Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: In recent years South African wines have been under the spotlight due to references in the international wine media to a distinctive ‘burnt rubber’ character. Many winemakers and wine experts argued that the peculiar character could be ascribed to winemaking errors linked to mismanaged fermentation. An alternative possible source of the taint was identified in the coal tar creosote used as a wood preservative in vineyard trellis systems. South African regulations allow for the use of creosoted utility poles in agricultural land, but in Europe and USA their usage has been highly regulated and even banned for certain applications. Contamination of foodstuff by polycyclic aromatic hydrocarbons (PAHs) is one of the major motives for the banning of creosote in Europe and USA. Some of the compounds in the creosote mixture display very strong odour characteristics and for this reason it became the focus of attention for the present study. The overall aim of this study was to determine if vines trellised with creosoted posts could accumulate or absorb the various malodorous compounds deriving from the wood treatment onto the grape berries. These compounds could then be extracted from the grape berries into the wine during alcoholic fermentation, creating quality and sensory problems. Chapter 2 of this thesis gives an overview of the extensive literature that deals with methods of analysis of PAHs and malodorous phenols using both Gas Chromatography (GC), as well as High Performance Liquid Chromatography (HPLC). New methods for sample preparation, as well as analysis of PAHs using HPLC-Diode Array Detector (DAD), were developed and the results reported in Chapter 3. It was demonstrated that Sauvignon blanc experimental wines contained only chrysene at very low levels. The concentrations of chrysene found in the experimental wines are within the prescribed parameters as established by The Commission of the European Communities. Since no other PAH compounds were found in the samples analysed, it was concluded that the experimental wines were safe for human consumption and complied with EU regulations. The effect of vineyard posts on the sensorial characteristics of wine is discussed in Chapter 4. Creosoted poles were found to be responsible for an off-flavour described as ‘burnt rubber’ and ‘tarry’ in Merlot wines produced from grapes grown in close proximity to the posts. Following some of the reported findings, new guidelines have been introduced in the Integrated Production of Wine certification, which advise against the use of creosoted poles for vineyard trellising. This preliminary but important guideline will bring the South African wine industry a step closer to the fulfilling the obligations for food safety as required by the legislation of our major export partners. Future investigations are recommended to completely understand and evaluate the cumulative effect of creosoted posts in a fully trellised vineyard. / AFRIKAANSE OPSOMMING: Oor die afgelope paar jaar is Suid-Afrikaanse wyne onder die soeklig geplaas as gevolg van verwysings in die internasionale wynmedia na ‘n duidelike ‘gebrande rubber’-karakter. Baie wynmakers en wyndeskundiges het aangedui dat hierdie besonderse karakter toegeskryf kan word aan wynbereidingsfoute wat verband hou met gisting wat wanbestuur is. ‘n Alternatiewe moontlike oorsprong van die smaak is geïdentifiseer in die koolteer wat as ‘n houtpreserveermiddel in wingerdopleistelsels gebruik word. Suid-Afrikaanse regulasies maak voorsiening vir die gebruik van kreosoteerde nutspale in landbougrond, hoewel hulle gebruik in Europa en die VSA hoogs gereguleerd en in sommige gevalle selfs verbied is. Die besmetting van kossoorte deur polisikliese aromatiese koolwaterstowwe (polycyclic aromatic hydrocarbons (PAHs)) is een van die vernaamste redes vir die verbanning van kreosoot in Europa en die VSA. Sommige van die verbindings in die kreosootmengsel het baie sterk geurkenmerke en daarom is dit die fokus van die huidige studie. Die oorhoofse doelwit van hierdie studie was om te bepaal of wingerde wat op kreosoteerde pale opgelei is, die verskillende onwelriekende verbindings afkomstig van die houtbehandeling in die druiwekorrels kan akkumuleer of absorbeer. Hierdie verbindings sou dan tydens alkoholiese gisting uit die druiwekorrels in die wyn geëkstraheer kon word, wat aanleiding sou gee tot kwaliteits- en sensoriese probleme. Hoofstuk 2 van hierdie tesis verskaf ‘n oorsig van die breedvoerige literatuur wat handel oor metodes om PAH’s en onwelriekende fenole met behulp van beide gaschromatografie (GC) en hoëdrukvloeistofchromatografie (HPLC) te analiseer. Nuwe metodes is ontwikkel om monsters voor te berei en om PAH’s met behulp van ‘n HPLC-diode array detector (DAD) te analiseer. Die resultate word in Hoofstuk 3 gerapporteer. Daar is aangetoon dat die eksperimentele Sauvignon blanc-wyne chriseen teen baie lae vlakke bevat het. Die konsentrasies van chriseen wat in die eksperimentele wyne gevind is, is binne die voorgeskrewe parameters van die Kommissie van die Europese Gemeenskap. Aangesien daar nie ander PAHverbindings in die geanaliseerde monsters gevind kon word nie, is daar tot die gevolgtrekking gekom dat die eksperimentele wyne veilig is vir menslike verbruik en aan die EG-regulasies voldoen. Die effek van wingerdpale op die sensoriese kenmerke van wyn word in Hoofstuk 4 bespreek. Kreosoteerde pale is gevind wat verantwoordelik is vir ‘n wangeur in Merlot-wyne afkomstig van druiwe wat naby die pale gegroei het en wat as ‘gebrande rubber’ en ‘teeragtig’ beskryf is. Op grond van sommige van die gerapporteerde bevindings, is nuwe riglyne ingesluit in die sertifisering vir die Geïntegreerde Produksie van Wyn, wat aanbeveel dat kreosoteerde pale nie vir wingerdoplei gebruik word nie. Hierdie voorlopige, maar belangrike riglyn sal die Suid-Afrikaanse wynbedryf al beter in staat stel om te voldoen aan die voedselveiligheid regulasies wat vereis word deur die wetgewing van ons belangrikste uitvoervennote. Toekomstige ondersoeke moet aangewend word om die kumulatiewe effek van kreosoteerde pale in volledig opgeleide wingerde ten volle te verstaan en te evalueer. / Indaba Scholarship and Winetech for their financial support

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Sauvignon blanc wines have become progressively more important in the commercial market. Extensive research is being done in various countries to gain more understanding about the aroma compounds found in Sauvignon blanc wines and the interactions between them. Sauvignon blanc wines often have either have a green or tropical style. The green style is caused by the methoxypyrazines while the volatile thiols are important contributing compounds to the tropical style. Various international studies have focussed on measuring the chemical composition of Sauvignon blanc wines. However, more research is required on South African Sauvignon blanc wines. Little is known of the volatile thiols content of South African Sauvignon blanc wines, although the methoxypyrazine content has been extensively reported on. Although methoxypyrazines and volatile thiols are seen as the most important aroma compounds contributing to Sauvignon blanc character, other compounds contribute as well. Esters, monoterpenes and phenols have been found to influence Sauvignon blanc aroma and interact with the methoxypyrazines and volatile thiols. The complex interaction between the compounds responsible for the aroma of Sauvignon blanc wines are still not fully understood and further research is thus needed. The first part of the current study investigated the interaction between a specific methoxypyrazine and volatile thiol. Five different concentrations of 2-isobutyl-3- methoxypyrazine (ibMP) and 3-mercaptohexan-1-ol (3MH) were spiked in dearomatize, neutral Sauvignon blanc wine. The single compounds as well as every possible combination of the range of concentrations were evaluated using sensory descriptive analysis. It was found using various statistical approaches that ibMP suppressed the tropical attributes associated with 3MH and that 3MH suppressed the green attributes that correlated with ibMP. The concentrations at which the suppression occurred and the degree of suppression was different for each attribute. The second part of the current study focussed on commercial South African Sauvignon blanc wines. Sensory descriptive analysis and chemical analysis were used to assess the wines and measure the volatile thiol and methoxypyrazine concentrations. The concentrations of volatile thiols and methoxypyrazines were found to be in line with international Sauvignon blanc wines. It was also shown for the first time that the mutually suppressive trend between the volatile thiols and methoxypyrazines can be seen in commercial Sauvignon blanc wines as well. Future work is needed to fully understand the complex interaction between the various compounds in Sauvignon blanc wines. Further research could focus on investigating the mechanism of interaction between the volatile thiols and methoxypyrazines as well as other aroma compounds. / AFRIKAANSE OPSOMMING: Sauvignon blanc-wyne word toenemend belangriker in die kommersiële mark. Omvattende navorsing word in etlike lande gedoen om meer begrip te ontwikkel van die aromaverbindings wat in Sauvignon blanc-wyne teenwoordig is, asook van die interaksies tussen hulle. Sauvignon blanc-wyne het in baie gevalle óf ’n groen óf ’n tropiese styl. Die groen styl word veroorsaak deur metoksipirasiene, terwyl die vlugtige tiole belangrike bydraende verbindings is wat aanleiding gee tot die tropiese style. Verskeie internasionale studies het reeds gefokus op die meet van die chemiese samestelling van Sauvignon blanc-wyne, maar meer navorsing is nodig oor Suid-Afrikaanse Sauvignon blanc-wyne. Min is bekend oor die inhoud van vlugtige tiole in Suid-Afrikaanse Sauvignon blanc-wyne, hoewel daar reeds op groot skaal oor die metoksipirasieninhoud verslag gedoen is. Hoewel metoksipirasiene en vlugtige tiole die belangrikste aromaverbindings is wat tot Sauvignon blanc-karakter bydra, is daar ook ander verbindings wat ’n bydrae maak. Esters, monoterpene en fenole het almal ’n invloed op Sauvignon blanc-aroma en reageer op die metoksipirasiene en vlugtige tiole. Die komplekse interaksie tussen die verbindings wat vir die aroma van Sauvignon blanc-wyne verantwoordelik is, word nog nie volledig begryp nie en verdere navorsing is nodig. Die eerste deel van die huidige studie het die interaksie tussen ’n spesifieke metoksipirasien en vlugtige tiol ondersoek. Vyf verskillende konsentrasies van 2-isobutiel-3-metoksipirasien (ibMP) en 3-merkaptoheksaan- 1-ool (3MH) is by ontgeurde, neutrale Sauvignon blanc-wyn gevoeg. Die enkel verbindings, asook elke moontlike kombinasie van die reeks konsentrasies, is deur middel van beskrywende sensoriese analise geëvalueer. Daar is met behulp van verskillende statistiese benaderings gevind dat ibMP die tropiese eienskappe wat verband hou met 3MH onderdruk het, terwyl 3MH die groen eienskappe wat verband hou met ibMP onderdruk het. Die konsentrasies waarteen die onderdrukking plaasgevind het en die vlak van onderdrukking het vir elke eienskap verskil. Die tweede deel van die studie het gefokus op kommersiële Suid-Afrikaanse Sauvignon blancwyne. Beskrywende sensoriese analise en chemiese analise is gebruik om die wyne te assesseer en die konsentrasies van vlugtige tiole en metoksipirasiene te meet. Die konsentrasies vlugtige tiole en metoksipirasiene was in lyn met dié van internasionale Sauvignon blanc-wyne. Daar is ook vir die eerste keer gewys dat die wedersyds onderdrukkende tendens tussen die vlugtige tiole en metoksipirasiene ook in kommersiële Sauvignon blanc-wyne gevind word. Toekomstige werk sou kon fokus op ’n begrip van die komplekse interaksie tussen die verskillende verbindings in Sauvignon blanc-wyne. Verdere navorsing sou ook kon fokus op ’n ondersoek van die meganisme van interaksie tussen die vlugtige tiole en metoksipirasiene, sowel as ander aromaverbindings. / The South African Wine Industry (Winetech), THRIP and the NRF for financial support

Thesis (MScAgric)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The WineScan FT120 is widely used in wine laboratories across South Africa. The WineScan FT120 uses Fourier transform infrared (FT-IR) spectroscopy with multivariate data analysis to correlate spectra with chemical compositional data. Ready-to-use, commercially available calibration models for a FT-IR spectroscopy instrument are an advantage for unskilled users and routine analysis. Introducing spirit products to this technology introduced new interferences, which necessitated vastly different calibrations models to compensate for the changes. Accuracy, precision and ruggedness of the reference methods validated during method validation, verified the suitability of the reference methods used to quantify the parameters in question before calibration model building was attempted. Various principal component analysis (PCA) were performed prior to the calibration step with the aim to identify outliers and inspect groupings. PCA models could identify samples with atypical spectra and differentiate between product types. Two tactics regarding data sets for calibration set-up was experimented with, all the products together and calibration models per product. Partial least squares (PLS) regression was used to establish the calibration models for ethanol, density, obscuration and colour. With all the calibration models, the calibration models based on the product specific data sets, achieved better predicting statistics. The best performing ethanol calibration models achieved Residual mean square error of prediction (RMSEP) = 0.038 to 0.106 %v/v and showed significant improvement on previously reported prediction errors by Lachenmeier (2007). The results for the density calibration showed a similar trend, with the product specific calibration models outperforming the calibration model when all samples were included into one calibration model. This study produced novel results for quantification of obscuration (RMSEP = 0.10 and 0.09 in blended brandies and potstill brandies, respectively) and colour (RMSEP < 2.286 gold units) of brandies and whiskies. The correlation coefficients (R²) between true and predicted values, for the four parameters tested, indicated good to excellent precision (0.8 < R² < 1.0). Minimising the variation between the samples of the data set, gave more accurate regression statistics, but this resulted in a lower residual predictive deviation (RPD) value (< 5) that indicated models were not suitable for quantification. Adding more samples per product will add more variability into a data set per product, increase the SD and result in an increase in the RPD. The results pave the way for the development of calibration models for the quantification of other parameters for specific products. Following the groupings of product types, further classifications of brandy brands were investigated. PCA plots showed clear separation between potstill brandies and blended brandies and some degree of clustering between some of the blended brands was observed. Classification of brandies were investigated using the Soft Independent Modeling of Class Analogy (SIMCA) approach resulting in a total correct classification rates between 81.25% and 100% for the various brandy brands. These preliminary results were very promising and highlight the potential of using FT-IR spectroscopy and multivariate classification techniques as a tool for rapid quality control and authentication of brandy brands. Using this work as base for further classification projects, this could be of great benefit to the alcoholic beverage industry of South Africa. Future work will involve the development of a database comprised of more products guaranteed authentic to expand the discriminating options. The results suggest FT-IR spectroscopy could be useful in authentication studies. / AFRIKAANSE OPSOMMING: Die WineScan FT120 is ‘n algemeen gebruikte instrument regoor Suid-Afrika. Die WineScan FT120 gebruik Fourier-transformasie-infrarooi (FT-IR) spektroskopie tesame met multiveranderlike statistiese metodes om spektra te korreleer met chemiese samestellingsdata. Die kommersieël beskikbare kalibrasiemodelle vir die FT-IR spektroskopie-instrument is ‘n voordeel vir onbedrewe gebruikers en roetine ontleding. Blootstelling van spiritusprodukte aan die tegnologie, het nuwe hindernisse bekend gestel en dus is verskillende kalibrasiemodelle genoodsaak om hiervoor te kompenseer. Akkuraatheid, presiesheid en ruheid van die verwysingsmetodes is geëvalueer tydens metodevalidasie. Die verwysingsmetodes is geskik verklaar vir die konstruksie van die kalibrasiemodel met geverifieërde akkurate verwysingsresultate. Verskeie multiveranderlike hoofkomponentanalise (MVK) was uitgevoer voor die kalibrasiestap met die doel om uitskieters te identifiseer en groeperings te inspekteer. MVK modelle kon monsters met atipiese spektra identifiseer en onderskei tussen verskillende produk tipes. Twee taktieke aangaande datastelsamestelling is getoets tydens kalibrasiemodel-opstelling, al die produkte saam en kalibrasiemodelle per produk soos met die MVK aangedui. Parsiële kleinste kwadraat (PKK)- regressie is gebruik vir die opstel van die kalibrasiemodelle vir etanol, digtheid, obskurasie en kleur. Met al die kalibrasiemodelle het die produk spesifieke kalibrasiemodelle beter regressiestatistiek gelewer. Die beste presterende etanol kalibrasiemodelle het ‘n standaardvoorspellingsfout (SVF) = 0.038 tot 0.106 %v/v bereik en het ‘n beduidende verbetering getoon op vorige gerapporteerde studies op spiritusprodukte (Lachenmeier, 2007). Die resultate vir die digtheidskalibrasiemodelle het ‘n eenderse tendens getoon soos die etanol, met die produk spesifieke kalibrasiemodelle wat beter presteer het. Hierdie studie was eerste in sy soort met die kalibrasiemodel vir obskurasie (SVF = 0.10 en 0.09 in gemengde brandewyne en potketel brandewyne, onderskeidelik) en kleur (SVF < 2.286 goud eenhede) van brandewyne en whiskies. Die bepalingskoëffisiënt (R²) vir die vier parameters, dui op goeie tot uitstekende presiesheid (0.8 < R² < 1.0). Vermindering van die variasie tussen die monsters in die datastel, het meer akkurate regressiestatistiek teweeg gebring, maar ‘n laer relatiewe voorspellingsafwyking (RVA) waarde (<5) tot gevolg gehad wat aan dui dat hierdie modelle nie geskik is vir sifting of kwantifisering nie. Die byvoeging van meer monsters per produk sal meer verskeidenheid in die datastel per produk bring, wat dan die standaardafwyking sal laat toeneem en uiteindelik die RVA laat toeneem. Die resultate het die fondasie gelê vir die ontwikkeling van kalibrasiemodelle vir die kwantifisering van ander parameters vir spesifieke produkte. As opvolg tot die groeperings van die produk tipe, waargeneem in die MVK modelle, was klassifikasie van brandewyn handelsmerke ondersoek. MVK modelle het duidelike skeiding gewys tussen potketel en gemengde brandewyne en tot ‘n sekere mate groepering tussen handelsmerke. Klassifikasie van brandewyne was ondersoek met behulp van the Soft Independent Modeling of Class Analogy (SIMCA) met die resultaat van ‘n totale korrekte klassifikasiekoers van tussen 81.25% en 100% vir die verskeie brandewyn handelsmerke. Hierdie voorlopige resultate toon belowend en beklemtoon die potensiaal van FT-IR spektroskopie en chemometrics tegnieke as toerusting vir die vinnige kwaliteitskontrole en egtheid van brandewyn handelsmerke studies. Met hierdie werk as basis vir verdere klassifikasie projekte, kan dit ‘n groot aanwins wees tot die alkoholiese drank industrie van Suid-Afrika. Toekomstige werk sal insluit die ontwikkeling van ‘n databasis saamgestel met meer gewaarborgde egte produkte om die klassifikasie uit te brei.

Thesis (MScAgric)--Stellenbosch University, 2014 / ENGLISH ABSTRACT: The objective of the study was to determine combined effects of irrigation and canopy management practices on grapevine water status, growth, yield and juice characteristics. The field study was carried out with Shiraz/110R grapevines in the Breede River Valley. Grapevines were drip irrigated at 30%, 60% and 90% plant available water (PAW) depletion, respectively. For each PAW level, grapevines had (i) suckered, vertical shoot positioned (VSP), (ii) non-suckered, VSP and (iii) sprawling canopies. Treatments were replicated three times in a randomised block design and applied during the 2011/12 and 2012/13 seasons. Irrigation applied at low PAW depletion levels, i.e. high frequency irrigation, required substantially higher irrigation volumes compared to high depletion levels, i.e. low frequency irrigation. Low frequency irrigation increased grapevine water constraints compared to high frequency irrigation. Sprawling canopy grapevines experienced more water constraints than VSP grapevines. Grapevines irrigated at 90% PAW depletion experienced strong water constraints. Low frequency irrigation seemed to accelerate berry ripening compared to high frequencies, probably due to smaller berries and lower yields. Sprawling canopies consistently enhanced berry ripening due to more sunlight interception by the leaves. Berry ripening of VSP grapevines was slower, but inconsistent between seasons. Level of PAW depletion and canopy management practice did not affect number of leaves per primary shoot. Low frequency irrigation reduced number of leaves per secondary shoot. Leaf number per shoot contributed more to total leaf area than leaf size. Level of PAW depletion did not affect number of shoots per grapevine. Suckering reduced number of shoots per grapevine. Low frequency irrigation reduced total leaf area per grapevine compared to high frequency irrigation. Effects of canopy management practice were more pronounced in the case of high frequency irrigation compared to low frequency irrigation. At pruning, primary cane length was not affected by level of PAW depletion or canopy management practice. Secondary cane mass and diameter were not affected by canopy management practice. Multiple linear regression showed that cane mass was a function of cane length and diameter. Low frequency irrigation reduced berry mass compared to high frequency irrigation, irrespective of canopy management practice. However, at harvest there was no difference in berry mass between 30% and 60% PAW depletion. Low irrigation The objective of the study was to determine combined effects of irrigation and canopy management practices on grapevine water status, growth, yield and juice characteristics. The field study was carried out with Shiraz/110R grapevines in the Breede River Valley. Grapevines were drip irrigated at 30%, 60% and 90% plant available water (PAW) depletion, respectively. For each PAW level, grapevines had (i) suckered, vertical shoot positioned (VSP), (ii) non-suckered, VSP and (iii) sprawling canopies. Treatments were replicated three times in a randomised block design and applied during the 2011/12 and 2012/13 seasons. Irrigation applied at low PAW depletion levels, i.e. high frequency irrigation, required substantially higher irrigation volumes compared to high depletion levels, i.e. low frequency irrigation. Low frequency irrigation increased grapevine water constraints compared to high frequency irrigation. Sprawling canopy grapevines experienced more water constraints than VSP grapevines. Grapevines irrigated at 90% PAW depletion experienced strong water constraints. Low frequency irrigation seemed to accelerate berry ripening compared to high frequencies, probably due to smaller berries and lower yields. Sprawling canopies consistently enhanced berry ripening due to more sunlight interception by the leaves. Berry ripening of VSP grapevines was slower, but inconsistent between seasons. Level of PAW depletion and canopy management practice did not affect number of leaves per primary shoot. Low frequency irrigation reduced number of leaves per secondary shoot. Leaf number per shoot contributed more to total leaf area than leaf size. Level of PAW depletion did not affect number of shoots per grapevine. Suckering reduced number of shoots per grapevine. Low frequency irrigation reduced total leaf area per grapevine compared to high frequency irrigation. Effects of canopy management practice were more pronounced in the case of high frequency irrigation compared to low frequency irrigation. At pruning, primary cane length was not affected by level of PAW depletion or canopy management practice. Secondary cane mass and diameter were not affected by canopy management practice. Multiple linear regression showed that cane mass was a function of cane length and diameter. Low frequency irrigation reduced berry mass compared to high frequency irrigation, irrespective of canopy management practice. However, at harvest there was no difference in berry mass between 30% and 60% PAW depletion. Low irrigation. / AFRIKAANSE OPSOMMING: Die doelwit van hierdie studie was om die gekombineerde effek van besproeiing en lowerbestuurspraktyke op wingerd waterstatus, groei, opbrengs en druiwesap eienskappe te bepaal. Die veld studie is uitgevoer met Shiraz/110R wingerdstokke in die Breede Rivier Vallei. Wingerdstokke was d.m.v. drupbesproeiing teen 30%, 60% en 90% plant beskikbare water (PBW) ontrekking, onderskeidelik besproei. Vir elke PBW ontrekkingspeil, was wingerdstokke (i) gesuier en vertikale lootposisionering toegepas, (ii) ongesuier en vertikale lootposisionering toegepas en (iii) geen lowerbestuur toegepas nie (lowers wat oophang). Behandelings is drie keer in ‘n ewekansige blokontwerp herhaal en tydens die 2011/12 en 2012/13 seisoene toegepas. Besproeiing wat teen ‘n lae PBW ontrekkingspeil toegedien is, d.w.s. hoë frekwensie besproeiing, vereis aansienlik hoër besproeiings volumes i.v.m. hoë besproeiing ontrekkingspeile, d.w.s. lae frekwensie besproeiing. Wingerdstokke wat oopgehang het meer watertekorte as vertikaal lootgeposisioneerde wingerdstokke ervaar. Wingerdstokke wat teen 90% PBW ontrekking besproei was, het sterk watertekorte ervaar. Dit het voorgekom of lae frekwensie besproeiing korrelrypwording versnel het i.v.m. hoë frekwensie besproeiing. Dit was heelwaarskynlik a.g.v. kleiner korrels en laer opbrengste. Wingerdstokke wat oophang het, het konsekwent korrelrypwording versnel a.g.v. meer sonligonderskepping deur die blare. Korrelrypwording van vertikaal lootgeposisioneerde wingerdstokke was stadiger, maar teenstrydig tussen die seisoene. Plant beskikbare water ontrekkingspeil en lowerbestuurspraktyke het geen invoeld gehad op die aantal blare per primêre loot nie. Lae frekwensie besproeiing het die aantal blare per sekondêre loot verminder. Die hoeveelheid blare per loot het ‘n groter bygedra gemaak i.v.m. blaar grootte. Plant beskikbare water ontrekkingspeil het geen invloed gehad op die aantal lote per wingerdstok nie. Suier verminder die aantal lote per wingerdstok. Lae frekwensie besproeiing verminder die totale blaar oppervlak i.v.m. hoë frekwensie besproeiing. Die effek van lowerebestuurspraktyke is duideliker sigbaar by hoë frekwensie besproeiing i.v.m. lae frekwensie besproeiing. Primêre lootlengte was nie deur PBW ontrekkingspeil of lowerbestuurspraktyke beïnvloed nie. Sekondêre lootmassa en -deursnit is nie deur lowerbestuurspraktyk beïnvloed nie. Meervoudige lineêre regressie het getoon dat lootmassa ‘n funksie van lootlengte en -deursnit was. Lae frekwensie besproeiing het korrelmassa verminder ongeag die lowerbestuurspraktyk i.v.m. hoë frekwensie besproeiing. Daar was egter geen verskil in korrelmassa by oes tussen 30% en 60% PBW ontrekking nie. Lae frekwensie besproeiing was geneig om suiker akkumulasie te versnel i.v.m. hoë frekwensie besproeiing. Wingerdstokke wat oopgehang het, het veral by lae frekwensie besproeiing korrelrypwording versnel i.v.m. vertikaal lootgeposisioneeide wingerdstokke. Suikerinhoud per korrel het geneig om toe te neem totdat dit ‘n plato bereik het. Hierdie plato was meer prominent by hoë frekwensie besproeiing i.v.m. lae frekwensie besproeiing. Wingerdstokke wat oopgehang het, het ook hierdie plato vroeër bereik i.v.m. vertikaal lootgeposisioneerde wingerdstokke. By oes was die totale titreerbare suur (TTS) hoër vir wingerdstokke wat vroeër geoes was. As gevolg van versnelde rypwording was TTS van wingerdstokke wat teen lae frekwensie besproei is hoër i.v.m. hoë frekwensie besproeiing. ‘n Ligter oeslading in verhouding tot ‘n hoër blaaroppervlak het ook gelei tot hoër TTS by oes. Plant beskikbare water ontrekkingspeil en lowerbestuurspraktyke het geen invloed op die pH gehad met oes nie. Die hoeveelheid trosse per wingerdstok het nie duidelike tendense gewys wat verbind kon word met watertekorte wat deur die stokke ervaar is nie. Gesuierde vertikaal lootgeposisioneerde wingerdstokke het die hoeveelheid trosse per stok verminder i.v.m. die ongesuierde vertikaal lootgeposisioneerde wingerdstokke en wingerstokke wat oopgehang het. Trosmassa het dieselfde tendense as korrels per tros gevolg. Lae frekwensie besproeiing het opbrengs aansienlik verminder i.v.m. hoë frekwensie besproeiing. Gesuierde vertikaal lootgeposisioneerde wingerdstokke het geneig om opbrengste te verminder i.v.m. ongesuierde vertikaal lootgeposisioneerde wingerdstokke. Hierdie effek het egter verdwyn waar wingerdstokke teen 90% PBW ontrekking besproei was. Druif skade a.g.v. suurvrot was meer prominent by hoë frekwensie besproeiing, veral vir ongesuierde vertikaal lootgeposisioneerde wingerdstokke. Total opbrengs verlies, uitgedruk as ‘n persentasie, was hoofsaaklik ‘n funksie van sonbrand eerder as ‘n funksie van suurvrot.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The yeast species, Saccharomyces cerevisiae and S. bayanus are well known for the key role they play during alcoholic fermentation in both wine and beer industries. These yeasts are available in pure active dried form and can be used to produce different wine styles and to manage quality. There are more than 200 commercial wine yeast strains on the market and include naturally isolated strains and hybrids. With all these commercial yeasts available, strain authenticity is very important to the manufacturer of active dried wine yeasts (ADWY) because it can prevent commercial losses and maintain market credibility. It is as important to the winemaker as it may impact wine quality. Various traditional and molecular techniques have been successfully applied to perform quality control of wine yeast strains. The aims of this study were to evaluate electrophoretic karyotyping (CHEF) and PCRbased methods to distinguish between Saccharomyces wine yeast strains and to establish a database containing molecular profiles of commercial strains. CHEF karyotyping was chosen because it is generally used in the wine industry to distinguish between wine yeast strains, but can be time-consuming. Alternatively, PCR-based methods are considered to be reliable and fast. These PCR methods included the evaluation of interdelta regions, multiplex-PCR of miniand microsatellites, MET2 gene RFLP analysis and the use of several species-specific primers. In this study, 62 commercial wine yeast strains, were randomly selected from various manufacturers of ADWY, and two reference strains, S. bayanus CBS 380 and S. cerevisiae CBS 1171, were evaluated. CHEF karyotyping could successfully differentiate between all 64 yeast strains. The two primer sets used for interdelta amplifications, delta1-2 and delta12-21, yielded 59 and 62 profiles, respectively. Yeast strains considered to be similar or identical according to interdelta amplification results, were resolved with CHEF karyotyping. CHEF karyotyping was proven to be more accurate than interdelta amplifications in distinguishing between commercial wine yeast strains. However, the results of interdelta amplifications were very useful and less time-consuming. The multiplex-PCR of mini- and microsatellite primers only succeeded in identifying a specific band within 55 of the 64 yeast strains including the S. cerevisiae reference strain, a possible indication of species specificity. However, oenological designation using MET2 gene RFLP analysis and species-specific primers indicated that all the commercial strains in this study had a S. cerevisiae ancestry. Restriction analysis of the MET2 gene with EcoRI also successfully identified AWRI Fusion and Zymaflore X5 as hybrid yeast strains. A wine yeast database was created and contains three libraries, i.e. CHEF karyotypes, delta1-2 and delta12-21 electrophoretic profiles. The database was proven to be functional and showed great accuracy in grouping and identifying test strains. The database has many possible applications, but there is still some optimisation and refinement needed. / AFRIKAANSE OPSOMMING: Die Saccharomyces sensu stricto kompleks, is bekend vir die belangrike rol wat hierdie giste speel tydens alkoholiese fermentasie in biede wyn en bier industrieë. Dit is om hierdie rede dat kelders rein aktief gedroogte wyngis gebruik vir die produksie van spesifieke wynstyle, asook kwaliteit. Daar is meer as 200 kommersiële wyngiste op die mark beskikbaar en dit sluit natuurlike isolate en hibriede in. Daarom is gisras verifikasie baie belangrik vir die vervaardiger van aktief gedroogde wyngiste asook die wynmaker om finansiële verliese te voorkom en mark vertrouenswaardigheid te handhaaf. Verskeie tradisionele en molekulêre metodes word suksesvol toegepas vir gehalte beheer van die gisrasse. Die doel van hierdie studie was om elektroforetiese kariotipering (CHEF) en PKR gebaseerde tegnieke se vermoë om tussen Saccharomyces wyngiste te onderskei, te ondersoek. Ook deel van die doelwitte was om ‘n databasis te skep wat die verskillende elektroforetiese profiele van die kommersiële gisrasse bevat. Tydens hierdie studie is 62 kommersiële gisrasse van verskeie vervaardigers ewekansig geselekteer. Saccharomyces bayanus CBS 380 en S. cerevisiae CBS 1171 is as verwysingsrasse gebruik. Elektroforetiese kariotipering (CHEF) is gekies omdat dit een van die mees algemeenste tegnieke is wat gebruik word om tussen wyngiste te onderskei, maar dit word as tydrowend en arbeidsintensief beskou. As ‘n alternatief is daar na PKR gebaseerde tegnieke gekyk. Hierdie tegnieke word as betroubaar en vinnig beskou. Verskeie PKR gebaseerde tegnieke is ondersoek, naamlik PKR van interdelta areas, multipleks-PKR van mini- en mikrosatelliete, MET2 geen RFLP analise en die gebruik van spesie-spesifieke inleiers. Interdelta amplifikasies en mini- en makrosatelliet inleiers is geselekteer as gevolg van hul vermoë om Saccharomyces wyngiste tot op spesie en ras vlak te onderskei. Die MET2 geen en spesie-spesifieke inleiers is geselekteer om die kommersiele wyngis as S. cerevisiae, S. bayanus of as hibriede te klassifiseer. CHEF kariotipering kon tussen al 64 giste onderskeid tref. Die twee stelle inleiers wat vir interdelta amplifikasie gebruik was, delta1-2 en delta12-21, het onderskeidelik 59 en 62 profiele gelewer. Gis rasse wat identiese profiele met die delta inleiers gelewer het, kon egter met CHEF kariotipering onderskei word. Die resultate het getoon dat CHEF kariotipering beter tussen die kommersiële wyngiste kon onderskei as die interdelta amplifikasies, maar dat die interdelta amplifikasies nogsteeds goeie onderskeiding toon en dat dit minder tydrowend is. Die multipleks-PKR van mini- en mikrosatelliete kon slegs ‘n enkele band in 55 van die 64 giste uit lig. ‘n Moontlike aanduiding van spesie spesifiekheid. Die oenologiese groepering volgens MET2 geen analise en spesies-spesifieke inleiers dui aan dat al die kommersiele wyngiste wat in hierdie studie gebruik is, moontlik van S. cerevisiae afkomstig is. Restriksie analise van die MET2 geen met EcoRI het ook AWRI Fusion en Zymaflore X5 as hibriede geïdentifiseer. Die CHEF kariotipes en interdelta elektroforetiese profiele is gebruik om ‘n databasis van die kommersiële Saccharomyces wyngiste op te stel. Die databasis is funksioneel en het die toets rasse akkuraat geïdentifiseer en korrek gegroepeer. Die databasis moet egter nog verdere optimisering en verfyning ondergaan.

Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--Stellenbosch University, 2008. / Although bananas are widely grown worldwide in many tropical and a few subtropical countries, banana beverages are still among the fruit beverages processed by use of rudimentary methods such as the use of feet or/and spear grass to extract juice. Because banana juice and beer remained on a home made basis, there is a research drive to come up with modern technologies to more effectively process bananas and to make acceptable banana juices and wines. One of the main hindrances in the production of highly desirable beverages is the pectinaceous nature of the banana fruit, which makes juice extraction and clarification very difficult. Commercial enzyme applications seem to be the major way forward in solving processing problems in order to improve banana juice and wine quality. The particular pectinolytic enzymes that were selected for this study are Rapidase CB, Rapidase TF, Rapidase X-press and OE-Lallzyme. In addition this study, investigate the applicability of recombinant yeast strains with pectinolytic, xylanolytic, glucanolytic and amylolytic activities in degrading the banana polysaccharides (pectin, xylan, glucan starch) for juice and wine extraction and product clarification. The overall objective of this research was to improve banana juice and wine by enzymatic processing techniques and to improve alcoholic fermentation and to produce limpid and shelf-stable products of clarified juice and wine. The focus was on applying the selected commercial enzyme preparations specifically for the production of better clarified banana juice and wine. This is because the turbid banana juice and beer, which contain suspended solids that are characterised by a very intense banana flavour, require a holistic approach to address challenges and opportunities in order to process pure banana beverages with desirable organoleptic qualities. The specific objectives of applying commercial enzymes in the processing of banana juice and wine, comparing with grape winemaking practices, use of recombinant yeast and analyses of various parameters in the juices and wines made have enabled generation of information that could be of help to prospective banana juice and wine processors. The research findings obtained could be used to establish a pilot plant or small-scale industry in the banana processing beverages producing large quantities,and finally the overall objective of obtaining limpid and shelf stable products would be achieved.

Thesis (PhD (Viticulture and Oenology. Wine Biotechnology))--Stellenbosch University, 2008. / The occurrence of sluggish and stuck fermentations continues to be a serious problem in the global wine industry, leading to loss of product, low quality wines, cellar management problems and consequently to significant financial losses. Comprehensive research has shown that many different factors can act either in isolation, or more commonly synergistically, to negatively affect fermentative activity of wine yeast strains of the species Saccharomyces cerevisiae. The individual factors most commonly referred to in the literature are various nutrient and oxygen limitations. However, other factors have been shown to contribute to the problem. Because of the mostly synergistic nature of the impacts, no single factor can usually be identified as the primary cause of stuck fermentation. In this study, several strategies to evolutionarily engineer wine yeast strains that are expected to reduce the occurrence of stuck and sluggish fermentations are investigated. In particular, the investigations focus on improving the ability of wine yeast to better respond to two of the factors that commonly contribute to the occurrence of such fermentations, nitrogen limitation and the development of an unfavorable ratio of glucose and fructose during fermentation. The evolutionary engineering strategies relied on mass-mating or mutagenesis of successful commercial wine yeast strains to generate yeast populations of diverse genetic backgrounds. These culture populations were then exposed to enrichment procedures either in continuous or sequential batch cultivation conditions while applying specific evolutionary selection pressures. In one of the stragegies, yeast populations were subjected to continuous cultivation under hexose, and especially fructose, limitation. The data show that the strains selected after this procedure were usually able to out-compete the parental strains in these selective conditions. However, the improved phenotype was not detectable when strains were evaluated in laboratory scale wine fermentations. In contrast, the selection procedure in continuous cultivation in nitrogen limiting conditions proved to be highly efficient for the generation of yeast strains with higher total fermentative capacity in low nitrogen musts. Furthermore, yeast strains selected after mutagenesis and sequential batch cultivation in synthetic musts with a very low glucose on fructose ratio showed a fructose specific improvement in fermentative capacity. This phenotype, which corresponds to the desired outcome, was also present in laboratory scale wine fermentations, where the discrepancy between glucose and fructose utilization of the selected strains was significantly reduced when compared to the parents. Finally, a novel strategy for the rectification of stuck fermentations was adjusted to industrial conditions. The strategy is based on the use of a natural isolate of the yeast species Zygosaccharomyces bailii, which is known for its preference of fructose. This process was successfully established and implemented in the wine industry.

Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Plants have developed sophisticated means of combating plant diseases. The events that prepare the plant for, and follow plant-pathogenic interactions, are extremely complex and have been the topic of intensive investigation in recent years. These interactions involve a plethora of genes and proteins, and intricate regulation thereof; from the host and pathogen alike. Studying the contribution of single genes and their encoded proteins to the molecular dialogue between plant and pathogen has been a focus of plant molecular biologists. To this end, a gene encoding a polygalacturonase-inhibiting protein (PGIP) was recently cloned from Vitis vinifera. These proteins have the ability to inhibit fungal endopolygalacturonases (ePGs), enzymes which have been shown to be required for the full virulence of several fungi on their respective plant hosts. The activity of PGIP in inhibiting fungal macerating enzymes is particularly attractive for the improvement of disease tolerance of crop species. The VvPGIP-encoding gene was subsequently transferred to Nicotiana tabacum for high-level expression of VvPGIP. These transgenic plants were found to be less susceptible to infection by Botrytis cinerea in an initial detached leaf assay. Also, it was shown that ePG inhibition by protein extracts from these lines correlated to the observed decrease in susceptibility to B. cinerea. This study expands on previous findings by corroborating the antifungal nature of the introduced PGIP by whole-plant, timecourse infection assays. Six transgenic tobacco lines and an untransformed wildtype (WT) were infected and the lesions measured daily from day three to seven, and again at day 15. The transgenic lines exhibited smaller lesions sizes from three to seven days post-inoculation, although these differences only became statistically significant following seven days of incubation. At this point, four of the six lines exhibited significantly smaller lesions than the WT, with reductions in disease susceptibility ranging between 46 and 69% as compared to the WT. Two of the lines exhibited disease susceptibility comparable to the WT. In these resistant plant lines, a correlation could be drawn between Vvpgip1 expression, PGIP activity and ePG inhibition. These lines were therefore considered to be PGIP-specific resistant lines, and provided ideal resources to further study the possible in planta roles of PGIP in plant defense. The current hypothesis regarding the role(s) of PGIP in plant defense is twofold. Firstly, PGIPs have the ability to specifically and effectively inhibit fungal ePGs. This direct inhibition results in reduced fungal pathogenicity. Alternatively, unhindered action of these enzymes results in maceration of plant tissue and ultimately, tissue necrosis. Subsequently, it could be shown that, in vitro, the inhibition of ePGs prolongs the existence of oligogalacturonides, molecules with the ability to activate plant defense responses. Thus, PGIPs limit tissue damage by inhibition of ePG; this inhibition results in activation of plant defense responses aimed at limiting pathogen ingress. Several publications reported reduced susceptibility to Botrytis in transgenic plant lines overexpressing PGIP-encoding genes. However, none of these publications could expand on the current hypotheses regarding the possible in planta roles of PGIP in plant defense. In this study we used transgenic tobacco lines overexpressing Vvpgip1 as resources to study the in planta roles for PGIP. Transcriptomic and hormonal analyses were performed on these lines and a WT line, both before and following inoculation with Botrytis cinerea. Transcriptomic analysis was performed on uninfected as well as infected tobacco leaf material utilizing a Solanum tuberosum microarray. From the analysis with healthy, uninfected plant material, it became clear that genes involved in cell wall metabolism were differentially expressed between the transgenic lines and the WT. Under these conditions, it could be shown and confirmed that the gene encoding tobacco xyloglucan endotransglycosylase (XET/XTH) was downregulated in the transgenic lines. Additionally, genes involved in the lignin biosynthetic pathway were affected in the individual transgenic lines. Biochemical evidence corroborated the indication of increased lignin deposition in their cell walls. Additionally, phytohormone profiling revealed an increased indole-acetic acid content in the transgenic lines. These results show that constitutive levels of PGIP may affect cell wall metabolism in the Vvpgip1-transgenic lines which may have a positive impact on the observed reduced susceptibilities of these plants. An additional role for PGIP in the contribution to plant defenses is therefore proposed. PGIP may directly influence defense responses in the plant leading to the strengthening of cell walls. This might occur by virtue of its structural features or its integration in the cell wall. These reinforced cell walls are thus “primed” before pathogen ingress and contribute to the decrease in disease susceptibility observed in lines accumulating high levels of PGIP. Transcriptional and hormonal analyses, at the localized response, were performed on Botrytis-infected leaf tissue of the transgenic lines and a WT line. Several Botrytis responsive genes were found to be upregulated in both the WT and the transgenic lines. Although limited differential expression was observed between the two genotypes, the analyses identified a gene which was upregulated two-fold in the transgenic lines, as compared to WT. This was confirmed by quantitative Real-Time PCR. This gene is involved in the lipoxygenase pathway, specifically the 9-LOX branch, leading to the synthesis of the divinyl ether oxylipins colneleic and colnelenic acid, which show inhibitory effects on Botrytis spore germination. Phytohormone profiling revealed that the transgenic lines accumulated more of the defense-related hormone pool of jasmonates. These are formed via the 13-LOX pathway and have been shown to be important for the restriction of Botrytis growth at the site of infection. Collectively, the results from the infection analyses indicate that in these transgenic lines, both branches of the lipoxygenase pathway are differentially induced at the level of the localized response to Botrytis infection. Similarly, an increased induction of the synthesis of the defense-related hormone salicylic acid could be observed, although this hormone did not accumulate to significantly higher levels. These results are the first report of differential induction of a defense-related pathway in pgip-overexpressing lines and substantiate the proposal that following ePG inhibition by PGIP, signaling which activates plant defense responses, takes place. Taken together, these results significantly contribute to our understanding of the in planta role of PGIP in plant defense responses. / AFRIKAANSE OPSOMMING: Plante het deur evolusie gesofistikeerde meganismes teen die aanslag van plantsiektes ontwikkel. Die gebeure wat die plant voorberei, asook dié wat op plant-patogeen interaksies volg, is uiters kompleks en vorm die kern van verskeie navorsingstemas die afgelope paar jaar. Etlike plant- én patogeengene en proteïene is by hierdie interaksies betrokke en aan komplekse reguleringsprosesse onderworpe. Die bestudering van die bydrae van enkelgene en hul gekodeerde proteïene tot die molekulêre interaksie tussen ‘n plant en patogeen is ‘n sterk fokus van plant-molekulêre bioloë. Met hierdie doel as fokus, is ‘n geen wat vir ‘n poligalakturonaseinhiberende proteïen (PGIP) kodeer, van Vitis vinifera gekloneer. Hierdie proteïene beskik oor die vermoë om fungiese endopoligalakturonases (ePG's), ensieme wat benodig word vir die virulensie van verskeie fungi op hul gasheerplante, te inhibeer. Die inhibisie van ePG's deur PGIP en die gepaardgaande verminderde weefseldegradasie is ‘n baie belowende strategie vir die verbetering van verboude gewasse se patogeentoleransie. Die VvPGIPenkoderende geen is gevolglik na Nicotiana tabacum oorgedra vir hoëvlakuitdrukking van VvPGIP. Daar is gevind dat hierdie transgeniese plante minder vatbaar vir Botrytis cinerea-infeksies was in ‘n inisiële antifungiese toets wat gebruik gemaak het van blaarweefsel wat van die moederplant verwyder is. Daar is ook ‘n korrelasie gevind tussen B. cinerea-siekteweerstand en ePG-inhibisie deur proteïenekstrakte van die transgeniese populasie. Die huidige studie bou voort op en bevestig vorige bevindinge betreffende die antfungiese aard van die heteroloë PGIP in die heelplant en oor tyd. Ses transgeniese tabaklyne en 'n ongetransformeerde wilde-tipe (WT) is geïnfekteer en die lesies is vanaf dag drie tot sewe, en weer op dag 15, gemeet. Die transgeniese lyne het in die tydperk van drie tot sewe dae ná-inokulasie kleiner lesies as die WT getoon, alhoewel hierdie verskille slegs statisties beduidend geword het na sewe dae van inkubasie. Op daardie tydstip het vier van die ses lyne aansienlik kleiner lesies as die WT getoon, en verlagings in siektevatbaarheid het, in vergelyking met die WT, van 46% tot 69% gewissel. Twee van die lyne het siektevatbaarheid getoon wat vergelykbaar was met dié van die WT. In die siekteweerstandbiedende plantlyne was daar 'n verband tussen Vvpgip1-ekspressie, PGIP-aktiwiteit en ePG-inhibisie. Hierdie plantlyne is dus as PGIP-spesifieke siekteweerstandslyne beskou en dien dus as ideale eksperimentele bronne vir die ontleding van die moontlike in plantafunksies van PGIP in plantsiekteweerstandbiedendheid. Die huidige hipotese betreffende die funksie(s) van PGIP in plantsiekteweerstand is tweeledig. Eerstens het PGIP die vermoë om fungusePG's spesifiek en doeltreffend te inhibeer. Hierdie direkte inhibisie veroorsaak ‘n vermindering in patogenisiteit van die fungus op die gasheer. Indien ePG's egter hulle ensimatiese aksie onverstoord voortsit, sal weefseldegradasie en uiteindelik weefselnekrose die gevolg wees. Daar kon ook bewys word dat die in vitroinhibisie van ePG's deur PGIP die leeftyd van oligogalakturoniede, molekules wat die vermoë het om die plantweerstandsrespons aan te skakel, kan verleng. PGIP het dus nie net die vermoë om ePG's, en dus weefseldegradasie, te inhibeer nie; maar hierdie inhibisie lei ook daartoe dat plantweerstandsresponse aangeskakel word met die oog op die vermindering van patogeenindringing. Verskeie publikasies het reeds gerapporteer oor verminderde Botrytisvatbaarheid in PGIP transgeniese plantlyne. Geeneen van hierdie publikasies kon egter uitbrei op die huidige hipotese aangaande die moontlike in planta-funksie van PGIP in plantsiekteweerstand nie. In hierdie studie is transgeniese tabaklyne wat PGIP ooruitgedruk gebruik om hierdie moontlike in planta-funksies vir PGIP uit te klaar. Transkriptoom- en hormonale analises is op hierdie plantlyne en ‘n WT voor en ná inokulasie met die nekrotroof Botrytis cinerea uitgevoer,. Transkriptoomanalises is uitgevoer op ongeïnfekteerde, sowel as geïnfekteerde tabakblaarmateriaal deur gebruik te maak van ‘n Solanum tuberosum-mikroraster. Die analises met gesonde, ongeïnfekteerde plantmateriaal het daarop gewys dat gene betrokke by selwandmetabolisme tussen die transgeniese lyne en die WT verskillend uitgedruk was. Dit kon bewys word dat, sonder infeksiedruk, die geen wat xiloglukaan-endotransglikosilase (XET) kodeer, in die transgeniese lyne afgereguleer was. Gene wat betrokke is in die lignien-biosintetiese pad was ook in die individuele transgeniese lyne beïnvloed. Biochemiese toetse het ook die aanduiding van verhoogde ligniendeposisie in die transgeniese lyne se selwande bevestig. Addisionele fitohormoonprofiele het getoon dat hierdie lyne ook beskik oor verhoogde vlakke van indoolasynsuur (IAA). Hierdie resultate wys daarop dat konstitutiewe vlakke van PGIP selwandmetabolisme in die Vvpgip1-transgeniese lyne moontlik kan beïnvloed, wat plantsiekteweerstand in dié lyne positief kan beïnvloed. Dit wil dus voorkom asof PGIP 'n bykomende funksie in plantsiekteweerstand het. Plantweerstandsreponse kan direk deur PGIP beïnvloed word, wat tot die versterking van plantselwande kan lei; dit kan geskied by wyse van die strukturele eienskappe van die proteïen of die integrasie daarvan in die selwand. Hierdie selwande is dus “voorberei” alvorens patogeenindringing plaasvind en kon bydra tot die verminderde siektevatbaarheid wat waargeneem is in lyne wat hoë vlakke van PGIP akkumuleer. Transkriptoom- en hormonale analises is ook uitgevoer op Botrytisgeïnfekteerde blaarmateriaal van beide die transgeniese lyne en ‘n WT. Verskeie Botrytis-responsgene is in beide die transgeniese lyne en die WT opgereguleer. Differensïele geenekspressie tussen die twee genotipes was taamlik beperk, maar in die analises kon ‘n geen geïdentifiseer word wat tweevoudig in die transgeniese lyne opgereguleer was in vergelyking met die WT. Hierdie resultaat is ook bevestig met behulp van die “Real-Time” Polimerasekettingreaksie (PKR). Hierdie geen is betrokke in die lipoksigenase (LOX) -pad (spesifiek die 9-LOXarm), wat tot die sintese van die diviniel-eter oksilipiene “colneleic-” en “colnelenic”-suur lei. Daar is al bewys dat hierdie twee verbindings Botrytisspoorontkieming kan inhibeer. Fitohormoonprofiele van die geïnfekteerde plante het gewys dat die transgeniese lyne verhoogde vlakke van die poel van jasmonate wat plantsiekteweerstands-hormone is, ná inokulasie akkumuleer. Hierdie hormone word in die 13-LOX-arm van die lipoksigenase pad gevorm en is belangrik vir die beperking van Botrytis by die infeksiesetel. Die resultate van die analises wat op Botrytis-infeksie volg, dui daarop dat beide arms van die lipoksigenasepad in die transgeniese lyne verskillend by die lokale respons geïnduseer word. ‘n Verhoogde induksie van ‘n ander plantsiekteweerstandshormoon, salisielsuur, kon ook opgemerk word, alhoewel die totaal geakkumuleerde vlakke nie beduidend hoër was as dié van die WT nie. Hierdie resultate is die eerste wat onderskeidende induksie van ‘n siekteweerstandspad in enige van die pgip-ooruitgedrukte plantlyne rapporteer. Daarmee ondersteun dit ook die hipotese dat, seintransduksie wat plantweerstandsresponse aanskakel, ná inhibisie van ePG deur PGIP plaasvind. Die resultate wat met hierdie studie verkry is, dra dus beduidend by tot die huidige kennis van die in planta-funksie van PGIP in plantsiekteweerstandsresponse.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2007. / Biogenic amines are nitrogenous compounds of low molecular weight found in most fermented foods, including wine. These biologically produced amines are essential at low concentrations for normal metabolic and physiological functions in animals, plants and micro-organisms. However, biogenic amines can have adverse effects at high concentrations and pose a health risk for sensitive individuals. Symptoms include nausea, hot flushes, headaches, red rashes, respiratory distress and fluctuations in blood pressure. A number of countries have implemented upper limits for histamine in food and wine. This development has already started to threaten commercial export transactions and may become more serious in the near future, especially in the competitive wine industry of today. The most important biogenic amines in wine include histamine, tyramine, putrescine, cadaverine and phenylethylamine which are produced from the amino acids histidine, tyrosine, ornithine, lysine and phenylalanine respectively. Biogenic amines are mainly produced in wine by microbial decarboxylation of the corresponding precursor amino acid. It may be produced by yeast during alcoholic fermentation, by lactic acid bacteria during malolactic fermentation, or potentially by spoilage microbes such as acetic acid bacteria and Brettanomyces. However, lactic acid bacteria are widely accepted as the main causative agents. Inoculation with commercial malolactic fermentation starter cultures that do not possess the relevant decarboxylase genes may inhibit the growth and activity of decarboxylase positive indigenous bacteria and as such control the production of biogenic amines in wine. In this study it was shown that co-inoculation of malolactic starter cultures together with alcoholic fermentation could reduce the incidence of biogenic amines in wine compared to conventional inoculation protocols; presumably because undesirable activities were restrained at an earlier stage during co-inoculation. It was also indicated in this work that in some cases the effect of co-inoculation on biogenic amine reduction may only be visible after a period of ageing. The frequency of biogenic amine occurrence in wines aged for a short period was generally higher in the presence of fermentation lees than in its absence. This work also included a preliminary investigation into the contribution of commercial wine yeast starter cultures to biogenic amine production. Diamines and polyamines (putrescine, spermidine and cadaverine) were produced to variable extents by all yeasts with very little or no production of physiologically important biogenic amines (histamine, tyramine and phenylethylamine). Another objective of this study was to evaluate the influence of common winemaking practices on biogenic amine production under winemaking conditions. We have shown that biogenic amine production by lactic acid bacteria could be influenced, amongst others, by the presence of precursor amino acids in the grape must or wine, the time of contact between juice or wine and grape skins, the time of contact between wine and yeast lees, the presence of microbial nutrients, wine pH, sulphite and ethanol levels, the phenolic composition of the wine and the number of decarboxylase positive lactic acid bacteria present in the wine. Lately, the wine industry is under increasing pressure to increase measures to ensure food safety and security and to eliminate any compound, present even in trace amounts that could reduce the wholesomeness of the wine. The need arises for a rapid and inexpensive method for quality control. In this study we investigated the potential to use Fourier transform infrared spectroscopy to rapidly screen for the presence of elevated levels of biogenic amines. This presents a novel method for the detection and quantification of total biogenic amines in wines.

Thesis (MSc (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2002. / Pathogen devastation of food products has been the topic of extensive research efforts worldwide. Fungal infections are foremost amongst these pests, contributing not only to losses in product yield, but also significantly affecting the quality thereof. It is not surprising then that producers of these foodstuffs and their derived products continually strive towards the highest possible product quality. Therefore, it remains imperative that satisfactory methods are implemented to control these fungal pathogens. The current strategies are all hampered by drawbacks, and severe crop losses are still experienced. New technologies are being explored; one such technology is the genetic transformation of plant species. This method has enabled scientists to introduce foreign genes, with known functions and predictable outcomes, into plants. Genes identified to be involved in disease resistance have become the focus of numerous research efforts concerned with the improvement of the plant's innate defence response. This study aimed to enhance disease resistance to fungal pathogens by means of the genetic transformation of two genes previously shown to be involved in disease resistance. These genes encode polygalacturonase-inhibiting proteins (PGIPs) from Phaseolus vulgaris and resveratrol synthase from Vitis vinifera. PGIPs specifically inhibit the action of fungal polygalacturonases (PGs), which are enzymes responsible for the hydrolytic breakdown of plant cell walls. These enzymes were also found to be the first enzymes that are secreted by fungal pathogens during infection of the host plant. Additionally, PGIP-PG interaction results in the existence of molecules involved in the activation of plant defence responses. Resveratrol, the product of resveratrol synthase, exerts its antifungal action by destruction of the microbial cellular membranes. These mentioned genes were transformed alone, and in combination, into Nicotiana tabacum and the resultant transgenic lines were evaluated for enhanced disease resistance and for possible synergistic effects between the transgenes. Several independent transgenic lines were regenerated with genes integrated into the tobacco genome. Almost all the plants harbouring only pgip or vst1 genes also expressed these genes at a high frequency. Some non-expressing lines were identified from the transgenic plants that had integrated both genes, but several lines were obtained expressing both transgenes. Good correlations were observed between transgene product activity and enhanced resistance to the fungus Botrytis cinerea in an antifungal in planta assay. Lines showing the highest PGIP activity and resveratrollevels were more resistant to the pathogen, leading to disease resistance of up to 80% seven days after inoculation in comparison to an untransformed control. These lines maintained their strong inhibition, even three weeks post-inoculation, showing a complete halt in disease development and fungal growth. These results provide good indications of the efficacy of these transgenes in the upregulation of plant defence. However, the study will have to be expanded to include even more transgenic lines to elucidate the possible synergistic effects of these genes. In an additional pilot study, genes encoding for precursors and for the formation of resveratrol were introduced into the yeast Saccharomyces cerevisiae. The resultant recombinant yeast strains were evaluated for their ability to produce the phenolic substance, resveratrol. This compound has been implicated in beneficial aspects relating to human health, including positive effects on atherosclerosis and platelet aggregation as a direct result of its antioxidant and anti-inflammatory activities. Recombinant yeast strains were constructed that expressed genes coding for coenzyme A ligase and resveratrol synthase. These strains were shown to be able to produce the phenolic compound resveratrol from the precursors present in the yeast as well as from the products introduced with the transformation. The resveratrol was complexed with an added glucose moiety. These results are extremely positive, considering the possibility of manipulating wine yeasts to produce resveratrol during the wine fermentation, thereby adding to the health aspects of both red and white wine. This is the first report of the production of this compound by the introduction of genes necessary for its biosynthesis in a foreign host. This study has confirmed the importance of PGIPs and resveratrol in the effort to enhance disease resistance in plants through genetic transformation technology. It has also shown that the health benefits of resveratrol could be exploited more optimally in the wine industry, by producing wine yeasts with the ability to synthesise this important antioxidant.