Dissertations / Theses on the topic 'Grapevines'

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, 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.

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 (MScAgric)--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: Petri grapevme decline, also known as black goo, slow die-back and Phaeoacremonium grapevine decline, causes significant losses of young vines worldwide. Species of Phaeoacremonium, Phaeomoniella chlamydospora and related genera are associated with this grapevine disease. This study investigates the Phaeoacremonium-complex and Phaeomoniella chlamydospora, focussing on the species isolated from grapevines. Fungicide sensitivity of Pa. chlamydospora and the possibility of employing molecular techniques for the detection of Pa. chlamydospora in grapevines were also investigated. In an overview of the literature on Petri grapevine decline the disease history and the relatedness of Petri grapevine decline to esca is discussed. Petri grapvine decline occurs in propagation material or young vines. Infected material can appear asymptomatic and therefore the possibilities of molecular techniques for identification were also investigated in the literature. In South Africa Pa. chlamydospora is the dominant organism causing Petri grapevine decline and therefore different fungicides were evaluated to control this fungus. Six isolates of Pa. chlamydospora, from Stellenbosch, Wellington, Somerset West and Malmesbury of Western Cape province, South Africa, were screened against twelve fungicides testing their effect on mycelial inhibition in vitro. These fungicides included benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole and thiram. Results provided the base-line sensitivity of South African isolates of Pa. chlamydospora. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride and tebuconazole were the most effective (with EC50 values ranging from 0.01 to 0.05 ug/ml) for inhibiting mycelial growth of Pa. chlamydospora in vitro. This in vitro test gave a good indication of which fungicides could be selected for further studies in glasshouses and nurseries. The molecular phylogeny of Phaeoacremonium and Phaeomoniella isolates from grapevines of South Africa, or isolates obtained from the Centraalbureau voor Schimmelcultures (CBS) in the Netherland, were investigated. Sequence data were created from the rONA region and partial B-tubulin gene of 33 of these isolates using the PCR technique. This sequence data were analysed with PAUP* version 4.Ob2a. An analysis of the sequence data confirmed the genus Phaeomoniella to be distinct from Phaeoacremonium (Pm.) based on DNA phylogeny. Although morphologically similar, the species status of Pm. aleophi/um and Pm. angustius was confirmed with DNA phylogeny and cultural characteristics. Pm. aleophilum has an optimum growth rate at 30°C and the ability to grow at 35°C, where as Pm. angustius has an optimum growth rate at 25°C and cannot grow at 35°C_ Pm. viticola was shown to be synonymous with Pm. angustius, and a new species, Pm. mortoniae, was newly described from grapevine occurring in California. Futhermore, Pm. aleophilum was newly reported from South Africa and grapevine isolates thought to be Pm. inflatipes were all re-identified as Pm. aleophilum. These findings therefore also shed some doubt on the possible role of Pm. inflatipes in Petri grapevine decline. It was confirmed that Pa. chlamydospora, Pm. aleophilum and Pm. angustius are the species involved in Petri grapevine decline. Pm. mortoniae was isolated from grapevines, but its pathogenicity should still be confirmed and the role of Pm. injlatipes in Petri grapevine decline remains unclear. Pa. chlamydospora has been routinely isolated from symptomless propagation and nursery material. Because the disease can take years to develop, it is crucial that healthy propagation material is used at planting. Pa. chlamydospora is a slowgrowing fungus, and positive identification from symptomless grapevine tissue can take up to 4 wks. The possibility of employing molecular techniques for the detection of Pa. chlamydospora in apparently healthy grapevines was investigated. Speciesspecific primers (PCLI and PCL2) based on the regions ITSI and ITS2 were designed for Pa. chlamydospora. These primers were highly sensitive and amplification was achieved from genomic DNA of Pa. chlamydospora from as low as 16 pg. Phaeoacremonium spp., related genera and common fungal taxa from grapevines were tested with these primers, but positive amplification was achieved for Pa. chlamydospora only. The presence of Pa. chlamydospora in symptomless grapevine tissue culture plants was confirmed by PCR within 24 hours. These primers therefore allow rapid and accurate identification of Pa. c~lamydospora. Testing on a larger scale with nursery material should be conducted to determine the feasibility of using these species-specific primers in the grapevine industry.
AFRIKAANSE OPSOMMING: Petri-terugsterwing van jong wingerde, ook algemeen bekend as "black goo" en Phaeoacremonium-terugsterwing, veroorsaak wêreldwyd groot geldelike verliese in die wingerdbedryf. Spesies van Phaeoacremonium, Phaeomoniella chlamydospora en verwante genera word met hierdie wingerdsiekte geassosieer. In die tesis word In oorsig gegee van die geskiedenis van hierdie siekte, die verwantskap tussen Petriterugsterwing en esca, en moontlike maniere van siektebestuur. Swamme wat by die siektekompleks betrokke is, kan in simptoomlose plantweefsel voorkom en daarom is die moontlikhede van die gebruik van molekulêre tegnieke vir swamidentifikasie in oënskou geneem. In Suid-Afrika is Pa. chlamydospora die dominante swam wat met Petriterugsterwing geassosieerword, gevolglik is verskillende fungisiedes vir die chemiese beheer van Pa. chlamydospora geëvalueer. Ses isolate van Pa. chlamydospora, versamel vanaf verskillende areas in die Wes-Kaap provinsie, is in dié studie gebruik. Benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole en thiram se effek op miselium inhibisie van Pa. chlamydospora is in vitro geëvalueer. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole was die mees effektiewe middels. Die effektiewe konsentrasie waarby 50% van die miselium groei geïnhibeer is (EKso),was tussen 0.01 en 0.05 ug/ml vir die mees effektiewe groep middels. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole het in vitro goeie potensiaal getoon, en verder toetse moet in vivo uitgevoer word. 'n Molekulêre studie is van Phaeoacremonium en Phaeomoniella isolate; verkry uit Suid-Afrikaanse wingerde, of vanaf die "Centraalbureau voor Schimmelcultures" (CBS) van Nederland; gedoen. Deur van die PKR tegniek gebruik te maak, is die basispaaropeenvolgingsdata van 33 isolate, van die ITSl, 5.8S, ITS2 rDNA area en die gedeeltelike B-tubullen geen verkry. Gekombineerde molekulêre data het die teorie ondersteun dat Phaeomoniella (Herpotrichiellaceae) gedistansieerd is van Phaeoacremonium (Magnaporthaceae). Pm. aleophilum en Pm. angustius was morfologies moeilik onderskeibaar, maar kon op grond van molekulêre data en kulturele eienskappe onderskei word. Pm. aleophilum se optimum groeitemperatuur was by 30°C en die swam besit die vermoë om by 35°C te groei. Pm. angus/ius se optimum groeitemperatuur was by 25°C, maar het nie by 35°C gegroei nie. 'n Studie van molekulêre en kulturele eienskappe het getoon dat Pm. angus/ius en Pm. viticola sinoniem is. 'n Nuwe spesie, Pm. mortoniae, wat uit wingerde van Kalifornie geïsoleer is, is beskrywe. Verder is Pm. aleophilum die eerste keer in Suid-Afrikaanse wingerde aangetref en Pm. tnflatipes isolate, wat vanuit wingerde geïsoleer is, is almal met molekulêre data gewys om Pm. aleophilum te wees. Hierdie bevindinge trek die rol van Pm. inflatipes in Petri-terugsterwing van wingerde in twyfel. Phaeomoniella chlamydospora IS m voortplantingsmateriaal en kwekerystokkies opgespoor. Omdat dit jare kan duur voordat siektesimptome ontwikkel, is dit belangrik om vroegtydig te weet of jong stokkies met Pa. chlamydospora geïnfekteer is. Pa. chlamydospora groei baie stadig en positiewe identifikasie van simptoomlose infeksies duur tot vier weke. Die toepassing van molekulêre tegnieke vir die vinnige identifikasie van Pa. chlamydospora in wingerde is dus ondersoek. Spesie-spesifieke oligonukleotiedes (PCU en PCL2) is vir Pa. chlamydospora ontwerp. Hierdie oligonukleotiedes is uiters sensitief en genomiese DNA van Pa. chlamydospora is van so laag as 16 pg geamplifiseer. Phaeoacremonium spp., verwante genera en algemene swamme vanuit wingerdmateriaal is met die oligonukleotiedes getoets, maar positiewe amplifikasie was slegs met Pa. chlamydospora moontlik. Die teenwoordigheid van Pa. chlamydospora is binne 24 uur in asimptomatiese wingerd weefselkultuurplantjies bevestig. Hierdie oligonukleotiedes identifiseer Pa. chlamydospora vinnig en akkuraat en toetsing op 'n groter skaal moet vervolgens met kwekerymateriaal onderneem word.

La sequera és una de les majors limitacions per a l’agricultura en general, factor que pot incrementar d’acord amb les prediccions del canvi climàtic. Per això, reduir l’ús de l’aigua en el reg i augmentar l’eficiència en l’ús de l’aigua (EUA) constitueix una de les majors prioritats per aconseguir una agricultura sostenible. L’EUA és un balanç entre guanys de biomassa i les despeses d’aigua. Aquesta Tesi s’ha centrat en l’estudi de 3 dels processos fisiològics que afecten a l’ús de l’aigua i a la producció de la planta en el cas de la vinya, i que per tant poden ser considerats factors potencials per millorar l’EUA: (1) conductància del mesòfil, (2) transpiració nocturna (3) respiració. Els resultats d’aquesta Tesi revelen que la conductància del mesòfil i la respiració són els principals components per millorar l’EUA permetent millorar l’assimilació de carboni o minimitzant les pèrdues del carboni fixat per la fotosíntesis

Thesis (MScAgric )--Stellenbosch University, 2004.
ENGLISH ABSTRACT: In an attempt to combat some of the pathogens that are associated with trunk diseases and disorders of grapevines, research in this thesis focused on the taxonomy and pathological aspects of Coniellai/Pilidiella, Botryosphaeria and Phomopsis spp. Previously, conidial pigmentation was used to separate Pilidiella from Coniella. Recently, however, the two genera have been regarded as synonymous, with the older name, Coniella, having priority. The most important species in the Coniellai/Pilidiella complex of grapevines is C. diplodiella (Speg.) Petr. & Syd., the causal organism of white rot of grapevines. Previous studies found it difficult to distinguish between C. diplodiella and C. fragariae (Oudem.) B. Sutton, which is known to occur in soil and caused leaf diseases of Fragaria and Eucalyptus. Both these species have previously been reported from South Africa. None of the reports on C. diplodiella could be scientifically substantiated; therefore it is still a quarantine organism. However, this status has been questioned. Based on sequence analyses of the internal transcribed spacer region (ITS 1, ITS 2), 5.8S gene, large subunit (LSU) and elongation factor 1- α gene (EF l- α) from the type species of Pilidiella and Coniella, Coniella was separated from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by species with hyaline to pale brown conidia (avg. length: width >1.5), with Coniella having dark brown conidia (avg. length: width ≤1.5). Pilidiella diplodiella, previously C. diplodiella, causal organism of white rot of grapevines, was shown to be an older name for C. petrakii. This fungus is present in South Africa and is therefore no longer of quarantine importance. Based on analyses of the histone (H3) gene sequences of isolates in the P. diplodiella species complex, P. diplodiella was separated from a newly described species, P. diplodiopsis. A new species, P. eucalyptorum, is proposed for isolates formerly treated as C. fragariae, associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens was newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. The genus Botryosphaeria Ces. & De Not. are known to be cosmopolitan, with broad host ranges and geographical distributions. Several saprotrophic species have been reported from grapevines, while others are severe pathogens of this host. These species include B. dothidea (Moug.: Fr.) Ces. & De Not., B. parva Pennycook & Samuels, B. obtusa (Schwein.) Shoemaker, B. stevensii Shoemaker, B. lutea A.J.L. Phillips and B. ribis Grossenb. & Duggar. Species reported from South Africa as grapevine pathogens are B. obtusa, B. dothidea, B. ribis and B. vitis (Schulzer) Sacco. In the present study, morphological, DNA sequence data (ITS 1, 5.8S, ITS 2 and EFI-α) and pathological data were used to distinguish 11 Botryosphaeria spp. associated with grapevines from South Africa and other parts of the world. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina and a Diplodia sp. were confirmed from grapevines in South Africa, while Diplodia porosum, Fusicoccum viticlavatum and F. vitifusiforme were described as new species. Although isolates of B. dothidea and B. stevensii were confirmed from grapevines in Portugal, neither of these species, nor B. ribis, were isolated in this study. All grapevine isolates from Portugal, formerly presumed to be B. rib is, are identified as B. parva based on EF1-α sequence data. Artificial inoculations on grapevine shoots showed that B. australis, B. parva, B. ribis and B. stevensii are more virulent than the other species studied. The Diplodia sp. collected from grapevine canes was identified as morphologically similar, but phylogenetically distinct from D. sarmentorum, while D. sarmentorum was confirmed as anamorph of Otthia spiraeae, the type species of the genus Otthia (Botryosphaeriaceae). A culture identified as O. spiraeae clustered within Botryosphaeria, and is thus regarded as a probable synonym. These findings confirm earlier suggestions that the generic concept of Botryosphaeria should be expanded to include genera with septate ascospores and Diplodia anamorphs. The genus Phomopsis (Sacc.) Bubak contains many species that are plant pathogenic or saprotrophic. Ten species are known from grapevines. However, only two have been confirmed as being pathogenic, namely P. viticola (Sacc.) Sacc., causal organism of Phomopsis cane and leaf spot and P. vitimegaspora Kuo & Leu (teleomorph Diaporthe kyushuensis Kajitani & Kanem.), causal organism of swelling arm disease of grapevines. P. amygdali (Delacr.) 1.1. Tuset & M.T. Portilla, a known pathogen from Prunus sp., was shown to be a possible pathogen of grapevines in a previous study. D. perjuncta Niessl. causes bleaching of dormant canes only and is therefore of little importance as a grapevine pathogen. Recently a number of Phomopsis isolates were obtained from grapevines in the Western Cape province of South Africa. Isolations were made from Phomopsis-like symptoms, pruning wounds and asymptomatic nursery plants. These isolates showed great variation in morphology and cultural characteristics. Earlier taxonomic treatments of Phomopsis, based species identification on host specificity, cultural characteristics and morphology. Recent studies have indicated that these characteristics can no longer be used to distinguish species of Phomopsis due to wide host ranges and morphological plasticity of some species. The use of anamorph/teleomorph relationships in species identification is also untenable, since Diaporthe teleomorphs have only been described for approximately 20% of the known Phomopsis species. In this study morphological data, DNA sequences (ITS-I, 5.8S, ITS-2) and pathogenicity data were combined to distinguish Phomopsis spp. from grapevines. Fifteen species of Phomopsis were delineated by phylogenetic analysis of ITS sequence data. Diaporthe helianthi, a sunflower pathogen, was reported from grapevines for the first time, with a further six, unknown species also distinguished. Three different clades contained isolates previously identified as D. perjuncta. Based on type studies, it appeared that the name D. viticola was available for collections from Portugal and Germany, a new species, D. australafricana, was proposed for South African and Australian isolates, formerly treated as D. perjuncta or D. viticola. An epitype specimen and culture were designated for D. perjuncta. This species was distinguished from D. viticola and D. australafricana based on morphology and DNA phylogeny. Artificial inoculations of green grapevine shoots indicated that, of the species tested, P. amygdali, a known pathogen of peaches in the USA, and P. viticola were the most virulent.
AFRIKAANSE OPSOMMING: In 'n poging om sommige patogene geassosieer met stamsiektes en syndrome, te beveg, het die navorsing in die tesis gefokus op die taksonomie en patologiese aspekte van ConiellaiPilidiella, Botryosphaeria en Phomopsis spp Voorheen is konidium pigmentasie gebruik om Pilidiella (hialien tot ligbruin konidia) van Coniella (donkerbruin konidia) te skei. Onlangs is hierdie twee genera egter as sinoniem beskou met die ouer naam, Coniella, wat voorkeur gekry het. Die belangrikste spesies in die ConiellaiPilidiella kompleks van wingerd is C. diplodiella (Speg.) Petr. & Syd., die veroorsakende organisme van witvrot van wingerd. Vorige studies het dit moeilik gevind om te onderskei tussen C. diplodiella en C. fragariae (Oudem.) B. Sutton, wat bekend is dat dit in grond voorkom en ook blaarsiektes van Fragaria en Eucalyptus veroorsaak. Beide hierdie spesies is tevore in Suid-Afrika aangemeld. Geen van die aanmeldings van C. diplodiella is egter wetenskaplik bewys nie en daarom is dit steeds 'n kwarantyn organisme. Hierdie kwarantyn status is egter bevraagteken. Op grond van DNS volgordes van die interne getranskribeerde spasieerder area ("ITS 1", "ITS2"), die 5.8S rRNS geen, die groot ribosomale subeenheid ("LSU") en die verlengingsfaktor 1-α geen ("EF-lα") van die tipe spesies van Pilidiella en Coniella, is Coniella van Pilidiella geskei, met die meerderheid van die taxa wat binne Pilidiella resorteer. Pilidiella word gekarakteriseer deur spesies met hialien tot ligbruin konidia (gem. lengte: breedte > 1.5), in teenstelling met die donkerbruin konidia van Coniella (gem. lengte: breedte ≤ 1.5). Daar is verder bewys dat Pilidiella diplodiella, voorheen C. diplodiella, veroorsakende organisme van witvrot van wingerd, die ouer naam van C. petrakii is. Hierdie swam is teenwoordig in Suid-Afrika en P. diplodiella is dus nie meer van kwarantyn belang nie. Op grond van analises van die histoon (H3) volgordes van spesies in die P. diplodiella spesies kompleks, is P. diplodiella geskei van 'n nuut beskryfde spesie, P. diplodiopsis. 'n Nuwe spesie, P. eucalyptorum, is ook voorgestel vir isolate voorheen beskou as C. fragariae, geassosieer met blaarvlek van Eucalyptus spp. Hierdie spesie het basaal van Pilidiella gegroepeer en mag moontlik nog 'n derde genus binne hierdie kompleks verteenwoordig. Pilidiella destruens is nuut as anamorf van Schizoparme destruens beskryf, wat geassosieer word met loot terugsterwing van Eucalyptus spp. in Hawaii. Die genus Botryosphaeria Ces. & De Not. is bekend as kosmopolitaans met 'n wye gasheerreeks en geografiese verspreiding. Verskeie saprofitiese spesies is aangemeld vanaf wingerd, terwyl ander ernstige patogene van hierdie gasheer is. Laasgenoemde spesies sluit in B. dothidea (Moug.: Fr.) Ces. & De Not., B. parva Pennycook & Samuels, B. obtusa (Schwein.) Shoemaker, B. stevensii Shoemaker, B. lutea A.1.L. Phillips en B. ribis Grossenb. & Duggar. Spesies aangemeld in Suid-Afrika as wingerdpatogene, is B. obtusa, B. dothidea, B. ribis en B. vitis (Schulzer) Sacco In hierdie studie is morfologiese, DNS volgorde data ("ITSl", "ITS2", 5.8S en "EF-Iα") en plantpatologiese data gebruik om II Botryosphaeria spesies, geassosieer met wingerde in Suid-Afrika en verskeie ander werelddele, te onderskei. Botryosphaeria australis, B. lutea, B. obtusa, B. parva, B. rhodina en 'n Diplodia sp. is bevestig van wingerde in Suid-Afrika, terwyl Diplodia porosum, Fusicoccum viticlavatum en F. vitifusiforme as nuwe spesies beskryf is. AIhoewel isolate van B. dothidea en B. stevensii bevestig is van wingerde in Portugal, is geen van hierdie spesies en ook nie B. ribis geïsoleer nie. AIle isolate vanaf wingerd in Portugal, voorheen beskou as B. rib is, is as B. parva op grond van hul "EF-lα" volgordes geïdentifiseer. Uit kunsmatige isolasies gemaak op wingerdlote is die gevolgtrekking gemaak dat B. australis, B. parva, B. ribis en B. stevensii meer virulent is as die ander spesies wat bestudeer is. Die Diplodia sp. versamel vanaf wingerdlote is geïdentifiseer as morfologies eenders, maar filogeneties verskillend van D. sarmentorum, terwyl D. sarmentorum bevestig is as die anamorf van Otthia spiraeae, die tipe spesie van die genus Otthia (Botryosphaeriaceae). 'n Kultuur wat as 0. spiraeae geïdentifiseer is, het binne Botryosphaeria gegroepeer, en word dus as 'n moontlike sinoniem beskou. Hierdie bevindinge bevestig vroeëre voorstelle dat die generiese konsep van Botryosphaeria uitgebrei behoort te word om genera met gesepteerde askospore en Diplodia anamorwe in te sluit. Die genus Phomopsis (Sacc.) Bubak bevat verskeie spesies wat as of plantpatogenies, of saprofities, beskryf is. Tien spesies is bekend op wingerd. Slegs twee is as patogenies bevestig, naamlik P. viticola (Sacc.) Sacc., veroorsakende organisme van loot-en-blaarvlek ("streepvlek") en P. vitimegaspora Kuo & Leu (teleomorf Diaporthe kyushuensis Kajitani & Kanem.), veroorsakende organisme van geswelde arm van wingerd. In 'n vroeëre studie is bevind dat P. amygdali (Delacr.) 1.1. Tuset & M.T. Portilla, 'n bekende patogeen van Prunus sp., moontlik ook 'n patogeen van wingerd mag wees. D. perjuncta Niessl. veroorsaak egter net verbleiking van dormante lote en is dus van min belang as 'n wingerd patogeen. Gedurende die afgelope twee jaar is verskeie Phomopsis isolate van wingerde in die Wes-Kaap provinsie van Suid-Afrika verkry. Isolasies is gemaak van Phomopsis-agtige simptome, snoeiwonde en asimptomatiese kwekeryplante. Die isolate verkry uit hierdie materiaal het groot variasie ten opsigte van morfologie en kultuureienskappe getoon. Vroeëre taksonomiese verhandelings van Phomopsis het spesies-identifikasie op gasheerspesifisiteit, kultuureienskappe en morfologie gebasseer. Onlangse studies het egter getoon dat, weens wye gasheerreekse en morfologiese plastisiteit van somnuge spesies, hierdie eienskappe me meer gebruik kan word om Phomopsis spesies te identifiseer nie. Die gebruik van anamorflteleomorf verwantskappe in die identifikasie van Phomopsis spesies ook onbruikbaar omdat Diaporthe teleomorwe vir slegs ongeveer 20% van die bekende Phomopsis spesies beskryf is. Die huidige studie het dus morfologiese data, DNS volgordes ("ITS 1", 5.8S, "ITS2") en patogenisiteitsdata gekombineer ten einde Phomopsis spp. vanaf wingerd te identifiseer. Vyftien Phomopsis spesies is deur die filogenetiese analise van die interne getranskribeerde spasieerder area ("ITS") volgordes geskei. Diaporthe helianthi, 'n bekende patogeen van sonneblomme, is vir die eerste maal op wingerd aangeteken, terwyl 'n verdere ses, tans onbekende spesies van Phomopsis ook geidentifiseer is. Drie verskillende groepe het isolate bevat wat voorheen as D. perjuncta geidentifiseer is. Gebasseer op studies van tipes, het dit voorgekom dat die naam D. viticola beskikbaar is vir isolate uit Portugal en Duitsland. 'n Nuwe spesie, D. australafricana, is voorgestel vir Suid-Afrikaanse en Australiese isolate wat voorheen behandel is as D. perjuncta of D. viticola. 'n Epitipe monster en kultuur is vir D. perjuncta benoem. Hierdie spesie is van D. viticola en D. australafricana onderskei op grond van morfologie en DNS filogenie. Kunsmatige inokulasies van groen wingerdlote het getoon dat P. amygdali, bekende perske patogeen, en P. viticola die mees virulent was.

Eutypa dieback of grapevines, caused by Eutypa lata, is a major cause of reduced longevity in vineyards worldwide. The fungus grows in the woody tissue of infected vines, producing translocatable toxins that cause foliar symptoms of the disease. By the time foliar symptoms are evident the pathogen may have become well established in the vine. One aim of this study was to develop DNA markers to allow rapid reliable identification of E. lata and to detect the pathogen in infected wood. The second aim was to analyse secondary metabolite production by E. lata in order to gain information on the compounds responsible for the foliar symptoms of the disease and to identify metabolites which could be used as markers to detect the early stages of the disease prior to the expression of foliar symptoms. In addition, genetic variation of the pathogen was assessed using RFLP and RAPD analysis. Two techniques were used to develop DNA markers; first, SCAR markers derived from RAPD fragments were developed and, second, an E. lata genomic DNA library was constructed, from which DNA fragments specific to E. lata were identified. These markers were used in either PCR- or Southern hybridisation-based assays to detect the pathogen in infected wood. PCR-based detection of the pathogen in infected wood was prone to inhibition by phenolic compounds, however, Southern hybridisation techniques were capable of detecting E. lata in wood. Genetic variation among 38 isolates of E. lata was assessed using six randomly selected clones from the genomic DNA library. A subset of 11 isolates was subjected to RAPD analysis using 10 random primers. Considerable genetic diversity, in terms of RFLP and RAPD profiles, was observed among isolates. There was no apparent correlation between grouping of isolates following neighbour joining analysis and either host species or geographic origin of isolates. The RAPD and RFLP profiles of two isolates differed significantly from the majority of the other isolates. These isolates, which were morphologically similar to all other isolates, were subsequently found not to be E. lata. Secondary metabolite production of 11 isolates was analysed by HPLC following growth on a range of media. A wider range of secondary metabolites was detected in E. lata than has previously been reported. Two of the secondary metabolites, eutypine and an unidentified compound with a retention time of 19.6 min, were produced by eight of nine isolates of E. lata. Neither of the non-E. lata isolates produced these compounds. It was concluded that the remaining isolate of E. lata may have lost the ability to produce these compounds following storage. Whilst a wider range of isolates needs to be screened before a candidate marker can be selected, these results suggest that certain compounds are present in the majority of E. lata isolates and, hence, may prove suitable markers for the detection of the pathogen prior to the expression of foliar symptoms. The molecular probes developed in this study will allow the rapid and reliable identification and detection of E. lata in grapevine cane or wood. These probes also have the potential to be used as a research tool to gather information on the epidemiology of the disease and to assess the efficacy of potential control agents against E. lata. Suitable control measures could then be applied to vines which have been shown by the use of chemical markers to have latent infection. Used in combination, therefore, the DNA and biochemical markers could facilitate improved management of eutypa dieback.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture and Wine, 2003.

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.

Bibliography: p. 261-273. The general hypothesis tested in this study was that: 'partial drying of the root system of grapevines will result in reduced vegetative growth with beneficial effects for fruit yield, fruit composition and water-use efficiency'. Previously published work has suggested that partial drying of the root system may give rise to non-dydraulic root-derived signals capable of changing leaf development and transpiration.

Thesis (MScAgric)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, and Botryosphaeria spp. are important trunk disease pathogens that cause premature decline and dieback of grapevine. Previous research has focused primarily on wine grapes and the incidence and symptomatology of these pathogens on table grapes were largely unknown. A survey was therefore conducted to determine the status and distribution of these pathogens and associated symptoms in climatically diverse table grape growing regions. Fifteen farms were identified in the winter rainfall (De Doorns, Paarl and Trawal) and summer rainfall (Upington and Groblersdal) areas. Samples were taken in July and August 2004 from Dan-ben-Hannah vineyards that were 8 years and older. Distal ends of arms were removed from 20 randomly selected plants in each vineyard. These sections were dissected and isolations were made from each of the various symptom types observed: brown or black vascular streaking, brown internal necrosis, wedge-shaped necrosis, watery necrosis, esca-like brown and yellow soft wood rot, as well as asymptomatic wood. Fungal isolates were identified using molecular and morphological techniques. Pa. chlamydospora was most frequently isolated (46.0%), followed by Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea and an undescribed Diplodia sp. (0.2% each), while E. lata was not found. Most of these pathogens were isolated from a variety of symptom types, indicating that disease diagnosis can not be based on symptomatology alone. Pa. chlamydospora was isolated from all areas sampled, although most frequently from the winter rainfall region. Pm. aleophilum was found predominantly in Paarl, while P. viticola only occurred in this area. Although B. obtusa was not isolated from samples taken in De Doorns and Groblersdal, it was the most commonly isolated Botryosphaeria sp., being isolated from Upington, Paarl and Trawal. B. rhodina occurred only in Groblersdal and B. parva in Paarl, Trawal and Groblersdal, while B. australis was isolated from Paarl only. The rest of the isolates (33%) consisted of sterile cultures, Exochalara, Cephalosporium, Wangiella, Scytalidium, Penicillium spp. and two unidentified basidiomycetes, which were isolated from five samples with yellow esca-like symptoms from the Paarl area. These findings clearly illustrate that grapevine trunk diseases are caused by a complex of fungal pathogens, which has serious implications for disease diagnosis and management. Protection of wounds against infection by any of these trunk disease pathogens is the most efficient and cost-effective means to prevent grapevine trunk diseases. However, previous research on the effectiveness of chemical pruning wound protectants has mostly focused on the control of Eutypa dieback only. Fungicide sensitivity studies have been conducted for Pa. chlamydospora, P. viticola and Eutypa lata, but no such studies have been conducted for the pathogenic Botryosphaeria species from grapevine in South Africa. Ten fungicides were therefore tested in vitro for their efficacy on mycelial inhibition of the four most common and/or pathogenic Botryosphaeria species in South Africa, B. australis, B. obtusa, B. parva and B. rhodina. Iprodione, pyrimethanil, copper ammonium acetate, kresoxim-methyl and boscalid were ineffective in inhibiting the mycelial growth at the highest concentration tested (5 μg/ml; 20 μg/ml for copper ammonium acetate). Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole were the most effective fungicides with EC50 values for the different species ranging from 0.36-0.55, 0.07-0.17, 0.07-1.15 and 0.04-0.36 μg/ml, respectively. These fungicides, except prochloraz manganese chloride, are registered on grapes in South Africa and were also reported to be effective against Pa. chlamydospora, P. viticola and E. lata. Results from bioassays on 1-year-old Chenin Blanc grapevine shoots indicated that benomyl, tebuconazole and prochloraz manganese chloride were most effective in limiting lesion length in pruning wounds that were inoculated with the Botryosphaeria spp after fungicide treatment. The bioassay findings were, however, inconclusive due to low and varied re-isolation data of the inoculated lesions. Benomyl, tebuconazole, prochloraz manganese chloride and flusilazole can nonetheless be identified as fungicides to be evaluated as pruning wound protectants in additional bioassays and vineyard trials against Botryosphaeria spp. as well as the other grapevine trunk disease pathogens.
AFRIKAANSE OPSOMMING: Phaeomoniella chlamydospora, Eutypa lata, Phomopsis, Phaeoacremonium, en Botryosphaeria spesies is die mees belangrikste stamsiekte patogene wat agteruitgang en vroeë terugsterwing van wingerd veroorsaak. Voorafgaande navorsing het hoofsaaklik gefokus op wyndruiwe en die voorkoms en simptomatologie van hierdie patogene op tafeldruiwe is dus grootliks onbekend. ‘n Opname is gevolglik gedoen in verskillende klimaaatsareas waar tafeldruiwe verbou word om die voorkoms en verspreiding, asook die simptome geassosieer met hierdie patogene, te bepaal. Vyftien plase is geïdentifiseer in die winter- (De Doorns, Paarl en Trawal) en somer-reënval (Upington en Groblersdal) streke. Wingerde (8 jaar en ouer) met die kultivar Dan-ben-Hannah is gekies vir opname en monsters is gedurende Julie en Augustus 2004 geneem. Die distale deel van ‘n arm is verwyder vanaf 20 lukraak gekose plante in elke wingerd. Hierdie dele is ontleed en isolasies is gemaak vanuit elke simptoomtipe wat beskryf is, naamlik bruin en swart vaskulêre verkleuring, bruin interne nekrose, wig-vormige nekrose, waterige nekrose, esca-geassosieerde bruin en geel sagte houtverrotting en asimptomatiese hout. Identifikasie van die swamagtige isolate is gedoen op grond van morfologiese eienskappe en molekulêre tegnieke. Pa. chlamydospora is die meeste geïsoleer (46.0%), gevolg deur Phaeoacremonium aleophilum (10.0%), Phomopsis viticola (3.0%), Botryosphaeria obtusa (3.0%), B. rhodina (2.2%), B. parva (2.0%), Fusicoccum vitifusiforme (0.6%), B. australis, B. dothidea en ‘n onbeskryfde Diplodia sp. (0.2% elk), terwyl E. lata nie geïsoleer is nie. Hierdie patogene is elk geïsoleer vanuit ‘n verskeidenheid simptoomtipes, wat daarop dui dat siektediagnose nie alleenlik op simptomatologie gebaseer kan word nie. Pa. chlamydospora is geïsoleer vanuit al die gebiede, alhoewel die patogeen opmerklik meer voorgekom het in die winter-reënval area. Pm. aleophilum het hoofsaaklik voorgekom in Paarl, terwyl P. viticola slegs in hierdie area voorgekom het. Alhoewel B. obtusa nie voorgekom het in die De Doorns en Groblersdal areas nie, was dit die mees algemeen geïsoleerde Botryosphaeria sp. en het in Upington, Paarl en Trawal voorgekom. B. rhodina het slegs in Groblersdal voorgekom, B. parva in Paarl, Groblersdal en Trawal en B. australis het slegs in Paarl voorgekom. Die res van die isolate (33%) het bestaan uit steriele kulture, Exochalara, Cephalosporium, Wangiella, Scytalidium, en Penicillium spesies asook twee onbekende basidiomycete isolate, geïsoleer vanuit vyf monsters met geel eska-geassosieerde simptome vanuit die Paarl area. Hierdie resultate illustreer dus die feit dat wingerdstamsiektes deur ‘n kompleks van swampatogene veroorsaak word, wat belangrike implikasies het vir die bestuur en diagnose van hierdie siektes. Wondbeskerming teen infeksie van enige van hierdie stamsiekte patogene is die mees doeltreffende en koste-effektiewe manier om wingerdstamsiektes te voorkom. Vorige navorsing aangaande die effektiwiteit van chemiese wondbeskermingsmiddels het egter slegs gefokus op die beheer van Eutypa terugsterwing. In vitro swamdoder sensitiwiteitstoetse is gedoen vir Pa. chlamydospora, P. viticola en Eutypa lata, maar geen studies is al gedoen ten opsigte van die patogeniese Botryosphaeria spesies op wingerd in Suid-Afrika nie. Tien swamdoders is dus getoets vir inhibisie van in vitro miseliumgroei van die vier mees algemene en/of patogeniese Botryosphaeria spesies wat in Suid-Afrika voorkom, naamlik B. australis, B. obtusa, B. parva en B. rhodina. Iprodione, pyrimethanil, koper ammonium asetaat, kresoxim-metiel en boscalid was oneffektief by die hoogste konsentrasies getoets (5 μg/ml; 20 μg/ml vir koper ammonium asetaat). Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool was die mees effektiewe swamdoders met EC50 waardes tussen 0.36-0.55, 0.07-0.17, 0.07-1.15 en 0.04-0.36 μg/ml, onderskeidelik vir die verskillende spesies. Hierdie fungisiedes, behalwe prochloraz mangaan chloried, is geregistreer op druiwe in Suid-Afrika en is ook effektief gevind teenoor Pa. chlamydospora, P. viticola en E. lata. Resultate van biotoetse op 1-jaar-oue Chenin Blanc wingerd lote het getoon dat benomyl, tebuconasool en prochloraz mangaan chloried die effektiefste was om die lengte van letsels in snoeiwonde, geinokuleer met Botryosphaeria spesies na die aanwending van swamdoder behandelings, te verminder. Die bevindinge was egter onbeslis as gevolg van die lae en variërende her-isolerings data. Benomyl, tebuconasool, prochloraz mangaan chloried en flusilasool kan egter geïdentifiseer word as swamdoders wat verder geevalueer kan word as snoeiwond beskermingsmiddels teen Botryosphaeria spesies asook ander wingerd stamsiekte patogene in verdere biotoetse en wingerdproewe.

Thesis (Ph.D.) -- University of Western Sydney, 2005.
"Thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy, Centre for Plant and Food Sciences, University of Western Sydney, Australia, November 2005." Includes bibliographical references and appendices.

This project was designed to correlate environmental variables with the development of Pierce’s Disease (PD), an infection caused by the gram negative bacterium Xylella fastidiosa (Xf), in grapes (Hopkins et al. 2002). PD is one of the most difficult crop pathogens to manage because it is vectored by insects and its continued presence in the vine is usually fatal. PD is influenced by the interaction of host, disease and vector, and probably many other environmental variables. The general objective was to study and identify the most important variables involved in the expression of Pierce’s Disease. Over a two year period, from a total of eight sites in northern and southern California data was collected on forty-five environmental variables including those relating to soil moisture, soil chemistry, soil nutritional status, vine nutritional status, vine water status, in-season and dormant season climate, incidence of Xf in adjacent vineyard vegetation, sharpshooter species and abundance at each location, and proximity of vineyard field sites to citrus or riparian areas. The environmental variables were analyzed with canonical correspondence analysis (CCA) to determine significance of each as they correspond with increased disease severity. The significance of environmental variables produced by CCA indicates increased soil moisture as the leading cause for increased PD incidence; several other environmental variables positively correlate with increased disease presence. Conversely, vineyard factors identified by CCA as not conducive to disease formation may play an inhibitory role in PD severity. We undertook polymerase chain reaction (PCR) to test for the presence of PD in vegetative samples, using a 733 base pair probe specific to Xf. These samples indicate alternative hosts in adjacent locations which act as reservoirs of Pierce’s Disease as well as verifying diseased vines within the vineyard locations.

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, 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

Pierce’s disease (PD) of grapevines, caused by Xylella fastidiosa, is devastating Texas vineyards. Two rapid diagnostic techniques, real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), were compared on the basis of cost, reliability, and their ability to quantify X. fastidiosa in diseased tissues. A high correlation was found between the two techniques for measuring bacterial titer in vitro. A similar relationship was not detected when applying the methods to diseased tissue. There was a 75% similarity between the techniques when used to diagnose PD in artificially infected grapevines. Where the two methods differed, real-time PCR was more successful in identifying plants known to be infected with the bacterium. In uninoculated grapevines, the two techniques were similar, where the positive rates were 7% and 4% for ELISA and real-time PCR respectively. In a second study, 3 grape cultivars, ‘Cynthiana’, ‘Cabernet Sauvignon’, and ‘Chardonnay’, were inoculated with 2 isolates of X. fastidiosa to measure disease development and colonization by the pathogen. The bacteria colonized similar distances from the inoculation point over a 25 week period in all three cultivars. Real-time PCR and ELISA absorbance values suggest that the concentrations of bacteria ranged between 104 and 106 cells/ml in a 1.27 cm section of grapevine cane. Concentrations of bacteria didn’t vary based on distance from the inoculation point. Marginal leaf-scorch symptoms were seen on ‘Cabernet Sauvignon’ and ‘Chardonnay’ grapevines 9 weeks post-inoculation. Leaf-scorch symptoms were not observed on ‘Cynthiana’. The vigor of all inoculated grapevines was reduced compared to negative control grapevines the season after initial infection. In a third study, a Texas vineyard planted in Viognier grapevines was surveyed for PD symptoms on 3 separate dates. In October 2003, 45/50 rows had significant aggregation of symptomatic grapevines according to Ordinary Runs Analysis. Aggregation of symptomatic grapevines was found down the row more often than across the row. The rapid rate of disease progress and mortality rate of vines in this vineyard suggest that vine-to-vine spread is occurring and that Viognier vines are highly susceptibly to PD.

The detection by serology of nematode-transmitted polyhedral viruses (nepoviruses) in grapevines is often unreliable. Nepoviruses were detected by enzyme-linked immunosorbent assay (ELISA) in tissue-cultured and field-grown grapevines. Nepovirus detection in in vitro plants (plantlets) was affected by virus distribution and growth room temperature. The reliability of virus detection in field-grown grapevines was improved when modified grinding buffers were used. Arabis mosaic virus (AMV) was detected by ELISA, for the first time, in in vitro grapevines initiated from field-and screenhouse-grown plants throughout the summer. The virus was not reliably and repeatedly detected in in vitro plantlets grown at 25°C. AMV and grapevine fanleaf virus (GFLV) distribution was not uniform throughout the plantlets. This distribution was affected by the culture room temperature. The best plant parts to sample for virus detection came from the zones of rapidly proliferating shoots. The viruses were sometimes not detected in samples taken from other tissues. Growth room temperature had an important effect on virus detection in plantlets. The highest virus titres were found in plants growing at 15°C. Temperature increases in 5°C steps to 30°C reduced virus titre. AMV became undetectable in nearly all plantlets growing at 30°C for as little as 30 days. Growth at 30°C reduced ELISA absorbance values by 76% after 8 days and after 21 days the values were at 4% of pre-treatment levels. The virus titre dropped below detectable levels in most plantlets. AMV could not be detected in plantlets or rooted explants after being placed in a 30°C treatment for 2 months. Tomato ringspot virus was detected by ELISA, for the first time, in in vitro grapevine plants. The virus was repeatedly detected in in vitro plants growing at 20°C. Under the typical summer conditions experienced at Sidney, B.C., modifying the standard ELISA grinding buffer (0.01 M phosphate buffered saline, pH 7.4, 0.05% Tween-20, 0.2% ovalbumin, 2% polyvinylpyrrolidone) was essential for reliable detection of AMV or GFLV. AMV was reliably detected by ELISA in foliar samples from field or screenhouse plants throughout the summer when the grinding buffer was modified by increasing the pH to at least 8.2 and adding 1% nicotine or 0.15 M phosphate buffered saline. The most reliable results with GFLV were obtained with the nicotine enhanced buffers. In comparison, because of the increased workload associated with growing plants in vitro and the unreliable detection of viruses in these plants, it remained preferable to detect nepoviruses in field plants by ELISA.
Land and Food Systems, Faculty of
Graduate

Thesis (MSc)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: Plants are constantly exposed to biotic and abiotic stress inducing factors that threaten their existence. Biotic factors such as pathogens are the cause of huge yield losses to crop plants worldwide with fungal pathogens debatably constituting the worst damage. Fungal pathogens such as Botrytis cinerea, which has a wide host range, release cell wall degrading enzymes called endopolygalacturonases (ePGs) during plant infection. These ePGs break down the pectin component of the cell wall, thus providing an entry route, as well as nutrients for the fungus. Plants have evolved mechanisms to counteract and suppress the action of the ePGs. This is achieved through the action of cell wall associated proteins called polygalacturonaseinhibiting proteins, PGIPs. PGIPs directly inhibit ePGs and their inhibitory action also prolongs the existence of longer chain oligogalacturonide residues which are believed to elicit a cascade of defence responses. In grapevine, a PGIP encoding gene, VvPGIP1, was previously isolated and characterised. VvPGIP1, as well as nine non-vinifera grapevine PGIPs have been expressed in tobacco and shown to be potent antifungal proteins that caused the transgenic tobacco to have strong resistance phenotypes against Botrytis in whole plant infection assays. Following on the tobacco study, two of the non-vinifera PGIPs were expressed in cultivars of the susceptible Vitis vinifera. Characterisation of the putative transgenic population showed that transgene integration was successful, the transgenes were being expressed and there were at least 29 transgenic lines with independent integration events. The transgenic lines were confirmed to have active PGIPs (transgene-derived) in their leaves. Crude protein extracts from 22 lines exhibited 100% inhibition against crude B. cinerea PGs (BcPGs). The plant lines with positive transgene integration, expression, independent integration events and exhibiting 100% transgene-derived PGIP activity were further selected for whole plant and detached leaf antifungal assays where they were challenged with B. cinerea. The whole plant infection assay showed that expression of the non-vinifera PGIPs in V. vinifera promotes susceptibility to B. cinerea, not resistance. This surprising result could perhaps be explained by a quicker and stronger recognition between the pathogen and the host and the stronger activation of defence responses in the host. A more active hypersensitive response in the host would benefit Botrytis being a necrotroph. The type of lesions and the onset and speed of lesion development observed on the transgenics lines versus the wild type support this possibility. Knowledge gaps with regards to the efficiency of the ePG inhibition by the nonvinifera PGIPs during infection of grapevine tissue; the potential changes that might be caused by expressing PGIPs in a grapevine host with a native PGIP with high homology to the transgenes (including potential gene silencing) and the potential impact on defence signalling and defence responses all provides further avenues of study to elucidate this very interesting phenotype further. Overall, this study provides a comprehensively characterised population of transgenic plants that provides useful resources for in vivo analysis of PGIP function in defence, where the host plant harbours a native copy of the PGIP encoding gene.
AFRIKAANSE OPSOMMING: Plante word voortdurend blootgestel aan biotiese en abiotiese faktore, wat stres veroorsaak en hul bestaan bedreig. Biotiese faktore, soos patogene, veroorsaak groot verliese in wêreldwye gewasopbrengste, met swampatogene wat moontlik die grootste skade veroorsaak. Swampatogene, soos Botrytis cinerea, wat ‘n wye reeks gasheerplante kan infekteer, stel selwand-afbrekende ensieme tydens plantinfeksie vry, wat as endo-poligalakturonases (ePG’s). bekend staan. Hierdie ePG’s breek die pektienkomponent van die selwand af, wat gevolglik as ‘n ingangspunt dien,asook voedingstowwe vir die swam verskaf. Plante het meganismes ontwikkel om die aktiwiteit van hierdie ePG’s te bekamp en te onderdruk. Die aktiwiteit van die selwand-geassosieërde proteïene, genaamd poligalakturonase-inhiberende proteïene (PGIP’s), speel hier ‘n rol. PGIP’s inhibeer ePG’s direk en hul inhiberende aktiwiteit verleng ook die bestaan van langketting oligogalakturoniedresidu’s, wat blykbaar ‘n kaskade van weerstandsreaksies kan inisieer. ‘n PGIP-koderende geen, VvPGIP1, is voorheen uit wingerd geïsoleer en gekarakteriseer. VvPGIP1, asook nege nie-vinifera wingerd-PGIP’s is voorheen in tabak uitgedruk en bevestig as proteïene met sterk anti-swamaktiwiteit, soos bevestig deur die bevinding dat die transgeniese tabak ‘n weerstandsfenotipe teen Botrytis in heelplant-infeksietoetse het. Ná die tabakstudie is twee van die nie-vinifera PGIP’s uitgedruk in vatbare V. vinifera-kultivars. Karakterisering van die vermeende transgeniese bevolking het getoon dat die transgeen-integrasie suksesvol was, dat die transgeen uitgedruk word en dat daar ten minste 29 transgeniese lyne met onafhanklike integrasie gebeurtenisse geskep is. Daar is verder bevestig dat die transgeniese lyne aktiewe PGIP’s (transgeen-afkomstig) in hul blare het. Ongesuiwerde proteïenekstrakte van 22 lyne het 100% inhibisie teen ‘n mengsel van ongesuiwerde B. cinerea PGs (BcPGs) getoon. Die plantlyne met positiewe transgeenintegrasie en -uitdrukking, asook onafhanklike integrasiegebeure en wat 100% transgeen-afkomstige PGIP-aktiwiteit getoon het, is verder aan heel-plant en verwyderde blaarswaminfeksies met B cinerea onderwerp. Die heelplantinfeksietoetse het getoon dat uitdrukking van nie-vinifera PGIP’s in V. vinifera ‘n toename, in plaas van ‘n afname, in vatbaarheid teen B. cinerea veroorsaak. Hierdie verbasende resultaat kan moontlik toegeskryf word aan ‘n vinniger en sterker herkenningsreaksie tussen patogeen en gasheer en die moontlike sterker stimulering van weerstandsreaksies in die gasheer. ‘n Meer aktiewe hipersensitiewe reaksie in die gasheer sal tot die voordeel van Botrytis, wat ‘n nektrotroof is, wees. Die tipe letsel, asook die aanvang en spoed van letselontwikkeling wat waargeneem is in transgeniese lyne teenoor die wilde-tipe ondersteun hierdie moontlikheid. Gapings in kennis ten opsigte van die doeltreffendheid van die ePG-inhibisie deur die nievinifera PGIP’s tydens infeksie van wingerdweefsel, die moontlike veranderinge (insluitend ‘n moontlike geenuitdowingseffek) wat veroorsaak kan word deur die uitdrukking van PGIP-gene in ‘n kultivar met ‘n inheemse en baie homoloë PGIP-geen, kon ‘n invloed op weerstandseine en weerstandsreaksies gehad het. Hierdie aspekte lewer verdere studiemoontlikhede om hierdie interessante fenotipe verder te verklaar.Algeheel lewer hierdie studie ‘n breedvoeriggekarakteriseerde bevolking trangeniese plante, wat dien as nuttige hulpbronne vir in vivoanalise van PGIP se funksie in siekteweerstandbiedendheid, veral waar die gasheerplant ‘n inheemse kopie van die PGIP-koderende geen huisves.

Bibliography: leaves 127-152. The aim of this work is to study viroids in grapevines, particularly their vertical transmission via seeds, during meristem culture and micropropagation. There was also an attempt to produce viroid-free vines by shoot apical meristem culture (SAMC).

Thesis (MScAgric)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Soilborne diseases of grapevines represent a complex problem with limited information available, both locally and internationally. Previous research in South Africa indicated that Phytophthora and Pythium spp. were the most widespread and devastating pathogens in grapevine nurseries and vineyards in the Western Cape province. The local grapevine industry is currently expanding; new cultivars, methods and agricultural chemicals are being used which can affect soilborne pathogens. It has therefore become necessary to reassess the status of soilborne pathogens in nurseries, since information in this regard is crucial for the development of disease management practices for the expanding local grapevine industry. Soilborne fungal genera associated with roots and crowns of declining nursery grapevines were assessed in surveys conducted at three different grapevine nurseries in the Western Cape province. Cylindrocarpon, Fusarium, Pythium, and Rhizoctonia spp. were consistently isolated from roots and crowns of declining nursery grapevines. Cylindrocladiella spp. and Phytophthora cinnamomi were infrequently isolated from diseased roots, crowns and soil whereas Pythium spp. were abundant in most of the soils. Results suggest that the status of soilborne fungal pathogens in grapevine nurseries in the Western Cape province has changed over the last 30 years. The DNA phylogeny and pathogenicity of the isolates of Cylindrocladiella were determined. Four species of Cylindrocladiella occur on grapevines in South Africa, namely C. lageniformis, C. parva, C. peruviana, as well as a new species, described in this study as C. viticola, which forms part of the C. infestans species complex. Pathogenicity trials were inconclusive. Ten Fusarium spp. were isolated from roots and crowns of declining nursery grapevines, namely F. acuminatum, F. anthophilum, F. chlamydosporum, F. equiseti, F. nygamai, F. oxysporum, F. proliferatum, F. scirpi, F. semitectum and F. solani. The dominant species was F. oxysporum, followed by F. proliferatum and F. solani. In pathogenicity trials F. oxysporum and F. solani significantly reduced root volume, root dry mass, length of new shoots, stem diameter and number of leaves, but increased the percentage of chlorotic leaves and root rot severity. Fusarium proliferatum also caused a significant reduction in new shoot growth, number of leaves and increased root rot severity compared to the controls. Fusarium so/ani seems to be more virulent than F. oxysporum, followed by F. pro/iferatum. This is the first report of F. oxysporum, F. pro/iferatum and F. so/ani as pathogens of grapevines in South Africa, and the first report of F. proliferatum as a pathogen of grapevines in the world. Phytophthora cinnamomi was isolated at low frequencies from declined grapevines, although present in the rhizosphere soil. It is possible that the extensive use of downy mildew chemicals in grapevine nurseries may protect grapevines from infection by P. cinnamomi. The effect of chemicals used to combat downy mildew on Phytophthora root rot of nursery grapevines was evaluated in a glasshouse. There was very little discernable effect of the chemicals tested relative to the control plants for the parameters measured and it was concluded that the inoculation technique needed refinement. However, plants treated with phosphorous acid tended to be taller and have more leaves, greater stem diameter and root volume than controls or plants treated with the other chemicals. The data obtained in this study are not conclusive, but indicated certain trends that more glasshouse trials and field trials would resolve. Results presented in this thesis indicate that a major shift has occurred in the status of soilborne fungi associated with roots and crowns of grapevines in nurseries in the Western Cape since the 1970s when Phytophthora and Pythium were predominant. The prevalence and role of soilborne fungi need to be determined so that new appropriate disease management strategies can be developed to limit losses in grapevine nurseries and ensure the sustainable production of healthy plants for the grapevine industry.
AFRIKAANSE OPSOMMING: 'N ONDERSOEK NA GRONDGEDRAAGDE SWAMME GEASSOSIEER MET WORTELS EN KRONE VAN WINGERD IN KWEKERYE Grondgedraagde siektes van wingerd is 'n komplekse probleem waaroor min inligting, beide plaaslik en internasionaal, beskikbaar is. Vorige navorsing in Suid-Afrika het aangedui dat swamme van die genera Phytophthora en Pythium die mees algemene en vernietigende grondgedraagde patogene in kwekerye en wingerde in die Wes-Kaap provinsie is. Die plaaslike wingerdbedryf brei huidiglik uit; nuwe kultivars, metodes en landbouchemikalieë word gebruik wat 'n invloed kan hê op grondgedraagde patogene. Gevolglik het dit noodsaaklik geword om die status van grondgedraagde patogene in wingerdkwekerye weer te bepaal, aangesien inligting in hierdie verband noodsaaklik is vir die ontwikkeling van siekte bestuurspraktyke vir die ontwikkelende plaaslike wingerdbedryf. Grondgedraagde swamgenera geassosieer met wortels en krone van terugsterwende wingerd in kwekerye is bepaal in opnames wat by drie verskillende wingerdkwekerye in die Wes-Kaap provinsie uitgevoer is. Cylindrocarpon, Fusarium, Pythium, en Rhizoctonia spp. is konstant vanuit wortels en krone van terugsterwende wingerdplante in kwekery geïsoleer, Cylindrocladiella spp. en Phytophthora cinnamomi is ongereeld vanuit siek wortels, krone en grond geïsoleer, terwyl Pythium spp. algemeen in meeste gronde voorgekom het. Resultate dui daarop dat die status van grondgedraagde swampatogene in wingerdkwekerye in die Wes- Kaap provinsie oor die laaste 30 jaar verander het. Die DNA filogenie en patogenisiteit van die isolate van Cylindrocladiella is bepaal. Vier spesies van Cylindrocladiella kom voor op wingerd in Suid-Afrika, naamlik C. lageniformis, C. parva, C. peruviana, sowel as 'n nuwe spesie, wat in hierdie studie as C. viticola aangedui is en wat deel is van die C. infestans spesie kompleks. Patogenisiteits proewe was onvoldoende om die patogeniese status van die swam me te bepaal. Tien Fusarium spp. is vanuit wortels en krone van terugsterwende wingerdplante in kwekery geïsoleer, naamlik F. acuminatum, F. anthophilum, F. chlamydosporum, F. equiseti, F. nygamai, F. oxysporum, F. proliferatum, F. scirpi, F. semitectum en F. solani. Die dominante spesies was F. oxysporum, gevolg deur F. proliferatum en F. solani. In pathogenisteitsproewe het F. oxysporum en F. solani gelei tot 'n betekenisvolle laer wortelvolume, droë massa van wortels, lengte en droë massa van nuwe groei en aantal blare, maar het die persentasie chlorotiese blare en graad van wortelvrot verhoog. Fusarium proliferatum het ook gelei tot 'n betekenisvolle afname in lengte en massa van nuwe groei, aantal blare en 'n verhoogde graad van wortelvrot in vergelyking met die kontrole behandelings. Dit wil voorkom asof Fusarium solani meer virulent is as F. oxysporum, gevolg deur F. proliferatum. Hierdie is die eerste aanmelding van F. oxysporum, F. proliferatum en F. solani as patogene van wingerd in Suid-Afrika, en die eerste aanmelding van F. proliferatum as 'n patogeen van wingerd in die wêreld. Phytophthora cinnamomi is konstant teen lae frekwensies vanuit terugsterwende wingerd in kwekerye geïsoleer, alhoewel dit in risosfeer gronde teenwoordig was. Dit is moontlik dat die ekstensiewe gebruik van chemikalieë teen donsskimmel in wingerdkwekerye die wingerdplante kan beskerm teen infeksie deur P. cinnamomi. Die effek van chemikalieë wat gebruik word teen donsskimmel op Phytophthora wortelverrotting van wingerd in kwekerye, is 'n glashuis geëvalueer. Die chemikalieë wat gestoets is, het vir die gemete parameters, tot baie min onderskeibare effek gelei relatief tot die kontrole plante, en daar is afgelei dat die inokulasie tegniek verbetering benodig. Plante wat met fosforiensuur behandel is, het egter geneig om langer te wees met meer blare, 'n groter stamdeursnee en wortelvolume as kontrole plante of plante behandel met ander chemikalieë. Data verkry vanuit die hierdie studie was onvoldoende, maar sekere neigings is aangedui wat deur verdere glashuis- en veldproewe verklaar sal word. Resultate wat in hierdie tesis weergegee is, het aangedui dat 'n algehele verskuiwing in die status van grondgedraagde swamme geassosieer met wortels en krone van wingerd in kwekerye vanaf die 1970s, toe Phytophthora en Pythium die dominante genera was, plaasgevind het. Die voorkoms en rol van grondgedraagde swamme moet bepaal word, sodat nuwe voldoende siektebestuurspraktyke ontwikkel kan word om verliese in wingerdkwekerye te beperk en sodoende die volhoubare produksie van gesonde plante vir die wingerdbedryf te verseker.

One red and one white cultivar of winegrapes grown in Southern Arizona was pruned to four different methods. The red cultivar was 'Merlot' and the white was 'Sauvignon Blanc'. The pruning methods were 2 bud spur, 4 bud spur, cane and basal buds only. The basal bud treatment was eliminated for 'Sauvignon Blanc'. The 4 bud spur method resulted in significantly greater yield when compared to the other methods. Fruit produced from the basal bud only treatment resulted in fruit that was significantly greater in pH and acid content. The 'Sauvignon Blanc' cultivar had significantly higher yield with cane pruning with no difference in fruit quality.

The leafhopper, Acia lineatifrons (Naude) was identified as a pest on grapevines near Tulbagh in 1978 and has since been reported on grapevines all over the South Western Cape. A. lineatifrons causes browning of the leaves which often results in the shedding of the discoloured leaves. Heavy defoliation before harvest can result in sunburn damage to the grapes, whilst premature leaf loss after harvest adversely affects the ripening of the canes and the accumulation of reserves. This project was aimed at obtaining basic information on the biology and population dynamics of A. lineatifrons as well as to identify priorities for future research. This information is needed to develop a reliable crop-linked predictive model, methods for monitoring pest populations as well as to develop efficient short- and long-term control measures and pest management strategies. The life cycle of A. lineatifrons was studied in the laboratory. At 26°C the mean incubation period of the eggs was nine to eleven days, the mean developmental period for the five nymphal instars was 15 days and the minimum pre-oviposition period five to ten days. This adds up to a mean generation time of 29 to 36 days at 26°C. At 20°C the mean nymphal development period was 25 days, confirming the strong influence of temperature on the development rate. Fecundity was determined in the laboratory as the number of nymphs produced per female. The mean of 8,5 nymphs per female recorded at 26°C is very low compared to that of other leafhopper species (see Appendix 2). The low fecundity measured was most likely due to sub-optimal environmental conditions in the laboratory, a reduction in the suitability of the host plant under these conditions and handling of the females. The seasonal occurrence of A. lineatifrons on grapevines was studied over three seasons. It was found that the leafhoppers overwinter in the adult stage on indigenous Rubus spp, and that they enter the vineyard from the end of October until the beginning of November. Peak populations occurred between the middle of February and the end of March after which the population declined steadily towards the end of the season as the vine leaves were shed. The sex ratio of the overwintering population on R. chrysocarpus was heavily female biased, possibly due to differential mortality of the sexes. During the growing season the sex ratio was slightly male biased and reached equality on several occasions, both on the Rubus and on the grapevines. The movement of A. lineatifrons between the Rubus and the grapevines was investigated, but no evidence of a directional migration from the Rubus to the grapevines was found. Furthermore, no evidence was found to indicate that morphologically distinct short- and long-distance fliers, as found in Cicadulina species by Rose (1972b), exist in the A. lineatifrons population. Host preference tests also showed that adult leafhoppers apparently have no significant preference for grapevines to Rubus or vice versa. It seems, therefore, that the leafhoppers' move onto the grapevines at the beginning of the growing season is not prompted by a host preference. Chaboussou (1971) suggested that certain organic fungicides may cause leafhopper outbreaks because they affect the suitability of the vines as host plants and alter leafhopper fecundity. The effect of Mikal-M (active ingredient Fosetyl AL/Mancozeb), a systemic dithiocarbanate fungicide, on A. lineatifrons was investigated. Laboratory experiments showed no significant effect on fecundity and leaf analysis of potted vines treated with Mikal-M indicated no significant difference in total leaf nitrogen compared to untreated control plants. However, the field experiment on the effect of Mikal-M on the population build-up of the leafhopper showed that significantly more leafhoppers occurred on the vines treated with Mikal-M than on those treated with a conventional inorganic fungicide, copper oxychloride. In view of the far-reaching implications this can have on the viticultural industry, further research on the effects of organic fungicides on leafhopper populations is recommended to confirm the generality of these results so that recommendations regarding the use of these fungicides may be made. The question as to why A. lineatifrons became a pest only recently was raised. Three possibilities were considered, namely (1) that A. lineatifrons is a species of tropical origin which moved down the continent and became established in the Western Cape only recently, (2) that is has been in the Western Cape at least as long as the grapevines, but required prolonged exposure to establish itself on the new host and (3) that is has been on the vines for some time, but was noticed only recently when outbreaks occurred. These outbreaks could have been caused by the introduction of organic fungicides or the depletion of natural enemies by insecticides used to control other insects in the vineyards. Due to the lack of evidence this question could not be answered conclusively. Other research priorities that were established are the development of methods for damage assessment and monitoring of leafhopper populations, determining if A. lineatifrons can transmit grapevine virusses, the development of an economic threshold level and the identification of natural enemies of A. lineatifrons to enable the development of efficient pest management strategies (Summary, p. 63-65)

Viticultural practices such as trunk girdling and shoot topping along with defoliation, shading and node number per vine treatments were used to alter the carbohydrate physiology of mature Chardonnay grapevines growing in the cool climate of Canterbury, New Zealand. The timing of vine defoliation in the season previous to fruiting decreased concentrations of over-wintering carbohydrate reserves (mostly starch) in both the trunks and roots of grapevines. Roots were particularly sensitive, with defoliation as early as 4 weeks after bloom in the previous season reducing starch concentrations to 1.5%Dwt at bud burst compared with 17%Dwt in non-defoliated vines. In contrast, partial vine defoliation as early as bloom in the previous season reduced root starch concentrations to 4-7%Dwt at bud burst compared with 15%Dwt in non-defoliated vines. Vine shading and trunk girdling treatments at bloom in the previous season, resulted in small reductions in root starch concentrations (16%Dwt) compared with non-shaded and non-girdled vines (19%Dwt), but shoot topping did not. Study across three growing seasons established that higher concentrations of over-wintering trunk and root carbohydrate reserves were associated with warmer and sunnier weather in the previous growing season. Individual shoot leaf removal at either the beginning or towards the end of the inflorescence initiation period, reduced shoot starch concentrations to 3-6%Dwt compared with 11 %Dwt for no leaf removal, such reductions persisted through to the following season. Shoot topping at the start of the initiation period had no effect on shoot carbohydrate accumulation, but trunk girdling temporarily increased shoot starch concentrations during the first 31 days after treatment. Reductions in over-wintering trunk and root carbohydrate reserves were associated with a reduction in inflorescences per shoot and flowers per inflorescence in the following season, the reduction as much as 50% compared with non carbohydrate stressed vines. While there were strong linear or curvilinear relationships between the concentration of starch in trunks and roots at bud burst and inflorescences per shoot and flowers per inflorescence, in case the of inflorescences per shoot, there was not an immediate cause and effect because inflorescences were initiated in the previous season. Individual shoot leaf removal during the inflorescence initiation period illustrated that leaf removal directly inhibited the initiation of inflorescences in latent buds. Shoot carbohydrate measurements showed a strong curvilinear relationship to the number of inflorescences per shoot, with a threshold starch concentration of 10-12%Dwt during the inflorescence initiation period required for a maximum number of inflorescences per shoot. Furthermore, examination of individual node positions emphasised the importance of the subtending leaf on the initiation of inflorescences within the latent bud. The number of inflorescences per shoot post bud burst was reduced on vines that were both carbohydrate reserve stressed (by previous season's defoliation) and had a high node (108) number retained per vine after winter pruning compared with little or no reduction in inflorescences per shoot on carbohydrate reserve stressed vines that had a low (20) node number per vine. The reduction in inflorescences per shoot on high node vines was associated with reduced carbohydrate reserves and reduced shoot vigour (thinner and lighter shoots). Flowers per inflorescence were reduced by as much 50% in response to lower overwintering carbohydrate reserves. Fewer flowers per inflorescence were attributed to a reduction in primary branching of the inflorescence and also a reduction in flowers per branch. Strong linear relationships between the concentrations of starch in trunks and roots and flowers per inflorescence indicate that the determination of flowers per inflorescence, unlike inflorescences per shoot, may be dependent on the level of overwintering carbohydrate reserves. This is most likely due to changes in branching of the inflorescence and individual flower formation occurring during the bud burst period. Per cent fruitset was not affected by reductions in carbohydrate reserves, so fewer inflorescences per shoot and flowers per inflorescence resulted in reduced vine yield. The findings of this thesis indicate that changes in the level of carbohydrate production and partitioning in response to a range of viticultural management practices and seasonal weather contribute to seasonal variation in grapevine flowering and yields in New Zealand's cool climate environment. The relationships between carbohydrate reserves and flowering illustrate the potential to use this information to predict grapevine flowering and forecast yields. The practical implications of this research illustrate that the viticulturist must manage grapevines not only for the current crop, but also for subsequent crops by maintaining sufficient carbohydrate reserves for balanced growth flowering and fruiting from season to season.

Thesis (MScAgric)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: The effect of shoot heterogeneity on vegetative and reproductive growth parameters, vine physiology and grape composition was investigated in a ShirazlRichter 99 vineyard. Comparisons between underdeveloped (typically shorter and less ripened at véraison) and normally developed shoots in both shaded (non-manipulated) and well-exposed (manipulated) canopies were made. Compared to underdeveloped shoots, normal shoots had a larger total leaf area, due to the higher occurrence of secondary shoots as well as larger leaves on primary and secondary shoots. Since the photosynthetic activity of the leaves from normal shoots was higher than those from underdeveloped shoots, higher levels of carbohydrates were produced and stored in the former. Starch was more evenly distributed over the length of the whole shoot in normally developed shoots compared to underdeveloped shoots. Normally developed shoots were longer and thicker in diameter than underdeveloped shoots. The larger clusters of the normally developed shoots are evidence of their more favourable total leaf area per gram berry mass. Berries from the normally developed shoots were smaller at five weeks after véraison than those from underdeveloped shoots, displaying a higher skin to pulp ratio and therefore higher anthocyanin and total phenolic extraction potential for winemaking. The smaller clusters and fewer berries per cluster found for the underdeveloped shoots indicate an imbalance between vegetative and reproductive growth initiated during the vegetative growth phase and continued during the ripening period. The peculiar absence of statistically significant differences in grape composition between normally and underdeveloped shoots indicates that assimilates needed for berry ripening of the latter originated in organs other than the leaves [e.g. from adjacent normal shoots and the rest of the permanent structure of the vine (cordon, trunk, roots)]. The larger differences in berry size that occurred between shoot types in the shaded compared to the well-exposed canopies may be evidence for this. The photosynthetic activity of shoots was lower in shaded than in exposed canopies. The total carbohydrate production of the normal shoots in shaded canopies seemed insufficient to supply in the ripening needs of the shoot itself, their own clusters as well as the ripening of stem tissue and clusters of the underdeveloped shoots in the canopy. This is illustrated by the lower levels of starch that accumulated in the normal shoots from shaded compared to that of exposed canopies. Vine shoot heterogeneity clearly leads to visible and physiological imbalances that would impact negatively on grape and wine quality as well as production costs and should therefore be avoided on any terroir.
AFRIKAANSE OPSOMMING: In hierdie studie is die effek van heterogene lootontwikkeling in die lower van 'n ShirazlRichter 99 wingerd ondersoek. Vergelykings is getref tussen normale en onderontwikkelde lote in beskadude en blootgestelde lowers ten opsigte van hul vegetatiewe en reproduktiewe groeieienskappe, fisiologiese aktiwiteit en druifsamestelling en -gehalte. 'n Groter totale blaaroppervlak het by die normaalontwikkelde lote voorgekom as gevolg van die groter aantal sekondêre lote en groter primêre en sekondêre blare. Aangesien die blare van die normaal ontwikkelde lote fotosinteties meer aktief was as dié van onderontwikkelde lote, het die eersgenoemde lote groter hoeveelhede koolhidrate geproduseer en gestoor. Styselopberging het meer eweredig oor die lengte van die normale lote plaasgevind. Laasgenoemde lote was ook heelwat langer en dikker in deursnee as die onderontwikkelde lote. Die gunstiger totale blaaroppervlak per korrelmassa verhouding van die normale lote is duidelik weerspieêl in die groter trosse, terwyl die kleiner korrels (en dus die groter dop:pulp verhouding) op 'n groter potensiaal vir kleur- en fenolekstraksie tydens die wynbereidingsproses dui. Die kleiner trosse en kleiner aantal korrels per tros wat by die onderontwikkelde lote gevind is, dui op 'n wanbalans tussen die vegetatiewe en reproduktiewe groei van die loot wat tydens die vegetatiewe groeifase van die wingerdstok geïnisieer is en tydens die rypwordingsperiode voortgesit is. Die vreemde afwesigheid van enige statisties betekenisvolle verskille in druifsamestelling tussen die normale en onderontwikkelde lote dui daarop dat die verbindings wat vir die rypmaking van trosse op onderontwikkelde lote aangewend is, waarskynlik van ander wingerdorgane (bv. naasliggende lote, kordonarms, stam, wortels) as die spesifieke loot se blare afkomstig was. Die waarneming dat die korrelgroottes van normale en onderontwikkelde lote meer van mekaar verskil het in die beskadude as blootgestelde lowers, kan moontlik as bewys hiervoor dien. Die fotosintetiese aktiwiteit van beide loottipes was laer in die skaduryke lowers. Die koolhidrate wat deur normaalontwikkelde lote in skadu-Iowers geproduseer is, was oênskynlik onvoldoende vir die rypmaking van die loot self, die spesifieke loot se trosse, asook die trosse en lootweefsel van naasliggende onderontwikkelde lote. Hierdie bewerings word gerugsteun deur die laer vlakke van styselakkumulasie wat by die normale lote in beskadude lowers gevind is. Aangesien heterogene lootontwikkeling en -groei duidelike sigbare en fisiologiese wanbalanse in die wingerdstok tot gevolg het wat negatief op druifsamestelling, wyngehalte en produksiekoste inwerk, behoort dit in kommersiêle wingerde vermy te word.

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Arbuscular mycorrhizal (AM) C-costs in grapevines were investigated. Since both dormant vines and AM colonisation rely on stored C for initial growth, AM colonisation costs would therefore compete with plant growth for available C reserves. The aims of this study were to assess the host C economy during AM development and the subsequent C-costs of N and P uptake, as well as the effects of C costs on host growth. This was evaluated in two separate experiments; one assessing the symbiotic influence on the C costs of fungal establishment and nutritional benefits, whilst the other one evaluated the effects of the symbiosis on host growth and nutrient productivities. This study has shown that AM acts as a C sink, competing with the host for available C. Past work on the AM sink effect has focused mainly on the movement of photosynthetic C below ground to support the AM fungus. This however, does not take into account the effect that stored C will have on the C economy of the plant and symbiosis. The role of stored C becomes even more crucial when working with deciduous plants that rely on stored C for new growth at start of a growing season. It has been reported that stored C in AM plants is remobilized at the start of a growing season and then the C reserves are refilled towards the end of the season, when the plants enter dormancy. The initial costs of AM fungal colonisation were borne by the above-ground C reserves, at the expense of new growth in host plants. These costs were offset once the plateau phase was reached, and the depleted reserves started to refill. Once established, the active symbiosis imposed a considerable below ground C sink on host reserves. In spite of these costs, the improved P nutrition of AM roots was achieved with a more efficient C-use. This concurs with other findings, that of the belowground C allocated to AM roots, a greater part is used by AM respiration and a smaller part for P uptake. The C costs of the AM fungal phase of rapid development can be seen as negative to root growth and shoot development. These negative effects may continue for a period of time, even during the plateau phase of fungal development. Once the AM symbiosis is fully established, the host growth and development is then improved to a greater extent than in non-AM plants. From this study it can be concluded that AM growth directly competes with host development, but the symbionts revert to a beneficial partnership once it is fully established.
AFRIKAANSE OPSOMMING: Die C koste van arbuskulêre mikorisa (AM) in wingerdstokke is ondersoek. Beide rustende wingerdstokke en AM koloniseering is afhanklik van gestoorde C vir aanvanklike groei. AM kolonisering sou dus met plantgroei kompeteer vir beskikbare C reserwes. Die doelstellings van hierdie ondersoek was eerstens om die C-ekonomie van die gasheer tydens AM ontwikkeling en die gevolglike Ckostes van N en P opname te bepaal en tweedens sowel as die invloed van C veranderings op gasheergroei vas te stel. Hierdie is in twee afsonderlike eksperimente ondersoek: een om die simbiotiese invloed op die C-kostes van swam-vestiging en voedingsvoordele te bepaal, terwyl die ander die uitwerking van simbiose op gasheergroei en voedings doeltreffenheid evalueer het. Die ondersoek het bewys dat AM, as ‘n C-sink, kompeteer met die gasheer vir beskikbare C. Vorige werk oor die AM sink-effek het hoofsaaklik gefokus op die afwaartse beweging van fotosintetiese C om die AM-swam ondergronds te ondersteun. Die werk neem egter nie in ag wat die effek van gestoorde C op die C-ekonomie van die plant en simbiose sou wees nie. Die rol van gestoorde C is selfs nog meer belangrik wanneer met bladwisselende plante gewerk word, omdat sulke plante op gestoorde-C vir nuwe groei aan die begin van die groeiseisoen staatmaak. Dit is op rekord dat gestoorde C in bladwisselende plante by aanvang van die groeiseisoen gemobiliseer word en dat die C-reserwes teen die einde van die seisoen wanneer die plante rustyd nader, weer hervul word. Die aanvanklike kostes van AM kolonisering is deur die bogronds C-reserwes, ten koste van nuwe groei van die gasheerplante, gedra. Hierdie kostes herstel sodra die plato-fase bereik is, waar die uitgeputte reserwes begin hervul het. As die aktiewe simbiose eers gevestig is, sal dit as ‘n onderg P-voeding van AM wortels verkry wordrondse C-sink vir gasheer optree.Hierdie C verbruik word egter as doeltreffend beskou aangesien verbeterde. Dit is bekend dat ‘n groter deel van die ondergrondse C geallokeer word aan AM-wortels, deur middel van AM respirasie en P-opname. Die C-kostes van die AM-fungus tydens die fase van vinnige ontwikkeling, kan ‘n negatiewe effek op wortel- en lootontwikkeling hê. Hierdie negatiewe uitwerking kan vir ‘n tydperk voortdeur, selfs gedurende die plato-fase van fungi-ontwikkeling. Sodra die AM-simbiose volledig gevestig is, word gasheergroei en ontwikkeling tot ‘n groter mate verbeter as in plante sonder AM-fungi. Hierdie ondersoek het bewys dat AM groei direk met gasheerontwikkeling kompeteer, maar dat die simbiose ‘n voordelige vennootskap vorm sodra dit volledig gevestig is.

Thesis (MSc)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Viruses are some of the most important pathogens of grapevines. There are no effective chemical treatments, and no grapevine- or other natural resistance genes have been discovered against grapevine infecting viruses. The primary method of grapevine virus control is prevention by biological indexing and molecular- and serological screening of rootstocks and scions before propagation. Due to the spread of grapevine viruses through insect vectors, and in the case of GRSPaV the absence of serological screening, these methods of virus control are not always effective. In the past several methods, from cross-protection to pathogen derived resistance (PDR), have been applied to induce plant virus resistance, but with inconsistent results. In recent years the application of post-transcriptional gene silencing (PTGS), a naturally occurring plant defense mechanism, to induce targeted virus resistance has achieved great success. The Waterhouse research group has designed plant transformation vectors that facilitate specific virus resistance through PTGS. The primary focus of this study was the production of virus specific transformation vectors for the introduction of grapevine virus resistance. The Waterhouse system has been successfully utilised for the construction of three transformation vectors with the pHannibal vector as backbone. Each vector contains homologous virus coat protein (CP) gene segments, cloned in a complementary conformation upstream and downstream of an intron sequence. The primary vector (pHann-SAScon) contains complementary CP gene segments of both GRSPaV and GLRaV-3 and was designed for the introduction of multiple-virus resistance. For the construction of the primary vector the GRSPaV CP gene was isolated from RSP infected grapevines. A clone of the GLRaV-3 CP gene was acquired. The second vector (pHann- LR3CPsas) contains complementary CP gene segments of GLRaV-3. The third vector (pHann-LR2CPsas) contains complementary CP gene segments of GLRaV-2. The cassette containing the complementary CP gene segments of both GRSPaV and GLRaV-3 was cloned into pART27 (pART27-HSAScon), and used to transform N tabacum cv. Petit Havana (SRI), through A. tumefaciens mediated transformation. Unfortunately potential transformants failed to regenerate on rooting media; hence no molecular tests were performed to confirm transformation. Once successful transformants are generated, infection with a recombinant virus vector (consisting of PYX, the GFP gene as screenable marker and the complementary CP gene segments of both GRSPaV and GLRaV-3) will be used to test for the efficacy of the vectors to induce resistance. A secondary aim was added to this project when a need was identified within the South African viticulture industry for GRSPaV specific antibodies to be used in serological screening. To facilitate future serological detection of GRSPaV, the CP gene was isolated and expressed with a bacterial expression system (pETI4b) within the E. coli BL2I(DE3)pLysS cell line. The expressed protein will be used to generate GRSPaV CP specific antibodies.
AFRIKAANSE OPSOMMING: Virusse is van die belangrikste patogene by wingerd. Daar bestaan geen effektiewe chemiese beheer nie, en geen wingerd- of ander natuurlike weerstandsgene teen wingerdvirusse is al ontdek nie. Die primêre metode van beheer t.o.v. wingerdvirusse is voorkoming deur biologiese indeksering, en molekulêre- en serologiese toetsing van onderstokke en entlote voor verspreiding. As gevolg van die verspreiding van wingerdvirusse deur insekvektore, en in die geval van GRSPa V die tekort aan serologiese toetsing, is dié metodes van virusbeheer nie altyd effektief nie. In die verlede is metodes soos kruis-beskerming en patogeen-afgeleide weerstand (PDR) gebruik om virusweerstand te induseer, maar met inkonsekwente resultate. In onlangse jare is post-transkripsionele geenonderdrukking (PTGS), 'n natuurlike plantbeskermingsmeganisme, met groot sukses toegepas om geteikende virusweerstand te induseer. Die Waterhouse-navorsingsgroep het planttransformasievektore ontwerp wat spesifieke virusweerstand induseer d.m.v. PTGS. Die vervaardiging van virus spesifieke tranformasievektore vir die indusering van wingerdvirusweerstand was die primêre doelwit van hierdie studie. Die Waterhouse-sisteem was gebruik vir die konstruksie van drie transformasievektore, met die pHannibal vektor as basis. Elke vektor bevat homoloë virus kapsiedproteïen (CP) geensegmente, gekloneer in 'n komplementêre vorm stroom-op en stroom-af van 'n intronvolgorde. Die primêre vektor (pHann-SAScon) bevat komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, en was ontwerp vir die indusering van veelvoudige-virusweerstand. Die CP-geen van GRSPa V was vanuit RSP-geïnfekteerde wingerd geïsoleer, vir die konstruksie van die primêre vektor. 'n Kloon van die GLRa V-3 CP-geen was verkry. Die tweede vektor (pHann-LR3CPsas) bevat komplementêre CP geensegmente van GLRaV-3. Die derde vektor (pHann-LR2CPsas) bevat komplementêre CP geensegmente van GLRa V-2. Die kasset bestaande uit die komplementêre CP geensegmente van beide GRSPaV en GLRaV-3, was gekloneer in pART27 (pART27-HSAScon), en gebruik om N tabacum cv. Petit Havana (SRI) te transformeer d.m.v. A. tumefaciens bemiddelde transformasie. Ongelukkig het potensiële transformante nie geregenereer op bewortelingsmedia nie; gevolglik was geen molekulêre toetse gedoen om transformasie te bevestig nie. Na suksesvolle transformante gegenereer is, sal infeksie met 'n rekombinante-virusvektor (bestaande uit PYX, die GFP geen as waarneembare merker en die komplementêre CP geensegmente van beide GRSPa V en GLRa V-3) gebruik word om die effektiwiteit van die vektore as weerstandsinduseerders te toets. 'n Sekondêre doelwit is by die projek gevoeg toe 'n behoefte aan GRSPaV spesifieke teenliggame binne die Suid-Afrikaanse wynbedryf geïdentifiseer is, vir gebruik in serologiese toetsing. Om toekomstige serologiese toetsing van GRSPa V te bemiddel, was die CP-geen geïsoleer en in 'n bakteriële uitdrukkingsisteem (PETI4b) uitgedruk, in die E. coli BL21(DE3)pLysS sellyn. Die uitgedrukte proteïne sal gebruik word vir die vervaardiging van GRSPa V CP spesifieke antiliggame.

Thesis (MSc (Plant Pathology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Esca is a disease affecting grapevines and is potentially devastating as there are economic losses due to a decrease in yield, wine quality and berry quality. Vineyards also need to be replaced earlier and therefore esca has a great impact on the wine, table grape and raisin industries. The disease is known to affect vineyards worldwide and has been studied extensively in Europe, but not in South Africa. Esca diseased grapevines were observed for the first time prior to 1981 in South African vineyards. The disease is primarily caused by Phaeoacremonium aleophilum, Phaeomoniella chlamydospora (both causing brown and black wood streaking) and white rot basidiomycete species such as Fomitiporia mediterranea which cause wood rot in the trunks and arms of generally older grapevines. Species of the Botryosphaeriaceae and Phomopsis (mainly Phomopsis viticola) and Eutypa lata have also been isolated from esca diseased vines, but their association with esca is unclear. Some of the symptoms associated with the disease on most grapevine cultivars include ‘tiger-stripe’ foliar symptoms, apoplexy and berry symptoms such as shriveling, discoloration and ‘black measles’. These external symptoms as well as internal symptoms are thought to be a result of toxin and enzyme production by the fungi involved. Symptom expression is erratic and varies from year to year making investigations into the causal fungi and the toxins and enzymes secreted in planta difficult. Vines with internal or external symptoms of esca were sampled in this study from table and wine grape cultivars in 37 towns in the Western Cape, Northern Cape and Limpopo provinces. The majority of sampled vines were over ten years of age, but vines as young as two to three years were also found to be infected. The external symptoms included dieback, tiger striped leaves, berry symptoms (shriveling, insufficient colouring and black spots) and apoplexy. These symptoms resembled those found on grapevines in Europe, Australia and the USA. The internal symptoms found were also similar to European symptoms and included white rot, black and brown wood streaking, brown necrosis within white rot, sectorial brown necrosis and central brown/ red/ black margin. The fungi mostly isolated from the white rot were the basidiomycetes. Black and brown wood streaking was primarily caused by Phaeomoniella chlamydospora. Brown necrosis within the white rot was caused by Phaeomoniella chlamydospora and less frequently by Phaeoacremonium spp., Eutypa lata, Botryosphaeriaceae and Pleurostomophora richardsiae. The sectorial brown necrosis and the central/ brown/ red/ black margin were dominated by Phaeomoniella chlamydospora. The fruiting bodies of the basidiomycetes were found on only a few grapevines. The fungal species associated with the internal wood symptoms were characterized on cultural growth patterns, morphology as well as phylogenetic inference. The gene areas sequenced included the internal transcribed spacers and the 5.8S rRNA gene for the basidiomycetes and Phomopsis isolates, the partial b-tubulin gene for Phaeoacremonium isolates and the partial translation elongation-1a gene for the Botryosphaeriaceae isolates. The basidiomycete isolates fell into ten taxa within the Hymenochaetales of which two could be linked to known genera, namely Fomitiporia and Phellinus. The ten basidiomycete taxa do not correspond to any published sequences. Eutypa lata, Diaporthe ambigua, Diplodia seriata, Neofusicoccum australe, Neofusicoccum parvum, Phomopsis viticola, Phomopsis sp. 1, Phaeomoniella chlamydospora and six species of Phaeoacremonium including P. aleophilum, P. alvesii, P. parasiticum, P. iranianum, P. mortoniae and P. sicilianum were also isolated of which the latter three are reported for the first time in South Africa. To understand the role of the basidiomycetes in the complex, toxin and enzyme analyses was determined for these fungi. Selected basidiomycete isolates were grown up in liquid broth and extractions performed to test for the presence of 4-hydroxy-benzaldehyde. All of the basidiomycete isolates were able to produce this toxin which is known to be phytotoxic. The basidiomycetes were then tested for the presence of certain wood degrading enzymes. All of the taxa were able to produce manganese peroxidase. Laccase was produced by all taxa, except Taxon 8. Lignin peroxidase was produced by Taxa 1, 2, 7, Fomitiporia sp. and the Phellinus sp. All the basidiomycete isolates were able to produce cellulose and none were able to produce xylanase. These enzyme tests showed that the basidiomycetes produce a wide variety of enzymes which are able to degrade cellulase and lignin which are both structural components of wood. Given the wide distribution of esca in the grape growing regions investigated in South Africa and the diverse amount of species found, this disease must surely be seen as a limiting factor to the productive lifespan of vineyards and quality of produce. Preventative measures such as sanitation and pruning wound protection contribute to the management of the disease, but many questions still remain about the synergy of the causal fungi, epidemiology and management of esca.
AFRIKAANSE OPSOMMING: Esca is ‘n wingerd siekte wat potensieel skade kan aanrig as gevolg van ekonomiese verliese weens verlaagde opbrengs, wyn kwaliteit en vrug kwaliteit. Wingerde moet ook vroeër vervang word en daarom het esca ’n groot impak op die wyn, tafeldryf en rosyne industrieë. Esca word wêreldwyd gevind op wingerd en is al intensief nagevors in Europa, maar nog nie in Suid-Afrika. Esca is vir die eerste keer in die 1980’s in Suid-Afrikaanse wingerde gerapporteer. Die primêre veroorsaakende organismes van esca is Phaeoacremonium aleophilum, Phaeomoniella chlamydospora wat bruin en swart vaatweefsel verkleuring veroorsaak en basidiomycete spesies soos Fomitiporia mediterranea wat wit verotting veroorsaak in die stam en arms van ouer wingerd. Spesies van die Botryosphaeriaceae en Phomopsis (hoofsaaklik Phomopsis viticola) en Eutypa lata is ook al vanaf esca simptome geïsoleer, maar hul assosiasie met die siekte is nie duidelik nie. Algemene simptome wat voorkom op die meeste wingerd kultivars met esca sluit in ‘tiger-stripe’ blaar simptome, apopleksie en vrug simptome soos verdroging, verkleuring en spikkels (black measles). Interne en eksterne simptome kan wees as gevolg van toksiene en ensiem produksie van die swamme wat betrokke is by esca. Eksterne simptoom uitdrukking is wisselvallig en varieer van jaar tot jaar. Dit bemoelik die bestudering van die swamme en die toksiene en ensieme wat afgeskei word in planta. Wingerd monsters met eksterne en interne simptome is versamel van tafel en wyndruif kultivars in 37 dorpe in die Wes-Kaap, Noord-Kaap en Limpopo provinsies. Die meerderheid monsters was ouer as tien jaar maar wingerde wat twee tot drie jaar oud was, was ook gevind. Die eksterne simptome wat op hierdie kultivars gevind is het terugsterwing, ‘tiger striped’ blare, vrug simptome (verkrimping en onvoldoende verkleuring) en apopleksie ingesluit. Hierdie simptome stem ooreen met soortgelyke simptome gevind op wingerd in Europa, Australië en die VSA. Interne simptome was ooreenstemmend met simptome wat gevind word in Europa. Die interne simptome het wit verotting, bruin en swart streepvorming, bruin nekrose met wit verotting, sektoriale bruin nekrose en sentrale bruin/ rooi/ swart kante ingesluit. Basidiomycete swamme is meestal uit die wit verotting gedeeltes geïsoleer. Swart en bruin hout streepvorming was meestal deur Phaeomoniella chlamydospora veroorsaak. Bruin nekrose binne die wit verotting was meestal deur Phaeomoniella chlamydospora veroorsaak en in ‘n mindere mate deur Phaeoacremonium spp., Eutypa lata, Botryosphaeriaceae en Pleurostomophora richardsiae. Phaeomoniella chlamydospora was die hoof veroorsakende organisme van sektoriale bruin nekrose en die sentrale bruin/ rooi/ swart kante. Vrugliggame van die basiodiomycete is op enkele wingerde gevind. Swam soorte wat geassosieer word met die interne hout simptome was verder gekarakteriseer op kultuur groei, morfologiese eienskappe, en filogenetiese analise. Die geen areas waarvan die basis paar volgorde bepaal was sluit in die interne getranskribeerde spasies en die 5.8S rRNA geen vir die basidiomycete en Phomopsis isolate, die gedeeltelike btubulien geen vir Phaeoacremonium isolate en die gedeeltelike translasie velenging-1a geen vir die Botryosphaericeae isolate. Die basidiomycete isolate was versprei oor tien taksons binne die Hymenochaetales waarvan twee genusse gekoppel kon word aan die genera Fomitiporia en Phellinus. Die tien basidiomycete taksons kom nie ooreen met enige gepubliseerde DNS volgordes. Eutypa lata, Phomopsis viticola, Phomopsis sp. 1, Diaporthe ambigua, Diplodia seriata, Neofusicoccum parvum, Neofusicoccum australe, Phaeomoniella chlamydospora en ses spesies van Phaeoacremonium insluitend P. aleophilum, P. alvesii, P. parasiticum, P. iranianum, P. mortoniae en P. sicilianum is ook geïsoleer. Hierdie is die eerste keer dat P. iranianum, P. mortoniae en P. sicilianum in Suid-Afrika gerapporteer word. Om die rol wat die basidiomycete in die siekte-kompleks speel beter te verstaan is toksien en ensiem analises uitgevoer. Geselekteerde basidiomycete isolate is gekweek in vloeibare groei medium en ekstraksies uitgevoer om te toets vir die teenwoordigheid van 4- hydroxy-benzaldehyde. Al die basidiomycete isolate kon 4-hydroxy-benzaldehyde, wat bekend is om fitotoksies te wees, produseer. Die basidiomycete isolate was verder getoets vir die produksie van spesifieke hout afbrekende ensieme. Al die basidiomycete taksons kon mangaan-peroksidase produseer. Lakkase was geproduseer deur al die taksons, uitsluitend Takson 8. Lignien-peroksidase was geproduseer deur Taksons 1, 2, 7, Fomitiporia sp. en die Phellinus sp. Al die basidiomycete isolate kon sellulose produseer, maar geen kon xilanase produseer. Die ensiem analises het gewys dat die basidiomycete wat moontlik betrokke is by esca ‘n wye reeks van ensieme kan produseer wat sellulose en lignien kan degradeer. Sellulose en lignien is beide strukturele komponente van hout. Weens die wye verspeiding van esca geaffekteerde wingerde in Suid Afrika en die wye reeks van spesies wat betrokke is by die siekte kompleks moet esca sekerlik gesien word as een van die beperkende faktore op die produktiewe leeftyd van wingerde en die kwaliteit van druiwe wat geproduseer word. Sanitasie en snoeiwond beskerming is voorkomende maatreëls wat ingestel kan word om die effek en verspreiding van esca te beperk maar daar is nog baie vrae wat antwoorde benodig oor die sinergie van die veroorsakende swamme, epidemiologie en bestuur van esca.

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.

Una de les principals preocupacions en l’agricultura és la incertesa deguda als efectes del canvi climàtic. En aquest aspecte, l’estudi i adopció de noves tècniques de maneig dels cultius i la gestió de l’aigua són elements essencials per tal de garantir la productivitat dels cultius. En la regió mediterrània, la vinya (Vitis vinifera L.) representa un dels cultius més importants, el qual es podria veure afectat especialment en aquelles zones on no hi ha disponibilitat d’aigua. A més, en els últims anys s’ha observat un avançament en la fenologia de la vinya, provocant que, entre d’altres, la fase de la post-verema esdevingui una fase crítica degut a la seva major durada i sota unes condicions més càlides i amb menor precipitació. Degut a que en els propers anys es preveu un increment en la freqüència i intensitat dels esdeveniments de sequera, a més d’un augment significatiu de les temperatures, els elements crítics per combatre els efectes del canvi climàtic són: la disponibilitat de models fenològics per predir els patrons estacionals de les fases fenològiques; i la selecció del moment més idoni per l’adopció d’estratègies de reg deficitari, per tal d’estalviar aigua i poder garantir uns bons rendiments productius de la verema amb la composició dels raïms adequada per a l’elaboració de vins i caves.
Una de las principales preocupaciones en la agricultura es la incertidumbre de los efectos del cambio climático. Por este motivo, el estudio y la adopción de nuevas técnicas de manejo de los cultivos y de la gestión del agua son elementos esenciales para garantizar la productividad de los cultivos. En la zona del mediterráneo, el viñedo (Vitis vinifera L.) representa uno de los cultivos más importantes, el cual se podría ver afectado especialmente en las zonas donde no hay disponibilidad de agua. Además, en los últimos años se ha observado un adelanto en la fenología de la viña, provocando que, entre otros, la fase de la post-vendimia esdevenga una fase crítica debido a su mayor duración y bajo unas condiciones más cálidas y con una menor pluviometría. Debido a que en los próximos años se prevé un incremento en la frecuencia e intensidad de los eventos de sequía, además de un aumento significativo de las temperaturas, los elementos críticos para combatir los efectos del cambio climático son: la disponibilidad de modelos fenológicos para predecir los patrones estacionales de las fases fenológicas; y la selección del momento más idóneo para la adopción de estrategias de riego deficitario, para el ahorro de agua y garantizar unos buenos rendimientos productivos de la vendimia con una composición de las bayas adecuada para la elaboración de vinos y cavas.
One of the main concerns in agriculture is the effect of climate change. As a result, studying the adoption of new management techniques and managing water use are essential for guaranteeing crop productivity. In the Mediterranean region, the grapevine (Vitis vinifera L.) is one of the most important crops affected by water availability, especially in areas without irrigation. Moreover, over the last years has been observed advancements in grapevine phenology, where the post-harvest period is becoming a critical stage due to their longer duration under warmer and dryer conditions. Due to the expected increases in the frequency and intensity of drought events and rising temperatures, the critical elements for combating the effects of climate change are: the availability of phenological models to predict seasonal patterns in the key phenological stages; and the selection of the most suitable moment at which to apply regulated deficit irrigation strategies. Their study is required to water savings and to guarantee good yields with the desired berry composition for wine and sparkling wine production.

Copies of author's previously published articles inserted. Bibliography: leaves 143-167. Temperature may affect fruit-set in grapevines through its effect on the development of the flowers up to flower opening and on pollination, be it on the germination of the pollen and the growth of the pollen tube, or on the post-fertilisation growth of the ovule.

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

Late-season Bunch Stem Necrosis (BSN) is observed as a necrosis of the cluster stem (rachis) that leads to shriveling of berries on the affected portion of the cluster. Field experiments were conducted over three years at two vineyards in northern Virginia to examine relationships between specific nutrients and the incidence of BSN of Cabernet Sauvignon grapevines. Nutrients, used alone or in combination, included nitrogen, magnesium, and calcium. During the 1997 and 1998 seasons at Leesburg vineyard, applications of nitrogen, magnesium, and calcium produced little change in bloom-time petiole mineral concentration. Fertilizer treatments appeared to have no effect on BSN incidence, but the incidence of BSN was less than or equal 1% in the control plots each year. During the 1996 season at Winchester vineyard, bloom-time leaf petiole and véraison rachis nitrogen concentration of unfertilized (control) vines were 0.80% and 1.16%, respectively. The corresponding control BSN incidence was 41% at harvest time. Application of nitrogen fertilizer at 112 kg/ha actual nitrogen increased bloom-time leaf petiole and véraison cluster stem nitrogen concentration to 1.85% and 2.18%, respectively. The corresponding BSN incidence was reduced to 14% at harvest time. BSN symptoms were not as pronounced during the 1997 season; however, all treatments, including the control plots, had elevated nitrogen levels in 1997. During the 1998 season, bloom-time leaf petiole and véraison rachis nitrogen concentration of unfertilized vines were 0.88% and 0.98%, respectively. The corresponding BSN incidence was 23% at harvest time. Application of nitrogen fertilizer again increased bloom-time leaf petiole and véraison rachis nitrogen concentration to 1.18% and 1.34%, respectively. Corresponding BSN was reduced to 3% at harvest time. Magnesium and calcium had no impact on BSN incidence; however, BSN symptoms were reduced when either was combined with nitrogen fertilizer. The relationship between mineral nutrition and BSN incidence at Leesburg was inconclusive. The BSN of Cabernet Sauvignon at Winchester was, however, positively associated with depressed bloom-time petiole total nitrogen concentrations. Véraison rachis analysis consistently revealed an increase in nitrogen concentration due to application of nitrogen fertilizer. Véraison tissue analysis may be a good diagnostic tool of vine nitrogen status. Magnesium and calcium appeared not to be involved in the disorder. The results illustrate that BSN-prone vineyards should be individually examined for nutrient imbalance or other stresses that may be contributing to BSN.
Master of Science

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.

Mestrado Vinifera EuroMaster - Instituto Superior de Agronomia
A study to compare the effects of hedging and curling the shoot tips (rolling) on the last wire of the trellising system was carried out in Saint Emilion (France) on Merlot grapevines in 2011. Vines were hedged or curled when shoots were 30 – 40 cm longer than the highest wire. The effects of the two canopy management strategies on vine performance, mainly vegetative growth, disease occurrence, phenology, water status, yield components and berry composition were compared. Curled, not hedged vines presented longer main shoots, more lateral shoots and higher potassium values on the petioles. Hedged plants had a higher percentage of shaded clusters and a higher leaf layer number at the cluster zone and ¾ of the canopy. Regarding leaf area, curled plants presented a bigger main leaf area but for lateral leaf area no differences were found. Although berries on hedged plants were prone to have a higher mass, no differences for berry composition were found. Hedging seems to be an appropriated technique for the Sain Emilion region as it is less time consuming, less expensive, possible to be mechanized and it has no detriment regarding quality of berries

Mestrado em Engenharia de Viticultura e Enologia / Instituto Superior de Agronomia. Universidade de Lisboa / Faculdade de Ciências. Universidade do Porto
The present work was aimed to elaborate a bibliographic review about the importance and effects, in a viticultural and enological point of view, of a new and innovating product, the LalVigneâ, that consists in inactive dry yeast (Saccharomyces cerevisiae) derivatives. It was discussed the ongoing challenges of climate change in wine industry, as the alterations of flowering, veraison and harvest, emphasizing the changes in the ripening process, the metabolism of the vine and also at sensorial level. It was referred in this work, some adaptation measures, being the application of LalVigneâ the most important and the one which was developed throughout the work. Elicitation topic went deeper, being referred what elicitor is and its mode of action. Bridging the gap between elicitors and its mode of action in vines, it was possible to develop the work about LalVigneâ. The LalVigneâ is an elicitor, consisting in inactive dry yeast derivatives, with foliar application. The characteristics of LalVigneâ, it’s mode of action, the advantages and disadvantages for viticulture and enology, as well as their side effects, were described. There are two types of LalVigneâ, Mature and Aroma, whose application is made in red and white grapes, respectively. Regarding the side effects, the use of LalVigneâ AROMA had: improvements in the skin thickness; developments in the aroma precursors; no impact in berry weight, Brix, pH or Tartaric acid; enhancements in the glutathione concentration; increases in thiolic varieties as 3MH and 3MHA; and reduction of herbaceous or aggressive character. The use of LalVigneâ MATURE had: improvements in the skin thickness; no impact in berry weight, Brix, pH or tartaric acid; a reduction of herbaceous or aggressive character; improvements in the concentration of extractable anthocyanins; improvements in the skin tannins; and developments in the degree of polymerization. Several trials were compared in this regard
N/A

Effects of trellising on water use, absorption of photosynthetically active radiation (PAR, 400 to 700 nm wavelength) by foliage and potentially by fruit, fruit composition, and yield were studied in 1988 under semi-arid conditions on field-grown Vitis vinifera L. 'Petite Sirah' grapevines in a mature vineyard. The positioning of shoots on trellises resulted in canopies that were (1) positioned vertically (vertical canopy), (2) positioned in a "V" pattern with sides inclined 60° from horizontal (inclined canopy), and (3) inclined toward the vineyard floor (standard canopy). Seasonal water use values determined from neutron scattering data were 393 ± 61, 554 ± 73, and 455 ± 57 mm for the standard, vertical and inclined canopies, respectively. On average, about 50% of seasonal water consumption occurred between fruit set and filling stages for each type of canopy. Average crop factors (ratio of actual to reference crop evapotranspiration) of 0.383, 0.540 and 0.444 were for the period bud burst to harvest for standard, vertical and inclined canopy systems, respectively. The diurnal water use patterns of the three trellising canopies were very similar when measured by either the heat pulse technique or by porometer. The average daily heat pulse velocity (HPV) for selected vertical, inclined and standard canopies for 6 days were 8.77, 7.58 and 6.85 cm h⁻¹, respectively. The HPV technique indicated that the average daily water use of the whole plant was 0.227, 0.192 and 0.137 kg/m² leaf area/d for standard, vertical and inclined canopies, respectively. The daily average transpiration rates as measured by the HPV technique were 32, 31, and 25% higher than the average transpiration rates estimated from porometer data for standard, vertical and inclined canopies, respectively. Stomatal conductances of the vertical and inclined canopies were 20 and 40%, respectively, below that of standard canopy. PAR absorption by foliage during mid-day was highest in the standard trellis, and lowest in the inclined trellis. PAR potentially available for absorption by fruits was lowest in the standard trellis, and highest in the inclined trellis. In both inclined and vertical canopies, the average sunfleck values were 26%, but was only 2% in the standard canopy. Analysis of fruit composition at harvest revealed that total dissolved solids (°Brix) was significantly higher in the inclined trellis than for the vertical trellis or the standard trellis. The inclined trellis resulted in the highest alcohol content of wine. Per vine yields did not differ significantly among the three trellis systems. Overall, the standard trellis was optimum because grapevines consumed less water and produced a shading which protected the fruit from direct solar radiation.

Partial rootzone drying (PRD) is an irrigation management technique designed to reduce water use in grapevines without a decline in yield, thereby increasing water–use efficiency (measured as t/ML) (WUE). The principle of PRD is to keep part of the root system at a constant drying rate to produce soil-derived signals to above–ground plant organs to induce a physiological response. Major PRD effects include a reduced canopy size and greatly increased WUE with possible improvements in fruit quality. Although we have a good understanding of the hormonal physiology of PRD, little is known on the effect of PRD on partitioning of C, N and inorganic ions such as K. This thesis broadens our knowledge on the effects of PRD on grapevine field performance, growth and dry matter accumulation as well as its effects on physiology and biochemistry. In field experiments over 3 seasons, PRD reduced water use in grapevines without a significant decline in yield. PRD effects included reduced shoot growth and greatly increased WUE. Field–grown Cabernet Sauvignon, where the PRD grapevines were irrigated at half the control rate, and Shiraz where the PRD grapevines were irrigated at same rate as controls, confirmed that PRD is not simply an irrigation strategy that applies less water, rather it alters the way in which the plant responds to its environment, e.g. PRD alters the sensitivity of the stomatal response to atmospheric conditions and significantly influence enzymes that regulate nutrient accumulation and partitioning. PRD did not change the total amount of carbon and nitrogen on a whole plant basis. However, it caused a significant partitioning of carbon and nitrogen towards trunk, roots and fruit at the expense of shoot growth. This change in partitioning occurred as a result of altered activity of the enzymes controlling the assimilation of carbon and nitrogen. PRD significantly reduced nitrate reductase (NR) activity in grapevine leaves, which catalyses the first step in the assimilation of nitrate irrespective of the amount of water applied. The reduction in NR activity is correlated with the development of the PRD cycle and the associated reduction in stomatal conductance. PRD also significantly altered grapevine sucrolytic enzyme activity that regulate source:sink relationships. PRD showed transient increases in leaf sucrose phosphate synthase (SPS) activity (formation of sucrose) compared to control, but significantly reduced leaf neutral invertase (sucrose cleavage) and leaf starch content in both field and potted experiments. This may indicate an increased photosynthetic capacity and a reduction in its sink strength for sucrose in favor of organs such as fruit and roots. This hypothesis was reinforced by the fact that berries showed significantly higher levels in glucose and fructose early in the season. Berry sugar content and Brix at harvest however was unaffected. Although PRD had no significant effect on berry characteristics at harvest such as Brix and pH, it occasionally reduced per berry K+ content and increased total amino acid concentration that may lead to positive outcomes for wine quality. PRD–treated grapevine roots on the 'wet'– and 'drying'–sides differed greatly in enzyme activity and osmolality. PRD significantly increased osmolality in both wet and drying roots by increasing total osmolyte concentration that may facilitate the movement of water from wet to dry roots. The increases in osmolality were also associated with increased free polyamine production (spermidine and spermine) in PRD roots that may be related to increased root growth and density.
Thesis (Ph.D.)--School of Agriculture and Wine, 2005.

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 (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

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