Hydraulic conductance of roots of the grapevine cultivar, Chardonnay, varies diurnally, peaking at 1400 h. The diurnal amplitude of hydraulic conductance between 600 and 1400 h was not altered when potted grapevines were water-stressed by withholding water for 8 days. However, the diurnal change was greatly reduced for water-stressed Grenache. If the diurnal change in root hydraulic conductance is a result of changes in aquaporin gene expression or activity, it suggests that aquaporins respond differently in water-stressed Chardonnay and Grenache roots. Both Chardonnay and Grenache demonstrated a reduction in hydraulic conductance in response to water stress, with Grenache exhibiting a larger reduction. Suberisation of the roots increased in response to water stress, with complete suberisation of the endodermis occurring closer to the root tip of Grenache compared to the more drought sensitive Chardonnay. The drought sensitive rootstock, 101-14 (V. riparia × V. rupestris) demonstrated a similar reduction in hydraulic conductance to Chardonnay, while drought tolerant 1103 Paulsen (V. berlandieri × V. rupestris) had a non-significant reduction when water-stressed compared to the large reduction observed for drought tolerant Grenache. Therefore, in this study the degree of reduction in hydraulic conductance did not relate to the drought tolerance of the four varieties examined. The impact of partial drying (watering only half the root system) on hydraulic conductance also differed between Chardonnay and Grenache. There was no change in the conductance of the whole root system of Chardonnay due to an increase in conductance of the roots in the wet half which compensated for the reduction on the dry side. In contrast, Grenache did suffer a reduction measured over the whole root system due to a much larger reduction on the dry side compared to Chardonnay. There was an increase in hydraulic conductance on the wet side but this could not compensate for the large reduction on the dry side. Two aquaporins (VvPIP1;1 and VvPIP2;2) were cloned from the roots of grapevine cultivar Chardonnay. The genes were expressed in Xenopus oocytes to determine their osmotic permeability. As has been shown in a number of plant species, VvPIP1;1 was only slightly permeable to water, whereas VvPIP2;2 did transport water. However, when VvPIP1;1 was injected into the oocytes with VvPIP2;2, there was a substantial increase in the osmotic permeability. There was no significant variation in the diurnal expression of VvPIP2;2, whereas VvPIP1;1 showed a peak in expression at 1000 h prior to the peak in hydraulic conductance and peaked again at 1800 h. VvPIP2;2 did not vary in transcript level in response to water stress or rewatering in Chardonnay or Grenache roots. The level of VvPIP1;1 doubled in water stressed Chardonnay roots and declined again when the vines were rewatered 24 h previously. This response to water stress did not occur in Grenache roots. The roots used were from the apical 5 cm. Similar roots were used to measure the water permeability of the cortical cell membranes using the cell pressure probe. Changes in cell membrane permeability in response to water stress corresponded to changes in VvPIP1;1 expression. An experiment to determine if shoot topping had an effect on root hydraulic conductance revealed a significant 50% decline. This response was also observed in soybean (Glycine max L.) and maize (Zea mays L.). A range of experiments have been performed to determine the reason for the decline. Possibilities included a response to final leaf area and reduced transpirational demand; loss of a carbohydrate sink; or hormonal signals such as abscisic acid, auxin and ethylene. At this stage the nature of the positive or negative signal that causes the change in root hydraulic conductance remains elusive. However, the signal did cause a reduction in the transcript level of VvPIP1;1, indicating the involvement of aquaporins in the response. The root hydraulic conductance of grapevines is variable and dependent on factors such as time of day, water-stress, transpiration rate and unknown signals from the shoot. A proportion of this variability is due to changes in aquaporin number or activity. There are also genotypic differences which may be beneficial for future breeding efforts to improve water use efficiency of grapevines. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311202

Hydraulic conductance of roots of the grapevine cultivar, Chardonnay, varies diurnally, peaking at 1400 h. The diurnal amplitude of hydraulic conductance between 600 and 1400 h was not altered when potted grapevines were water-stressed by withholding water for 8 days. However, the diurnal change was greatly reduced for water-stressed Grenache. If the diurnal change in root hydraulic conductance is a result of changes in aquaporin gene expression or activity, it suggests that aquaporins respond differently in water-stressed Chardonnay and Grenache roots. Both Chardonnay and Grenache demonstrated a reduction in hydraulic conductance in response to water stress, with Grenache exhibiting a larger reduction. Suberisation of the roots increased in response to water stress, with complete suberisation of the endodermis occurring closer to the root tip of Grenache compared to the more drought sensitive Chardonnay. The drought sensitive rootstock, 101-14 (V. riparia × V. rupestris) demonstrated a similar reduction in hydraulic conductance to Chardonnay, while drought tolerant 1103 Paulsen (V. berlandieri × V. rupestris) had a non-significant reduction when water-stressed compared to the large reduction observed for drought tolerant Grenache. Therefore, in this study the degree of reduction in hydraulic conductance did not relate to the drought tolerance of the four varieties examined. The impact of partial drying (watering only half the root system) on hydraulic conductance also differed between Chardonnay and Grenache. There was no change in the conductance of the whole root system of Chardonnay due to an increase in conductance of the roots in the wet half which compensated for the reduction on the dry side. In contrast, Grenache did suffer a reduction measured over the whole root system due to a much larger reduction on the dry side compared to Chardonnay. There was an increase in hydraulic conductance on the wet side but this could not compensate for the large reduction on the dry side. Two aquaporins (VvPIP1;1 and VvPIP2;2) were cloned from the roots of grapevine cultivar Chardonnay. The genes were expressed in Xenopus oocytes to determine their osmotic permeability. As has been shown in a number of plant species, VvPIP1;1 was only slightly permeable to water, whereas VvPIP2;2 did transport water. However, when VvPIP1;1 was injected into the oocytes with VvPIP2;2, there was a substantial increase in the osmotic permeability. There was no significant variation in the diurnal expression of VvPIP2;2, whereas VvPIP1;1 showed a peak in expression at 1000 h prior to the peak in hydraulic conductance and peaked again at 1800 h. VvPIP2;2 did not vary in transcript level in response to water stress or rewatering in Chardonnay or Grenache roots. The level of VvPIP1;1 doubled in water stressed Chardonnay roots and declined again when the vines were rewatered 24 h previously. This response to water stress did not occur in Grenache roots. The roots used were from the apical 5 cm. Similar roots were used to measure the water permeability of the cortical cell membranes using the cell pressure probe. Changes in cell membrane permeability in response to water stress corresponded to changes in VvPIP1;1 expression. An experiment to determine if shoot topping had an effect on root hydraulic conductance revealed a significant 50% decline. This response was also observed in soybean (Glycine max L.) and maize (Zea mays L.). A range of experiments have been performed to determine the reason for the decline. Possibilities included a response to final leaf area and reduced transpirational demand; loss of a carbohydrate sink; or hormonal signals such as abscisic acid, auxin and ethylene. At this stage the nature of the positive or negative signal that causes the change in root hydraulic conductance remains elusive. However, the signal did cause a reduction in the transcript level of VvPIP1;1, indicating the involvement of aquaporins in the response. The root hydraulic conductance of grapevines is variable and dependent on factors such as time of day, water-stress, transpiration rate and unknown signals from the shoot. A proportion of this variability is due to changes in aquaporin number or activity. There are also genotypic differences which may be beneficial for future breeding efforts to improve water use efficiency of grapevines. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1311202

[Truncated abstract] Nematodes are known to significantly affect productivity of grapevines worldwide. Although major surveys have been carried out on nematodes infesting roots of grapevines elsewhere, only a preliminary survey has been carried out in Western Australia (W.A.). This study on the effect of irrigation systems on pathogenicity of nematodes on vines commenced with a survey of nematodes in two major grapegrowing regions of W.A. In this survey, soil samples were taken from 5 vineyards from Margaret River and 7 vineyards from Swan Valley regions of the state. Root-knot nematode (Meloidogyne spp.) was found to be the dominant genus in both major grape growing regions. Meloidogyne spp. occurred 76% and 75% of total soil sample in Margaret River and Swan Valley. The highest density of Meloidogyne spp. was 7 nematodes/g soil in Margaret River and 3.17 nematodes/g soil in Swan Valley. In both regions, other plant parasitic nematodes were recorded that included the root lesion (Pratylenchus spp.), dagger (Xiphinema spp.) and stubby (Trichodorus spp.) nematodes. Paratylenchus spp. were found in a few soil samples from Margaret River region, and Helicotylenchus spp. were found only in Swan Valley region, but was widespread. Some vineyards have established only resistant cultivars (Schartzman, Ramsey and 34 EM) resistant to nematodes. In these vineyards total nematode population was lower than most of other vineyards. However, in comparison of nematode numbers between cultivars, there were lower number of nematodes in some susceptible cultivars than in the resistant cultivars. Most common nematode taxa were Meloidogyne, Pratylenchus and Xiphinema in both regions. Root-knot and root lesion nematodes were the most widespread and economically important genera. These two genera are known to have different life cycle and feeding habits.

Thesis (MScAgric)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 226-244).

Thesis (MSc Food Sc )--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Highly valued Sauvignon blanc wines, with the distinctive cultivar-typical aromas, reminiscent of grassy, green pepper or asparagus-like, are produced in some South African regions. Quite often, however, neutral and sulphur-like, low quality Sauvignon blanc wines are produced and this phenomenon is of great concern to wine producers and consumers, and affects our competition on overseas markets, negatively. The aim of this study was to investigate the effect of region, ascorbic acid/S02 treatments and yeast strain on Sauvignon blanc wine aroma and quality. Wines were produced from grapes obtained from the warmer Robertson and the relatively cooler Stellenbosch regions (1998 season). The juices were treated with different combinations of ascorbic acid/S02 treatments [commercially available ascorbic acid/meta preparate, S02 (control), pure ascorbic acid/S02] and Saccharomyces cerevisiae yeast strains (Vin 13, VL3C, NT 116). The wines were analysed for esters, higher alcohols, monoterpenes and 2-methoxy-3-isobutylpyrazine (ibMP). The wines were also sensorially evaluated for wine aroma intensities (fruity/ester, sulphur-like, grassy/green pepper) and overall quality. Additionally, the synergistic action of ibMP and the sulphur-containing component, 4-mercapto-4-methylpentan-2- one (MMP), considered to be the most important impact components of Sauvignon blanc, was studied. The two components were added, separately and in combinations at increasing concentrations, to different media. The nuances perceived, varied from dusty, grassy to green pepper for ibMP and from guava, sulphur-like to cat urine or "conifer" for MMP. Significant differences were observed between the wines treated with the different combinations of ascorbic acid/S02 treatments and fermented with different yeast strains, irrespective of region. The highest quality, cultivar-typical Sauvignon blanc wines were produced from the pure ascorbic acid/S02 treatment in combination with yeast strains Vin 13 and NT 116. This coincided with high ester and low higher alcohol concentrations, which did not overpower the typical Sauvignon blanc character. The treatments had, in some cases, a significant effect on monoterpene levels, but it was concluded that these differences were not big enough to affect wine quality. Levels of ibMP were too low and could not be reliably measured. Low quality wines, with prominent, undesirable sulphur-like aromas, were produced from juices, treated with the commercially available ascorbic acid/meta preparate and the French yeast strain, VL3C. Techniques, followed to identify the aroma components causing the sulphur-like offflavours, as MMP or as other sulphur-containing components, were gas chromatography/mass spectrometry, solid phase microextraction and sniffing. However, these tests were not successful and studies to identify these off-flavours should be continued. It was succeeded in this study to produce Sauvignon blanc wines without the undesirable, sulphur-like aromas. Although this investigation showed that a newly developed, commercially available ascorbic acid/meta preparate did not yield any sulphur-like off-flavours, quite often Sauvignon blanc wines with such off-flavours are still produced. Further research is needed to clarify this phenomenon. / AFRIKAANSE OPSOMMING: Hoë kwaliteit Sauvignon blanc wyne, met kenmerkende kultivar-tipiese, gras-, groenrissie- of aspersie-agtige aromas, word in sekere streke van Suid Afrika geproduseer. Die gereelde produksie van neutrale, en swawelagtige lae kwaliteit Sauvignon blanc wyne, wek egter nie net groot kommer by wynprodusente en verbruikers nie, maar het ook 'n negatiewe impak op kompetisie met oorsese markte. Die doel van hierdie studie was dus om die effek van streek, askorbiensuur/Sóbehandelings en gisras op Sauvignon blanc wynaroma en kwaliteit vas te stel. Wyne is berei met druiwe wat verkry is van die warmer Roberston en relatief koeler Stellenbosch streke (1998 seisoen). Verskillende kombinasies askorbiensuur/So, behandelings [kommersieel-beskikbare askorbiensuur/meta preparaat, S02 (kontrole), suiwer askorbiensuur/So-l en Saccharomyces cerevisiae gisrasse (Vin 13, VL3C, NT 116) is gebruik tydens die wynbereidingsproses. Spesifieke ester-, hoër alkohol-, monoterpeen- en 2-metoksi-3-isobutiel metoksipirasienkonsentrasies (ibMP) is in die wyne bepaal. Die wyne is ook sensories vir wynaroma intensiteite (vrugtig/ester, swaweiagtig, gras/groenrissie-agtig) en algehele kwaliteit geëvalueer. Die sinergistiese aksie van ibMP en die swawelbevattende komponent, 4-merkapto- 4-metielpenta-2-oon (MMP), wat beskou word as die belangrikste impakkomponente in Sauvignon blanc, is addisioneel bestudeer. Hierdie komponente is in toenemende konsentrasies, individueel en in kombinasies, tot verskillende media gevoeg. Die nuanses wat waargeneem is, het van stowwerig, gras-, groenrissie-agtig vir ibMP, tot koejawelagtig, swaweiagtig, katurine, konifeer vir MMP, gevarieer. Ongeag streke, is betekenisvolle verskille tussen die wyne wat berei is met die verskillende kombinasies van askorbiensuur/So- behandelings en gisrasse, waargeneem. Hoër kwaliteit, kultivar-tipiese Sauvignon blanc wyne is berei met suiwer askorbiensuur/So- in kombinasie met gisras Vin 13 of NT 116. Alhoewel die hoë ester- en lae hoër alkohol- konsentrasies, hierdie resultate bevestig het, is die tipiese Sauvignon blanc karakter nie hierdeur oorheers nie. Sommige behandelings het wel 'n betekenisvolle invloed of monoterpeenkonsentrasies gehad het, maar was te min om 'n effek op wynkwaliteit uit te oefen nie. Die konsentrasievlakke van ibMP swawelagtige aromas, het egter voorgekom in wyne wat met die kommersieelbeskikbare askorbiensuur/meta preparaat en die Franse gisras, VL3C, berei is. Verskeie tegnieke soos gaschromatografie/massa spektrometrie, soliede fase mikroekstraksie en "sniffing", is gebruik om die komponent, MMP, wat moontlik verantwoordelik vir hierdie ongewensde swawelagtige wangeure was, te identifiseer. Die identifikasie hiervan was egter onsuksesvol, en verdere studies is nodig om die komponent(e) verantwoordelik vir hierdie wangeure, te identifiseer. Die suksesvolle produksie van Sauvignon blanc, sonder ongewensde swaweiagtig aromas, was wel moontlik. Alhoewel hierdie studie ook duidelik getoon het dat daar geen swawelagtige wangeure in die wyne wat met die nuut ontwikkelde, kommersieel-beskikbare askorbiensuur/meta preparaat berei was, voorgekom het nie, vind die produksie van Sauvignon blanc wyne met van hierdie wangeure nog steeds plaas. Verdere navorsing rakende hierdie aspek is nodig.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / Grapevine Vitivirus A (GVA), genus Vitivirus, family Flexiviridae is a well characterised single-stranded RNA virus that has been implicated in the grapevine diseases, Kober stem grooving and Shiraz disease. The virus infects both its host, Vitis vinifera and the experimental model plant, Nicotiana spp.. Biological studies performed on the virus in its herbaceous host, Nicotiana benthami- ana, revealed that many divergent variants of the virus exists in South Africa and can induce di erent symptoms in the model plant. Further molecular analysis divided the variants into three molecular groups based on molecular heterogeneity and nucleotide identity. The establishment of an infectious full-length cDNA clone of GVA contributed towards the elucidation of gene functions for 4 of the 5 open reading frames (ORF's), and indicated ORF5 as the pathogenicity determinant within the genome. Further studies also showed that ORF5 encodes for a nucleic acid binding protein that exhibits suppression activity of a plants' natural virus silencing mechanism. Many proteins that have previously been identi ed as the pathogenicity determinant within a viral genome have been found to encode for suppression activity. Although suppression activity has been elucidated within the ORF5 of the Italian cDNA clone of GVA, IS 151, no such study has yet been performed on the divergent South African variants of GVA. Three variants, GTR1-1, GTR1- 2 and GTG11-1, which represent each of the molecular groups (Group III, II and I), were selected for this study. The aim of this study was to visually elucidate suppression activity of RNA transgene silencing by the ORF5's of GTR1-1, GTR1-2 and GTG11-1 in a transient expression assays in transgenic N. benthamiana (line 16c). Pathogenicity studies for these variants were also performed. The ORF5 of the infectious full-length clone, GVA118, which can also serve as an expression vector, was deleted and provided with restriction enzyme sites into which the respective ORF5s and the marker genes, GFP and GUS could be cloned directionally. Infectivity, symptom development and systemic movement were compared between the di erent full length clones after co-in ltration in N. benthamiana. Preliminary results obtained in this study failed to visually indicate any suppression activity encoded by the ORF5 of GTR1-1, GTR1-2 and GTG11-1. The deletion of ORF5 within GVA118 was successful and rendered the infectious full length clone asymptomatic. Directional cloning of the ORF5 of GTR1-1 into the unique restriction enzymes provided previously, resulted in much milder symptoms than those observe for GTR1-2 and GTG11-1. No GFP and GUS accumulation could be detected. This study has established an infectious full-length cDNA clone, pBINSN-e35SGVA118 ORF5-1-1-pA, that can possibly induce much milder symptoms in the herbaceous host, N. benthamiana. This construct can be further characterised as a possible expression vector of foreign proteins in herbaceous hosts and grapevine.

Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / An infectious clone of a viral RNA genome is one that can be used, either as an in vitro transcript or as cDNA, to produce an infection in a susceptible plant. Infectious clones serve as a tool to study viral RNA genomes at a molecular level to gain deeper insight into genome organization, viral gene function, presence of regulatory sequences and gene expression. In the Western Cape (and elsewhere) a new crippling grapevine disease, known as Shiraz disease, is emerging of which the aetiology and pathogenic agents involved are not yet fully understood. Grapevine virus A (GVA), genus Vitivirus, family Flexiviridae, is thought to be the associated with this disease. The aim of this study was to construct a full-length infectious cDNA clone of GVA, which will aid in the molecular study of the viral genome. This clone could ultimately be used to investigate GVA’s involvement in Shiraz disease, which could lead to the unravelling of the aetiology and control of the disease. A full-length clone of GVA, named GVA-IC2/T7-2972-3, was constructed in several steps using restriction digestion/ligation and primer overlap extension PCR. Grapevine virus A cDNA fragments were obtained from GVAinfected Nicotiana benthamiana and Vitis vinifera plants using three different techniques, of which the Rapid direct-one-tube RT-PCR was most successful. A 5’ T7 promoter and a 3’ poly-A tail were incorporated and the full-length clone was cloned into pBluescript II SK (+). Full-length sequencing of the clone, revealed two significant frameshift mutations. The first mutation was a single base pair insertion (one G) in a slippery site of 6 G’s at position 1380 – 1385 in open reading frame one (ORF 1) of the viral genome. This mutation was corrected by PCR-based site-directed mutagenesis, which resulted in pSK-GVA-mutagen-3 and pSK-GVA-mutagen-4. The second mutation was a single base pair deletion (one G) at position 6959 in ORF4, which coded for the coat protein (CP). Several techniques were attempted to correct this mutation, but none were successful. Even though the second mutation could not be corrected, in vitro transcriptions were performed on three clones followed by subsequent infections of N. benthamiana plants. The three clones included pSK-GVA-mutagen-3, pSKGVA- mutagen-4 (both hosting the mutation at position 6959) and GVA-IC2/T7-2972-3 (hosting both mutations). At 21 days post-inoculation no significant visual symptoms were observed in plants infected with in vitro RNA or in plants infected with wild type GVA. Rapid direct-one-tube RT-PCR results revealed the presence of viral RNA in infected leaves and apical leaves of infected plants, and provided preliminary evidence that the mutated clones were still capable of systemic infection and viral movement. These results are still inconclusive, and several post-infection studies will have to be performed to confirm these findings. Koch's postulates will also have to be proved in order to confirm the infectious nature of the clones. The effect of the two mutations in the constructed clones will be investigated further and post-infection analysis performed to deduce whether the viral progeny are devoid of the mutations. Three full-length GVA cDNA clones (hosting mutations) seemingly capable of systemic infection in N. benthamiana plants were constructed in this study and have laid the foundation for molecular and mutational analysis of the GVA genome. This could lead to the study of pathogen-host interactions in order to unravel the aetiology of Shiraz disease in the future.

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Ribosome inactivating proteins (RIPs) are potent toxins produced by a wide range of evolutionarily diverse plants. These toxins cause cell death by physically dismantling ribosomal RNA and shutting down protein synthesis. They also have a strong antiviral activity. Some believe that the antiviral property of RIPs is a function of ribosomal inactivation, others believe that the two properties are unrelated. RIPs are non-specific in their antiviral activity. Transgenic RIPexpressing plants are resistant to a wide spectrum of viruses. Many different viruses threaten grapevine. It is not practical to design individual remedies for each of these viruses. In this study, we screen the grapevine genome for the presence of a RIP gene using degenerate PCR primers. If a RIP gene does exist in grapevine, it is not being expressed in a useful way. We also clone several well-documented RIP genes from various plants into pGEM-T Easy: dianthin from Dianthus caryophyllus; p-Iuffin from Luffa octandra and mirabilis antiviral protein (MAP) from Mirabilis jalapa. These isolated genes are then subcloned into a selection of expression vectors: dianthin into pKK223-3, a bacterial expression vector; p-Iuffin into pCambia3301, a plant expression vector; and MAP into pFLAG, a yeast expression vector. The constructs prepared in this project may be used for the synthesis of RIP molecules. The exogenous application of RIPs has been shown to protect plants from viruses. Transformation of grapevine with the RIP-containing plant expression vector may result in a variety of vine that is resistant to a wide range viruses. This thesis describes preliminary work in an attempt to impart broad-spectrum antiviral resistance to grapevine. / AFRIKAANSE OPSOMMING: Ribosomale-inaktiverende proteïne (RIPs) is kragtige toksienes wat deur 'n wye verskeidenheid evolusionêr diverse plante verskaf word. Hierdie toksienes veroorsaak die dood van die selle deur fisies die ribosomale RNA af te breek en proteïensintese stop te sit. Hulle toon ook 'n sterk antivirale aktiwiteit. Sommige voel dat die antivirale eienskap van RIPs 'n funksie van ribosomale inaktivering is, terwyl ander glo dat die twee eienskappe onafhanklik optree. RIPs is in hul antivirale aktiwiteit onspesifiek. Transgeniese RIP-weergewende plante toon weerstand teen 'n wye spektrum virusse. Wingerd word deur baie verskillende virusse aangeval. Dit is onprakties om spesifieke teenmiddels vir elk van die virusse te ontwerp. In hierdie studie word die wingerdgenoom vir die voorkoms van 'n RIP-geen ondersoek, deur die gebruik van degeneratiewe PKR primers. As daar wel 'n RIP-geen in wingerd voorkom, word dit nie in 'n nuttige manier uitgedruk nie. Ons het ook 'n groep goedgedokumentêre RIP-gene vanuit verskeie plante in pGEM- T Easy gekloneer: dianthin vanuit Dianthus caryophyllus; p-Iuffin vanuit Luffa octandra; en mirabilis antivirale proteïen (MAP) vanuit Mirabilis jalapa. Hierdie geïsoleerde gene is toe in verskeie uitdrukkingsvektore gesubkloneer: dianthin in pKK223-3, 'n bakterïele uitdrukkingsvektor; p-Iuffin in pCambia3301, 'n plant uitdrukkingsvektor; en MAP in pFLAG, 'n gis uitdrukkingsvektor. Die constructs wat in hierdie projek voorberei is, kan gebruik word vir die sintese van RIP molekules. Dit is gevind dat die eksogeniese toepassing van RIPs plante teen virus-infeksie beskerm. Die transformasie van wingerd met die RIP-bevattende plant ekspressievektor kan 'n wingerd wat teen 'n wye verskeidenheid virusse bestand is tot stand bring. Hierdie tesis beskryf die voorlopige werk in 'n poging om breë-spektrum antivirale weerstand in wingerd deelagtig te maak.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Grapevine virus E (GVE) is a newly identified virus that has been detected in an established vineyard in South Africa. This virus is a member of the genus Vitivirus, family Flexiviridae. Members of this genus are known to infecte grapevine and are associated with various disease complexes, such as the Rugose wood complex (RWC) and Shiraz disease (SD). However, the role and impact of GVE in South African vineyards are still unknown. It is important to study these viruses to determine how they infect and the possible impact they may have on vine health. The accurate and early detection of grapevine viruses is the first important step in disease management. In this study, reverse transcription-polymerase chain reaction (RT-PCR), double antibody sandwich enzyme linked immunesorbent assay (DAS-ELISA) and quantitative (q)RT-PCR were used for the detection of GVE in the vineyard (Vitis vinifera cv Merlot) where GVE was first identified in South Africa. Reverse transcription-PCR was used for detection and determining the incidence of GVE. The incidence was as low as 3% in the vineyard surveyed. All the GVE positive plants were co-infected with GLRaV-3 and no disease association could therefore be made. Evaluation of the Bioreba Grapevine virus A (GVA) DAS-ELISA kit showed that it did not detect GVE. No cross-reactivity occurred with epitopes of GVE, confirming this kit to be a valid and specific assay for GVA infection. The relative virus titer of GVE was calculated over the growing season of 2010/2011, using qRT-PCR. No fluctuation in virus titer was observed during that growing season. Transmission experiments were performed in an attempt to transfer GVE from grapevine to an alternative host. Three different transmission buffers as well as nine different herbaceous plant species, that have shown to be susceptible to several plant viruses in previous studies, were evaluated. In these experiments, GVE could not be transmitted to any of the herbaceous species. To further characterize GVE, chimeric clones were constructed with GVA. The ORF2 and ORF5 of GVE were cloned into previously constructed GVA ORF2 and ORF5 deletion mutants. Construction of the chimeric clones, 35S-GVA-GR5-ΔORF2-GVE-ORF2 and 35S-GVA-118-ΔORF5-GVE-ORF5 were successful and they were evaluated for their infectivity in N. benthamiana. The 35S-GVA-GR5-ΔORF2-GVE-ORF2 chimera was able to infect and replicate in these plants and disease symptoms such as yellowing of veins and leaf curling were observed. Virus, derived from this vector, was detected by TPIA, RT-PCR and DAS-ELISA. The 35S-GVA-118-ΔORF5-GVE-ORF5 chimeric vector was not able to infect N. benthamiana as no disease symptoms were observed in any of the infiltrated plants and virus was not detected with serological analysis and RT-PCR. This study was aimed at further characterizing the recently identified virus GVE. Here, insight is given into the prevalence of this virus in the vineyard where it was first identified and attempts to biologically characterize GVE were made. / AFRIKAANSE OPSOMMING: Grapevine virus E (GVE) is „n nuut geïndetifiseerde virus wat onlangs in „n gevestigde wingerd in Suid Afrika opgespoor is. Hierdie virus vorm deel van die genus Vitivirus, familie Betaflexiviridae. Spesies in hierdie genus is bekend vir wingerdinfeksies en word met „n verskeidenheid wingerd siektes geassosieer, soos bv. Rugose wood complex (RWC) en Shiraz siekte (SD). Die rol en impak van GVE is nog onbekend. Dit is dus belangrik om die virus te bestudeer om te bepaal hoe dit infekteer en of dit enige impak het op wingerd gesondheid. Akkurate en vroeë opsporing van virusse is die eerste belangrike stap vir virussiekte beheer. In hierdie studie word tru-transkripsie (TT) – polimerase ketting reaksie (PKR), dubbel teenliggaam (DAS) -ensiem gekoppelde immuno-absorberende analise (ELISA) en qTT-PKR gebruik vir die opsporing van GVE in die wingerd (Vitis vinifera cv Merlot) waar dit vroeër in Suid Afrika geïdentifiseer was. Vir opsporing en bepaling van verspreiding is TT-PKR gebruik. Daar is bepaal dat 3% van die wingerd met GVE geïnfekteer is. Al die GVE-positiewe stokke het ook positief getoets vir GLRaV-3 en geen assosiasie met siekte simptome kon gemaak word nie. Evaluering van die Bioreba GVA DAS-ELISA met GVE positiewe stokke het nie GVE opgespoor nie. Geen kruisreaktiwiteit het plaasgevind met epitope van GVE nie en dus is die DAS-ELISA ʼn betroubare toets vir GVA infeksie. Die relatiewe virus titer van GVE was ook bepaal oor die groeiseisoen van 2010/2011 deur qTT-PKR te gebruik. Geen fluktuasie in virus titer gedurende die groeiseisoen is waargeneem nie. Transmissie eksperimente is gedoen om GVE vanaf wingerd na ʼn alternatiewe gasheer oor te dra. Drie verskillende transmissie buffers en tien verskillende sagteplant spesies, wat voorheen vatbaarheid vir plantvirusse getoon het, is gebruik. In die transmissie eksperimente kon GVE nie na enige van die sagteplante oorgedra word nie. Om GVE verder te karakteriseer is hibried-virusse met GVA gemaak. Die leesraam (ORF) 2 en ORF5 van GVE gekloneer in GVA ORF2 en -ORF5 delesie konstrukte, 35S-GVA-GR5-ΔORF2 en 35S-GVA-118-ΔORF5, onderskeidelik (Blignaut, 2009; Du Preez, 2010). Klonering van die hibried konstrukte, 35S-GVA-GR5-ΔORF2-GVE-ORF2 en 35S-GVA-118-ΔORF5-GVE-ORF5, was suksesvol en is in N. benthamiana geëvalueer. Virus afkomstig van die 35S-GVA-GR5-ΔORF2-GVE-ORF2 hibried konstruk, kon plante suksesvol infekteer en kon repliseer binne hierdie plante. Siektesimptome soos vergeling van die are en rolblaar is ook waargeneem in plante geïnfekteer met hierdie hibried konstruk. Plante is getoets met weefsel afdruk immuno analise (TPIA), TT-PKR en DAS-ELISA en is positief gevind vir virus afkomstig van hierdie konstruk. Die 35S-GVA-118-ΔORF5-GVE-ORF5 hibried kon nie N. benthamiana infekteer nie en geen siektesimptome is waargeneem in enige van die plante geïnfiltreer met hierdie konstruk. Serologiese analise en TT-PKR het ook nie virus in die N. benthamiana plante opgespoor nie. Die doel van hierdie studie was om GVE te karakteriseer. In hierdie studie word insig gegee oor die verspreiding van hierdie virus in Suid Afrika en pogings is gemaak om GVE biologies te karakteriseer.