characterised by relatively homogenous topography, climate, geological substrate and soil. A total of 1389 NTUs were identified in the Stellenbosch Wine of Origin District (84 537 ha). The identified NTUs were homogenous with respect to terrain morphological unit, altitude, aspect and soil type. Each of the identified units was further described with respect to the extent of the expected sea breeze effect and, for certain of the soil types, the associated parent material. As a second phase of data acquisition, a network of plots of Sauvignon blanc and Cabernet Sauvignon were delimited in commercial vineyards in proximity to weather stations and their viticultural and oenological response monitored for a period of seven years. Regression tree analyses were performed on the complete data set and the relative importance of the environmental and management related variables determined for each dependent variable. Excepting for scion clone, which had a high relative importance for bunch mass of Sauvignon blanc and yield to pruning mass index of Cabernet Sauvignon, no other non-environmental variable included in the analyses appeared to have a strong effect on grapevine performance and wine character. The performance of Cabernet Sauvignon was affected by the potassium content of the subsoil and the climate of the season. The performance of Sauvignon blanc appeared to be related to soil texture, wind exposure and temperature, both during the green berry growth stage and the month prior to ripening. From the results presented, it appears that environmental parameters have an overriding effect on the performance of both Cabernet Sauvignon and Sauvignon blanc, but that these two cultivars react differently to environmental stimuli. A knowledge-driven model used the rules generated in the regression tree analyses to directly classify natural terroir units with respect to expected response of Cabernet Sauvignon and Sauvignon blanc in the Stellenbosch Wine of Origin District. The natural terroir units were thus grouped into terroir units that were homogenous with respect to predicted viticultural and oenological response for each cultivar. The use of representative sites to determine the response of the grapevine to its environment is time consuming and costly and limits terroir studies to research related investigations. Vineyard managers were therefore surveyed with respect to the functioning of established Sauvignon blanc vineyards in the Stellenbosch Wine of Origin District in an attempt to obtain the necessary data. Comparison of the data generated with these questionnaires to measured data in commercial vineyards suggested that the vineyard managers were able to characterise the performance of vineyards with respect to vigour, signs of drought stress and yield. Each vineyard was mapped and the responses were linked to modelled environmental variables. Classification and regression trees were used to construct decision trees, which could be applied to environmental data in a geographic information system to determine viticultural terroirs for production of Sauvignon blanc. These terroirs, although fewer, were comparable to those generated using field data. Data gathered during terroir studies, and the identified viticultural terroirs for Cabernet Sauvignon and Sauvignon blanc, were used to revisit the boundaries of the Stellenbosch Wine of Origin District and the Simonsberg-Stellenbosch ward. Modifications were proposed based on expected wine characteristics. Boundaries for two new wards in the Helderberg basin were proposed. It was also possible to identify vineyards within a ward for the production of terroir specific wines. / AFRIKAANSE OPSOMMING: Toenemende verbruikersaanvraag vereis produkte waarvan die etikette nie net die oorsprong aandui nie, maar ook kan dien as ‘n waarborg vir die produk se kwaliteit en kenmerkendheid. Hierdie tendens verklaar die toenemende wereldwye fokus op afgebakende areas van oorsprong. Die integriteit van die afgebakende areas van oorsprong sowel as hul verdedigbaarheid kan gewaarborg word deur terroirs as basis vir afbakening te gebruik. Die doelstellings van die studie was om die oorheersende omgewingseienskappe wat wingerdprestasie en wynkarakter van twee belangrike cultivars, naamlik Cabernet Sauvignon en Sauvignon blanc, in die Stellenbosch Wyn van Oorsprongsdistrik bemvloed, te bepaal; tweedens om ‘n toepaslike metodologie te gebruik om terroirs in die die distrik te bepaal wat gebaseer is op die geidentifiseerde omgewingseienskappe met die gebruik van ‘n geografiese inligtingstelsel; en, ten slotte, om hierdie terroirs vir wingerbou te gebruik om afgebakende areas van oorsprong in dieselfde omgewing te identifiseer. ‘n Terroir kan gedefinieer word as ‘n samestelling van homogene omgewingseenhede, of natuurlike terroir-eenhede (NTE), wat gebaseer word op die kenmerkende eienskappe van die produkte wat daaruit verkry word. Identifisering en karakterisering van terroirs sal afhang van kennis van die omgewingsparameters, die funksionering van die wingerdstok en die eienskappe van die finale produk. Veldstudies waaruit puntdata verkry word, is noodsaaklik om die funksionering van die wingerdstok te ondersoek. Dit is egter noodsaaklik om eers hierdie inligting in ruimtelike konteks te plaas alvorens die inligting vir soneringstudies gebruik kan word. As 'n eerste fase van datagenerering, was die Stellenbosch Wyn van Oorsprongsdistrik gekarakteriseer en NTE’s geidentifiseer deur gebruik te maak van bestaande digitale data en ‘n geografiese inligtingstelsel. ‘n NTE kan gedefinieer word as ‘n landseenheid wat gekarakteriseer word deur ‘n relatiewe homogene topografie, klimaat, geologiese substraat en grondtipe. ‘n Totaal van 1389 NTE’s is geidentifiseer in die Stellenbosch Wyn van Oorsprongsdistrik (84 537 ha). Die geidentifiseerde NTE’s was homogeen met betrekking tot die terrein morfologiese eenheid, hoogte bo seespieSI, hellingsaspek en grondtipe. Elk van die geidentifiseerde eenhede was verder beskryf volgens die omvang van die seewindinvloed en, vir toepaslike grond tipes, die geassosieerde moedermateriaal. As ‘n tweede fase van datagenerering is ‘n netwerk van persele van Cabernet Sauvignon en Sauvignon blanc afgebaken binne bestaande kommersiele wingerde in die nabyheid van weerstasies. Hul wingerd- en wynkundige respons is vir ‘n periode van sewe jaar gemonitor. Regressieboomanalises is gebruik om die volledige stel data te analiseer en om die relatiewe belang van omgewings- en bestuurspraktykverbonde veranderlikes te bepaal. Die bostokkloon (wat 'n hoe relatiewe belang vir die trosmassa van Sauvignon blanc en die oes- tot snoeimassa verhouding van Cabernet Sauvignon het) is die enigste van die nie-omgewingsparameter wat ‘n sterk invloed op wingerdprestasie of wynkarakter blyk te he. Die prestasie van Cabernet Sauvignon is beTnvloed deur die kaliuminhoud van die ondergrond sowel as die seisoensklimaat. By Sauvignon blanc het dit voorgekom of die prestasie verband hou met grondtekstuur, windblootstelling en temperatuur tydens die groen fase van korrelgroei sowel as die maand voor rypwording. Alhoewel dit blyk uit die resultate dat omgewingsparameters 'n oorheersende invloed op die prestasie van beide Cabernet Sauvignon en Sauvignon blanc uitoefen, reageer die twee cultivars verskillend op omgewingsprikkels. ‘n Kennisgedrewe model waarvan die riglyne uit resultate van die regressieboomanalise saamgestel is, word gebruik om NTE direk te klassifiseer ten opsigte van die verwagte respons van Cabernet Sauvignon en Sauvignon blanc in die Stellenbosch Wyn van Oorsprongsdistrik. Die NTE is dus gegroepeer om terroir eenhede te vorm wat homogeen was ten opsigte van die verwagte wingerd- en wynkundige respons vir elke cultivar. Die gebruik van verwysingspersele om die respons van die wingerdstok teenoor sy onmiddelike omgewing te bepaal, is tydrowend en duur en beperk sodoende terroir studies tot navorsing. Gevolglik is ‘n opname onder wingerdbestuurders gemaak om inligting oor die prestasie van Sauvignon blanc in die Stellenbosch Wyn van Oorsprongsdistrik in te win en sodoende die nodige data te verkry. Na vergelyking van die ingewinde data, wat uit die opname verkry is, met gemete data vanaf kommersiele wingerde, kon afgelei word dat wingerdbestuurders by magte is om die prestasie van wingerde ten opsigte van groeikrag, tekens van droogtestres en opbrengs te karakteriseer. Elke wingerd is gekarteer en die respons is gekoppel aan die gemodeleerde omgewingsparamters. Klassifikasie en regressiebome is gebruik om besluitnemingsmodelle saam te stel wat toegepas kon word op omgewingsdata in ‘n geografiese inligtingstelsel om terroirs vir die produksie van Sauvignon blanc te bepaal. Hierdie terroirs, alhoewel minder, was vergelykbaar met die wat gegenereer was met behulp van veldstudies. Data verkry met terroirstudies, sowel as gei'dentifiseerde terroir-eenhede, was gebruik om die grense van die Stellenbosch Wyn van Oorsrongsdistrik en die Simonsberg-Stellenbosch wyk te herbepaal. Voorgestelde modifiserings was gebaseer op verwagte wyneienskappe. Grense vir twee nuwe wyke in die Helderbergkom is voorgestel. Dit was ook moontlik om wingerde binne ‘n wyk te identifiseer vir die produksie van terroir-spesifieke wyne.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: During winemaking, a number of biochemical changes occur as a result of the metabolic activity of wine lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). The latter process, which occurs mostly after alcoholic fermentation by wine yeasts, involves the conversion of L-malate to L-lactate and CO2, thus resulting to wine acidity reduction, microbiological stabilization and alterations of wine organoleptic quality. Although Oenococcus oeni is predominantly the most preferred species suitable for carrying out MLF in wine owing to its desirable oenological properties, Lactobacillus plantarum has also been considered as a potential candidate for MLF induction. Other species in the genera of Lactobacillus and Pediococcus are often associated with wine spoilage. These microorganisms induce wine spoilage by producing off-flavours derived from their metabolic activity. It is therefore of paramount importance to understand the mechanism by which wine microbiota cause spoilage. The purpose of this study was to investigate the presence of genes encoding enzymes of oenological relevance in wine-associated LAB strains. In order to achieve this, different sets of specific primers were designed and employed for a wide-scale genetic screening of wine LAB isolates for the presence of genes encoding enzymes involved in various metabolic pathways, such as citrate metabolism, amino acid metabolism, hydrolysis of glycosides, degradation of phenolic acids as well as proteolysis and peptidolysis. PCR detection results showed that the majority of the tested strains possessed most of the genes tested for. It was also noted that, among the O. oeni strains tested for the presence of the pad gene encoding a phenolic acid decarboxylase, only two strains possessed this gene. None of the O. oeni strains has previously been shown to possess the pad gene, and this study was the first to report on the presence of this gene in O. oeni strains. In an attempt to genetically characterize this putative gene, DNA fragments from the two positive O. oeni strains were sequenced. The newly determined sequences were compared to other closely related species. Surprisingly, no match was found when these sequences were compared to the published genomes of three O. oeni strains (PSU-1, ATCC BAA-1163 and AWRI B429). This reinforced a speculation that the pad gene in these two strains might have been acquired via the horizontal gene transfer. In addition, it remains to be further determined if the presence of this gene translates to volatile phenol production in wine. In this study, a novel strain isolated from South African grape and wine samples was also identified and characterized. The identification of this strain was performed through the 16S rDNA sequence analysis, which indicated that this strain belongs to Lactobacillus florum (99.9% sequence identity). A novel PCR assay using a species-specific primer for the rapid detection and identification of Lb. florum strains was also established. For further characterization, this strain was also investigated for the presence of genes encoding enzymes of oenological relevance. PCR detection results indicated that the Lb. florum strain also possess some of the genes tested for. In addition to genetic screening of wine LAB isolates for the presence of different genes, this study was also aimed at evaluating the regulation of the mleA gene encoding malate decarboxylase in three oenological strains of O. oeni. The regulation of this gene was tested in a synthetic wine medium under various conditions of pH and ethanol. From the expression analysis, it was observed that the mleA gene expression was negatively affected by high ethanol content in the medium. On the other hand, low pH of the medium seemed to favour the expression of this gene as the mleA gene expression was more pronounced at pH 3.2 than at pH 3.8. The findings from this study have shed more light on the distribution of a wide array of enzyme-encoding genes in LAB strains associated with winemaking. However, it remains unknown if the enzymes encoded by these genes are functional under oenological conditions, given that wine is such a hostile environment encompassing a multitude of unfavourable conditions for the enzymes to work on. Evaluating the expression of these genes will also help give more insights on the regulation of the genes under winemaking conditions. / AFRIKAANSE OPSOMMING: Gedurende wynmaak, sal 'n aantal biochemiese veranderinge plaasvind as gevolg van die metaboliese aktiwiteit van wyn melksuurbakterieë (MSB) wat betrokke is by appelmelksuurgisting (AMG). Die laasgenoemde proses, wat meestal na alkoholiese fermentasie deur wyngiste plaasvind, behels die omskepping van L-malaat na L-laktaat en CO2, om sodoende die wyn se suur te verminder, mikrobiologiese stabiliteit en verandering van wyn organoleptiese kwaliteit. Alhoewel Oenococcus oeni hoofsaaklik die mees gewenste spesies is wat geskik is vir die uitvoering van AMG in wyn weens sy geskikte wynkundige eienskappe, Lactobacillus plantarum word ook beskou as 'n potensiële kandidaat vir AMG induksie. Ander spesies in die genera Lactobacillus en Pediococcus word dikwels geassosieer met wynbederf. Hierdie mikro-organismes veroorsaak wynbederf deur die produksie van wangeure as gevolg van hul metaboliese aktiwiteite. Dit is dus van kardinale belang dat die meganisme van die wynbederf verstaan word. Die doel van hierdie studie was om die teenwoordigheid van koderend ensieme gene van wynkundige belang in wynverwante MSB stamme te ondersoek. Ten einde dit te bereik, was verskillende stelle van spesifieke peilers ontwerp en toegepas vir 'n groot skaal se genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van ensiemkoderende gene betrokke by verskeie metaboliese paaie, soos sitraat metabolisme, aminosuur metabolisme, hidrolise van glikosiede, agteruitgang van fenoliese sure sowel as proteolise en peptidolise. PKR opsporings resultate het getoon dat die meerderheid van die stamme getoets, die meeste van die gene getoets voor besit. Dit is ook opgemerk dat, onder die O. oeni stamme getoets vir die teenwoordigheid van die pad geen, slegs twee stamme hierdie geen besit. Geen O. oeni stamme het voorheen gewys dat hul die pad geen besit, en hierdie studie was die eerste bewys oor die teenwoordigheid van hierdie geen in O. oeni stamme. In 'n poging om die geen geneties te karakteriseer, is DNA-fragmente van die twee positiewe O. oeni stamme se sekwens volgorde bepaal. Die DNA volgorde is vergelyk met ander nouverwante spesies. Verrassend, was geen passende DNA volgorde gevind met die gepubliseerde genome van drie O. oeni stamme (PSU-1, ATCC BAA-1163 en AWRI B429) nie. Dit versterk die spekulasie dat die pad geen in hierdie twee stamme via die horisontale geen-oordrag verkry is. Verder moet dit nog bepaal word of die teenwoordigheid van hierdie geen lei na vlugtige fenol produksie in wyn. In hierdie studie, is ongeïdentifiseerde stam geïsoleerd van Suid-Afrikaanse druiwe en wyn monsters ook geïdentifiseer en karakteriseer. Die identifisering van hierdie stam is uitgevoer deur middel van die 16S rDNA volgorde analise, wat aangedui het dat hierdie stam behoort aan Lactobacillus florum (99.9% volgorde identiteit). PKR toetse met behulp van die spesie-spesifieke peiler vir die vinnige opsporing en identifikasie van Lb. florum stamme is ook ontwikkel. Vir verdere karakterisering, was hierdie stam ook ondersoek vir die teenwoordigheid van koderende ensiem gene van wynkundige belang. PKR opsporings resultate het aangedui dat die Lb. florum stam ook oor 'n paar van die gene getoets voor besit. Bykomend tot genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van verskillende gene, het die studie ook die evaluering van die regulering van die mleA geen, kodering malaatdekarboksilase in drie wyn stamme van O. oeni. Die regulering van hierdie geen was getoets in die sintetiese wynmedium onder verskillende pH en etanol kondisies. Van die uitdrukkingsresultate, is daar waargeneem dat die mleA geenuitdrukking is negatief geraak deur hoë etanol-inhoud in die medium. Aan die ander kant, in die lae pH medium was die uitdrukking van hierdie geen bevoordeel by pH 3.2 as by pH 3.8. Die bevindinge van hierdie studie het meer lig gewerp op die verspreiding van die wye verskeidenheid van ensiem-koderende gene in MSB stamme wat verband hou met wynmaak. Dit bly egter steeds onbekend of die ensieme gekodeer deur hierdie gene funksioneel is onder wynkondisies, gegewe dat wyn so 'n vyandige omgewing is menigte ongunstige toestande vir die werking van ensieme. Evaluering van die uitdrukking van hierdie gene sal ook help om meer insigte gee oor die regulering van die gene onder wynmaak toestande.

Thesis (PhD (Wine Biotechnology))--University of Stellenbosch, 2011. / Includes bibliography. / ENGLISH ABSTRACT: Alcoholic fermentation, and especially wine fermentation, is one of the most ancient microbiological processes utilized by man. Yeast of the species Saccharomyces cerevisiae are usually responsible for most of the fermentative activity, and many data sets clearly demonstrate the important impact of this species on the quality and character of the final product. However, many aspects of the genetic and metabolic processes that take place during alcoholic fermentation remain poorly understood, including the metabolic processes that impact on aroma and flavour of the fermentation product. To contribute to our understanding of these processes, this study took two approaches: In a first part, the initial aim had been to compare two techniques of transcriptome analysis, DNA oligo-microarrays and Serial Analysis of Gene Expression (SAGE), for their suitability to assess wine fermentation gene expression changes, and in particular to assess their potential to, in combination, provide combined quantitative and qualitative data for mRNA levels. The SAGE methodology however failed to produce conclusive data, and only the results of the microarray data are shown in this dissertation. These results provide a comprehensive overview of the transcriptomic changes during model wine fermentation, and serve as a reference database for the following experiments and for future studies using different fermentation conditions or genetically modified yeast. In a second part of the study, a screen to identify genes that impact on the formation of various important volatile aroma compounds including esters, fatty acids and higher alcohols is presented. Indeed, while the metabolic network that leads to the formation of these compounds is reasonably well mapped, surprisingly little is known about specific enzymes involved in specific reactions, the genetic regulation of the network and the physiological roles of individual pathways within the network. Various factors that directly or indirectly affect and regulate the network have been proposed in the past, but little conclusive evidence has been provided. To gain a better understanding of the regulations and physiological role of this network, we took a functional genomics approach by screening a subset of the EUROSCARF strain deletion library, and in particular genes encoding decarboxylases, dehydrogenases and reductases. Thus, ten genes whose deletion impacted most significantly on the aroma production network and higher alcohol formation were selected. Over-expression and single and multiple deletions of the selected genes were used to genetically assess their contribution to aroma production and to the Ehrlich pathway. The results demonstrate the sensitivity of the pathway to cellular redox homeostasis, strongly suggest direct roles for Thi3p, Aad6p and Hom2p, and highlight the important role of Bat2p in controlling the flux through the pathway. / AFRIKAANSE OPSOMMING: Alkoholiese fermentasie, en veral die maak van wyn, is een van die vroegste mikrobiologiese prosesse wat deur die mensdom ingespan is. Die gisspesie Saccharomyces cerevisiae is gewoonlik grotendeels verantwoordelik vir die fermentasie and verskeie vorige studies het gedemonstreer dat hierdie spesie ‘n baie belangrike rol speel in die uiteindelike kwaliteit en karakter van die voltooide produk. Nieteenstaande die feit is daar steeds baie aspekte van beide die genetiese en metaboliese prosesse wat plaasvind tydens alkoholiese fermentatsie wat nog swak verstaan word, insluitende metaboliese padweë wat ‘n impak het op die smaak en aroma van die fermentasie produk. Om ons kennis van die veld uit te brei het die studie twee aanslae geneem: In die eerste geval is gepoog om twee tegnieke van transkriptoom analiese, nl. DNA oligomikro- arrays en Serial Analysis of Gene Expression (SAGE) te bestudeer vir hul vermoë om geen ekspressie veranderinge tydens wynfermentasie te ondersoek en meer spesifiek om hul potensiaal om ‘n kombinasie van kwantitatiewe sowel as kwalitatiewe data met betreking to mRNA vlakke te produseer. Die SAGE metode kon egter geen betroubare resultate produseer nie en dus word slegs die resultate van die mikro-array eksperimente in die tesis bespreek. Die resultaat is ‘n geheeloorsig oor die geenekspressie veranderinge wat so ‘n wyngis tydens alkoholiese fermentasie ondergaan en dien as ‘n verwysingsraamwerk vir toekomstige studies met geneties gemodifiseerde gis of selfs verskillende fermentasieparameters. Die tweede deel van die studie het gefokus op die identifikasie van gene wat ‘n impak het op die vorming van belangrike, vlugtige aroma komponente, o. a. Esters vetsure en hoër alkohole d.m.v. ‘n siftingseksperiment. Alhoewel daar redelik baie inligting is oor die onderligende metaboliese netwerke wat lei tot die vorming van die verbindings, is daar min kennis van die genetiese regulasie van die netwerk en die fisiologiese rol van individuele padweë wat die netwerk vorm. Verskeie faktore – wat of die netwerk direk of indirek affekteer – is al voorgestel, meer met min konkrete bewyse. Dus het ons gepoog om meer lig op die onderwerp te laat m.b.v. ‘n funksionele genoom aanslag deur ‘n siftingseksperiment te doen op ‘n subgroep (spesifiek gene wat kodeer vir dekarboksilase, dehidrogenase en reduktase ensieme) van die EUROSCARF delesiebiblioteek. Dus is tien gene geïdentifiseer – die delesie waarvan ‘n merkbare effek het op die aroma produksie netwerk en spesifiek die van hoër alkohole. Ooruitdrukkings en enkel en meervoudige delesie rasse van die tien gene is gemaak om d.mv. genetiese analiese, hulle rol in aroma produksie en die Ehrlich padweh uit te pluis. Die resultate toon dat hierdie padweg sensitief is teenoor die sellulêre redoks balans en dui op direkte rolle vir Thi3p, Aad6p en Hom2p, asook dat Bat2p ‘n baie belangrike rol speel in die werking van die padweg.

Thesis (PhD(Agric))--Stellenbosch University, 2014. / ENGLISH ABSTRACT: This study focussed on the effect of oxygen on the chemical and sensory evolution of Sauvignon blanc wine under controlled oxidation conditions. The sensorial interactive effects between Sauvignon blanc varietal aroma compounds and compounds that typically arise during oxidation of white wines were also investigated. In the first research chapter the sensorial interactive effects of Sauvignon blanc impact compounds with aldehydes typically originating from oxidation of white wines were investigated. Four compounds, 3‐ mercaptohexan‐1‐ol (3MH), 3‐isobutyl‐2‐methoxypyrazine (IBMP), 3‐(methylthio)‐propionaldehyde (methional) and phenylacetaldehyde were added together in a model wine medium at varying concentrations. The concentrations chosen were according to those reported in literature to occur in Sauvignon blanc wines. The sensory effects of these compounds were profiled using a trained sensory panel. Compounds were first profiled individually and results showed that the change in comcpound concentration not only led to a change in intensity ratings but also in some cases a change in the descriptor. All four compounds in the same sample showed complicated interactive effects. Data were statistically analysed using relatively novel techniques such as statistical networks that allowed deeper insights into the interactions involved. Various observations were made such as the contribution of 3MH to the ‘green’ character of the wine, the potent suppressing abilities of methional on 3MH and IBMP and the additive effect of methional and IBMP contributing to the ‘cooked’ character of the sample. In the second research chapter the effect of repetitive oxidation on a fresh and fruity style Sauvignon blanc wine was investigated. Results showed the progress and evolution of aromatic and non‐aromatic compounds during an oxidative aging period. A large range of chemical analyses were conducted together with extensive sensory profiling. Results showed a decrease in volatile thiols responsible for the fruity nuances and an increase in oxidation‐related compounds, such as acetaldehyde, during the course of the oxidation. Sensory profiling showed the evolution of the wine aroma during oxidation. The wine evolved from a fresh and fruity wine to slight oxidation and then developed extreme oxidative characteristics. The Control samples (no oxygen added) developed a ‘cooked’ character which could indicate the formation of reductive compounds in these wines. Conversely, the wines that received a single dose of oxygen did not develop this flavour and were perceived to be more fresh and fruity than the Control samples. The evolution of the wine colour was also monitored using a spectrophotometer as well as a sensory panel and results suggest that the colour of the wine evolved before the disappearance of the pleasant aroma. The advantages and disadvantages of oxygen exposure to this type of wine style are discussed as well as the complexity of the wine matrix and sensory interactions occurring in the specific wine. The aim of the third research chapter was again to investigate further sensory interactions between Sauvignon blanc varietal aroma compounds and an oxidation‐related compound. After the observations reported in the second research chapter, the inclusion of acetaldehyde in an interaction study seemed to be of critical importance. Acetaldehyde can reach significant concentrations during oxidation and can have a detrimental effect on wine aroma. This interaction study included three compounds, 3MH, IBMP and acetaldehyde. Acetaldehyde was able to effectively mask the ‘green’ character of the sample, while it also enhanced certain fruity nuances when present at specific concentrations. 3MH was able to suppress the oxidising character of acetaldehyde when present at sufficient concentrations. The results from this study clearly showed the complexity of the wine medium and the interactions involved. It also highlighted the importance of performing these types of sensory studies in a simple medium opposed to a complex matrix such as wine. The effect of oxygen on various aspects of the wine was investigated and the combination of chemical and sensory data delivered some interesting conclusions also involving interactions that occurred. This study paved the way for future investigations on the sensory relationships of Sauvignon blanc aroma compounds and the role of proper oxygen management in the production of quality wines. / AFRIKAANSE OPSOMMING: Die doelwit van die studie was om die effek van gekontroleerde oksidasie op die ontwikkeling en verloop van verskeie chemiese komponente asook die ontwikkeling van die sensoriese profiel van ‘n Sauvignon blanc wyn te ondersoek. Bykomende studies ondersoek die interaksie/wisselwerking tussen aromakomponente in ‘n eenvoudige matriks. Interaksies tussen spesifieke Sauvignon blanc kultivargeassosieerde aromakomponente en komponente wat normaalweg tydens die oksidasie van witwyne ontwikkel, word ondersoek. Die eerste navorsingshoofstuk het ten doel om die interaksie tussen tipiese Sauvignon blanc aromakomponente (of impakkomponente) met aldehiede wat normaalweg tydens oksidasie van witwyne ontwikkel, na te vors. Vier komponente is in ‘n modelwyn gevoeg teen verskeie konsentrasies, wat oor die algemeen in die literatuur gerapporteer is om voor the kom in Sauvignon blanc wyne. Die komponente wat ondersoek is, is: 3‐merkaptoheksanol (3MH), 3‐isobutiel‐2‐metoksiepirasien (IBMP), 3‐ (metieltio)‐propionaldehied (methional) en fenielasetaldehied. Die sensoriese effekte van die komponente is deur ‘n opgeleide sensoriese paneel geëvalueer. Komponente is eers individueel geanaliseer en die resultate het getoon dat dat die intensiteit van die spesifieke aroma verander namate die konsentrasie verander. In sommige gevalle het die beskrywende woord vir die aroma ook verander. Deur al vier komponente in dieselfde monster te voeg word die ondersoek gekompliseer. Die data is statisties geanaliseer deur gebruik te maak van relatiewe nuwe tegnieke soos statistiese netwerke wat dieper insig in die betrokke interaksies bewerkstellig. Verskeie waarnemings word gerapporteer onder andere die bydrae van 3MH tot die ‘groen’ karakter van die wyn, die kragtige onderdrukkingsvermoë van methional op 3MH en IBMP asook die opbouende effek van methional en IBMP wat bydra tot die ‘gekookte’ karakter van die monster. Die tweede navorsingshoofstuk is daarop gemik om die effek van herhalende oksidasie op 'n vars en vrugtige styl Sauvignon blanc wyn te ondersoek en om die vordering en ontwikkeling van aromatiese en nie‐aromatiese komponente gedurende hierdie tydperk te analiseer. ‘n Wye reeks chemiese komponente is geanaliseer tesame met omvangryke sensoriese analise. ‘n Afname in die vlugtige tiole, wat verantwoordelik is vir die vrugtige geure, is gevind tesame met ‘n toename in oksidasie‐verwante komponente (soos asetaldehied). Sensoriese ondersoeke toon ook die evolusie van die wynaroma tydens oksidasie. Die wyn het ontwikkel van ‘n vars en vrugtige styl na effense oksidasiegeure waarna ekstreme oksidasiekarakters waargeneem is. Die Kontrole monsters het ‘n ‘gekookte’ karakter ontwikkel wat ‘n aanduiding van die ontwikkeling van ‘reduktiewe’ komponente in hierdie wyn kan wees. Aan die ander kant het wyne wat een suurstofdosering ontvang het, geen van hierdie geure ontwikkel nie en die wyn is as varser en vrugtiger beskryf in vergelyking met die Kontrole monsters. Die ontwikkeling van die wynkleur is ook gemonitor deur gebruik te maak van ‘n spektrofotometer asook 'n sensoriese paneel. Resultate stel voor dat die kleur van die wyn ontwikkel voor die aangename geure begin verdwyn. Die voor‐ en nadele van suurstofblootstelling aan hierdie tipe wynstyl word bespreek asook die kompleksiteit van die wynmatriks en sensoriese interaksies wat in hierdie spesifieke wyn voorkom. Die derde navorsingshoofstuk is weereens daarop gemik om die sensoriese interaksie tussen tipiese Sauvignon blanc kultivar‐geassosieerde aromakomponente en nog ‘n oksidasie‐geassosierde aromakomponent te ondersoek. Die resultate vanuit die tweede navorsingshoofstuk het die insluiting van asetaldehied in die interaksiestudie genoodsaak. Asetaldehied kan betekenisvolle konsentrasies tydens oksidasie behaal en kan ook nadelige effekte op wynaroma hê. Hierdie interaksiestudie het die volgende drie komponente ingesluit: 3MH, IBMP en asetaldehied. Asetaldehied het die ‘groen’ karakter van IBMP effektief gemaskeer terwyl dit die waarneming van die vrugtige aroma ondersteun en selfs verhoog het wanneer dit teen sekere konsentrasies teenwoordig was. 3‐Merkaptoheksanol het die oksidasiekarakter van asetaldehied onderdruk wanneer dit teen genoegsame konsentrasies teenwoordig was. Die kompleksiteit van wyn as ‘n navorsingsmedium is duidelik vanuit die studie veral in die ondersoeking van interaksie‐effekte tussen komponente. Die belangrikheid van die gebruik van 'n eenvoudige medium teenoor ‘n komplekse medium vir soortgelyke studies is dus duidelik. Die effek van suurstof op verskeie aspekte van witwyn is ondersoek en die kombinasie van chemiese en sensoriese data het interessante gevolgtrekkings gelewer. Die studie het die pad vir toekomstige studies gebaan in terme van sensoriese interaksies met betrekking tot Sauvignon blanc aroma. Die belangrikheid van oordeelkundige suurstofbestuur tydens die produksie van kwaliteit wyne is ook uitgelig.

Thesis (MSc)--Stellenbosch University, 2011 / ENGLISH ABSTRACT: South African Chenin blanc wine is characterised by its style diversity and has been receiving more industry limelight over the past few years. Three dry Chenin blanc wine styles are currently described by the wine industry as Fresh and Fruity (FF), Rich and Ripe unwooded (RR-unwooded) and, Rich and Ripe wooded (RR-wooded). According to the South African wine industry, however, consumers seem to be somewhat confused by these various Chenin blanc wine styles, which have not yet been clearly defined and classified. During this study we investigated the following perspectives of this wine style classification confusion: Currently, no specified sensory method is being used to differentiate between wine styles of a single wine cultivar, such as Chenin blanc. The sorting method can be used as a more cost and time effective classification and profiling method than general quantitative descriptive analysis (QDA). It was therefore investigated whether both uninstructed and instructed sorting, with consumers and wine experts, can be used to classify and describe the Chenin blanc wine styles similarly to QDA. The increase in young wine consumers, Generation Y, introduces new wine marketing opportunities for the wine industry which need to be investigated. Young consumers are not aware of the various Chenin blanc wine styles, which may influence their wine preferences. The influence of wine style knowledge on these consumers’ wine style preferences were investigated during blind and wine style informed preference test. Any segmentation within this Generation Y group was also investigated with regards to their Chenin blanc wine style preferences and/or exploratory data. The following scientific and industrial findings were made: QDA was successfully substituted by instructed and uninstructed sorting tasks. Although the sorting tasks easily differentiated between wooded and unwooded wines, QDA differentiated more successfully between the unwooded wines. Wine tasting experience did not significantly influence the sorting results of the consumers versus the wine experts, but more experienced panellists consistently used fewer descriptors than untrained consumers. All panellists sorted products slightly better when they were provided with wine style sorting instructions. However, there were no significant differences between the instructed and uninstructed sorting task results, i.e. a wine style descriptor list did not improve panellists’ abilities to describe their sorted wine groups. South African Chenin blanc wines can definitely be divided into wooded and unwooded wine styles, i.e. two wine style groups, but the unwooded Chenin blanc wine spectrum formed a wine-style-continuum. This finding is apparent from both the sorting and QDA methods. The unwooded continuum wines can further be differentiated from one another with the use of a fresh-to-mature scale and/or a light-to-full body scale during QDA. Generation Y wine consumers preferred all the Chenin blanc wine styles equally and moderately during the blind tasting, but their knowledge of the wine style concepts significantly increased their preferences for the wines during the informed wine style tasting. The cognitive influence of wine style knowledge is therefore powerful in terms of influencing a consumer’s wine preferences. Other interesting correlations were also made between Generation Y consumers’ wine style preferences and their exploratory data. / AFRIKAANSE OPSOMMING: Suid-Afrikaanse Chenin blanc wyn word gekarakteriseer deur styl diversiteit en is die afgelope paar jaar meer in die kollig. Drie droë Chenin blanc wynstyle word tans deur die wynbedryf beskryf as: Vars en Vrugtig, Ryk en Ryp ongehout en, Ryk en Ryp gehout. Volgens die Suid- Afrikaanse wynbedryf is daar heelwat verwarring is tussen hierdie Chenin blanc wynstyle weens die feit dat hierdie wynstyle nog nie duidelik gedefinieer en geklassifiseer is nie. Tydens die huidige studie het ons die volgende perspektiewe van hierdie wynstyl-verwarring ondersoek: Huidiglik word daar geen gespesifiseerde sensoriese metode gebruik om te onderskei tussen verskillende wynstyle van ’n enkele wyn kultivar, soos Chenin blanc, nie. Die sorteringsmetode kan gebruik word, instede van kwantitatiewe beskrywende analise, as ’n meer koste- en tyd-effektiewe produk klassifikasie en profileringsmetode. DIt was dus ondersoek of beide geïnstrukteerde en ongeïnstrukteerde sorteringstake, met verbruikers en wynkundiges, gebruik kan word om die verskeie Chenin blanc wynstyle kan profileer en klassifiseer soortgelyk soos kwantitatiewe beskrywende analise. Die toename in jong verbruikers, Generasie Y, skep nuwe wynbemarkingsgeleenthede wat ondersoek moet word. Jong verbruikers se onbewustheid van die verskeidenheid Chenin blanc wynstyle mag hulle voorkeure vir hierdie wyne beïnvloed. Die invloed van wynstyl kennis op hierdie jong verbruikers se wynstyl voorkeure was ook ondersoek gedurende ’n blinde en ’n wynstyl-ingeligte voorkeur toets. Enige segmentasie binne hierdie Generasie Y verbruikersgroep was ook ondersoek in verband met hul Chenin blanc wynstyl voorkeure en/of verkennende data. Die volgende wetenskaplike en industriële bevindings is gemaak: Kwantitatiewe beskrywende analise kan suksesvol vervang word deur beide geïnstrukteerde en ongeïnstrukteerde sorteringsmetodes. Alhoewel die sorteringsmetodes maklik gebruik kon word om te onderskei tussen gehoute en ongehoute wyne, kan kwantitatiewe beskrywende analise fyner onderskeid tref tussen die individuele ongehoute wyne. Die wynproe-ervaringsvlak het geen invloed op die sorteringsresultate gehad nie, maar meer ervare paneellede het konsekwent gebruik gemaak van minder beskrywende terme as onopgeleide verbruikers wat baie meer en verskillende soorte terme gebruik het. Beide panele het effens beter gesorteer wanneer hulle spesifieke wynstyl sorteringsinstruksies ontvang het. Daar was egter geen beduidende verskille tussen die geïnstrukteerde en ongeïnstrukteerde sorteringsresultate nie, wat daarop dui dat sorteringsinstruksies en ’n lys van wynstyl beskrywings nie die paneellede se wyse van sortering verander het nie. Suid-Afrikaanse Chenin blanc wyne kan definitief verdeel word in gehoute en ongehoute wynstyle, d.w.s twee wynstyl groepe, maar die ongehoute Chenin blanc wyn spektrum vorm ’n wynstyl-kontinuum. Laasgenoemde is bevind tydens beide die sorteringstake asook kwantitatiewe beskrywende analise. Die ongehoute kontinuum wyne kan verder van mekaar onderskei word met behulp van ‘n vars-tot-volryp en/of ‘n ligte-tot-volmond skaal tydens kwantitatiewe beskrywende analise. Alle jong wyn verbruikers het ewe veel van al die wynstyle gehou tydens die blinde wynstyl voorkeur toets, maar tydens die wynstyl-ingeligte voorkeur toets het hulle voorkeure vir al die wynstyle drasties verbeter. Die kognitiewe invloed van Chenin blanc wynstyl kennis kan dus lei tot hoër voorkeure vir die wynstyle. Ander interessante korrelasies is ook bevind tussen hierdie Generasie Y wyn verbruikersgroep se wynstyl voorkeure en hul verkennende data.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006. / The production of premium quality wine is dependant on excellent management of the total wine production value chain. To achieve this we need rapid and reliable analytical tools. Over the last decade Fourier transform infrared (FT-IR) spectroscopy has made a significant contribution to wine research and in the last five years South African institutions have also exploited the use of this technology not only for research, but also in industrial cellars. The FT-IR apparatus is equipped with global calibrations and therefore we first investigated the validity of these for South African conditions. To achieve this new calibration sets for pH, titratable acidity and °Brix were made and compared to the global calibrations with statistical methods. Results obtained between the °Brix calibrations displayed high correlation and the global calibration can therefore be implemented. However, the new TA calibration was more accurate than the global calibration. Results were inconclusive for the new pH calibration sample set and therefore needs to be enlarged before it can be validated as the possibility of being more accurate exists. It was concluded that FT-IR spectroscopy in the simultaneous measurement for °Brix, pH and TA in grape must showed potential for accurate analysis and quality control purposes in an industrial cellar. Rapid analysis of these parameters will lead to higher throughput of grape must samples in the laboratory as well as adhering to good laboratory practices by validation. The importance of correct sample preparation in the laboratory was illustrated when using FT-IR spectroscopy for one-step analysis and adjustments to global calibrations. Results obtained showed that grape parameters such as °Brix, nitrogen content were not influenced by the two sample preparation methods (hand pressed vs. homogenised), but pH, TA, colour index, anthocyanins and polyphenols were influenced. Important key factors were identified in the quality control chain from vineyard to the cellar. Numerous grape loads had an increase in microbial populations after harvesting the vineyard and transport to the weighbridge. Transport is critical especially for the vineyards in the Lutzville area (had the highest yeast population), which are situated the furthest from the cellar. Sauvignon blanc had the highest acetic acid bacteria and lactic acid bacteria populations compared to the other cultivars. Gluconic acid, glycerol and arabitol was highly correlated to each other. High populations of acetic acid bacteria and lactic acid bacteria also had high levels of gluconic acid and 2,3-butanediol in the grape juice. Meso-inositol differed significantly between the vineyard and weighbridge and it had a high standard deviation compared to the mean value of all the samples between the vineyard and weighbridge. Temperature of grape loads delivered to the cellar ranged from 14 to 36ºC, which had a major impact on the grape quality and the resultant wine. It can be concluded for this study that management of the total value chain is of critical importance to ensure that A-grade grapes results in good quality wine that merits the effort of the grape producer.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2006. / Resveratrol is a phytoalexin that is produced in the leaves and skins of grape berries in response to biotic and abiotic factors. Substitution and polymerisation of resveratrol units produce an array of compounds which form part of the active disease defence mechanism in grapevine. Wine is one of the major sources of resveratrol in the human diet. Resveratrol is one of the phenolic compounds present in wine that mediates protective effects on human health. It has been shown to prevent the development of cardiovascular disease, cancer and pathogenesis related to inflammation. Red wines contain higher levels of resveratrol than white wines owing to extended maceration times during fermentation on the skins. During white wine vinification skin contact is limited as skins are removed prior to fermentation. Thus, the extraction of resveratrol into white wines is minimal. The principal focus of our research is the development of a wine yeast strain capable of resveratrol production during grape must fermentation. It is proposed that red and white wines produced with such a resveratrol-producing yeast will contain elevated levels of resveratrol, and that added health benefits may be derived from their consumption. Initial work done in our laboratory established that expressing multiple copies of the genes encoding coenzyme A ligase (4CL216) and resveratrol synthase (vst1) in laboratory yeast enabled the yeast to produce resveratrol, conditional to the supplementation of the growth medium with p-coumaric acid. This study focused on the optimisation of resveratrol production in Saccharomyces cerevisiae. It involved the integration and constitutive expression of 4CL216 from hybrid poplar and vst1 from grapevine. Integration and expression of these genes in three laboratory strains was confirmed by Southern and Northern blot analyses. The evaluation of resveratrol production by yeast required the initial optimisation of the analytical techniques. We optimised the method for sample preparation from the intracellular fraction of yeast and devised a procedure for the assay of the extracellular fractions. The LCMSMS method was further developed to encompass detection and quantification of other compounds related to resveratrol production in yeast. Comparison of resveratrol production in three different yeast genetic backgrounds indicated that the onset of production and the resveratrol yield is yeast strain dependent. Precursor feeding studies indicated that p-coumaric acid availability was a factor limiting maximal resveratrol production. Early indications were obtained that endogenously-produced resveratrol may have an impact on yeast viability during extended culture periods. This study has broadened our understanding of the resveratrol production dynamics in S. cerevisiae and provided important indications as to where further optimisation would be beneficial in order to optimally engineer a wine yeast for maximal resveratrol production.

Thesis (MSc (Wine Biotechnology))--Stellenbosch University, 2008. / In the wine industry, the importance of selecting an appropriate yeast strain, generally of the species Saccharomyces cerevisiae, to ensure reliable fermentation and to achieve a desired level of quality has been well established. As a consequence, the demand for new starter cultures with improved or new oenological characteristics is increasing. Appropriately selected starter cultures can reduce the occurrence of stuck fermentations, impart specific aroma profiles and reduce the development of offflavours. Using standard breeding and selection procedures, several wine yeast strains that would be less likely than currently existing strains to experience stuck fermentation have previously been developed at the Institute for Wine Biotechnology. The target of these projects had been to develop strains with improved nitrogen efficiency [defined as the amount of fermented hexoses for a given amount of free amino nitrogen (FAN)], improved fructose utilization and ethanol tolerance. These three parameters are known contributors to stuck fermentation. Two of the strains that had been isolated in these projects, strain 116 for nitrogen efficiency and strain 38-1 for efficient fructose utilization, were chosen as parental strains for the current study. The aim was to further improve and possibly combine these traits in yeast strains by using hybridization followed by various enrichment and directed evolution procedures in a continuous fermentation setup. The strategy was to sequentially subject the population of mass-mated hybrids to a number of selective environments for a large number of generations. The yeasts were subjected to a high fructose/glucose ratio for 12 generations, followed by selection in an environment with a limited supply of nitrogen for 54 generations and finally to high ethanol stress. After each round of enrichment, individual strains were analysed to assess the results. For the hybrid strains selected after enrichment in a medium with a high fructose/glucose ratio, no general improvement could be discerned. However, one of the hybrids, hybrid strain 331, fermented fructose better than the parental strains and other hybrid strains. These results may suggest that the selection pressure was not applied for a sufficient number of generations and may not have been sufficiently strong. In addition, the parental strain may already performing at a rate that may render further improvement more difficult in this genetic background. The next aim of this study was to enhance fermentation performance of wine yeast hybrid strains in low nitrogen and high sugar conditions. Several hybrid strains 331, RR03 and 05R generated in this study showed improvement in efficiency of nitrogen utilization when compared to the parental strains, indicating a successful selection strategy. Several strains also showed higher ethanol tolerance, and some strains possessed] combinations of the traits to be improved. Future research will evaluate these hybrids regarding the production of aromatic compounds and of the sensory profile produced. Such strains would help the wine industry to control the occurrence of stuck fermentations and to produce quality wines.

Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2007. / Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria and are active against other bacteria, either in the same species (narrow spectrum) or across genera (broad spectrum). The application of bacteriocins during the vinification process might help to prevent the production of undesired compounds by inhibiting the indigenous bacterial microflora and allowing malolactic fermentation to be conducted by a selected bacterial strain. Furthermore, the use of bacteriocins might allow reducing the total sulphur dioxide amount in wine. The purpose of this study was the selection of lactic acid bacteria (LAB) belonging to the genera Oenococcus, Lactobacillus and Pediococcus with the ability to produce bacteriocins, with respective biological activity against undesired indigenous wine LAB and the capability to complete malolactic fermentation. The first objective of this study was the screening of LAB isolated from South African red wines for the production of bacteriocins. Only 27 strains out of 330 wine isolates, belonging to the species Lb. plantarum, Lb. paracasei, Lb. hilgardii and O. oeni, showed activity towards various wine-related and non wine-related indicator strains with the colony-overlay method. It is the first time that bacteriocin activity is reported in O. oeni. The second objective was the detection and identification of known structural bacteriocin genes of Lb. plantarum wine strains. Furthermore, the web server BAGEL was used to in silico analyse putative bacteriocin-encoding genes in the genome of O. oeni and primers were designed to amplify four possible bacteriocin-encoding genes. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Moreover, PCR analysis rendered positive results with all four chosen putative bacteriocin-encoding genes in the eight tested O. oeni strains with antimicrobial activity. The latter genes of O. oeni were heterologously expressed in different Escherichia coli host strains, but no antimicrobial activity could be detected. The third objective of this study was the transformation and expression of the heterologous bacteriocin genes nisin A and pediocin PA-1 in two selected Lb. plantarum strains. To enhance their antimicrobial activity a plasmid containing the nisin A gene was successfully cloned into the two strains. Indeed, an enhanced antimicrobial activity could be detected, but the transformed plasmid was not stable. The fourth objective in this project was the evaluation of bacteriocin production in liquid media. A co-culture experiment with a plantaricin producing Lb. plantarum strain and an Enterococcus faecalis strain as indicator was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 hours. The last objective was the evaluation of the impacts of phenolic compounds on the activity of nisin and pediocin. The short term influence of two phenolic acids, two flavan-3-ols, grape tannins and oak tannins on the activity of nisin and pediocin PA-1 was investigated. No influence on the activity was detected. Furthermore, synergistic effects on bacterial growth inhibition were observed. This study confirms the potential use of either bacteriocin additives or bacteriocin-producing LAB in order to control the bacterial microflora during the vinification process.

Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Agriculture worldwide is under great pressure to produce enough food in order to sustain the ever-growing world population. Among the many challenges faced by food producers, crop losses and damage caused by fungal plant pathogens is a major problem. The study of fungal pathogens and the interaction between plants and fungi is therefore essential, and has been carried out for many years. Much has been learned in this time, but the full mechanisms of the various modes of fungal attack and plant defence have still not been elucidated. Many fungi rely on the action of cell-wall degrading enzymes (CWDEs) to breach the plant cell wall and facilitate access to the nutrients within. CWDEs are among the very first enzymes to be secreted at the start of fungal attack, and many of them are considered to be essential pathogenesis factors. Endopolygalacturonases (ePGs) are CWDEs that cleave the homogalacturonan stretches of the plant cell wall and are vital virulence factors for a number of fungi, including Botrytis cinerea. An important defence mechanism of plants involves the inhibition of CWDEs in order to halt or slow down the fungal attack. Plant polygalacturonaseinhibiting proteins (PGIPs) are cell wall associated CWDE-inhibiting proteins that specifically act on fungal ePGs. Many different PGIPs from a number of diverse plant species have been described to date. They are known to have differential inhibition capabilities that often result from only a few key amino acid changes within the leucine-rich repeat (LRR) active domains. Previously, the first grapevine PGIP was isolated and characterised from Vitis vinifera cultivar Pinotage (Vvpgip1). This Vvpgip1 gene was overexpressed in the tobacco species Nicotiana tabacum, and was shown to be very effective in reducing the susceptibility of tobacco towards B. cinerea. The combined results confirmed transgene overexpression, increased PGIP activity and a strong resistance response against Botrytis, leading to the characterisation of these lines as having PGIP-specific resistance phenotypes. In a subsequent transcriptomic analysis of these lines it was found that they display differential expression of cell wall metabolism genes and biochemical characteristics that might indicate possible cell wall strengthening compared to wild-type tobacco under uninfecting conditions. The V. vinifera cultivars are all very susceptible to fungal attack, whereas other grapevine species, specifically the North American Vitis species, are known for their strong resistance and even immunity against many fungal pathogens. Thirty seven PGIPs have previously been isolated from these more resistant species. The amino acid sequences of the active domains of these PGIPs were previously aligned with that of VvPGIP1, and the proteins were found to be highly homologous with each other and with VvPGIP1. The different nonvinifera PGIPs separated into 14 subgroups based on their active domain sequences. For this study, one PGIP from each group was selected for functional analysis in tobacco. The selected PGIP-encoding genes were transformed into tobacco by means of Agrobacterium tumefaciens. Analyses of the putatively transformed plantlets were performed to test for transgene presence, transgene expression, and PGIP activity: final transgenic tobacco populations consisting of three to twelve individually transformed lines of nine different nonvinifera PGIPs were obtained. A subset of the resultant transgenic lines was infected with B. cinerea in two independent whole plant infections over 11-14 days in order to investigate the disease resistance afforded by the various PGIPs towards this fungus. A line from the previously characterised VvPGIP1 population was included as reference; all the infections were contrasted to the WT tobacco. All the infected lines overexpressing the non-vinifera PGIPs displayed very strong disease reduction in comparison to the WT control: after initial primary lesion formation, the spread of fungal infection was contained and halted in these lines, while wild-type tobacco plants were severely affected. Although the VvPGIP1 line displayed the characteristic PGIP-defense response, the non-vinifera PGIP plants displayed smaller lesions, indicating very strong resistance phenotypes. The characterised non-vinifera PGIP overexpressing lines, together with the VvPGIP1 line and the WT control were also used to further evaluate the previous observation that overexpression might lead to changes in expression of cell wall genes. Analysis of the expression of a xyloglucan endotransglycosylase (xth) gene in the transgenic population showed that this gene was down-regulated in healthy uninfected tissue from all the transgenic lines tested. This confirmed previous results and have confirmed in all grapevine PGIP overexpressing lines tested so far that this gene is downregulated. XTH is typically involved in cell wall metabolism and specifically in controlling the strength and elasticity of the plant cell wall. From previous work it is known that downregulation of this gene leads to strengthening of the wall. The results obtained in this study showed that the PGIP-specific resistance phenotype seen for VvPGIP1-overexpressing tobacco could be confirmed in transgenic tobacco overexpressing non-vinifera PGIPs from more resistant grapevine species as well. The fact that these PGIPs lines all performed even better than the VvPGIP1 lines in conferring resistance towards B. cinerea provides an interesting angle for further investigation into the structural differences between the non-vinifera PGIPs and VvPGIP1. The transgenic lines are also excellent material to study the in vivo functions of PGIPs further in the context of plant-pathogen interactions. / AFRIKAANSE OPSOMMING: Die landboubedryf is wêreldwyd onder groot druk om genoeg voedsel te produseer vir die groeiende wêreldbevolking. Een van die grootste probleme wat die bedryf ondervind, is die groot skade wat aan gewasse aangerig word deur patogeniese swamme. Dit is dus noodsaaklik om swamme en die interaksie tussen plante en swamme te bestudeer, en dit word al vir jare gedoen. Hoewel daar al baie geleer is in hierdie tydperk, is die volle meganismes van die verskeie maniere hoe swamme aanval en hoe plante hulleself verdedig, nog nie bekend nie. Verskeie swamme maak staat op die aktiwiteit van selwand-afbrekende ensieme (SWAEe) om deur die plantselwand te breek en sodoende toegang tot voedingstowwe in die plantsel te fasiliteer. SWAEe is van die eerste ensieme wat tydens die begin van patogeniese aanval deur swamme afgeskei word en verskeie SWAEe word as noodsaaklike patogeniese faktore beskou. Endopoligalakturonases (ePGs) is SWAEe wat die homogalakturoniese dele van die plantselwand verteer en is noodsaaklike virulensie faktore vir ‘n aantal swamme, onder andere Botrytis cinerea. ‘n Belangrike weerstandsmeganisme van plante behels die inhibering van swam SWAEe om sodoende die patogeen-aanval te stop of te vertraag. Die poligalakturonase-inhiberende proteïne (PGIPs) van plante is selwand-geassosieerde SWAEinhiberende proteïne wat spesifiek teen swam ePGs optree. Verskeie verskillende PGIPs vanuit verskillende plantspesies is tot dusver beskryf. Dit is bekend dat hulle differensiële inhiberende vermoëns het wat dikwels toegeskryf kan word aan slegs ‘n paar belangrike aminosuurvolgordeverskille in die leusien-ryke herhalende (LRH) aktiewe areas. Die eerste wingerd PGIP is vantevore geïsoleer vanuit Vitis vinifera kultivar Pinotage (Vvpgip1) en gekarakteriseer. Hierdie Vvpgip1 geen is ooruitgedruk in die tabakspesie Nicotiana tabacum en was baie effektief om die weerstand van tabak teen die swam Botrytis cinerea te verhoog. Die ooruitdrukking van die transgeen, verhoogde PGIP aktiwiteit en goeie weerstand teen Botrytis cinerea is bevestig, en het gelei daartoe dat die transgeniese VvPGIP1 plantlyne geklassifiseer is as lyne met PGIP-spesifieke weerstandsfenotipes. ‘n Daaropvolgende transkriptomiese analise van die plantlyne het gewys dat hulle differensiële uitdrukking van selwand-geassosieerde gene het, asook biochemiese eienskappe, wat ‘n moontlike selwandversterking aandui in vergelyking met wilde-tipe tabak in die afwesigheid van infeksie. Die V. vinifera kultivars is hoogs vatbaar vir swamme, terwyl ander wingerdspesies, spesifiek die Noord-Amerikaanse spesies, bekend is vir hoë weerstand en selfs immuniteit teenoor verskeie patogeniese swamme. Sewe-en-dertig PGIPs is vantevore geïsoleer vanuit hierdie meer weerstandbiedende spesies. Die aminosuurvolgordes van die aktiewe areas van hierdie PGIPs is vantevore vergelyk met die van VvPGIP1 en dit is gevind dat hierdie proteïne hoogs homoloog is aan mekaar, sowel as aan VvPGIP1. Die verskillende nie-vinifera PGIPs het in 14 groepe verdeel na aanleiding van die homologie van hulle aktiewe areas. Vir hierdie studie is een PGIP vanuit elkeen van hierdie groepe gekies vir verdere funksionele analise in tabak. Die 14 nie-vinifera PGIP-koderende gene is stabiel oorgedra na tabak deur middel van Agrobacterium tumefaciens. Die vermeende transgeniese plante is geanaliseer vir die teenwoordigheid van die transgeen, die uitdrukking daarvan en PGIP aktiwiteit: bevestigde transgeniese tabak populasies wat wissel van drie tot 12 individuele getransformeerde lyne kon verkry word vir nege van die verskillende nie-vinifera PGIPs. ‘n Aantal van die transgeniese lyne is geïnfekteer met B. cinerea in twee onafhanklike heelplantinfeksies vir 11-14 dae om die siekteweerstand van hierdie PGIPs teenoor die swam te evalueer. ‘n Plantlyn van die VvPGIP1-populasie is as ‘n verwysing ingesluit en al die infeksies is vergelyk met die wilde-tipe tabak. Al die geïnfekteerde lyne wat die nie-vinifera PGIPs ooruitdruk het ‘n baie sterk afname in siektesimptome getoon in vergelyking met die wilde-tipe kontrole: na aanvanklikle primêre lesies gevorm het, is die verspreiding van die infeksie ingeperk en gestop in hierdie lyne, terwyl die wilde-tipe plante baie erg geaffekteer is. Terwyl die VvPGIP1 lyn ook die tipiese PGIPweerstandsrespons getoon het, het die nie-vinifera PGIPe kleiner lesies ontwikkel, wat dui op baie sterk weerstandsfenotipes. Die gekarakteriseerde nie-vinifera PGIP ooruitdrukkende lyne, asook die VvPGIP1 lyn en die wilde-tipe kontrole, is gebruik om die vorige waarneming dat die ooruitdrukking kan lei tot veranderinge in selwandgeen-uitdrukking verder te ondersoek. Analise van die uitdrukking van ‘n xiloglukaan-endotransglikosilase (xth) geen in die transgeniese populasie het getoon dat hierdie geen afgereguleer is in gesonde, oninfekteerde weefsel van al die transgeniese lyne wat getoets is. Dit het vorige resultate bevestig en het ook bevestig dat hierdie geen afgereguleer is in alle wingerd PGIP-ooruitdrukkende lyne wat tot dusver getoets is. XTH is tipies betrokke by selwandmetabolisme, spesifiek by die beheer van selwandsterkte en selwandelastisiteit. Dit is uit vorige werk bekend dat die afregulering van hierdie geen lei tot versterking van die plantselwand. Die resultate verkry tydens hierdie studie het gewys dat die PGIP-spesifieke weerstand fenotipe van VvPGIP1-ooruitdrukkende tabak ook bevestig kon word in transgeniese tabak wat nie-vinifera PGIPs vanuit meer weerstandbiedende wingerdspesies ooruitdruk. Die feit dat hierdie PGIP lyne almal selfs beter weerstand teen B. cinerea bied as VvPGIP1 lyne is ‘n interessante invalshoek vir opvolgende ondersoeke na die belang van strukturele verskille tussen die nie-vinifera PGIPs en VvPGIP1. Hierdie transgeniese lyne is ook uitstekende hulpbronne om die in vivo funksies van PGIPs verder te bestudeer in die konteks van plantpatogeen interaksies.