Fungal diseases pose significant challenges for grapevine producers in Kentucky due to the region’s abundant moisture and relative humidity. Methods to reduce fungicide application frequency would prove both economically and temporally valuable to producers. A field experiment was established in Bowling Green, KY in 2017 to investigate Bacillus mycoides isolate J (LifeGard) as a supplement to a fungicide program for systemic acquired resistance (SAR). Three fungicide treatment regimens were implemented consisting of a program modelled from the Midwest Fruit Pest Management Guide (2017) and an identical program supplemented with 140 g ha-1 LifeGard per application (both applied on 14 day intervals), a reduced frequency application every 28 days supplemented with 140 g ha-1 LifeGard, and an untreated control. Treatments were applied to 9-year-old French-Hybrid grapevines (cv. Chambourcin); each treatment was replicated 3 times in a randomized complete block design. All treatments were applied with a backpack sprayer delivering 150 L ha-1 at 2 Bar pressure. Canopy management, fertility, herbicide, and insect management were standardized across treatments and no supplemental irrigation was applied. Data collected included fruit yield, pH, ºBrix, and titratable acidity (TA). Data were analyzed with SAS PROC GLIMMIX; differences in means were determined at  < 0.05. Plots supplemented with B. mycoides had lower fruit pH than untreated plots but higher fruit pH than the traditional fungicide program. Treatment regime did not influence Brix, TA, or total yield; however, all treated plots yielded more high quality fruit than the untreated control.

American Viticultural Areas (AVAs) are susceptible to small variations in climate and microclimates and are found within a narrow latitudinal range of prime climate conditions. These AVAs are geographically determined based on the best soil, climate, precipitation and temperature combinations for specific winegrape regions. As climate change continues to alter the local weather and the greater climate region of the Western United States, winegrape growing regions in Oregon are being affected. In an effort to determine what the pattern of change is, and compare previous studies of climate change using climate indices, a comparative study based in part on prior research was conducted. Using 800 meter resolution Parameter-elevation Regressions on Independent Slopes Model (PRISM) climate datasets, four individual climate indices were analyzed for statistical correlation with the climate data. These climate indices are: growing degree-days (GDD), the average growing season temperatures (GST), Huglin Index (HI) and the biologically effective degree-day (BEDD). Based on currently available data for this research, these climate indices were statistically analyzed during the years 2000 to 2010. A further avenue of research included a statistical analysis of the reported winegrape production, although this data was available only at an aggregated county-level. Results show that all four climate indices exhibit statistical significance, although the inclusion of the winegrape production data exhibited no statistical significance for many of the analyses, most likely due to subjective and aggregated data, few did result in significance with the climate indices. The research discussed here confirms the accuracy of the four climate indices and suggest that a longer time frame, coupled with less aggregated and subjective winegrape production data could produce interesting results in future research on the results of climate indices in winegrape growing regions.

A two-year study was conducted at a commercial vineyard in California’s Templeton Gap AVA to evaluate the effect of vine age on viticultural, enological, and sensory attributes of Vitis vinifera L. cv. Zinfandel grapes and wines. The experimental block was dry farmed, conventionally managed, with own-rooted Zinfandel vines that, when determined unproductive, were replaced with new vines of genetically identical scion plant material grafted onto St. George (Vitis rupestris Scheele) rootstock. Treatments included Young vines (5 to 12 years old), Control (representative proportion of young to old vines in the block), and Old vines (40 to 60 years old). Results indicated Young vines progressed more slowly during berry formation and more rapidly during berry ripening than Old vines. Due to variation in the timing of sugar accumulation, Old vines were harvested 21 days after Young vines in 2019, and 9 days after in 2020. Old vines produced, on average between both seasons, 3.7 kg more fruit per vine than Young vines. Old vines also produced, on average between both seasons, 22.8 more clusters per vine than Young vines (5.41 tons/acre and 2.64 tons/acre, respectively). The larger vine capacity observed was attributed to Old vines having more arm, spur and dormant bud positions per vine than Young vines, in addition to larger trunk circumference and diameter. Vine age also had an effect on vine vegetative growth, with Old vines producing shorter internodes (25.5% decrease) and smaller shoot diameters (29.3% decrease) compared to Young vines. Young vines had higher mid-day stomatal conductance and tended to have higher mid-day photosynthetic rates, although no differences in corresponding pre-dawn measurements were found. While root architecture was similar between age groups, Old vines displayed greater rooting depths. Young vine wines had lower pH and titratable acidity than Old vine wines. Old vine wines were defined by a wider array and intensity of aromatics, including raisin, orange peel, black fruit and spices relative to Young vine wines which were defined by wet topsoil and pomegranate aromas. These results suggest the potential for greater yield and improved wine quality when extending the longevity of Zinfandel vineyards.

Descriptive sensory analysis is essential to understanding how differing varietals, viticultural techniques, enological techniques, vintage, regions, etc. impact the sensory characteristics found in wines. These studies analyzed the sensory impacts of vine age, whole cluster and stem addition influences, the frequency of cap management regimes, and selected fermentation variables, on 2019 Zinfandel, 2020 Syrah, 2020 Tannat, 2020 Cabernet Sauvignon, and 2021 Graciano wines respectively, from the Central Coast of California, USA. The investigation of Old vine Zinfandel indicated that these wines were defined as complex due to the wide variety of descriptors used to characterize them, having high color saturation and long length, while the Young vine wines were described as considerably less aromatically diverse, yet being defined by high intensity wet topsoil and pomegranate aromas. Acknowledging the absence of literature on classifying Californian Tannat, descriptors such as high color saturation, purple hue, cooked blue fruit, violet, raisin, bitterness, and astringency may serve as descriptors to these uncommon wines. Regarding stem addition, Dried stem wines exhibited the highest color saturation, purple hue, and astringency intensities in both the Syrah and Tannat wines. Generally, the Green stem wines were less lifted, however the presence of stems added a sense of freshness. The Whole cluster and Control wines generally opposed each other. With reference to cap management, as punch down (PD) increased, perceptions of the overall astringency, bitterness, and velvety characteristics increased. Concerning the various fermentation variables, dirty (unwashed grapes prior to fermentation) wines were described by the earthy aroma, washed (washed grapes prior to fermentation) wines by the floral aroma, and spontaneous (uninoculated) wines by the fruity aromas; however, fruity was the predominant aroma across most wines.

A two-year study was conducted to assess the effects of cofermentation on red wine varietals. During the winemaking process, wines can be made from two or more varieties by picking, crushing and fermenting them together, a practice known as cofermentation. They can also be blended either after the completion of alcoholic fermentation or after malolactic fermentation. In the first year of the study, two grape varieties, Merlot (Mer), and Malbec (Mal) were cofermented. On the second year, a third varietal, Petite Sirah (PS) was also studied. Cofermented wines containing every possible binomial combination of the varietals was made and one trinomial on 2019. The cofermented wines were compared to monovarietal wines and also to wines that were produced by blending either after alcoholic fermentation or after malolactic fermentation. The phenolic profile of the wines was followed from the onset of fermentation up to 36 months of bottle aging for the 2018 vintage and in the case of the 2019 vintage, up until 250 days after crushing. In 2018, cofermented wines and wines that were blended after malolactic fermentation had an anthocyanin profile that was more similar to Malbec than to Merlot, while the tannin profile was more resemblant of Merlot. In 2019 cofermentation improved the anthocyanin content when compared to post alcoholic and post malolactic blend only when the three varietals were cofermented. A sensory analysis with 10 trained individuals was conducted on the 2018 vintage. It was demonstrated that Malbec wines had a higher amount of red fruit aromas while Merlot wines were perceived as being more astringent. Cofermented and post malolactic fermentation blended wines were indistinguishable to panelists and blending after alcoholic fermentation produced wines that highlighted the individual varietal character.

Thesis (MScAgric (Viticulture and Oenology))--Stellenbosch University, 2008. / Soil is a component of the environment and sustains growth of several plants and animals. It forms part of the biosphere and can be described as the interface between the atmosphere and the lithosphere. The interaction between soil, climate and topography and the resulting agricultural aptitude forms the concept of terroir. This relationship is complex and it is difficult to quantify the contribution of each. Grapevines are exposed to an array of soil types. Soils have varying colours, which can be ascribed to their origin from different parent materials and pedogenetic factors. Historical and experimental evidence points to the key role that soil physical conditions play in determining grape berry composition, but other soil related factors may also play a role. This study was conducted to investigate the effect of soil surface colour on the vegetative and reproductive growth characteristics of Cabernet Sauvignon. The aim was to determine whether a relationship exists between soil colour, reflective light quality below and inside the grapevine canopy, vegetative growth of the grapevine and the berry and wine composition. The reflected light from soils was measured in three positions of the canopy and across the light spectrum (300–2500 nm) for three different soil surface treatments (black, red and grey). The effect of soil colour on vegetative parameters, yield and berry composition and wine quality was investigated. Soil surface colour resulted in differences in the reflected light quality below and in the canopy. The differences in the light quality were associated with differences in vegetative parameters such as mean main leaf, with grey soils inducing higher values. Potassium levels of the grapes and berry number per bunch appeared to be influenced by soil surface colour throughout berry development with red and black soils having higher levels of potassium and berry number per bunch than grey soils. Grape ripening parameters were not influenced by soil surface colour, but the grey treatment had a significantly more intense grape colour measured at 520 nm (red pigments). It is assumed that the importance of soil colour is its association with the physical and the pedogenetic properties that contribute to the grapevine water balance. From these results it can be concluded that soil surface colour appeared to have a direct effect on some aspects of vegetative and reproductive growth, and berry composition, but the contribution of different wavebands and mechanism of their effect deserves further study.

Thesis (MscAgric (Viticulture and Oenology))--University of Stellenbosch, 2007. / The rapid expansion of the world wine industry has increased the pressure on wine producers to produce high quality, distinguishable wines. The use of sensory evaluation alone as a tool to distinguish between wines is limited by its subjective nature. Chemical characterisation using analytical methods and data analysis techniques are increasingly being used in conjunction with sensory analysis for comprehensive profiling of wine. Analytical chemistry and chemometric techniques are important and inextricable parts of the chemical characterisation of wine. Through this process insight into the inherent composition of wines, be it in a general sense or related to a particular wine category is gained. Data generated during chemical characterisation are typically compiled into electronic databases. The application of such information towards wine quality control includes the establishment of industry benchmarks and authentication. The current project is part of The South African Young Wine Aroma Project, a long term research initiative funded by the South African Wine Industry with the ultimate aim to establish a comprehensive, up-to-date, database of the volatile composition of young wines. The data generated during this thesis represent the first contribution towards realising this ambition. Three clearly defined aims were set for this project, the first of which is the chemical characterisation of South African young wines in terms of selected volatile and non-volatile compounds and Fourier transform infrared spectra, with particular focus on the volatile composition. FTMIR spectra are information rich and non-specific instrumental signals that could provide invaluable information of the inherent composition of the wines. The second aim is the evaluation of the analytical methods used to generate the data and in the last instance, the optimisation of FTMIR spectroscopy for rapid quantification of major wine parameters and volatile compounds. The concentrations of 27 volatile compounds in South African young wines were determined by gas chromatography coupled to flame ionisation detection (GC-FID) using liquid-liquid extraction of the analytes. Wine samples of the 2005 and 2006 vintages produced from six of the most important cultivars in the South African wine industry, namely Sauvignon blanc, Chardonnay, Pinotage, Cabernet Sauvignon, Merlot and Shiraz were used. The producing cellars were from four major South African wine producing regions, namely Stellenbosch, Paarl, Robertson and Worcester. The data captured made a significant contribution to the establishment of the Aroma Project Database. Univariate statistics showed wide variations in the chemical composition of the wines. Red wines were generally characterised by high levels of higher alcohols and white wines by high levels of esters. Most of the differences between vintages were cultivar dependent and phenological differences between cultivars were suggested as a possible cause. Fusel alcohols, iso-acids and esters of fusel alcohols were particularly responsible for differences between red wines. A combination of fatty acids and higher alcohols were responsible for differences between production regions. However, using univariate statistics alone was limited in identifying characteristic features of the chemical composition of the wines. In order to explore the correlations between the volatile components, FTMIR spectra and nonvolatile components the data were further investigated with multivariate data analysis. Principal component analysis was successfully employed to distinguish between wines of different vintages and cultivars. The role of the volatile composition was more influential in the separation of vintage and red wine cultivar groupings than the non-volatile components or the FTMIR spectra. Almost all the individual volatile components contributed to the separation between the vintages and cultivars, thereby highlighting the multivariate nature required to establish the distinguishing features pertaining to each of these categories. The FTMIR spectra and the non-volatile components were more important than the volatile components to characterise the differences between the white cultivars. It was not surprising that both the volatile components and the FTMIR spectra were needed to distinguish between both red and white cultivars simultaneously. It was of interest the full spectrum, including all wavenumbers were required for a powerful classification model. This finding supports the initial expectation that the non-selective but information rich signal captured in the FTMIR spectra is indispensable. No distinction could be made between the production regions, which was not surprising since the wines used in this study was not of guaranteed origin. Furthermore, no clear correlation could be established between the chemical composition or the FTMIR spectra and the quality ratings of the wines. Limitations in the dataset were pointed out that must be taken into account during further investigations in the future. The liquid-liquid extraction method used during the analysis of the volatile components was evaluated for precision, accuracy and robustness. Generally good precision and accuracy were observed. There were slight indications of inconsistencies in the recoveries of analytes between the red and white wine matrices. Certain parameters of the protocol, namely sample volume, solvent volume, sonication temperature and sonication time, were identified as factors that had a major influence on quantification. The results obtained in this study made a major contribution towards establishing this technique for routine GC-FID analysis in our environment. Due to the high sample throughput in wine laboratories, the use of rapid quantitative analytical methods such as FTMIR spectroscopy is becoming increasingly important. Enzymatic-linked spectrophotometric assays and high performance liquid chromatography (HPLC) methods were evaluated for their suitability to serve as reference methods for optimising and establishing FTMIR calibrations for glucose, fructose, malic acid, lactic acid and glycerol. Pigmented and phenolic compounds were identified as sources of interference in the determination of organic acids in red wines with both enzymatic assays and HPLC. The use of fining treatments for the decolourisation of red wine samples was investigated. Activated charcoal was more efficient in terms of colour removal than polyvinyl polypyrrolidone (PVPP), but neither were compatible with the specific enzymatic method used in this study. Solid phase extraction (SPE), a method commonly used during sample clean-up prior to HPLC analysis of organic acids in wine, and PVPP fining were evaluated as sample preparation methods for HPLC analysis to optimise the quantification of organic acids in red wine. Four different types of SPE cartridges were evaluated and the SPE method was optimised in order to recover the maximum amount of organic acids. However, low recoveries, in some instance less than 50%, for the organic acids in wine were reported for the optimised SPE method. In this respect one was the worst. On average, excellent recoveries were observed for the organic acids using the PVPP method that were in excess of 90%. This method therefore provides a very valuable and simple alternative to SPE for sample-cleanup prior to HPLC analysis. One aspect that still needs to be investigated is the reproducibility of the method that should still be optimised. In general, enzymatic analysis was more suitable for the determination of glucose and fructose, while HPLC analysis were more suitable for the quantification of organic acids. Efficient glycerol quantification was observed with both enzymatic and HPLC analysis, although a lower measurement error was observed during the HPLC analysis. Apart from reliable reference methods, successful FTMIR calibrations also rely on the variability present in the reference sample set. The reference sample set used to establish FTMIR calibrations must ideally be representative of the samples that will be analysed in the future. Commercial, or so-called global, FTMIR calibrations for the determination of important wine parameters were evaluated for their compatibility to a South African young wine matrix. The prediction pH, titratable acidity, malic acid, glucose, fructose, ethanol and glycerol could be improved by establishing a brand new FTMIR calibration, thereby clearly indicating that the South African young wine matrices were significantly different from the samples of European origin that were used to establish the commercial calibrations. New preliminary calibration models were established for a young wine sample matrix and were validated using independent test sets. On average the prediction errors were considered sufficient for at least screening purposes. The effect of wavenumber selection was evaluated. Relatively successful models could be established for all the compounds except glucose. Wavenumber selection had an influence on the efficiency of the calibration models. Some models were more effective using a small amount of highly correlated wavenumbers, while others were more effective using larger wavenumber regions. Preliminary FTMIR calibration models for the screening of volatile compound groups in young wines were evaluated. Compound groups were compiled based on chemical similarity and flavour similarity. Good linearity were observed for the “total alcohol”, “total fatty acids”, “esters” models while an interesting polynomial trend was observed for the “total esters” model. Relatively high prediction errors indicated the possibility of spectral interferences, but the models were nevertheless considered suitable for screening purposes. These findings are a valuable contribution to our environment where fermentation flavour profiles must often be examined. The important role sound and validated analytical methods to generate high quality analytical data, and the subsequent application of chemometric techniques to model the data for the purpose of wine characterisation has been thoroughly explored in this study. After a critical evaluation of the analytical methods used in this study, various statistical methods were used to uncover the chemical composition of South African young wines. The use of multivariate data analysis has revealed some limitations in the dataset and therefore it must be said that wine characterisation is not just reliant on sophisticated analytical chemistry and advanced data analytical techniques, but also on high quality sample sets.

Thesis (MscAgric (Viticulture and Oenology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Little scientific information regarding the effect of different geological parent materials on grapevine performance is currently available in South Africa. This aspect is of special significance for the Helderberg area, where parent material may change from granite to shale over a short distance. This results in shale- as well as granite-derived soils often occurring within the same vineyard. The objectives of this study were to (i) quantify the nutritional status and other soil properties of different parent materials (shale and granite) and overlying soils (ii) investigate the impact of geological differences in the soil on the vine nutritional status and certain vine parameters. This study was done over two seasons (2006/2007 and 2007/2008). Two Sauvignon blanc and two Cabernet Sauvignon vineyard blocks were selected at two different localities for each cultivar in the Helderberg area, South Africa. Shale- and granitederived soils were identified within each block. Kaolinite was the dominant mineral, whereas quartz and feldspar were sub-dominant. Traces of mica were also present in some shale- and granite-derived soils. Granite- contained significantly higher contents of coarse sand than shale-derived soils, whilst the opposite occurred in terms of fine sand. These differences affected the water holding capacity, in general making it higher in shale- than granite-derived soils. Shale-derived soils had higher concentrations of total K but granite-derived soils had a higher ability to release K as they contained higher concentrations of soluble K. The Q/I parameters, potential buffering capacity of K (PBCK) and equilibrium activity ratio of K (ARK) showed no consistent responses to geological differences. Potassium concentrations were higher in the leaf blades (obtained before harvest in 2007) from Sauvignon blanc grapevines on granite- than on shale-derived soils. Potassium concentrations in the Cabernet Sauvignon juice (obtained in 2007) tended higher in juice from granite- than from shale-derived soils. In 2008, K concentrations tended higher in juice from shale- than from granite-derived soils for both cultivars. The pH of the Sauvignon blanc juice (obtained in 2008) tended higher in juice from shale-than from granite-derived soils, thus corresponding with the K concentrations in the juice in this season. Nitrogen concentrations were higher in Cabernet Sauvignon juice (obtained in 2007) and Sauvignon blanc juice (obtained in 2008) from shalethan from granite-derived soils. In terms of vine water status, vines on granite-derived soils appeared more stressed than those on shale-derived soils in both seasons at one of the vineyards. In these Sauvignon blanc and Cabernet Sauvignon vineyards, the K nutritional status was not affected by geology in a consistent manner but there were some noticeable tendencies for a specific cultivar during certain seasons. On account of vines being planted on shale- and granite-derived soils within the same block, soil preparation was done similarly for both soils, and they were exposed to similar irrigation schedules, canopy management strategies and climatic conditions. Therefore, there is a high probability that all these practices may have negated the effect of geology on the K status of soils and especially on juice K concentration at the time of harvest. It was clear that seasonal differences and fertilisation affected the nutritional status of the vines more than geology. / AFRIKAANSE OPSOMMING: In Suid-Afrika is daar tans min wetenskaplike inligting oor die effek van verskillende geologiese moedermateriale op die prestasie van wingerd beskikbaar. Hierdie aspek is veral van belang in die Helderberg-area, waar moedermateriaal oor ‘n baie kort afstand van graniet na skalie kan wissel. Dit lei daartoe dat skalie-, sowel as granietgronde, dikwels binne dieselfde wingerd voorkom. Die doelwitte van die studie was om: (i) die voedingstatus en ander grondkundige eienskappe van die verskillende moedermateriale (skalie en graniet) en oorliggende gronde te kwantifiseer (ii) die impak van geologiese verskille in die grond op wingerd se voedingstatus en sekere wingerdkundige parameters, te ondersoek. Hierdie studie is oor twee seisoene (2006/2007 en 2007/2008) gedoen. Twee Sauvignon blanc en twee Cabernet Sauvignon wingerdblokke is geselekteer by twee verskillende lokaliteite vir elke kultivar in die Helderbergarea, Suid-Afrika. Beide skalie- en granietgrond is binne elke blok geïdentifiseer. Kaoliniet was die dominante mineraal, met kwarts en veldspaat sub-dominant, terwyl spore van mika ook in beide skalie- en granietgronde gevind is. Granietgronde het betekenisvol hoër hoeveelhede growwe sand bevat, terwyl skaliegronde meer fyn sand bevat het. Hierdie verskille het ‘n effek op waterhouvermoë gehad en daartoe gelei dat waterinhoude oor die algemeen hoër was vir skaliegronde. Skaliegronde het groter hoeveelhede totale K bevat, maar granietgronde se vermoë om K vry te stel was hoër, omdat hulle ‘n hoër konsentrasie oplosbare K bevat het. Die Q/I parameters, potensiële buffervermoë vir K (PBCK) en ewewig aktiwiteitsverhouding vir K (ARK), is nie op ‘n konsekwente wyse deur geologiese verskille beïnvloed nie. Vir die Sauvignon blanc wingerde was kalium konsentrasies in blaarskywe (gemonster voor oes in 2007) hoër vir graniet- as vir skaliegronde. Kalium konsentrasies in die sap vanaf Cabernet Sauvignon (gemonster in 2007) het hoër geneig vir granietgronde. In 2008 het die kalium konsentrasies, vir beide kultivars, hoër geneig in sap vanaf skaliegronde. Gedurende dié seisoen het die pH van sap ook hoër geneig vir Sauvignon blanc vanaf skaliegronde, wat dus ooreenstem met die K inhoud van die sap. Stikstof konsentrasies was hoër in sap vanaf skaliegronde vir Cabernet Sauvignon (2007) en vir Sauvignon blanc (2008). In terme van die wingerde se waterstatus, het stokke op die granietgrond, by een van die lokaliteite, geneig om gedurende beide seisoene onder groter stremming te wees op graniet as op skaliegrond. In hierdie Sauvignon blanc en Cabernet Sauvignon wingerde, is K voedingstatus nie op ‘n konsekwente wyse deur geologie geaffekteer nie, maar gedurende sommige seisoene was daar wel duidelike tendense vir ‘n spesifieke kultivar. Omdat die stokke binne dieselfde blok op skalie- en graniet gronde geplant is, was grondvoorbereiding eenders vir die twee grondtipes terwyl besproeiingskedule, lowerbestuur en klimaatstoestande ook identies was. Daar is dus ‘n hoë waarskynlikheid dat al hierdie faktore daartoe kon bygedra het dat die effek van geologie op die K status van van gronde versluier is, veral die effek op die K inhoud van sap teen oestyd. Dit was duidelik dat seisoenale klimaatsverskile en bemestingspraktyke ’n groter effek as geologie op die voedingstatus van die wingerd gehad het.

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 (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Necrotrophic fungi infect many economically important crop plants. This results in great losses in the agricultural sector world-wide. Understanding the nature by which plants respond to pathogens is imperative for genetically enhancing disease resistance in plants. Research tools have significantly contributed to our understanding of how the plant responds to pathogen attack, identifying an array of defence mechanisms used by plants upon attack. Many fungal pathogens secrete endopolygalacturonases (endoPGs) when infecting plants. These hydrolytic enzymes are inhibited by polygalacturonase-inhibiting proteins (PGIPs) associated with plant cell walls. PGIPs are well characterised and their current known functions are all linked to endoPG inhibition and the subsequent upregulation of plant defence pathways. Work on grapevine PGIPs have shown that apart from being efficient antifungal proteins, leading to protection of the plant against Botrytis cinerea when overexpressed, PGIPs might also have additional functions linked to cell wall strengthening. This working hypothesis formed the motivation of this study where a cinnamyl alcohol dehydrogenase (CAD) (1.1.1.195) gene was targeted for functional analysis in tobacco (Nicotiana tabacum). Some previous work and genetic resources obtained is relevant to this study, specifically previously characterized transgenic tobacco lines overexpressing the Vitis vinifera pgip1 (Vvpgip1) gene. These lines have confirmed PGIP-specific resistance phenotypes against B. cinerea, as well as increased levels of CAD transcripts in healthy plants. Moreover, preliminary evaluations indicated increased lignin levels as well as differential expression of several other cell wall genes in these overexpressing lines (in the absence of infections). In this study we generated a transgenic tobacco population, overexpressing the native CAD14 gene, via Agrobacterium transformations. The transgene was overexpressed with the Cauliflower Mosaic Virus promoter (CaMV 35Sp). The CAD transgenic population was analyzed for transgene integration and expression and showed active transcription, even from leaves that normally don’t express CAD to high levels. These lines, together with the untransformed control, and a representative transgenic VvPGIP1 tobacco line previously characterized with elevated expression of CAD were used for all further analyses, specifically CAD activity assays of stems and leaves, as well as whole plant infections with B. cinerea. CAD enzyme activity assays were performed on healthy uninfected plant lines, without inducing native CAD expression or resistance phenotypes (i.e. without Botrytis infection). CAD activity was detected in leaves and stems, but a statistically sound separation between the CAD population and the untransformed control was only observed in the stems. The CAD assays also confirmed previous results that indicated that CAD transcription was upregulated in the PGIP line in the absence of infection. Overall, in all plant lines the stems exhibited 10-fold higher levels of CAD activity than the leaves, but the transgenic VvPGIP1 line showed a further 2-3-fold increase in CAD activity in the stems, when compared to the untransformed control and the majority of the CAD overexpressing lines. Disease assessment by whole plant infections with B. cinerea of the CAD transgenic plants revealed reduced disease susceptibility towards this pathogen. A reduction in disease susceptibility of 20 – 40% (based on lesion sizes) was observed for a homologous group of transgenic lines that was statistically clearly separated from the untransformed control plants following infection with Botrytis over an 11-day-period. The VvPGIP1 transgenic line displayed the strongest resistance phenotype, with reduction in susceptibility of 47%. The reduction in plant tissue maceration and lesion expansion was most pronounced in the VvPGIP1 line compared to the CAD transgenic plants, while the CAD transgenic plants showed more reduction than the untransformed control. In combination, the data confirms that CAD upregulation could lead to resistance phenotypes. Relating this data back to the previously observed upregulation of CAD in the VvPGIP1-overexpressing lines, the findings from this study corroborates that increased CAD activity contributes to the observed resistance phenotypes, possibility by strengthening the cell wall. In conclusion, this study yielded a characterized transgenic population overexpressing the CAD14 gene; this overexpression contributed to increased RNA transcription compared to the untransformed control plant, increased CAD activity (most notably in the stems) and a disease resistance phenotype against Botrytis. These findings corroborates the current working hypothesis in our group that PGIPs might have a role in preparing the plant cell for attack by contributing to specific cell wall changes. The exact mechanisms are still currently unknown and under investigation. The transgenic lines generated in this study will be invaluable in the subsequent analyses where these various phenotypes will be subjected to profiling and accurate cell wall analyses. / AFRIKAANSE OPSOMMING: Nekrotrofiese swamme infekteer en beskadig verskeie ekonomies belangrike gewasse. Dit lei wêreldwyd tot groot verliese vir die landbousektor. Dit is noodsaaklik om te verstaan hoe plante reageer teenoor patogene, sodat die siekteweerstand van plante verbeter kan word. Navorsingshulpbronne het beduidend bygedra tot die kennis van plantreaksies tydens patogeniese aanvalle, en het sodoende ‘n reeks van weerstandmeganismes, wat die plant inspan tydens ‘n aanval, geïdentifiseer. Verskeie patogeniese swamme skei endopoligalakturonases (endoPGs) af tydens plantinfeksie. Hierdie hidrolitiese ensieme word geïnhibeer deur poligalakturonase-inhiberende proteïene (PGIPs) wat met die plantselwand geassosieerd is. PGIPs is goed gekarakteriseerd en al hulle huidiglik bekende funksies is gekoppel aan endoPG inhibisie en die daaropvolgende opregulering van plant weerstandspaaie. Navorsing op wingerd PGIPs het gewys dat, afgesien van die feit dat PGIPs goeie antifungiese proteïene is wat lei tot beskerming van die plant teen Botrytis cinerea wanneer dit ooruitgedruk word, PGIPs ook moontlik addisionele funksies verrig wat verwant is aan selwandversterking. Hierdie werkshipotese vorm die motivering vir hierdie studie waarin ‘n sinnamiel alkohol dehidrogenase (SAD) (1.1.1.195) geen geteiken is vir funksionele analise in tabak (Nicotiana tabacum). Vorige navorsing en genetiese hulpbronne daardeur verkry is belangrik vir hierdie studie, spesifiek die gekarakteriseerde transgeniese tabaklyne wat die Vitis vinifera pgip1 (Vvpgip1) geen ooruitdruk. Hierdie lyne besit bevestigde PGIP-spesifieke weerstandsfenotipes teen B. cinerea, sowel as hoër vlakke van SAD transkripte in gesonde plante. Voorlopige analises het ook aangedui dat hierdie ooruitdrukkende lyne hoër lignien vlakke het, asook differensiële uitdrukking van verskeie ander selwandgene (in die afwesigheid van infeksie). In hierdie studie is ‘n transgeniese tabakpopulasie gegenereer wat die natuurlike tabak SAD14 geen ooruitdruk, deur middel van Agrobacterium transformasie. Die transgeen is ooruitgedruk deur die Blomkool Mosaïek Virus promoter (CaMV 35Sp). Die SAD transgeniese populasie is geanaliseer vir transgeen integrasie en uitdrukking en het aktiewe transkriptering getoon, selfs in blare waar daar normaalweg nie hoë vlakke van SAD uitgedruk word nie. Hierdie lyne, die ongetransformeerde wilde-tipe kontrole sowel as ’n verteenwoordigende transgeniese VvPGIP1 tabaklyn wat vooraf gekarakteriseerd was met hoë SAD uitdrukking, is gebruik vir alle verdere analises, spesifiek SAD aktiwiteitstoetse in stingels en blare, asook heelplantinfeksies met B. cinerea. Aktiwiteitsanalises van die SAD ensiem is gedoen op gesonde ongeinfekteerde plantlyne, sonder om natuurlike tabak SAD uitdrukking of weerstandsfenotipes te induseer (dus sonder Botrytis infeksie). SAD aktiwiteit is waargeneem in beide die blare en stingels, maar ‘n statisties betekenisvolle skeiding is slegs gevind tussen die SAD populasie en die ongetransformeerde kontrole in die stingels. Die SAD toetse het ook vorige resultate bevestig wat aangedui het dat SAD transkripsie opgereguleer word in die PGIP lyn in die afwesigheid van infeksie. Die stingels het oor die algemeen ‘n 10-voudige vermeerdering in SAD aktiwiteitsvlakke getoon in vergelyking met die blare, maar die transgeniese VvPGIP1 lyn het ‘n verdere 2-3-voudige verhoging in SAD aktiwiteit gehad in die stingels ,in vergelyking met die ongetransformeerde kontrole en die meerderheid van die SADooruitdrukkende lyne. Siekteweerstand ondersoeke deur middel van heelplantinfeksies met B. cinerea van die SAD transgeniese plante, het verminderde vatbaarheid aangedui ten opsigte van hierdie patogeen. ‘n Afname in siekte-vatbaarheid van 20 – 40% (gebaseer op wondgroottes) is waargeneem vir ‘n homoloë groep transgeniese lyne wat statisties betekenisvol geskei kon word van die ongetransformeerde kontrole plante na infeksie met Botrytis in ‘n infeksietoets wat 11 dae geduur het. Die VvPGIP1 transgeniese lyn het die mees weerstandbiedende fenotipe gehad, met ‘n afname in siekte-vatbaarheid van 47%. Die afname in plantweefselafbreking en wondgrootte was die duidelikste in die VvPGIP1 lyn in vergelyking met die SAD transgeniese plante, terwyl die SAD transgeniese plante ‘n groter afname aangedui het as die ongetransformeerde kontrole. In kombinasie het die data bevestig dat SAD opregulasie kan lei tot weerstandbiedende fenotipes. Hierdie data, in vergelyking met die vorige bevinding van opregulasie van SAD in die VvPGIP1-ooruitdrukkende lyne, bevestig dat hoër SAD aktiwiteit bydra tot die waargenome weerstandbiedende fenotipes, moontlik deur versterking van die plantselwand. Ter afsluiting, hierdie studie het ‘n gekarakteriseerde transgeniese populasie wat die SAD14 geen ooruitdruk gelewer; hierdie ooruitdrukking het bygedra tot hoër RNA transkripsie in vergelyking met die kontrole, verhoogde SAD aktiwiteit (veral in die stingels) en siekteweerstandbiedende fenotipes teenoor Botrytis. Hierdie bevindinge ondersteun die huidige werkshipotese in ons groep dat PGIPs moontlik ‘n rol speel in die voorbereiding van die plantsel teen infeksie deur spesifieke selwandveranderinge te veroorsaak. Die spesifieke meganismes is steeds onbekend en word verder ondersoek. Die transgeniese lyne wat tydens hierdie studie gegenereer is, sal baie belangrik wees in opvolgende analises waar hierdie verskillende fenotipes gebruik kan word om die profiel van selwandkomponente, maar ook die akkurate selwandsamestelling te bestudeer.