Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Metagenomic studies that make use of next-generation sequencing (NGS) generate large amounts of sequence data, representing the genomes of multiple organisms of which no prior knowledge is necessarily available. In this study, a metagenomic NGS approach was used to detect multiple novel mycoviral sequences in grapevine phloem tissue. Individual sequencing libraries of doublestranded RNA (dsRNA) from two grapevine leafroll diseased (GLD) and three shiraz diseased (SD) vines were sequenced using an Illumina HiScanSQ instrument. Over 3.2 million reads were generated from each of the samples and these reads were trimmed and filtered for quality before being de novo assembled into longer contigs. The assembled contigs were subjected to BLAST (Basic Local Alignment Search Tool) analyses against the NCBI (National Centre for Biotechnology Information) database and classified according to database sequences with which they had the highest identity. Twenty-six putative mycovirus species were identified, belonging to the families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae and Totiviridae. Two of the identified mycoviruses, namely grapevine-associated chrysovirus (GaCV) and grapevine-associated mycovirus 1 (GaMV-1) have previously been identified in grapevine while the rest appeared to be novel mycoviruses not present in the NCBI database. Primers were designed from the de novo assembled mycoviral sequences and used to screen the grapevine dsRNA used for sequencing as well as endophytic fungi isolated from the five sample vines. Only two mycoviruses, related to sclerotinia sclerotiorum partitivirus S and chalara elegans endornavirus 1 (CeEV-1), could be detected in grapevine dsRNA and in fungus isolates. In order to validate the presence of mycoviruses in grapevine phloem tissue, two additional sequencing runs, using an Illumina HiScanSQ and an Applied Biosystems (ABI) SOLiD 5500xl instrument respectively, were performed. These runs generated more and higher quality sequence data than the first sequencing run. Twenty-two of the putative mycoviral sequences initially detected were detected in the subsequent sequence datasets, as well as an additional 29 species not identified in the first HiScanSQ sequence datasets. The samples harboured diverse mycovirus populations, with as many as 19 putative species identified in a single vine. This indicates that the complete virome of diseased grapevines will include a high number of mycoviruses. Additionally, the complete genome of a novel endornavirus, for which we propose the name grapevine endophyte endornavirus (GEEV), was assembled from one of the second HiScanSQ sequence datasets. This is the first complete genome of a mycovirus detected in grapevine. Grapevine endophyte endornavirus has the highest sequence similarity to CeEV-1 and is the same virus that was previously detected in fungus isolates using the mycovirus primers. The virus was detected in two fungus isolates, namely Stemphylium sp. and Aureobasidium pullulans, which is of interest since mycoviruses are not known to be naturally associated with two distinctly different fungus genera. Mycoviral sequence data generated in this study can be used to further investigate the diversity and the effect of mycoviruses in grapevine. / AFRIKAANSE OPSOMMING: Metagenomiese studies, wat gebruik maak van volgende-generasie volgordebepalingstegnologie, het die vermoë om die genetiese samestelling van veelvoudige onbekende organismes te bepaal deurdat dit groot hoeveelhede data genereer. Die bogenoemde tegniek was in hierdie studie aangewend om aantal nuwe mikovirusse in die floëem weefsel van wingerd te identifiseer. Dubbelstring-RNS was gesuiwer vanuit twee druiwestokke met rolbladsiekte en drie met shirazsiekte en Illumina HiScanSQ instrument is gebruik om meer as 3.2 miljoen volgorde fragmente te genereer van elk van die monsters. Lae-kwaliteit volgordes was verwyder en die oorblywende kort volgorde fragmente was saamgestel om langer konstrukte te vorm wat met behulp van BLAST soektogte teen die NCBI databasis geïdentifiseer kon word. Ses-en-twintig mikovirus spesies, wat aan die families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae en Totiviridae behoort, was geïdentifiseer. Twee van die geïdentifiseerde mikovirusse, naamlik grapevine-associated chrysovirus (GaCV) en grapevine-associated mycovirus 1 (GaMV-1), was voorheen al in wingerd gekry terwyl die res nuwe mikovirusse is wat tans nie in die NCBI databasis voorkom nie. Inleiers was ontwerp vanaf die saamgestelde mikovirus basisvolgordes en gebruik om wingerd dubbelstring-RNS sowel as swamme wat vanuit die wingerd geïsoleer is te toets vir die teenwoordigheid van hierdie mikovirusse. Slegs twee mikovirusse, wat onderskeidelik verwant is aan sclerotinia sclerotiorum partitivirus S en chalara elegans endornavirus 1 (CeEV-1), kon deur middel van die inleiers in wingerd en swam isolate geïdentifiseer word. Twee addisionele volgordebepalingsreaksies, wat gebruik gemaak het van die Illumina HiScanSQ en ABI SOLiD 5500xl volgordebepalingsplatforms, was gebruik om die teenwoordigheid van mikovirusse in wingerd te bevestig. Groter hoeveelheid volgorde fragmente was geprodusser wat ook van hoër gehalte was as dié van die eerste volgordebepalingsreaksie. Twee-en-twintig mikovirus spesies kon weer geïdentifiseer word, sowel as 29 spesies wat nie in die eerste HiScanSQ basisvolgorde datastelle gevind was nie. Die wingerdstokke wat in hierdie studie ondersoek was, het hoë diversiteit van mikovirusse bevat aangesien daar tot 19 mikovirus spesies in enkele wingerdstok geïdentifiseer was. Dit is aanduiding dat volledige virus profiele van siek wingerdstokke aantal mikovirusse sal insluit. Die vollengte genoomvolgorde van voorheen onbekende endornavirus was saamgestel vanuit een van die tweede HiScanSQ volgorde datastelle. Dit is die eerste mikovirus wat in wingerd gevind word waarvan die volledige genoomvolgorde bepaal is en ons stel die naam grapevine endophyte endornavirus (GEEV) voor vir hierdie virus. Grapevine endophyte endornavirus is die naaste verwant aan CeEV-1 en is dieselfde virus wat voorheen in wingerd dubbelstring-RNS en swam isolate gevind was deur middel van die mikovirus inleiers. Swam isolate waarin GEEV gevind is, was geïdentifiseer as Stemphylium sp. en Aureobasidium pullulans. Dit is van belang dat GEEV in twee swam isolate gevind is wat aan verskillende genusse behoort aangesien hierdie verskynsel nog nie voorheen in die natuur gevind is nie. Mikovirus nukleiensuurvolgordes wat in hierdie studie bepaal was kan gebruik word in toekomstige studies om die verskeidenheid en impak van mikovirusse in wingerd verder te ondersoek. / National Research Foundation (NRF) / Stellenbosch University

In order to aid the booming wine industry in the state of Virginia, U.S.A., we developed a series of studies to provide a deeper understanding of the viruses and vectors for management of virus diseases and development of better tools for grapevine virus diagnostics. A statewide survey for 14 different grapevine viruses between 2009 and 2014 was conducted: 721 samples were collected from 116 vineyards in the period. Among the 12 viruses identified, Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine rupestris stem-pitting associated virus (GRSPaV), and Grapevine red blotch-associated virus (GRBaV) were most commonly present. A new real-time PCR method for the detection of the V2 gene of GRBaV was developed. The resulting method takes less time for more accurate diagnostics than conventional PCR. Evaluation of insecticide effectiveness on GLRaV-3 vectors (mealybugs) and the spread of GLRaV-3 were examined: Four trials conducted from 2012 to 2014 revealed that despite successful control of mealybugs, GLRaV-3 is spread at a very rapid rate. A new sampling technique for efficient nucleic acid storage and testing was developed: the nitrocellulose membrane-based method allows simpler extraction of nucleic acid and provides a storage medium that can hold viable RNA/DNA at room temperature for up to 18 months. An investigation of multiple virus-infected vines and the impact of these co-infections on grapevine fruit chemistry was conducted. GLRaV-3, GRBaV, GRSPaV, and co-infections of the 3 all negatively impacted Brix, pH, titratable acidity, and anthocyanin levels. / Ph. D.

Grapevine leafroll-associated virus-2 (GLRaV-2), GLRaV-3, and grapevine fleck virus (GFkV) are widespread in grapes around the world. These viruses can cause significant crop loss and affect wine quality by reducing sugar accumulation and compromising skin color. Mealybugs are vectors of grapevine leafroll-associated viruses (GLRaVs). A statewide survey of commercial and wild grapevines in Virginia was conducted during 2009 through 2011. Also, vector management options were tested in two field studies. GLRaV-2, GLRaV-3, and GFkV were detected in 8%, 25%, and 1%, respectively, of over 1,200 vine samples (41 wine grape varieties) from 77 locations, and 64% of vineyards were positive for at least one of the tested viruses. All 100 wild grapevines tested were free of these three viruses, indicating that they are not alternative hosts. The majority of infected vines from commercial vineyards were planted prior to the 1990\'s; however, some new plantings were also found to be positive, indicating movement of the viruses among vineyards and also potential infection prior to planting. The high frequency of virus-infected vines emphasizes the importance of clean plant materials, as well as management of vector insects. The insecticide trials resulted in promising vector control with dinotefuran and spirotetramat; however, acetamiprid and pryrethroid resulted in an increase in mealybug population. This study is the first to examine multiple grape viruses in VA. It will aid in developing better strategies aimed at controlling mealybugs to restrict the movement of viral diseases. / Master of Science in Life Sciences

Thesis (PhD(Agric))--Stellenbosch University, 2012. / ENGLISH ABSTRACT: A lack of defects is required for successful table grape marketing, which pre-suppose optimal vine performance, berry development and post-harvest quality. The supply of mineral nutrients affects vine development, physiology and berry quality. Despite a vast amount of research conducted over decades, there remain many unresolved issues regarding table grape vine nutrition to ensure optimal table grape quality and shelve-life. Unjustified fertilisation practices often include excessive applications of nitrogen (N), potassium (K) and calcium (Ca). A four-year field trial was therefore conducted on a sandy soil in the Paarl district of South Africa, using grafted on Ramsey, and trained to a gable trellis system. Nitrogen, potassium and calcium were applied, singular or in combination, at rates up to 300% the calculated annual nutritional requirement. The effect of these excessive applications on table grape performance under typical South African cultivation conditions was investigated for Vitis vinifera L. cv. Prime Seedless, a very early seedless table cultivar that is produced with minimum berry diameter of 18mm, with special reference to 1) vegetative growth, 2) expression of grapevine nutrient availability through foliar analyses, 3) berry nutrient accumulation patterns of this early cultivar, 4) manipulation of berry nutrient content through soil and bunch directed applications and 5) the effect of berry nutrient content on its quality. No definite vegetative growth responses (expressed as shoot length, leaf surface area and shoot mass) and leaf chlorophyll content differences were obtained for all the treatments. These results were obtained in a vineyard on a sandy soil where excessive N fertilisation caused a reduction of soil pH to detrimentally low levels and where the excessive N, K and Ca applications reduced mutual concentrations and that of Mg, in the soil. A lack of stimulation in vegetative growth may therefore be ascribed to the combined negative effect of these excessive applications on soil pH and vine nutrition. Although the N content of petioles was higher for treatments where N was applied, consistent significant increases in petiole N with N fertilisation were not observed. Petiole N concentration showed a decreasing trend throughout the season. Petiole K concentrations were significantly increased by the K fertilisation at all phenological stages. None of the K fertilisation treatments, however, succeeded to raise petiole K concentrations above the accepted maximum norms and petiole K concentration at a specific sampling stage varied significantly between the four seasons. A general decrease in petiole K concentration was found for all seasons. Calcium fertilisation did not increase soil Ca content, resulting in a lack of differences in petiole Ca concentrations between treatments. An increase in petiole Ca concentration towards harvest was obtained. Correlations between petiole nutrient concentration and berry mineral content at harvest were poor. The only way of knowing the mineral content of berries would seem to be by measuring it directly instead of deducing it from the results of leaf or petiole analyses. The dynamics of berry growth impacted on berry nutrient concentration. Early rapid berry growth, predominantly due to cell division and cell growth, was associated with the most rapid decreases in N, P and Ca concentration. Due to mobility of K and Mg in the plant, that exceeds other nutrients, the decrease in concentration of these two mineral elements was not as pronounced as that of the others. Nutrient accumulation was most rapid during the pre-véraison period, but only Ca showed a definite termination during the early ripening period. The continued inflow of N, P, K and Mg, albeit at slower rates immediately after véraison, should be taken into consideration when fertilisation is applied. As a table grape, total accumulation of each nutrient in Prime Seedless berries also far exceeded that of other cultivars studied thus far. A particular difference is that the berry flesh:skin ratio is much higher than that of previously studied cultivars, leading to higher levels of nutrient accumulation in the flesh. Slightly larger berry size was obtained for N applications and is ascribed to slight increases in early vegetative growth, allowing a better response to GA3 treatments. The use of GA3 for berry enlargement is also considered the reason why K fertilisation, resulting in increased berry K levels, did not affect berry size, as is often found for wine grapes. Higher available NO3 - in the soil on account of excessive N applications resulted in higher levels of berry N, despite sub-optimal soil pH regimes that were created by these treatments. Berry K concentration and content were increased by K fertilisation. Rapid vine K uptake and translocation to the berries seem to negate the reduced vine nutritional status as observed in petioles for situations of over-fertilisation with N. Berry Ca levels were not increased by Ca fertilisation or by bunch applied Ca. The rapid rates of berry growth, together with low rates of berry Ca uptake and Ca uptake that terminates at the onset of ripening, are assumed to be the main reasons for this result. Low levels of decay as well as a lack of consistently increased decay were obtained for N containing treatments. Nitrogen levels in the berries above which their susceptibility to fungal infection is increased, should be established. Information on specific N compounds that may lead to more susceptibility is required. Potentially increased berry browning on account of high rates of K fertilisation needs to be further investigated; indications that this may occur were observed. Neither soil applied Ca nor bunch applied Ca improved berry quality, although Ca treatments seemed to reduce decay during the only season that significant differences were obtained. The negative effect of excessive fertilisation on soil chemistry of sandy soils has again been highlighted by this study. This annuls the fertilisation, leading to inefficient fertilisation and a lack of the desired responses. As indicator of vine nutrient availability, petiole analysis, was proven unreliable and should be evaluated in parallel with soil analyses, taking seasonal variation into consideration. The danger of being only guided by published norms for leaf nutrient concentrations when establishing fertilisation practices has again been highlighted by this study. This research indicated that for a very early cultivar like Prime Seedless, nutrient accumulation dynamics can already start to change during the pre-véraison period in some seasons. This is due to different edaphic and climatic conditions as well as berry size, which leads to much higher flesh:skin ratios. Future research on table grapes would need to develop an understanding of the various factors and dynamics that determine berry nutrient concentration and accumulation of early ripening, large berry sized, seedless table grape cultivars. / AFRIKAANSE OPSOMMING: Suksesvolle bemarking van tafeldruiwe is ten nouste afhanklik van die beskikbaarheid van druiwe sonder defekte, wat ‘n direkte verband met optimale wingerdprestasie, korrelontwikkeling en na-oes kwaliteit inhou. Voorsiening van minerale voedingstowwe beïnvloed die stok se groei, fisiologie en korrelgehalte. Ten spyte van ‘n oorweldigende hoeveelheid navorsing wat oor dekades reeds gedoen is, is daar steeds onopgeloste kwessies aangaande bemesting van tafeldruiwe vir optimale druifgehalte en houvermoë. Die gevolg is onoordeelkundige bemestingspraktyke wat o.a. aanleiding gee tot oorbemesting met stikstof (N), kalium (K) en kalsium (Ca). ‘n Vier-jaar-lange veldproef is gevolglik op ‘n sandgrond in die Paarl distrik (Suid-Afrika) onderneem deur gebruik te maak van Vitis vinifera L. cv. Prime Seedless geënt op Ramsey en op ‘n dubbel-gewel prieelstelsel opgelei is. Stikstof, K en Ca is alleen, of in kombinasie, toegedien teen hoeveelhede gelykstaande aan 300% van die wingerd se jaarlikse behoefte. Die effek van hierdie oormatige toedienings op tafeldruif prestasie onder Suid-Afrikaanse verbouingstoestande is ondersoek, met spesiale verwysing na 1) vegetatiewe groei, 2) uitdrukking van voedingstofbeskikbaarheid deur blaarontledings, 3) die voedingstof akkumulasie patrone van korrels van hierdie vroeë kultivar, 4) manipulasie van korrel voedingstofinhoud deur grond en trosgerigte toedienings en 5) die effek van korrel voedingstofinhoud op kwaliteit. Die doel van die proef was om bemestinspraktyke van Prime Seedless, ‘n baie vroeë pitlose tafeldruifkultivar met ‘n minimum korrelgrootte van 18 mm, te verfyn. Deur die akkumulasie patrone van die druiwe uit te klaar is daar ook ondersoek ingestel of oestyd en na-oes gehalte deur oormatige toediening van voedingstowwe affekteer word. Geen duidelike verskille betreffende vegetatiewe groeireaksies (uitgedruk as lootlengte, blaaroppervlaktes en lootmassas) asook verskille in blaar chlorofilinhoud is vir die behandelings verkry nie. Hierdie resultate is verkry in ‘n wingerd op ‘n sandgrond, waar oormatige N-bemesting aanleiding gegee het tot grond pH verlagings tot die peil van nadelige vlakke. Verder het die oormatige N, K en Ca toedienings wederkerige verlagings in konsentrasies, asook op dié van Mg, in die grond teweeggebring. Die tekort aan vegetatiewe groeiresponse op die behandelings kon dus toegeskryf word aan ‘n gekombineerde effek van die oormatige toedienings op grond pH en voedingstofbalanse. Hoewel die N-inhoud van bladstele hoër was vir behandelings wat N toediening ingesluit het, was daar nie konstante toenames in die vlakke verkry nie. Bladskyf N-konsentrasie het afgeneem deur die loop van die groeiseisoen. Vir alle fenologiese stadiums was bladskyf Kkonsentrasies betekenisvol verhoog deur K-bemesting. Nie een van die Kbemestingsbehandelings het egter daarin geslaag om bladskyf K inhoud vir enige monstertyd bo die algemeen aanvaarde maksimum norms te lig nie. Verder het bladskyf K inhoud by ‘n spesifieke fenologiese stadium ook betekenisvol tussen seisoene verskil. Die K-inhoud van bladskywe het afgeneem met verloop van die seisoen. Kalsiumbemesting het nie die grond se Ca inhoud deurgans verhoog nie, wat dus die tekort aan verskille in Ca konsentrasies tussen die behandelings verklaar. ‘n Toename in Ca konsentrasie en korrel Ca inhoud is vanaf set tot oes waargeneem. Swak korrelasies tussen bladskywe se voedingstofinhoude en korrels se voedingstofinhoude is verkry. Die enigste manier waarop korrels se voedingstofinhoude dus afgelei kan word, blyk te wees deur direkte bepaling daarvan. Voedingstofinhoude van korrels is deur groeipatrone daarvan beïnvloed. Vroeë korrelgroei, hoofsaaklik a.g.v. seldeling en selgroei, het met die vinnigste afnametempo van N, P en Ca gepaard gegaan. As gevolg van die hoër beweeglikheid van K en Mg in die plant in vergelyking met ander voedingstowwe, was die afname in konsentrasie van hierdie twee elemente nie so groot soos vir die ander nie. Voedingstofakkumulasie was die vinnigste in die periode voor deurslaan. Slegs Ca het ‘n beeïndiging van opname aan die einde van hierdie periode getoon. Die voortgesette opname van N, P, K en Mg, alhoewel stadiger kort na deurslaan, moet in ag geneem word wanneer bemesting toegedien word. Vir hierdie kultivar het die totale opname van elke bemestingstof dié van die ander kultivars wat tot hede bestudeer is, ver oorskry. ‘n Spesifieke verskil is ‘n baie hoër vleis:dop verhouding as wat vir ander kultivars verkry is. Dit gee aanleiding tot baie hoër vlakke van voedingstofakkumulasie in die vleis. Effens groter korrelgroottes is verkry waar N toedienings gemaak is. Dit word toegeskryf aan klein toenames in vroeë vegetatiewe groei, wat dus beter reaksie op GA3 behandelings tot gevolg gehad het. Die gebruik van GA3 vir korrelvergroting word ook beskou as die rede waarom K-bemesting, wat tot hoër vlakke van K in die korrels aanleiding gegee het, nie korrelgrootte, soos by wyndruiwe, bevorder het nie. Hoër NO3 - in die grond (water), na aanleiding van N toedienings, het aanleiding gegee tot hoer vlakke van N in die korrels. Dit het plaasgevind ten spyte van sub-optimale grond pH wat deur die oormatige N toedienings veroorsaak is. Korrel K konsentrasie en -inhoud is deur K-bemesting verhoog. Vinnige opname en translokasie van K na die korrels het ook geblyk die rede te wees waarom die verlaagde voedingstatus van die stokke a.g.v. oorbemesting met N nie die korrels se K inhoud geaffekteer het nie. Die vinnige groeitempo van die korrels, tesame met lae vlakke van Ca opname, asook korrels se Ca opname wat tydens rypwording ophou, word as die redes vir die tekorte aan behandelingseffekte beskou. Lae vlakke van bederf, asook ‘n tekort aan betroubare tendense dat bederf deur Nbemesting verhoog word, is verkry. Daar moet vasgestel word of daar N vlakke in die korrels is waarbo hul vatbaarheid vir swaminfeksies verhoog word, en of daar spesifieke N verbindings is wat die korrels meer vatbaar maak vir bederf. Indikasies dat K-bemesting interne verbruiningsvlakke verhoog het, regverdig verdere ondersoek. Korrelkwaliteit is nie deur grond- of trosgerigte toedienings bevoordeel nie. Die negatiewe effek van oormatige bemesting op die chemiese samestelling van sandgronde is weer deur hierdie navorsing uitgelig. Dit lei tot oneffektiewe bemesting en ‘n tekort aan die verlangde effekte. Blaarontledings blyk onbetroubaar te wees as aanduiding van voedingstof beskikbaarheid. Dit moet evalueer word saam met grondontledings en ook seisoenale variasie in ag neem. Die gevaar om slegs deur gepubliseerde norme gelei te word wanneer bemestingspraktyke bepaal word, is weer deur hierdie navorsing uitgelig. Voorst is daar in hierdie navorsing gevind dat voedingstof akkumulasiepatrone van ‘n baie vroeë kultivar soos Prime Seedless alreeds voor deurslaan begin verander a.g.v. omgewingstoestande en korrelgroei wat tot ‘n veel hoër vleis:dop verhouding aanleiding gee. Toekomstige navorsing op tafeldruiwe behoort die faktore en dinamika wat voedingstofkonsentrasie en -akkumulasie in korrels van vroeë, groot korrel, pitlose tafeldruifkultivars beïnvloed verder te ondersoek.

Vitis vinifera subsp. vinifera L., les principales espèces de raisins sont cultivées pour la production de fruits et la production de vin dans le monde est un hôte naturel d'une grande variété de micro-organismes procaryotes et eucaryotes qui interagissent avec la vigne, ayant des effets bénéfiques ou phytopathogènes. Ils pourraient également jouer un rôle majeur dans le rendement des fruits, la qualité du raisin, la protection des plantes et, finalement, dans le modèle de la fermentation du raisin et la production de vin. La phyllosphère (constituée des parties aériennes de la plante) est l'un des habitats microbiens les plus répandus sur terre et est un milieu assez négligé, en particulier dans les vignes et de nombreuses questions liées à cet habitat microbien sont toujours sans réponse.Cette thèse est un effort pour répondre à une question fondamentale en écologie microbienne: quels sont les facteurs qui déterminent le microbiome dans la phyllosphère de la vigne? Les communautés microbiennes de la phyllosphère (PMCs) vivent à l'interface plante-climat et sa capacité à s'établir, prospérer et se reproduire sur la surface des feuilles ou des fruits dépend de plusieurs caractéristiques fonctionnelles microbiennes, comme la capacité de se fixer sur la cuticule et d'utiliser la foliaire. nutriments ainsi que les conditions climatiques dominantes comme la température, l'humidité de l'air et la pluie. La chimie des feuilles ou des fruits, la physiologie et la structure morphologique diffèrent selon le génotype et l'espèce puisque tous ces traits ont une base génétique, et cette variation peut mener à une combinaison différente d'assemblage de PMC parmi les génotypes de plantes. Ainsi, le premier objectif de notre travail était d'évaluer les impacts des cultivars de vigne (variétés de Vitis vinifera L) et des espèces de vigne (espèces Vitis entièrement différentes) sur l'assemblage du microbiome dans la phyllosphère à un endroit géographique particulier (pour minimiser les effets environnementaux) . Plus tard, les impacts de certains cultivars et terroirs de vigne commercialement importants (représentés par trois zones climatiques françaises) ont également été évalués et comparés. Les impacts de la saison et des organes extérieurs de la plante (feuilles et baies) sur la structuration des taxons microbiens dans la phyllosphère ont également été évalués et présentés dans ce travail. De plus, des impacts spécifiques à l'espèce sur le microbiome de la phyllosphère ont également été testés et représentés.Dans l'ensemble, notre étude a évalué et comparé les nombreuses facettes des facteurs qui peuvent influencer structure du microbiome dans la phyllosphère avec un accent particulier sur la pression de sélection relative exercée par le génotype de la vigne et son interaction avec différentes conditions climatiques (ou terroir), ce qui peut améliorer nos chances de trouver des gènes contrôlant les PMCs sur la phyllosphère. les gènes sont réellement importants dans des environnements réalistes et probablement ces gènes nous donneraient de nouvelles idées pour la sélection de nouveaux cépages sains présentant de meilleurs caractères sur leur phyllosphère. De plus, considérant que les PMC végétales jouent un rôle crucial dans la santé et la forme des plantes car elles peuvent moduler la susceptibilité foliaire aux infections, cette étude pourrait également être utile pour développer des méthodes de biocontrôle innovantes et naturelles ou phytostimulation contre les pathogènes de la vigne. de variétés résistantes innovantes. / Vitis vinifera subsp. vinifera L., the main grape species are grown for fruit and wine production over the world is a natural host of a wide variety of prokaryotic and eukaryotic microorganisms that interact with grapevine, having either beneficial or phytopathogenic effects. They could also play a major role in fruit yield, grape quality, plant protection and, ultimately, in the pattern of grape fermentation and wine production. Phyllosphere (consists of the aerial parts of the plant) is one of the most prevalent microbial habitats on earth and is quite a neglected milieu, especially in grapevines and many questions related to this microbial habitat, are still unanswered.This thesis is an effort to answer a very fundamental question in microbial ecology- what are the drivers that shape the microbiome in the grapevine's phyllosphere? The phyllosphere microbial communities (PMCs) live at the plant-climate interface and its ability to establish, thrive and reproduce on the leaf or fruit surface depends on several microbial functional traits, such as the ability to attach to the cuticle and to use the foliar nutrients as well as well as to the prevailing climatic conditions like temperature, air humidity and rain. Leaf or fruit chemistry, physiology, and morphological structure differ among plant genotype and species as all these traits have a genetic basis, and this variation may lead to a different combination of PMCs assemblage among plant genotypes. Hence, the first objective of our work was to assess the impacts of grapevine cultivars (varieties of Vitis vinifera L) and grapevine species (entirely different Vitis species) on microbiome assemblage in the phyllosphere at a particular geographic location (to minimize the environmental effects). Later on, impacts of some commercially important grapevine cultivars and terroirs (represented by three French climate zones) were also assessed and compared. Impacts of the season and exterior plant organs (leaf and berries) on microbial taxa structuring in the phyllosphere was also assessed and presented in this work. Furthermore, species-specific impacts on phyllosphere microbiome were also tested and represented.Overall our study assessed and compared the many facets of the factors that may influence themicrobiome structure in the phyllosphere with a special focus on relative selection pressure exerted by grapevine genotype and its interaction with different climatic conditions (or terroir), which may improve our chances to find genes that controls PMCs on phyllosphere, and simultaneously increase our confidence that those genes are actually important in realistic environments and probably those genes would give us new insights for breeding new and healthy grape varieties displaying better traits on their phyllosphere. Moreover, considering that the plant PMCs plays a crucial role in plant health and fitness as it can modulate leaf susceptibility to infection, this study could also be helpful to develop innovative and natural biocontrol methods or phytostimulation against grapevine pathogens or rethink breeding schemes for the creation of innovative resistant varieties.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Cabernet Sauvignon is the most planted red cultivar in South Africa and the second most planted red cultivar in the Olifants River region. The cultivar is prone to vigorous growth with low yields. Excessive irrigation could accentuate these cultivar characteristics. Considering the foregoing, the aim of the study was to describe how Cabernet Sauvignon will react to climate, soil type (texture) and irrigation within the Lower Olifants River wine region to enable growers to make the right decisions regarding long term as well as short term cultivation practices. This study is part of a project carried out by the ARC Infruitec-Nietvoorbij at Stellenbosch to determine the effects of soil type and atmospheric conditions on yield and wine quality of Cabernet Sauvignon in different grape growing regions of South Africa. Similar studies are being carried out in the Orange River, Stellenbosch and Swartland regions of South Africa. The Lower Olifants River region could be divided into three climatic regions. Furthermore, two climatic regions is evident regarding the formation of grape wine colour and aromas. Proximity to the Atlantic Ocean would play an important role in a cultivar establishment policy. The variation in stem water potential (ΨS) could be related to soil water status expressed in terms of matric potential (ΨM). In the case of sandy soils, ΨS decreased substantially more as the ΨM decreased compared to the sandy loam soils. The reason could be that the unsaturated hydraulic conductivity in sandy soils decreased more rapidly as the ΨM decreased compared to the heavier soils. Thus could explain why the grapevines in the sandy soils experienced more water stress than the ones in the sandy loam soils at a given ΨM. Climate had a strong influence on grapevine water status with grapevines nearer to the ocean experiencing less water stress compared to the ones further inland. This was especially true for grapevines in the sandy soils. Vegetative growth and yield of grapevines in the sandy soils were more sensitive to water deficits compared to the ones in the sandy loam soils. For deficit irrigated grapevines in the sandy soils, vegetative growth and yield decreased by ca. 30% when ca. 55% less water was applied from flowering to harvest. Yield reduction were ca. 15% with no or very little influence on vegetative growth with ca. 80% reduction in water applied from flowering to harvest for grapevines in the sandy loam soils. The influence of soil texture on wine quality and style were evident under intensive irrigation as well as over different climatic regions. Overall sensorial potential wine quality of grapevines in sandy soils tended to be higher compared to the ones in the sandy loam soils. Deficit irrigation tended to increase wine colour intensity, irrespective of soil texture. Furthermore, deficit irrigation in sandy loam soils tended to increase wine fullness and the berry characteristics of the wine. Berry characteristics of wines from the sandy soils tended to be higher compared to the ones from the sandy loam soils. Too severe water deficits in sandy soils could be detrimental to wine quality. Climate tended to have an influence on wine style of grapevines in the sandy soils with wines produced further away from the ocean tended to have higher berry characteristics. Irrigation management could be a powerful tool to manipulate the grapevine in sandy soils. For grapevines the sandy loam soils in addition to regulated deficit irrigation, additional canopy management practices could be needed to improve wine quality. / AFRIKAANSE OPSOMMING: Cabernet Sauvignon is die mees aangeplante rooidruif kultivar in die Suid-Afrikaanse wynbedryf. In die Olifantsriver streek is dit naas Shiraz, die tweede mees aangeplante rooidruif kultivar. Cabernet Sauvignon is bekend as ‘n groeikragtige skaamdraer. Indien oorbesproei word, kan hierdie potensiële nadelige eienskappe nog meer na vore tree. Die doel van die studie is om die invloed van die klimaat, grond en besproeiing op Cabernet Sauvignon se vegetatiewe groei, produksie en wyngehalte in die Benede Olifantsrivier streek te bepaal. Hierdie inligting kan produsente help om ingeligte kortsowel as langtermyn besluite te maak rakende die verbouing van Cabernet Sauvignon. Hierdie studie vorm deel van ‘n breër studie in die Suid-Afrikaanse wynbedryf, gedryf deur die Landbou Navorsingsraad (LNR) Infruitec-Nietvoorbij, Stellenbosch om die invloed van atmosferiese toestande en grond op die produksie en wyngehalte van Cabernet Sauvignon te bepaal. Soortgelyke projekte word uitgevoer in die Oranjerivier, Stellenbosch en Swartland wynstreke. Die Benede Olifantsrivier streek kan verdeel word in drie klimaatstreke op grond van temperatuurdata. In terme van die ontwikkeling van druifkleur en aromas, kan die streek verdeel word in twee klimaatstreke. Die afstand vanaf die Atlantiese Oseaan kan ‘n belangrike rol speel in die ontwikkeling van ‘n kultivarriglynplan vir die streek. Grondwaterstatus, uitgedruk as die matrikspotensiaal (ΨM), kan aanleiding gee tot variasie in middag blaarwaterpotensiaal (ΨS) lesings. Die ΨS van die sand gronde verlaag vinniger soos die ΨM verlaag invergelyke met die sandleem gronde. Dit kan moontlik wees as gevolg van die verskil in die grond onversadigde hidroliese konduktiwiteit. Sand gronde se hidroliese konduktiwiteit verlaag vinniger soos die ΨM verlaag, invergelyke met sandleem gronde. Dit verklaar waarom wingerde in sand gronde by dieselfde ΨM, meer waterspanning ondervind as wingerde in sandleem gronde. Klimaat het ‘n invloed op die waterstatus van die wingerdstok. Wingerde nader aan die see het minder waterspanning ondervind invergelyke met wingerde wat verder in die binneland geleë is. Dit was veral die geval met wingerde in die sand gronde. Vegetatiewe groei en produksie van wingerde in die sand gronde is meer sensitief vir waterspanning as wingerde in die sandleem gronde. Tekortbesproeiing in die sand gronde het die groei asook produksie met ongeveer 30% verlaag deur ongeveer 55% minder water toe te dien vanaf blom tot oes. In teenstelling daarmee is die produksie van wingerde in die sandleem gronde met ongeveer 15% verlaag met geen tot baie min verlaging in die groeikrag. Ongeveer 80% minder water is toegedien vanaf blom tot oes. Grondtekstuur kan wyngehalte en -styl beïnvloed ten spyte van intensiewe besproeiing en klimaatsverskille. Sensoriese potensiële wyngehalte van wingerde in die sand gronde was beter invergelyke met dié van die sandleem gronde. Die wyne vanaf die sand gronde het ook geneig om oor meer bessie intensiteit te beskik as wyne vanaf die sandleem gronde. Tekortbesproeiing neig om die wynkleur intensiteit te verhoog, ongeag van grondtekstuur. Tekortbesproeiing in die sandleem gronde kan ook die volheid van die wyne verbeter, asook die bessie intensiteit van die wyn verhoog. Te hoë waterspanning in die sand gronde kan wyngehalte nadelig beïnvloed. Klimaat kan ook die wynstyl vanaf sand gronde beïnvloed met wyne verder vanaf die see wat oor meer bessie intensiteit beskik as wyne nader aan die see. Beheerde tekortbesproeiing kan as ‘n kragtige hulpmiddel gebruik word om wingerde in die sand gronde te manipuleer. Vir wingerde in die sandleem gronde, addisioneel tot beheerde tekortbesproeiing en normale loofbestuurspraktyke, kan ekstra loofbestuurspraktyke bv. die verwydering van sylootlote, dalk nodig wees om wyngehalte te verbeter.

Disputats. Universität Hohenheim, 2002. / Haves kun i elektronisk udg. Thesis (Ph.D.). 41 ill., 5 tables; approx. 200 ref. Summaries (En, Ge).

Downy mildew, caused by the biotrophic Oomycete Plasmopara viticola, is one of the most important diseases of grapevines world wide. It is particularly destructive in temperate viticultural regions that experience warm wet conditions during the vegetative growth of the vine (Wong et al., 2001). The disease is not normally a problem in mediterranean climates where the growing season tends to be hot and dry (Mullins et al., 1992; Sivasithamparam, 1993). Grape downy mildew is however a major disease in Australian viticulture (McLean et al., 1984; Magarey et al., 1991). Grape downy mildew was first reported in Europe in 1878 (Viennot-Bourgin, 1981). In Australia, it was recorded for the first time in 1917 at Rutherglen in Victoria (Vic) (de Castella, 1917). The first recorded outbreak of the disease in Western Australia (WA) occurred in 1997 in a small planting of vines in the far north of the state. In the subsequent year, it was detected in widespread commercial viticulture in the Swan Valley production area, near Perth (McKirdy et al., 1999). The pathogen has since been found in all grape growing regions of WA. Since its introduction into European vineyards in the 1880?s, P. viticola has become one of the world?s most investigated grapevine pathogens. Many aspects its basic biology however remain unknown (Wong et al., 2001). Due to the recent detection of P. viticola in WA, little is known of the nature of strains of the pathogen in the state and their response to local environmental conditions. Much of the research concerning the influence of environmental factors on the development of P. viticola has been conducted in Europe e.g. parts of France and Germany. Due to significant differences in climatic conditions and a shorter selection time on the pathogen in WA, much of the information described in European studies may not be directly applicable to the grape downy mildew disease situation in WA. The focus of this thesis was to examine epidemiological aspects of P. viticola in the mediterranean climate of WA. The environmental conditions that could favour the development of epidemics by strains of the pathogen that have been detected in the state were determined. The existence of P. viticola ecotypes and genetic variation among strains from WA and the Eastern states of Australia was also investigated.

A videira é uma das plantas mais antigas cultivadas pela humanidade, sendo que no Brasil a uva é a terceira fruta com maior volume de produção, atrás apenas do cultivo das bananas e das laranjas. Apesar da produção rentável, principalmente aos pequenos produtores, o parreiral é susceptível a várias doenças cujo manejo compromete até 59% dos gastos do produtor. No estado de São Paulo, dentre as doenças, três têm destaque: a antracnose (causada pelo Sphaceloma ampelinum), o míldio da videira (causado pelo Plasmopara viticola) e a ferrugem (causada pelo Phakopsora euvitis). Os produtores utilizam controle químico de forma intensa e preventiva, chegando a 100 aplicações de fungicidas em um ciclo de até 120 dias. Os principais fungicidas utilizados são os inibidores da quinona oxidase (QoI), que agem impedindo o transporte de elétrons do citocromo b ao citocromo c1 na cadeia respiratória da mitocôndria. Porém, existem relatos de resistência ao fungicida aplicado no campo em diversos países. As substituições G143A, G137R e F129L na sequência da proteína citocromo b impedem que o fungicida se ligue ao seu sítio alvo. As mutações que levam às substituições estão localizadas em uma das regiões chamada hotspot do gene citocromo b (cytB). Visto que, pela carência de estudos, a resistência genética a esses fungicidas nunca foi relatada no Brasil, o objetivo principal desse trabalho foi sequenciar e caracterizar a região hotspot em isolados de míldio, ferrugem e antracnose provenientes de parreirais do estado de São Paulo. Foram selecionados 35 isolados de 11 locais diferentes; desses, 11 isolados de míldio foram considerados geneticamente resistentes, pois apresentam a mutação para o resíduo alanina na posição 143, e 4 isolados foram considerados geneticamente sensíveis. Os dois isolados de ferrugem selecionados também foram considerados geneticamente sensíveis. Pela estratégia de Genome Walking foi possível sequenciar 65% do gene cytB de um dos isolados brasileiros de P. viticola; foram encontrados poucos polimorfismos e nenhum íntron na sequência analisada. Os resultados obtidos com esse estudo podem servir de suporte para a tomada de decisões de manejo mais adequadas para a realidade da viticultura brasileira; além disso, são importantes para futuros estudos sobre a evolução do patógeno com a pressão seletiva exercida pelos fungicidas. / Grapevine is one of the most ancient cultivated plants and its fruit, grape, is notably important in Brazil, since it is the third most produced, only behind banana and citrus. Although it is rentable especially to smallholders, the vineyard is often attacked by several pathogens and the damages induced by them can compromise up to 59% of the producers\' expenses in order to keep the diseases under control. In Sao Paulo state there are three important diseases that attack vineyards: anthracnose (caused by Sphaceloma ampelinum), downy mildew (caused by Plasmopara viticola) and rust (caused by Phakopsora euvitis). Pest management practices used by the producers relies on intensive and preventive use of fungicides, in which the culture is sprayed 100 times per vineyard\'s growth cycle (that last approximately 120 days). One of the most used fungicides are the quinone oxidase inhibitors (QoI), that act by blocking the electron transport chain at the mitochondria binding at the Qo site of the cytochrome b (cytB) complex. However, there are several reports of the presence of resistant strains in different countries. Resistance is caused by the aminoacids substitutions F129L, G137R and G143A in the cytochrome b protein sequence, that prevent the fungicide molecule binding to its target site. The mutations in the cytB gene that lead to these substitutions are harbored in a region called hotspot for fungicide resistance. Since this type of study was never reported in Brazil, the main purpose of this work was to sequence and characterize the hotspot region of different isolates from anthracnose, downy mildew and rust. Thirty five isolates from eleven different locations were choosen for the study. Eleven of them harbored the mutation that lead to the substitution G143A; these were then considered genetically resistant to the QoI fungicides. On the contrary, four downy mildew and the two rust isolates were considered sensitive to the QoI fungicides, since none of the aminoacids substitutions were observed. Also, by using a technique named Genome Walking it was possible to sequence 65% of cytB gene from a Brazilian downy mildew isolate. In this sequence were found few polymorphisms and none intron. These study findings are unique for Brazilian isolates and might be useful to provide reliable support for the pest management decisions regarding the reality that is found at the vineyards in Brazil. Furthermore, the results presented here are important to the comprehension of pathogen\'s evolution when suffering from a selective pressure caused by the intensive use of fungicides.

A videira é uma das plantas mais antigas cultivadas pela humanidade, sendo que no Brasil a uva é a terceira fruta com maior volume de produção, atrás apenas do cultivo das bananas e das laranjas. Apesar da produção rentável, principalmente aos pequenos produtores, o parreiral é susceptível a várias doenças cujo manejo compromete até 59% dos gastos do produtor. No estado de São Paulo, dentre as doenças, três têm destaque: a antracnose (causada pelo Sphaceloma ampelinum), o míldio da videira (causado pelo Plasmopara viticola) e a ferrugem (causada pelo Phakopsora euvitis). Os produtores utilizam controle químico de forma intensa e preventiva, chegando a 100 aplicações de fungicidas em um ciclo de até 120 dias. Os principais fungicidas utilizados são os inibidores da quinona oxidase (QoI), que agem impedindo o transporte de elétrons do citocromo b ao citocromo c1 na cadeia respiratória da mitocôndria. Porém, existem relatos de resistência ao fungicida aplicado no campo em diversos países. As substituições G143A, G137R e F129L na sequência da proteína citocromo b impedem que o fungicida se ligue ao seu sítio alvo. As mutações que levam às substituições estão localizadas em uma das regiões chamada hotspot do gene citocromo b (cytB). Visto que, pela carência de estudos, a resistência genética a esses fungicidas nunca foi relatada no Brasil, o objetivo principal desse trabalho foi sequenciar e caracterizar a região hotspot em isolados de míldio, ferrugem e antracnose provenientes de parreirais do estado de São Paulo. Foram selecionados 35 isolados de 11 locais diferentes; desses, 11 isolados de míldio foram considerados geneticamente resistentes, pois apresentam a mutação para o resíduo alanina na posição 143, e 4 isolados foram considerados geneticamente sensíveis. Os dois isolados de ferrugem selecionados também foram considerados geneticamente sensíveis. Pela estratégia de Genome Walking foi possível sequenciar 65% do gene cytB de um dos isolados brasileiros de P. viticola; foram encontrados poucos polimorfismos e nenhum íntron na sequência analisada. Os resultados obtidos com esse estudo podem servir de suporte para a tomada de decisões de manejo mais adequadas para a realidade da viticultura brasileira; além disso, são importantes para futuros estudos sobre a evolução do patógeno com a pressão seletiva exercida pelos fungicidas. / Grapevine is one of the most ancient cultivated plants and its fruit, grape, is notably important in Brazil, since it is the third most produced, only behind banana and citrus. Although it is rentable especially to smallholders, the vineyard is often attacked by several pathogens and the damages induced by them can compromise up to 59% of the producers\' expenses in order to keep the diseases under control. In Sao Paulo state there are three important diseases that attack vineyards: anthracnose (caused by Sphaceloma ampelinum), downy mildew (caused by Plasmopara viticola) and rust (caused by Phakopsora euvitis). Pest management practices used by the producers relies on intensive and preventive use of fungicides, in which the culture is sprayed 100 times per vineyard\'s growth cycle (that last approximately 120 days). One of the most used fungicides are the quinone oxidase inhibitors (QoI), that act by blocking the electron transport chain at the mitochondria binding at the Qo site of the cytochrome b (cytB) complex. However, there are several reports of the presence of resistant strains in different countries. Resistance is caused by the aminoacids substitutions F129L, G137R and G143A in the cytochrome b protein sequence, that prevent the fungicide molecule binding to its target site. The mutations in the cytB gene that lead to these substitutions are harbored in a region called hotspot for fungicide resistance. Since this type of study was never reported in Brazil, the main purpose of this work was to sequence and characterize the hotspot region of different isolates from anthracnose, downy mildew and rust. Thirty five isolates from eleven different locations were choosen for the study. Eleven of them harbored the mutation that lead to the substitution G143A; these were then considered genetically resistant to the QoI fungicides. On the contrary, four downy mildew and the two rust isolates were considered sensitive to the QoI fungicides, since none of the aminoacids substitutions were observed. Also, by using a technique named Genome Walking it was possible to sequence 65% of cytB gene from a Brazilian downy mildew isolate. In this sequence were found few polymorphisms and none intron. These study findings are unique for Brazilian isolates and might be useful to provide reliable support for the pest management decisions regarding the reality that is found at the vineyards in Brazil. Furthermore, the results presented here are important to the comprehension of pathogen\'s evolution when suffering from a selective pressure caused by the intensive use of fungicides.