Grapevine Leafroll disease (GLD), one of the most destructive diseases of grapevines, has been found in every country where grapevines are grown. Grapevine Leafroll associated virus type 3 (GLRaV-3), one of several viruses associated with GLD globally, is the most prevalent virus in South African grapevines and therefore control of GLRaV-3 takes high priority in any strategy aimed at control of GLD. GLD can be controlled through the use of an integrated strategy which includes using certified plant material, controlling insect vectors through use of systemic insecticides and the removal of infected vines by roguing. Infected individuals are identified each autumn, using either symptom display (in red cultivars, where infected individuals display interveinal reddening and downward rolling of leaves) or ELISA (in symptomless white cultivars). ELISA is laborious, time consuming and relatively insensitivity compared to molecular techniques and a simpler, more rapid and more sensitive means of indentifying GLRaV-3 infected vines is required. A simple RNA extraction procedure combined with a single-tube reverse transcriptase loop-mediated amplification (RT-LAMP) has been developed which allows for the rapid, simple detection of GLRaV-3. Using RT-LAMP, a viral target can be amplified in 2 hours under isothermal conditions. This GLRaV-3 specific RTLAMP uses hydroxy napthol blue (HNB), a colourimetric indicator that changes from violet to sky blue only where a positive RT-LAMP reaction has occurred, making results quick and easy to interpret. The sensitivity of this technique was compared to ELISA and nested PCR by pooling samples at varying ratios of healthy to infected plants. Using nested PCR and RT-LAMP 1 infected sample could be detected amongst 50 healthy individuals while ELISA could only detect 1 amongst 30 infected making RT-LAMP more sensitive than ELISA. Further RT-LAMP could be performed in 2 hours compared to nested PCR and ELISA’s 8 and 48 hours respectively. Based on these results, RT-LAMP is viable alternative for ELISA for the detection of GLRaV-3 in the field. RT-LAMP was also tested for its ability to detect GLRaV-3 in grapevine rootstocks where, due to low viral titres and erratic distribution, it is notoriously difficult to detect. The rootstocks which were used for testing of GLRaV-3 had been tested in a previous study and it was found that only 28% of samples tested positive after 33 months (post inoculation). Using RT-LAMP, 78% of samples tested positive for GLRaV-3. Although further testing must be done, RT-LAMP may also be a viable alternative for testing grapevine rootstocks for GLRaV-3 infection. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Microbiology and Plant Pathology / unrestricted

Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Phytoplasma diseases have caused disastrous effects in vineyards around the world. Therefore, the recent discovery of phytoplasmas in South African vineyards could be highly detrimental to the local wine industry. Antimicrobial peptides (AMPs) are small molecules expressed by almost all organisms as part of their non-specific defence system. These peptides can offer protection against a wide variety of bacterial and fungal pathogens in plants. Due to the fact that phytoplasmas lack an outer membrane and cell wall, AMPs are considered to be perfect candidates to confer resistance to this phytopathogen. The current study intends to explore the in planta activity of AMPs against the grapevine pathogen aster yellows phytoplasma (AYp) through Agrobacterium-mediated transient expression. The AMPs, Vv-AMP1, D4E1 and Snakin1 (isolated from potato and grapevine) were selected to be tested for their in planta effect against AYp. Cauliflower mosaic virus 35S expression vectors containing four different AMP-encoding sequences were therefore constructed. As an alternative method to observe the effect Vv-AMP1 might have on AYp in planta, grafting of Vv-AMP1 transgenic Vitis vinifera cv "Sultana‟ plant material was used. To allow assumptions about AMP efficacy in this transient expression system, attempts were made to describe the spatial distribution and pathogen titre of AYp in V. vinifera cv "Chardonnay‟ material. Additionally, transmission experiments were carried out to infect Catharanthus roseus and Nicotiana benthamiana with AYp through the insect vector Mgenia fuscovaria. Material was screened for AYp infection by a nested-PCR procedure using universal primers described by Gundersen and Lee (1996). For quantification of AYp infection, a semi-quantitative real-time PCR (qPCR) protocol was optimized, using the SYBR Green-based system. In total, 86 V. vinifera cv "Chardonnay‟ plantlets were screened for AYp infection two-, three-, four-, seven- and eleven weeks after introduction into in vitro conditions. No AYp infection could however be detected and plantlets displayed a "recovery phenotype‟. To examine the distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ plant, leaf and the corresponding node material from five canes were screened by a nested-PCR procedure. It can be concluded, that AYp was found predominantly in the nodes when compared to leaf material in the late season of the year. It is also highly unlikely for leaf material to show phytoplasma infection, if in the corresponding node no AYp could be detected. As AYp-infected grapevine material could not be maintained in vitro, the effect of VvAMP-1 transgenic grapevine against AYp could not be tested. Infection of C. roseus and N. benthamiana plants with AYp was successfully achieved by insect vector transmission experiments. Transient expression assays were conducted on AYp-infected N. benthamiana material. Quantification of phytoplasma in this material showed a decrease of AYp in both the AMP treatment groups and the control groups. This study optimized a qPCR procedure to detect and quantify AYp in infected plant material. The Agrobacterium-mediated transient expression system used during this study was not reliable, as no significant effect of the AMPs on AYp titre could be observed. This study showed, that AYp cannot be established and maintained in in vitro cultured V. vinifera cv "Chardonnay‟ material, and tissue culture itself might therefore be a way to eradicate AYp in this cultivar. To our knowledge, this study is the first to report on the spatial distribution of AYp in canes of an infected V. vinifera cv "Chardonnay‟ vine. / AFRIKAANSE OPSOMMING: Fitoplasma siektes veroorsaak ramspoedige gevolge in wingerde oor die hele wêreld. Dus kan die onlangse ontdekking van fitoplasma in Suid-Afrikaanse wingerde baie nadelige gevolge vir die plaaslike wynbedryf beteken. Antimikrobiese peptiede (AMPe) is klein molekules wat in amper al organismes as deel van hulle nie-spesifieke verdedegingsstelsel tot uitdruk kom. Hierdie peptiede kan beskerming aanbied teen ʼn wye verskeidenheid van bakteriële en swampatogene in plante. As gevolg van die feit dat fitoplasmas geen buitenste membraan en selwand het nie, word AMPe oorweeg as middle om weerstand te verleen teen hierdie fitopatogene. Die huidige studie beoog om die in planta aktiwiteit an AMPe teen die wingerdstok patogeen aster geel fitoplasma (AYp) deur middle van Agrobakteriumbemiddelde tydelike uitdrukkingssiteme, te ondersoek. Die AMPe, Vv-AMP1, D4E1 en Snakin1 (geïsoleer van aartappel en wingerd plante) is gekies om getoets te word vir hul in planta effek teen AYp. Blomkoolmosaïek-viruse 35S uitdrukkingsvektore met vier verskillende AMP-kodering rye, is dus ontwikkel. As ʼn aternatiewe method om die moontlike effek van Vv-AMP1 op AYp in planta in ag te neem, is oorplantings van die Vv-AMP1 transgeniese Vitis vinifera cv "Sultana‟ plantmateriaal gebruik. Om voorsiening te maak vir AMPe se doeltreffenheid in hierdie tydelike uitdrukkingsvektore, is pogings aangewend om die ruimlike verspreiding en patogeen konsentrasie van AYp in V. vinifera cv "Chardonnay‟ te beskryf. Addisioneel is transmissie eksperimente uitgevoer om Catharanthus roseus en Nicotania benthamiana te besmet met AYp deur die insekvektor, Mgenia fuscovaria. Plantmateriaal is getoets vir AYp deur van ʼn PCR protokol gebruik te maak met universele inleiers (grondlae) soos beskyf deur Grundersen en Lee (1996). Vir kwantifiseering van die AYp infeksie, is n semi-kwantitatiewe qPCR protokol geoptimaliseer, met hulp van die SYBR Groen-gebaseerde stelsel. In geheel is 86 V. vinifera cv "Chardonnay‟ planties getoets vir AYp infeksie – twee-, drie-, vier-, sewe- en elf weke na die bekendstelling aan die in vitro voorwaardes. Geen AYp infeksie kon egter opgespoor word en die plante het “herstel fenotipe‟ vertoon. Om die verspreiding van AYp in stingelknope van ʼn besemtte V. vinifera cv "Cardonnay‟ plant, blaar en ooreenstemmende stingelknope uit vyf stingels te ondersoek, is hulle getoets deur ʼn PCR protokol. Daar kon afgelei word dat AYp hoofsaaklik in die stingelknop in vergelyking met die blaarmaterial laat in die season, gevind word. Dit is hoogs oonwaarskynlik om fitoplasma infeksies in blaarmaterial te vind, as in die ooreenstemmende stingelknop daar geen AYp oopgespoor kon word nie. As gevolge daarvan dat die AYp-geinfekteerde wingerdmateriaal nie in vitro gegroei kon word nie, kon die effek van VvAMP-1 transgeniese wingerd teen AYp nie getoets word nie. Infeksies van C. roseus en N. benthamiana plante met AYp is suksesvol bereik deur transmissie eksperiemente. met ʼn insekvektor. Tydellike uitdrukkingvektore toetse is uitgevoer op die AYp besmette N. benthamiana material. Kwantifisering van fitoplasma in hierdie material het die afname van AYp in altwee, die AMP behandelings groepe en die kontrole groepe getoon. Hierdie studie het ʼn qPCR geoptimaliseer om besmette plantmaterial met AYp op te spoor en dit te kwantifiseer. Die Agrobacterium-bemiddelde tydelike uitdrukingsvektore wat in hierdie studie gebruik is, was nie vertroubaar genoeg, want geen beduidelike effek van die AMPe op AYp konsentrasie kon waargeneen word nie. Hierdie studie het bewys dat AYp nie vasgestel is en in stand gehou kan word deur in vitro aankweeking van V. vinifera cv "Chardonnay‟ material nie, en weefselkulture kan dus ʼn manier wees om AYp in hierdie kultivar uit te roei. Tot kennis, is hierdie studie die eerste studie om die ruimtelike verspreiding van AYp in stingelknope van ʼn besmette V. vinifera cv "Chardonnay‟ wingerstok te rapporteur. / Winetech and DAAD

Both the physiological and biochemical control of budburst in the grapevine, Vitis Vinifera L. were investigated. It was found that the accuracy of a predictive model for grapevine budburst based on ambient temperature was limited under the experimental conditions. There was a significant correlation of 4.7 ± 0.3 days between the days of maximal xylem exudation and budburst over the 3 years of investigation. The co-relationships between daily xylem exudate volume and a range of environmental parameters were considered. It was found that soil temperature was highly correlated against daily xylem exudation. Ambient temperature and soil moisture were significantly correlated with xylem exudation, however the coefficients of correlation were much lower than that of soil temperature. Rainfall showed only a very limited correlation with daily xylem exudate flow. Seasonal variations in the pH and the carbohydrate and inorganic nutrient concentrations of xylem exudate were investigated. Exudate carbohydrate concentrations fell from 660 µM before the day of maximal xylem exudation to zero levels within 4 weeks. Xylem exudate pH was found to consistently fall to a minimum at the time of maximal exudate flow. Exudate concentrations of the metallic cofactors Ca, K, Mg, Mn and Zn varied directly with daily exudate flow, suggesting some sort of flow-dependent mobilisation of these nutrients. A growth promontory oligosaccharide fraction was prepared by partial acid hydrolysis of grapevine primary cell wall material. This fraction significantly increased control growth of the Lemna minor L. bioassay over a limited ‘window’ of bioactivity. A growth inhibitory oligosaccharide fraction, similar in activity to abscisic acid was isolated from grapevine xylem exudate prior to budburst. The exudate concentration or efficacy of this substance declined after budburst such that there was no apparent growth inhibition. A model is proposed for grapevine budburst whereby an oligosaccharide growth inhibitor is gradually removed from the xylematic stream under the effects of soil temperature, allowing the surge of metabolic activity and vegetative growth that constitute budburst.

This project investigated the physiological responses of grapevine to deficit irrigation strategies including partial rootzone drying (PRD) and regulated deficit irrigation (RDI). The principle objectives of the project were to (1) establish if the response to partial rootzone drying (PRD) is as a consequence of irrigation amount or a unique vine physiological response to PRD; (2) to investigate the effect of limiting environmental conditions on vine responses to PRD and deficit irrigation; (3) to investigate the effect of PRD and deficit irrigation on berry metabolism and maturation; and (4) effect of PRD and deficit irrigation on vine water-use. The experiment used field-grown Shiraz grapevines in a commercial vineyard in the Strathbogie Ranges in north-eastern Victoria. The experiment ran from season 2001/2002 through to 2003/2004 in a medium vigour, warm climate vineyard, with soils of low water holding capacity. (For complete abstract open document)

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Within-vineyard variability in vigour and water status commonly occurs in South African vineyards. Different soil types found over short distances are probably the main cause of vigour variability, while differences in grapevine water status are commonly induced by lateral water flow in the vineyard, blocked irrigation emitters and differences in soil water-holding capacity. These factors can cause heterogeneous ripening and differences in fruit quality between different parts of the vineyard, an aspect that needs to be avoided as far as possible in order to produce quality wines. Measurements of carbon isotope discrimination (CID) have proved to be a tool to assess grapevine physiology in order to study the effects of environmental parameters on leaf carbon dioxide (CO2) gas exchange and stomatal conductance (gs). Grapevine water deficit stress/strain in reaction to these environmental conditions can then be determined by observing the amount of 13C absorbed by plant material after discrimination of 13C has taken place, and this is influenced by the grapevine stress condition and can indicate water-use efficiency. In this study, the variability of grapevine water status and vigour was determined in order to quantify these parameters in different parts of the vineyard. Two separate trials were conducted, the first at Wellington, South Africa, where different irrigation regimes resulted in variability in grapevine water status between plots. The second trial was at Stellenbosch, South Africa, where plots were divided among different vigour classes and irrigation was applied in different quantities for different irrigation treatments. Within-vineyard variability in water status (Wellington and Stellenbosch) and vigour (Stellenbosch) were then quantified and the effects on some grapevine physiological parameters and berry composition were measured. The treatments in the Wellington trial led to differences in grapevine water status, which could be quantified by measurements of stem water potential (SWP) and leaf water potential (LWP). Soil variability also led to differences in grapevine vigour, which were quantified by measurements of pruning mass, leaf area and shoot length. The effect of the variability in grapevine water status on grapevine physiology was assessed by measuring CID, which was the main focus of the study. Other physiological measurements, such as gs and leaf and canopy temperature, were also conducted. The effect of these conditions on grape berry composition was also studied. In the Stellenbosch trial, soil water content, plant water status measurements (SWP, predawn LWP and LWP), physiological measurements (CID and gs) and berry size measurements were used to classify plots into water status treatments (“wet” and “dry” treatments). The effect of vigour differences was analysed separately from these treatments by using pruning mass as a covariate in the statistical analyses. The effect of vigour variability on the measurements was studied by looking at the effect of the covariate on the measurements, while shoot growth rate, shoot length and leaf area measurements were conducted as vegetative growth measurements. Differences in measurements were then studied between the treatments and between the vigour levels of the different plots. In the Wellington trial, plant water status was determined by irrigation, showing increased stress for treatments that received less irrigation. The differences in plant water status then caused differences in grapevine physiology between the treatments, leading to increased gs for increased irrigation. This of course influenced leaf internal CO2 and therefore CID, although CID was also clearly influenced by berry development. Berry size was influenced by irrigation, with larger berries found in wetter treatments, while berry chemical composition was influenced by the irrigation regime, with increased irrigation leading to increased pH and leading to trends showing increased total soluble solids and malic acid, and reduced total and tartaric acid and colour intensity. In the Stellenbosch trial, plots with higher vigour had increased shoot growth rate, longer shoots and increased leaf area, although topping influenced this. Wet treatment vines also showed slightly longer shoots and larger leaf areas. There were differences in soil water content between the wet and dry treatments, and this led to differences in plant water status. Vigour also influenced pre-dawn LWP, especially in the 2007 season, as higher-vigour vines struggled more to rehydrate through the night. Differences in plant water potential led to differences in grapevine physiology, with increased gs for vines from the wet treatment, while higher-vigour vines had slightly increased gs. The differences in gs led to gas exchange differences and therefore differences in CID, meaning that water status and vigour influenced CID. CID measurements illustrated the long term effect of water status on plant physiology, while measurements such as SWP illustrated the short term effects. CID measurements therefore proved to be accumulative over the season, in contrast to SWP measurements that were much more dependent on the current state of grapevine water status. Other physiological measurements showed that wet-treatment vines had higher photosynthetic rates and evapotranspiration and lower leaf temperatures, while higher-vigour vines had slightly increased evapotranspiration and decreased leaf temperatures. Wet-treatment vines had larger berries, while a higher vigour also led to slightly larger berries. Berry composition was influenced by treatment, where wet-treatment vines had increased pH and total soluble solids, while higher-vigour vines had increased juice pH and, in the 2008 season, decreased total soluble solids. Extremely stressed conditions did not show significant effects on plant water potential, but SWP measurements indicated slightly higher stress for the extremely stressed vines and LWP showed slightly less stressed conditions for these vines. Measurements of gs showed slightly lower values for the extremely stressed vines, while measurements of CID showed large significant differences, with the extremely stressed vines having measurements showing high stress. The measurement therefore indicated highly stressed conditions accurately, while other physiological measurements, such as photosynthetic rate, evapotranspiration and leaf temperatures, only showed trends and no significant differences. Measurements of stomatal conductance reacted to plant water status measurements throughout the diurnal measurement days, while CID only reacted slightly with gs changes during these days and was perhaps influenced more by berry chemical composition and development at this early stage of the season. Vigour and water status therefore influenced grapevine physiology, with a more direct effect by water status and an indirect effect by vigour due to microclimatic differences. This also influenced berry composition and therefore quality. In future studies, CID measurements should be done on juice from which organic acids have been removed in order to eliminate the effect of seasonal berry composition on the measurement. Measurements of CID proved to be an integrative, but sensitive, indicator of grapevine stress, especially at the end of the season. It might at best be useful as a post-harvest management tool for producers or grape buyers, especially for irrigation control, as has also been stated by Van Leeuwen et al. (2007). / AFRIKAANSE OPSOMMING: Binne-wingerd variasie in groeikrag en waterstatus is algemeen in Suid-Afrikaanse wingerde. Verskillende grondsoorte wat na aan mekaar voorkom, is seker een van die vernaamste oorsake van variasie in groeikrag, terwyl verskille in wingerdwaterstatus algemeen deur laterale watervloei in die wingerd, verstopte besproeiingspuite en verskille in grond waterhouvermoë geïnduseer word. Hierdie faktore kan aanleiding gee tot heterogene rypwording en verskille in vrugkwaliteit tussen verskillende dele van die wingerd, ‘n aspek wat so ver moontlik vermy moet word om kwaliteitwyne te kan produseer. Die meting van koolstof-isotoopdiskriminasie (KID) is bewys om as gereedskap te kan dien vir die assessering van wingerdfisiologie om die effekte van omgewingsparameters op blaar koolstofdioksied (CO2) - gasuitruiling en stomatale geleiding (gs) te bestudeer. Die stres/stremming as gevolg van ‘n watertekort in die wingerd in reaksie op hierdie omgewingstoestande kan dan bepaal word deur te kyk na hoeveel 13C deur die plantmateriaal geabsorbeer word ná 13C-diskriminasie plaasgevind het, en dít word deur die wingerdstrestoestande beïnvloed en kan ‘n aanduiding verskaf van die doeltreffendheid van waterverbruik. In hierdie studie is die variasie in wingerdwaterstatus en groeikrag bepaal om hierdie parameters in verskillende dele van die wingerd te kwantifiseer. Twee afsonderlike proewe is uitgevoer, die eerste by Wellington, Suid-Afrika, waar verskillende besproeiingsregimes gelei het tot verskille in die wingerdwaterstatus tussen persele. Die tweede proef was by Stellenbosch, Suid-Afrika, waar persele tussen verskillende groeikragklasse verdeel is en besproeiing in verskillende hoeveelhede vir verskillende besproeiingsbehandelings toegepas is. Binne-wingerd variasie in waterstatus (Wellington en Stellenbosch) en groeikrag (Stellenbosch) is toe gekwantifiseer en die effekte op sekere wingerd-fisiologiese parameters en korrelsamestelling is gemeet. Die behandelings in die Wellington-proef het gelei tot verskille in wingerdwaterstatus, wat deur metings van stamwaterpotensiaal (SWP) en blaarwaterpotensiaal (BWP) gekwantifiseer kon word. Grondverskille het ook gelei tot verskille in wingerdgroeikrag, wat deur metings van snoeimassa, blaaroppervlak en lootlengte gekwantifiseer is. Die effek van die variasie in wingerdwaterstatus op wingerdfisiologie is deur metings van KID bepaal wat die hooffokus van hierdie studie was. Ander fisiologiese metings, soos gs en blaar- en lowertemperatuur, is ook gedoen. Die effekte van hierdie toestande op die samestelling van die druiwekorrels is ook bestudeer. In die Stellenbosch-proef is grondwaterinhoud, metings van plantwaterstatus (SWP, voorsonopgang SWP en BWP), fisiologiese metings (KID en gs) en metings van korrelgrootte gebruik om die persele in waterstatusbehandelings (“nat” en “droë” behandelings) te verdeel. Die effek van verskille in groeikrag is apart van hierdie behandelings geanaliseer deur snoeimassa as ‘n kovariaat in die statistiese analises te gebruik. Die effek van groeikragvariasie op die metings is bestudeer deur ondersoek in te stel na die effek van die kovariaat op die metings, terwyl lootgroeitempo-, lootlengte- en blaaroppervlakmetings as metings van vegetatiewe groei uitgevoer is. Verskille in metings tussen die behandelings en tussen die groeikragvlakke van die verskillende persele is toe bestudeer. In die Wellington-proef is plantwaterstatus deur besproeiing bepaal, met verhoogde stres in behandelings waar daar minder besproeiing toegedien is. Die verskille in plantwaterstatus het dan verskille in wingerdfisiologie tussen die behandelings veroorsaak, wat gelei het tot ‘n verhoogde gs in die geval van verhoogde besproeiing. Dit het natuurlik ‘n effek op die interne CO2 van die blaar en dus op KID gehad, hoewel KID ook duidelik deur korrelontwikkeling beïnvloed is. Korrelgrootte is deur besproeiing beïnvloed, met groter korrels in die natter behandelings, terwyl die chemiese samestelling van die korrel deur besproeiingsregime beïnvloed is. Verhoogde besproeiing het pH verhoog en gelei na tendense wat verhoogde totale oplosbare vaste stowwe en appelsuur, en verminderde totale suur, wynsteensuur en kleurintensiteit getoon het. In die Stellenbosch-proef het persele met hoër groeikrag ook verhoogde lootgroeitempo, langer lote en verhoogde blaaroppervlak getoon, hoewel dit deur top beïnvloed is. Wingerdstokke van die nat behandeling het ook effe langer lote en groter blaaroppervlakke getoon. Daar was verskille in grondwaterinhoud tussen die nat en droë behandelings en dit het verskille in plantwaterstatus veroorsaak. Groeikrag is ook deur voor-sonopgang BWP beïnvloed, veral in die 2007-seisoen, aangesien stokke met hoër groeikrag meer gesukkel het om in die nag te rehidreer. Verskille in plantwaterpotensiaal het gelei tot verskille in wingerdfisiologie, met ‘n verhoogde gs vir stokke in die nat behandeling, terwyl stokke met hoër groeikrag ‘n effens verhoogde gs getoon het. Die verskille in gs het gelei tot verskille in gasuitruiling en dus verskille in KID, wat beteken dat waterstatus en groeikrag ‘n invloed op KID het. KID was meer verteenwoordigend van die langtermyneffekte van water status op plantfisiologie, terwyl metings soos SWP die korttermyneffekte weerspieël het. KID metings was dus akkumalatief oor die seisoen, terwyl SWP metings meer ‘n weerspieëling was van die huidige toestand van plantwaterpotensiaal. Ander fisiologiese metings het getoon dat stokke in die nat behandeling ‘n hoër fotosintesetempo en evapotranspirasie sowel as laer blaartemperature ondervind het, terwyl die stokke met hoër groeikrag effe verhoogde evapotranspirasie en verminderde blaartemperature getoon het. Stokke in die nat behandeling het groter korrels gehad, terwyl hoër groeikrag ook effens groter korrels veroorsaak het. Korrelsamestelling is deur die behandelings beïnvloed, met stokke in die nat behandeling wat verhoogde pH en totale oplosbare vaste stowwe getoon het, terwyl stokke met hoër groeikrag verhoogde pH van die sap en verminderde totale oplosbare vaste stowwe (laasgenoemde in die 2008-seisoen) gehad het. Uitermate toestande van stres het geen beduidende effekte op plantwaterpotensiaal getoon nie, hoewel SWP-metings effens hoër stres vir die uitermate gestresde wingerde getoon het en BWP effens minder gestresde toestande vir hierdie stokke getoon het. Metings van gs het effens laer waardes vir die uitermate gestresde stokke getoon, terwyl metings van KID groot noemenswaardige verskille getoon het, met die metings vir die uitermate gestresde wingerde wat hoër stres aangedui het. Dié meting het dus hoogs gestresde toestande akkuraat aangedui, terwyl ander fisiologiese metings, soos tempo van fotosintese, evapotranspirasie en blaartemperature net tendense en nie beduidende verskille aangedui het nie. Metings van stomatale geleiding het dwarsdeur die dae waarop daaglikse metings gedoen is op plantwaterstatusmetings gereageer, terwyl KID net effens met gs-veranderinge op hierdie dae gereageer het en moontlik meer deur die chemiese samestelling en ontwikkeling van die korrel in hierdie vroeë stadium van die seisoen beïnvloed is. Groeikrag en waterstatus het dus wingerdfisiologie beïnvloed, met ‘n meer direkte effek deur waterstatus en ‘n indirekte effek deur groeikrag as gevolg van mikroklimaatsverskille. Dit het ook korrelsamestelling en dus kwaliteit beïnvloed. In toekomstige studies moet KID-metings gedoen word op sap waarvan die organiese sure verwyder is om die effek van seisoenale korrelsamestelling op die meting uit te sluit. Metings van KID is getoon om ‘n integrerende, maar gevoelige, aanduider van wingerdstres te wees, veral aan die einde van die seisoen. Dit is ten beste miskien bruikbaar as naoesbestuursgereedskap vir produsente of druiwekopers, veral vir besproeiingsbeheer, soos ook reeds deur Van Leeuwen et al. (2007) aangedui is.

Thesis (MScAgric (Plant Pathology))--Stellenbosch University, 2009. / ENGLISH ABSTRACT: Sufficient fungicide deposition on the target site is an essential requirement for effective chemical management of fruit- and foliar diseases such as grey mould of grapevines. Control failure is often attributed to insufficient quantitative deposition on susceptible grapevine tissue. However, in high disease pressure situations control failure might also be attributed to poor qualitative deposition. The primary objective of spray technology is to optimise deposition, of which the plant surface is a critical component in the spray application process, specifically in the retention of spray droplets. Adjuvant technology is reported to improve the wettability and spread of droplets by surface-acting-agents on the target surface and thereby improve deposition and retention of the fungicide active ingredient. However, this relatively new spray technology on viticulture and horticultural crops, and possible effects of adjuvants on epicuticular wax affecting plant disease development, needs to be investigated. Moreover, the development of useful prescriptions for adjuvants by determining water volumes and adjuvant dosages is required for different pesticide tank mixes. The aims of this study were, firstly to determine the effect of selected adjuvants on quantitative and qualitative spray deposition on grapevine leaves and subsequent biological efficacy of a fungicide, and secondly to evaluate selected adjuvants under field conditions and determine the effects of adjuvant dosage and spray volume on deposition. Leaves were sprayed under similar laboratory conditions to pre-run-off with 1 mL of a mixture of fenhexamid (Teldor® 500 SC, Bayer) at recommended dose, a fluorescent pigment (SARDI Fluorescent Pigment, 400 g/L EC; South Australian Research and Development Institute) at 0.2 L/100 L, as well as 15 selected commercial adjuvants to manipulate the deposition quality of a given quantity of deposited spray. Spray deposition on leaves was illuminated under black light (UV-A light in the 365 nm region) and visualised under a stereo microscope (Nikon SMZ800) at 10× magnification. Photos of sprayed leaf surfaces were taken with a digital camera (Nikon DMX 1200). Digital images were quantitatively and qualitatively analysed with Image-Pro Discovery version 6.2 for Windows (Media Cybernetics) software, to determine spray deposition. The sprayed leaves were inoculated with 5 mg dry airborne conidia of Botrytis cinerea in a spore settling tower and incubated for 24 h at high relative humidity (≥ 93%). Leaf discs were isolated onto Petri dishes with paraquat-amended water agar and rated 11 days later for development of B. cinerea from isolated leaf discs. B. cinerea incidence on the upper and lower surfaces of water sprayed leaves averaged 90.4% and 95.8%, respectively. Despite full spray cover of leaves, applications with fenhexamid alone did not completely prevent infection and resulted in 34.6% and 40.8% B. cinerea incidence on the upper and lower surfaces of leaves, respectively. Through the addition of certain adjuvants, B. cinerea incidences were significantly lower (2.9-17.1% and 10.0-30.8%, respectively), while some adjuvants did not differ from the fungicide-only treatment, even though they might have improved spray deposition. The effects of Hydrosilicote and Solitaire alone and in combination with fenhexamid on germinating Botrytis conidia on leaf surfaces were studied in a histopathology study using epifluorescence microscopy. Distinct differences were observed in conidium mortality, germination and germ tube lengths between adjuvants alone and in combination with the fungicide, which might be attributed to indirect effects of the adjuvant mode of action on B. cinerea. The laboratory study clearly demonstrated the potential of adjuvants to improve the bio-efficacy of a fungicide directly through improved deposition on grapevine leaf surfaces. For the vineyard evaluations, the same fluorometry, photomicrography and digital image analysis protocol were used to assess quantitative and qualitative spray deposits under varying adjuvant dosage and volume applications. The Furness visual droplet-rating technique was initially included to determine optimum spray volume with a STIHL SR400 motorised backpack mistblower by assessment of pigment deposition on Chardonnay leaves under illuminated black light. Both assessment protocols showed that quantitative spray deposition increased with increasing spray volume applications of 40 L/ha to 750 L/ha, but decreased at 900 L/ha, possibly due to run-off. The addition of selected adjuvants at recommended dosage and at 600 L/ha demonstrated the potential of adjuvants to increase quantitative and qualitative deposition significantly on upper and lower leaf surfaces. Agral 90, BB5, Nu-film-P, and Solitaire significantly improved deposition on upper and lower leaf surfaces compared with the fenhexamid only and water sprayed control. Break-thru S 240 and Villa 51 did not improve quantitative deposition, although remarkably better qualitative deposition was obtained. An adjuvant dosage effect (within the registered dosage range) was evident, especially those retained on the upper leaf surfaces. Agral 90 and Nu-film-P affected significant improvement of spray deposition at the higher, but not at the lower dosage tested. Solitaire improved deposition at the lower dosage tested, whereas reduced deposition at the higher dosage was attributed to excessive spray run-off. No significant improvement of spray deposition was observed for both dosages tested with Villa 51. Spray mixtures with adjuvants Agral 90 and Solitaire yielded similar deposition values at 600 L/ha compared with the fenhexamid only control at 900 L/ha, but reduced deposition at the higher spray volume, possibly due to spray run-off. This study clearly demonstrated the potential of adjuvants to improve quantitative and qualitative deposition, but highlights the necessity to match adjuvant dosages and application volumes on the spray target to achieve maximum spray deposition. / AFRIKAANSE OPSOMMING: Effektiewe beheer van vrug- en blaarsiektes soos vaalvrot op wingerde benodig voldoende deponering van die swamdoder op die teikenoppervlak. Verlies aan beheer word gewoonlik aan onvoldoende kwantitatiewe deponering op vatbare wingerddele toegeskryf. Onder ‟n hoë siektedruk kan mislukte beheer ook moontlik toegeskryf word aan swak kwalitatiewe deponering. Die primêre doelwit van spuittegnologie is om deponering te optimaliseer met die plantoppervlak as ‟n belangrike komponent in die spuittoedieningsproses, spesifiek in die retensie van spuitdruppels. Byvoemiddel tegnologie het bewys dat oppervlak-aktiewe-agente verbeterde benatting en verspreiding van druppels op die teiken oppervlakte tot gevolg kan hê, en verder ook die deponering en retensie van die aktiewe fungisied bestanddele kan verbeter. Hierdie relatiewe nuwe spuittegnologie op wingerd- en hortologiese verbouing, asook die moontlike effekte van byvoegmiddels op epikutikulêre waks om siekte ontwikkeling te beïnvloed, moet ondersoek word. Verder word nuttige aanbevelings benodig vir byvoegmiddel toedienings by verskillende spuitvolumes en dosisse van die betrokke spuitmengsel. Die doelwit van hierdie studie was, eerstens om die effek van sekere byvoegmiddels op kwantitatiewe en kwalitatiewe spuitbedekking van wingerdblare te bepaal en dan te vergelyk met die biologiese effektiwiteit van ‟n fungisied, en tweedens om van die byvoegmiddels onder veldtoestande te evalueer, asook die effek van byvoegmiddel dosisse en spuitvolumes te bepaal. Blare is onder dieselfde laboratorium toestande tot net voor-afloop met 1 mL van ‟n spuitmengsel, bestaande uit fenhexamied (Teldor® 500 SC, Bayer) teen die aanbevole dosis, ‟n fluoreserende pigment (400 g/L EC; Suid Australiese Navorsing en Ontwikkeling Instituut) teen 0.2 L/100 L, sowel as 15 geselekteerde kommersiële byvoegmiddels gespuit om die kwalitatiewe deponering, vir ‟n gegewe kwantiteit van spuitdeponering, te manipuleer. Die fluoreserende pigment is op die blaaroppervlak belig met ‟n swart lig (UV-A ligbron in die 365 nm golflengte) en deponering is onder ‟n stereo mikroskoop (Nikon SMZ800) teen 10× vergroting waargeneem. Die gespuite blaaroppervlaktes is op die manier met ‟n digitale kamera afgeneem (Nikon DMX 1200), waarna die digitale foto‟s kwantitatief en kwalitatief deur die gebruik van „Image-Pro Discovery version 6.2 for Windows (Media Cybernetics)‟ sagteware geanaliseer is om spuitbedekking te bepaal. Na elke blaarspuit is die blare met 5 mg droë konidia van B. cinerea in ‟n inokulasietoring geïnokuleer en daarna vir 24 h onder hoë relatiewe humiditeit (≥ 93%) geïnkubeer. ‟n Aantal skyfies vanuit elke blaar is op Petri bakkies met paraquat medium geïsoleer en 11 dae later is die persentasie van B. cinerea ontkieming bepaal. Die gemiddelde voorkoms van B. cinerea op die blare wat slegs met water gespuit is, was 90.4% op die boonste en 95.8% op die onderste blaaroppervlaktes. Spuitbehandelings met slegs fenhexamied, ongeag goeie blaarspuitbedekking, kon nie die B. cinerea infeksie ten volle voorkom nie, en infeksie van gemiddeld 34.6% en 40.8% is onderskeidelik op die boonste- en op die onderste blaaroppervlaktes waargeneem. Met die byvoeging van sekere byvoegmiddels het die voorkoms van B. cinerea betekenisvol verminder (2.9-17.1% en 10.0-30.8%, onderskeidelik), terwyl ander byvoegmiddels nie van die fenhexamied behandeling verskil het nie, hoewel hierdie middels meestal wel spuitdeponering verbeter het. Die effek van slegs Hydrosilicote en Solitaire, en in kombinasie met fenhexamied op ontkiemende Botrytis conidia, is bestudeer in ‟n histopatologiese studie deur middel van die gebruik van epifluoresensie mikroskopie op die blaaroppervlak. Duidelike verskille in die aantal dooie konidia, ontkiemingpersentasies en kiembuislengtes is tussen die byvoegmiddels en in kombinasie met fenhexamied waargeneem, waar sommige waarnemings moontlik aan die indirekte effek van die byvoegmiddel op B. cinerea toegeskryf kan word. Hierdie laboratoriumstudie wys duidelik dat byvoegmiddels oor goeie potensiaal beskik om die bio-effektiwiteit van die fungisied te verbeter deur die direkte verbetering van deponering op die wingerdblaaroppervlak. Dieselfde fluorometrie, fotomikrografie en digitale foto-analise protokol is in ‟n wingerd evaluasie om die kwantitatiewe en kwalitatiewe spuitdeponering van verskillende byvoegmidel dosisse and spuitvolumes te bepaal, gebruik. Die Furness visuele druppel meting tegniek is aanvanklik ingesluit om die optimale spuit volume met ‟n „STIHL SR400 motorised backpack mistblower‟ te bepaal deur visuele meetings van gedeponeerde pigment op Chardonnay blare onder ‟n swart ligbron. Beide protokolle wys dat kwantitatiewe spuitbedekking met ‟n toename in spuit volumes 40 L/ha tot 750 L/ha verbeter het, maar afgeneem het teen 900 L/ha, moontlik as gevolg van druppel-afloop. Die byvoeging van ‟n byvoegmiddel teen die aanbevole dosis en 600 L/ha wys uitstekende potensiaal om kwantitatiewe en kwalitatiewe deponering betekenisvol op boonste en onderste blaaroppervlaktes te verbeter. Agral 90, BB5, Nu-film-P, en Solitaire het deponering betekenisvol op boonste en onderste blare in vergelyking met die fenhexamied alleen en die water kontrole verbeter. Break-thru S 240 en Villa 51 het nie kwantitatiewe deponering verbeter nie, alhoewel verbeterde kwalitatiewe bedekking met hierdie produkte waargeneem is. ‟n Byvoegmiddel dosis effek (binne die registreerde dosis reeks) was duidelik waarneembaar, veral vir druppel retensie op die boonste oppervlak van blare. Agral 90 and Nu-film-P verbeter die spuit deponering betekenisvol met die hoër getoetste dosis, maar nie teen die lae dosis nie. Solitaire verbeter egter die deponering teen die laer dosis, maar minder deponering teen ‟n hoër dosis kan moontlik toegeskryf word aan oormatige druppel-afloop. In die geval van Villa 51 was geen betekenisvolle verbetering van spuitdeponering vir beide die behandelingsdosisse waargeneem nie. Spuitmengsels met byvoegmiddels, Agral 90 en Solitaire, het soortgelyke deponerings gelewer teen 600 L/ha in vergelyking met die fenhexamied kontrole teen 900 L/ha, maar deponering neem af teen hoër spuitvolumes met byvoegmiddels moontlik as gevolg van druppel-afloop. Hierdie studie wys duidelik die uitstekende potensiaal van Byvoegmiddels om kwantitatiewe en kwalitatiewe deponering te verbeter, maar beklemtoon die noodsaaklikheid van die korrekte gebruik van byvoegmiddel dosis en volume om die maksimum spuitdeponering op die teiken te verkry.

Thesis (MScAgric (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The Vitis vinifera L. carotenoid cleavage dioxygenase 1 gene (VvCCD1) is a member of a structurally conserved gene family encoding enzymes that cleave multiple carotenoid substrates to form apocarotenoids. Carotenoid pigments are synthesised in the chloroplast where they are primarily involved in light harvesting and photo-protection during photosynthesis while apocarotenoids fulfill diverse roles that range from pollinator attractants to phytohormones. CCD1 cleaves carotenoids at specific double bond sites producing volatile apocarotenoids. These CCD1-derived apocarotenoids typically possess a fruity and floral aroma, thus making them desirable targets for metabolic engineering. CCD1 orthologues are highly homologous and have been isolated and characterised from a number of plant species, including Arabidopsis, tomato, rose, petunia, and grapevine. VvCCD1 is localised to the cytosol and has been shown in vitro to cleave zeaxanthin and lutein resulting in 3-hydroxy-β-ionone. Expression of VvCCD1 increases during berry ripening, peaking at véraison. Due to the impact that VvCCD1 potentially has on the flavour and aroma of grape berries and therefore wine, this study aimed to characterise the specific enzyme action as well as the biological role that this enzyme plays in grapevine. Expression of VvCCD1 in carotenoid-accumulating Escherichia coli strains demonstrated cleavage of β-carotene at the 9,10 (9’,10’) position forming β-ionone; and lycopene at the 5,6 (5’,6’) and 9,10 (9’,10’) position, forming 6-methyl-5-hepten-2-one and pseudoionone, respectively. A transgenic grapevine population with modified VvCCD1 expression was generated and genetically and metabolically characterised. The transgenic population consisted of lines in which VvCCD1 was either overexpressed or silenced. Expression analysis of stable transformants showed a 12-fold range of VvCCD1 expression relative to the wild-type. HPLC analysis of the photosynthetic pigment content of the transgenic population necessitated the development and optimisation of a method for the extraction of pigments, specifically from grapevine. A number of parameters were identified and optimised, resulting in a method that provides accurate quantification of photosynthetic pigments from grape berries and leaves. Absolute quantification of the following major photosynthetic pigments present in grapevine is now possible: chlorophyll a, chlorophyll b, lutein, -carotene, zeaxanthin, antheraxanthin, violaxanthin and neoxanthin. Data suggest that various levels of molecular control regulate carotenoid cleavage and apocarotenoid biosynthesis. The majority of lines stably transformed with a VvCCD1 overexpression cassette exhibit post-transcriptional gene silencing. Expression analysis in these lines demonstrated that, despite the additional contribution of transgene-derived VvCCD1 transcripts, the total VvCCD1 transcript levels were not significantly higher than in wild-type lines. In lines where transgenic manipulation of VvCCD1 expression was successful, subsequent analysis of carotenoids and apocarotenoids in leaf tissue showed no correlation between the measured metabolites and gene expression. The in planta action of VvCCD1 is presumably distinct from the observed in vitro activity due to the strict compartmentalisation required in photosynthetic leaf tissue preventing access of cytosolic VvCCD1 to the chloroplastic carotenoids. Future studies on reproductive organs (grape berries) from the transgenic lines generated in this study will be of great importance in further elucidation of the in planta function of VvCCD1. / AFRIKAANSE OPSOMMING: Die Vitis vinifera L. “carotenoid cleavage dioxygenase” 1 geen (VvCCD1) behoort aan ‘n geenfamilie wat struktureel gekonserveerd is en kodeer vir ensieme wat verskeie karotenoïed substrate afbreek om apokarotenoïede te vorm. Karotenoïed pigmente word in die chloroplaste gesintetiseer waar hulle primêr betrokke is by lig-insameling, sowel as beskerming tydens fotosintese, terwyl apokarotenoïede diverse funksies in die plant verrig wat strek van aantrekking van stuifmeelverspreiders tot phytohormone. CCD1 breek karotenoïede by spesifieke dubbelbindingsetels af om vlugtige apokarotenoïede te vorm. Die apokarotenoïede wat van CCD1 afkomstig is besit tipies vrugtige en blomagtige aromas wat hul gesogte teikens maak vir metaboliese manipulering. CCD1 ortoloë is hoogs homoloog en is al geїsoleer en gekarakteriseer vanuit ‘n verskeidenheid plantspesies wat Arabidopsis, tamatie, roos, petunia en wingerd insluit. VvCCD1 is in die sitosol gelokaliseer en dit is vantevore gewys dat dit beide zeaxanthin en lutein in vitro kan afbreek om 3-hidroksi-b-ionoon te vorm. Die uitdrukking van VvCCD1 vermeerder tydens korrel rypwording en bereik ‘n maksimum tydens véraison. Weens die potensieële invloed vanVvCCD1 op die geur en aroma van druiwe, en dus wyn, is hierdie studie gerig op die karakterisering van die spesifieke ensiematiese aksie, sowel as die biologiese rol van hierdie ensiem in wingerd. Uitdrukking van VvCCD1 in Escherichia coli rasse wat karotenoïede versamel het getoon dat β-karoteen by die 9,10 (9’,10’) posisie afgebreek word om β-ionoon te vorm, en likopeen by die 5,6 (5’,6’) en 9,10 (9’,10’) posisie om onderskeidelik 6-metiel-5-hepteen-2-oon en pseudo-ionoon te vorm. ‘n Transgeniese wingerd populasie is gegenereer met gewysigde VvCCD1 uitdrukking en is geneties en metabolies gekarakteriseer. Die transgeniese populasie het bestaan uit lyne waar VvCCD1 óf ooruitgedruk óf afgereguleer is. Uitdrukkings analise van die stabiele transformante het ‘n 12-voudige reeks van VvCCD1 uitdrukking getoon, relatief tot die wilde tipe. HPLC analise van die fotosintetiese-pigment inhoud van die transgeniese populasie het die ontwikkeling en optimisering van ‘n wingerd-spesifieke metode vir die ekstraksie van pigmente genoodsaak. ‘n Aantal parameters is geïdentifiseer en geoptimiseer, en het gelei tot ‘n metode wat akkurate kwantifisering van fotosintetiese pigmente in druiwe en wingerdblare kan lewer. Absolute kwantifisering van die volgende belangrike fotosintetiese pigmente aanwesig in wingerd is nou moontlik: chlorophyll a, chlorophyll b, lutein, -karoteen, zeaxantien, anteraxantien, violaxantien en neoxantien. Data dui aan dat verskeie vlakke van molekulêre beheer die afbreking van karoteen en die biosintese van apokarotenoïede reguleer. Die meerderheid van die lyne wat stabiel getransformeer is met ‘n VvCCD1 ooruitdrukkingskasset het na-transkripsioneleafregulering van die geen getoon. Uitdrukking analise van die lyne het gewys dat ten spyte van die addisionele transgeniese VvCCD1 transkripte, die totale VvCCD1 transkripvlakke nie beduidend hoër was as dié van die wilde-tipe lyne nie. In die lyne waar transgeniese manipulasie van VvCCD1 uitdrukking wel suksesvol was, het verdere analise van die karotenoïed en apokarotenoïed vlakke in blaarweefsel geen korrelasie getoon tussen die metaboliete en VvCCD1 uitdrukking nie. Die in planta aktiwiteit van VvCCD1 is vermoedelik anders as die in vitro aktiwiteit weens die streng kompartementalisering benodig in fotosintetiese blaarweefsel, wat verhoed dat die sitosoliese VvCCD1 toegang het tot die chloroplastiese karotenoïede. Toekomstige bestudering van die reproduktiewe organe (druiwe) van die transgeniese lyne wat in hierdie studie gegenereer is sal belangrik wees in die verdere verduideliking van die in planta funksie van VvCCD1.

Thesis (MSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The importance of small antimicrobial peptides in the innate immune system of plants became increasingly apparent over the past decade. Antimicrobial peptides are unique and diverse molecules that are found in many tissue types in a variety of invertebrate, plant and animal species. Many of these peptides, such as plant defensins, have been found to be ubiquitous throughout the plant kingdom and have been isolated from flowers, leaves, roots, seeds, seedlings, pods, tubers and bark. The growing relevance of antimicrobial peptides (including plant defensins) in research can be largely attributed to their broad-spectrum antifungal activity. This makes them promising potential targets, both as therapeutic agents and for their use in crop protection and disease resistance. The continuing discovery of novel antimicrobial peptides has advanced the development of strategies to overexpress these genes in plants to attempt to enhance the plant’s natural ability to resist pathogenic attack. The first grapevine antifungal peptide, Vv-AMP1, was isolated and characterized and was shown to be tissue specific and developmentally regulated, being expressed only in berries at the onset of berry ripening. The peptide showed strong antifungal activity against a number of plant pathogenic fungi in vitro. In this study, the biological role of the Vv-AMP1 peptide was further investigated, both within its native host (Vitis vinifera) and under in vitro conditions against a panel of grapevine-specific pathogens. As a first step, recombinant production of Vv-AMP1 using an existing bacterial expression system was evaluated and the heterologous production of the Vv-AMP1 peptide improved. Specific optimizations targeting both production and purification of the peptide showed to improve the yield of Vv-AMP1. Steps in the production process targeted for improvement included induction conditions of peptide production by the bacterial culture as well as a number of purification steps, such as lysate preparation, binding conditions, column washing, elution conditions and thrombin protease cleavage. The optimized purification method produced up to 3 mg of pure Vv-AMP1 peptide from 1.6 L of overnight culture. While production was markedly improved, the resultant purified Vv-AMP1 proved biologically inactive and structurally unstable. This is uncharacteristic of the peptide, suggesting that an important aspect necessary for peptide activity, such as folding or the presence of specific co-factors might not be supported in this non-host prokaryotic production system. The study also entailed the characterization and evaluation of the Vv-AMP1 peptide against a panel of grapevine-specific pathogens that are culturable to sporulating cultures using in vitro antifungal assays and microscopy analysis. Vv-AMP1 showed strong inhibitory activity against all pathogens tested, inhibiting the growth of Diplodia seriata and Cylindrocarpon liriodendri by 50% at concentrations between 4.8 μg/ml and 9.6 μg/ml. Phaemoniella chlamydospora and Phomopsis viticola proved particularly sensitive, with IC50 values of 5.5 μg/ml and 4.0 μg/ml respectively. Microscopy analysis of the effect of the Vv-AMP1 peptide on P. viticola showed a severe inhibition on fungal germination and growth. The peptide did not induce morphological changes in fungal hyphae but compromises the fungal membranes, supporting the theory that the peptide induces membrane permeabilization. Functional analysis of a transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was included in this study to provide the opportunity to study the in planta role of the peptide in its native host. The genetic characterization of the putative population included confirming gene integration and copy number through PCR and Southern blot analysis as well as gene expression through northern blot analysis. A confirmed transgenic population was evaluated for improved disease resistance against Botrytis cinerea as a first test organism in an attempt to link the overexpression of the Vv-AMP1 gene to a disease resistance phenotype. Observations of lesion type, average lesion size and further statistical analysis concluded that the transgenic population showed a definite, albeit slight, improved resistance when compared to the untransformed control lines. In conclusion, the study determined that Vv-AMP1 had a strong antifungal action against grapevine-specific pathogenic fungi when tested in vitro. A definite link could be established between the overexpression of Vv-AMP1 and a mild resistance phenotype within its native host plant. The characterized transgenic population is important for further work to evaluate the in planta activity of the peptide against more grapevine pathogens such as the stem pathogens that were proven sensitive and specifically those that cannot be cultured and are obligate pathogens, such as the downy and powdery mildews. / AFRIKAANSE OPSOMMING: Die belang van klein antimikrobiese peptiede in die ingebore immuunstelsel van plante het tydens die afgelope dekade toenemend duidelik geraak. Antimikrobiese peptide is unieke en diverse molekules wat in verskeie weefseltipes in ‘n verskeidenheid van invertebraat-, plant- en dierspesies gevind word. Baie van hierdie peptiede, soos bv. “plant defensins”, word bevind om alomteenwoordig in die plantryk te wees en is reeds geïsoleer vanuit blomme, blare, wortels, sade, saailinge, peule, knolle en bas. Die toenemende belang van antimikrobiese peptiede (insluitend “plant defensins”) in navorsing kan grootliks toegeskryf word aan hul breë-spektrum antifungiese aktiwiteit. Hierdie eienskap maak hul belowende potensiële teikens, beide as terapeutiese middels asook vir gebruik in gewasbeskerming en siekteweerstand. Die voortdurende ontdekking van nuwe antimikrobiese peptiede bevorder tans die ontwikkeling van strategieë om hierdie gene in plante uit te druk in ‘n poging om die plant se natuurlike vermoeë om patogeniese aanval teen te staan te verbeter. Die eerste wingerd antifungale peptied, Vv-AMP1, is geïsoleer en gekarakteriseer as ‘n ontwikkelings-gereguleerde peptied wat slegs uitgedruk word in korrels, tydens die aanvang van bessie rypwording. Die peptied het tydens in vitro toetse sterk antifungale aktiwiteit getoon teen ‘n verskeidenheid plant-patogeniese swamme. In hierdie studie word die biologiese rol van die Vv-AMP1 peptied verder ondersoek, beide binne sy natuurlike gasheerplant, (Vitis vinifera) asook onder in vitro kondisies teen ‘n paneel van wingerd-spesifieke patogene. As ‘n beginpunt is rekombinante produksie van Vv-AMP1 met behulp van ‘n bakteriële ekspressie sisteem evalueer en die hetereloë produksie van die Vv-AMP1 peptied stelselmatig verbeter. Spesifieke optimerings het gefokus op beide die produksie en suiwering van die peptied en het die algehele opbrengs van Vv-AMP1 verhoog. Spesifieke stappe wat in die produksieproses vir verbetering geteiken is sluit beide induksietoestande van peptiedproduksie deur die bakteriële kultuur in sowel as ‘n aantal suiweringsstappe, soos lisaatvoorbereiding, bindingskondisies, kolom wasstappe, eluasie kondisies en “thrombin” protease snyding in. Die optimale suiweringsmetode het tot 3 mg suiwer Vv-AMP1 peptied opgelewer vanaf ‘n 1.6 L oornag bakteriële kultuur. Hoewel die produksie van die peptide noemenswaardig verbeter is, was die gesuiwerde Vv-AMP1 beide onaktief en struktureel onstabiel. Dit is buitengewoon vir hierdie peptied, wat daarop dui dat belangrike aspekte benodig vir antifungiese aktiwiteit, soos korrekte vou of die teenwoordigheid van spesifieke kofaktore, moontlik ontbreek in hierdie nie-gasheer prokariotiese produksiesisteem. Die studie het ook die karakterisering en evaluering van die Vv-AMP1 peptied teen 'n paneel van wingerd-spesifieke patogene wat kultureerbaar is en sporuleer, insluitend in vitro antifungale toetse en mikroskopiese analise, behels. Vv-AMP1 toon sterk inhiberende aktiwiteit teen alle patogene getoets. Dit inhibeer die groei van Diplodia seriata en Cylindrocarpon liriodendri met 50% teen konsentrasies tussen 4.8 μg/ml en 9.6 μg/ml. Phaemoniella chlamydospora en Phomopsis viticola was besonders sensitief, met IC50 waardes van 5.5 μg/ml en 4.0 μg/ml, onderskeidelik. Mikroskopiese analise van die effek van die Vv-AMP1 peptied op P. viticola het 'n ernstige inhibisie op swam ontkieming en groei aangedui. Die peptied het geen morfologiese veranderinge in swam hifes veroorsaak nie maar het wel die swam membraan beskadig. Hierdie bevinding ondersteun die teorie dat die peptied membraan permeabilisasie induseer. Funksionele analise van ‘n transgeniese V. vinifera (cv. Sultana) populasie wat die Vv-AMP1 geen ooruitdruk is by die studie ingesluit om ‘n geleentheid te bied om die in planta rol van die peptide binne sy natuurlike gasheerplant te bestudeer. Die genetiese karakterisering van die vermeende transgeniese bevolking het die bevestiging van beide geenintegrasie en kopiegetal deur PKR en Southern-klad analise ingesluit, sowel as geenuitdrukking d.m.v. noordelike-klad analise. ‘n Bevestigde transgeniese bevolking is evalueer vir potensiële verbeterde weerstand (in vergelyking met die wilde tipe) deur infeksie met Botrytis cinerea as ‘n eerste toetsorganisme in ‘n poging om ‘n weerstandbiedende fenotipe met die ooruitdrukking van Vv-AMP1 te assosieer. Waarnemings van letsel tipe, letsel grootte en verdere statistiese analise het tot die gevolgtrekking gelei dat die transgeniese bevolking ‘n definitiewe (dog geringe) verbeterde weerstand toon in vergelyking met die ongetransformeerde lyne. Ten slotte bepaal die studie dat Vv-AMP1 ‘n sterk antifungale effek teen wingerdspesifieke patogene toon tydens in vitro toetse. ‘n Definitiewe korrelasie is vasgestel tussen die ooruitdrukking van Vv-AMP1 in wingerd en ‘n weerstandsfenotipe in die transgeniese bevolking. Die gekarakteriseerde transgeniese bevolking is uiteraard belangrik vir toekomstige werk om die in planta aktiwiteit van die peptied te evalueer teen verdere wingerdpatogene soos bv. die stampatogene wat sensitief getoets het teen die peptide, asook patogene wat nie kultureerbaar is nie, insluitend verpligte patogene soos dons- en poeierskimmel.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The latest strategies in the protection of crops against microbial pathogens are rooted in harnessing the natural, highly complex defense mechanisms of plants through genetic engineering to ultimately reduce the application of chemical pesticides. This approach relies on an in-depth understanding of plant-pathogen interactions to develop reasonable strategies for plant improvement. Among the highly specialized defense mechanisms in the plant’s arsenal against pathogen attack, is the de novo production of proteinaceous antimicrobial peptides (AMPs) as part of the plant’s innate immunity. These AMPs are small, cysteine-rich peptides such as plant defensins that are known for their broad-spectrum of antifungal activity. These plant defensin peptides have been found to be present in most, if not all plant species and the defensin encoding genes are over-represented in plant genomes. Most of these defensins are generally the products of single genes, allowing the plant to deliver these molecules relatively rapidly and with minimal energetic expense to the plant. These factors contribute to establishing AMPs as excellent candidates for genetic engineering strategies in the pursuit of alternative crop protection mechanisms. The first antimicrobial peptide identified and isolated from grapevine, Vv-AMP1, was found to be developmentally regulated and exclusively expressed in berries from the onset of ripening. Recombinantly produced Vv-AMP1 showed strong antifungal activity against a wide range of plant pathogenic fungi at remarkably low peptide concentrations in vitro, however, no in planta defense phenotype could thus far be linked to this peptide. In this study, the antifungal activity of Vv-AMP1 constitutively overexpressed in its native host (Vitis vinifera) was evaluated against grapevine-specific necrotrophic and biotrophic fungi. Firstly, a hardened-off genetically characterised transgenic V. vinifera (cv. Sultana) population overexpressing Vv-AMP1 was generated and morphologically characterized. In order to evaluate the in planta functionality of Vv-AMP1 overexpressed in grapevine, this confirmed transgenic population was subjected to antifungal assays with the necrotrophic fungus, B. cinerea and the biotrophic powdery mildew fungus, Erysiphe necator. For the purpose of infection assays with a biotrophic fungus, a method for the cultivation and infection with E. necator was optimized to generate a reproducible pathosystem for this fungus on grapevine. Detached leaf assays according to the optimized method with E. necator revealed programmed cell death (PCD) associated resistance linked to overexpression of Vv-AMP1 that can be compared to that of the highly resistant grapevine species, Muscadinia rotundifolia. Contrastingly, whole-plant infection assays with B. cinerea revealed that Vv-AMP1 overexpression does not confer V. vinifera with elevated resistance against this necrotrophic fungus. An in silico analysis of the transcription of defensin-like (DEFL) genes previously identified in grapevine was included in this study. This analysis revealed putative co-expression of these DEFL genes and other genes in the grapevine genome driven by either tissue- or cultivar specific regulation or the plant’s response to biotic and abiotic stress stimuli. In conclusion, this study contributed to our knowledge regarding Vv-AMP1 and revealed an in planta defense phenotype for this defensin in grapevine. In silico analysis of the DEFL genes in grapevine further revealed conditions driving expression of these genes allowing for inferences to be made regarding the possible biological functions of DEFL peptides in grapevine. / AFRIKAANSE OPSOMMING: Die nuutste strategieë wat deel vorm van die beskerming van plant gewasse teen mikrobiese patogene het hul oorsprong in die inspanning van die natuurlike, hoogs gekompliseerde verdedigingsmeganismes van die plant deur middel van genetiese enginieurswese ten einde die gebruik van chemiese plaagdoders te verlaag. Hierdie benadering maak staat op ‘n in-diepte begrip van plant-patogeen interaksies om verstandige strategieë vir plantverbetering te kan ontwikkel. Van hierdie hoogs-gespesialiseerde verdedigingsmeganismses in die plant se arsenaal teen patogeen aanvalle sluit die de novo produksie van proteinagtige antimikrobiese peptiede (AMPs) in as deel van die plant se ingebore immuunstelsel. Hierdie AMPs is klein, sisteïen-ryke peptiede soos die plant “defensins” en is bekend vir hul breë-spektrum antifungiese aktiwiteit. Hierdie plant defensinpeptiede word aangetref in meeste, indien nie alle plant spesies nie en die defensin koderende gene word oor-verteenwoordig in plant genome. Meeste van hierdie defensins is gewoonlik die produkte van enkele gene wat die plant in staat stel om hierdie molekules relatief spoedig en met minimale energie verbruik in die plant te vorm. Hierdie faktore dra by tot die vestiging van AMPs as uitstekende kandidate vir genetiese ingenieursstrategieë as deel van die strewe na alternatiewe gewasbeskermingsmeganismes. Die eerste antimikrobiese peptied wat geïdentifiseer en geïsoleer is uit wingerd, Vv-AMP1, word beheer deur die ontwikkelingsstadium en word eksklusief uitgedruk in korrels vanaf die aanvang van rypwording. Rekombinant-geproduseerde Vv-AMP1 het sterk antifungiese aktiwiteit getoon teen ‘n wye reeks plantpatogeniese swamme teen merkwaardige lae peptied konsentrasies in vitro, alhoewel geen in planta verdedigingsfenotipe tot dusver gekoppel kon word aan hierdie peptied nie. In hierdie studie was die antifungiese aktiwiteit van Vv-AMP1 wat ooruitgedruk is in sy natuurlike gasheerplant (Vitis vinifera) ge-evalueer teen wingerd-spesifieke nekrotrofiese- en biotrofiese swamme. Eerstens is ‘n afgeharde geneties-gekarakteriseerde transgeniese V. vinifera (cv. Sultana) populasie wat Vv-AMP1 ooruitdruk gegenereer en morfologies gekarakteriseer. Om die in planta funksionaliteit van Vv-AMP1 ooruitgedruk in wingerd te evalueer is hierdie bevestigde transgeniese populasie blootgestel aan antifungiese toetse met die nekrotrofiese swam, B. cinerea en die biotrofiese swam, Erysiphe necator. Vir die doel om infeksiestudies uit te voer met ‘n biotrofiese swam is ‘n metode geoptimiseer vir die kweek en infeksies met E. necator wat gelei het tot ‘n herhaalbare patosisteem vir hierdie swam op wingerd. Blaarstudies, volgens die pas-verbeterde metode vir E. necator infeksies het ‘n geprogrammeerde seldood-geassosieërde weerstand, gekoppel aan die ooruitdrukking van Vv-AMP1 onthul, wat vergelyk kan word met dié van die hoogs-weerstandige wingerdspesie, Muscadinia rotundifolia. Hierteenoor het heel-plant infeksie studies met B. cinerea onthul dat Vv-AMP1 ooruitdrukking geen verhoogde weerstand teen dié nekrotrofiese swam aan V. vinifera bied nie. ‘n In silico analise van die transkripsie van defensin-agtige (DEFL) gene wat vroeër in wingerd geïdentifiseer is, is by hierdie studie ingesluit. Hierdie analise het vermeende gesamentlike uitdrukking van hierdie DEFL gene en ander gene in die wingerd genoom onthul wat aangedryf word deur weefsel- of kultivar-spesifieke regulering of die plant se reaksie tot biotiese en abiotiese stress stimuli. Ten slotte, hierdie resultate het bygedra tot ons kennis in verband met Vv-AMP1 en het ‘n in planta verdedigingsfenotipe vir hierdie defensin in wingerd onthul. In silico analiese van die DEFL gene in wingerd het verder toestande onthul wat die uitdrukking van hierdie gene aandryf wat ons toelaat om aannames te maak ten opsigte van die moontlike biologiese funksies van DEFL peptiede in wingerd en ondersteun die opstel en toets van hipoteses vir die rol en megansimes van aksie van die wingerd defensin familie.

Bunch stem necrosis (BSN) is a physiological disorder in grapes. It results in shrivelled berries with poor quality attributes such that wine produced from grapes with high BSN incidence is of compromised quality. Past research has proposed many different hypotheses to explain the disorder. Literature indicates that conditions during certain stages of development may predispose berries to BSN but results are not consistent as to which stage is the critical one or which factors have the most impact. This study was designed to resolve these points of uncertainty. Treatments that either enhanced or decreased vine vigour, or manipulated the light environment around the fruit zone were applied to field grown 'Cabernet Sauvignon' vines over three seasons. Treatments included root pruning, heading back of canes by 50%, laying down a reflective mulch and two 50% shade treatments applied for three weeks either pre- or post-full bloom (FB). A strong positive correlation was found between vine vigour and the incidence of BSN. Three weeks post-FB, during both the current and previous season, was identified as the critical period within which factors predispose bunches to BSN. Plant growth regulators, including GA3, IAA and NPA, were applied to bunches on a different group of field grown vines immediately after FB. Application of GA3 during the critical period, tended to reduce the incidence of BSN, while the effects of IAA and NPA application were less clear and require further research. In a controlled environment (CE) trial, pot-grown vines were placed in CE rooms during one of three development stages. Results showed that treatments applied during the critical threeweek period after FB increased the incidence of BSN three fold compared with no change in BSN incidence for vines that were placed in the CE rooms immediately prior to FB or prior to veraison. Collective results from these studies clearly demonstrate that the period immediately following FB is the most critical time in the predisposition of bunches to BSN. It is suggested that competitive dominance of vegetative growth over the developing inflorescence and bunch for assimilates and/or nutrients may be the predisposing factor/s influencing this disorder.