"April, 2004" / "List of figures" - inside back cover. / Bibliography: leaves 83-93 / 93, [8] leaves : ill., plates (some col.), photos ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The specific objectives of this work include: 1. Completion of the GLRaV-1 genome sequence and bioinformatic analysis of the viral open reading frames ; 2. Production of an appropriate GLRaV-1 certification protocol addressing the shortcomings of the current tests for leafroll detection ; 3. Intracellular localisation of the GLRaV-1 gene products via generation of green fluorescent protein (GFP)-fusion constructs, in an attempt to further characterise the function of these proteins. / Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Wine and Horticulture, 2004

Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Wine and Horticulture, 2004. / "April, 2004" "List of figures" - inside back cover. Bibliography: leaves 83-93.

In the last decade, several studies demonstrated that plants have developed a tight partnership with the edaphic microbial communities, mainly bacteria, fungi, and archaea. Such microbiome accomplishes essential functions and ecological services complementary to the functions encoded by the host plant, conferring adaptive advantages to the plant, particularly during stressful conditions. The interaction between microbial communities and their host plants in the natural ecosystem are complex and the mechanisms regulating these mutualistic associations are not fully elucidated. Several biotic and abiotic factors have been shown to be important during this process, including the plant properties (species, age, stage, etc.), the soil type and agronomic practices, the geo-climate conditions, and the biotic interaction. In this context, the vineyard ecosystems represent a unique biogeography model to study and disentangle microbial biodiversity patterns (compositional diversity and potential functionality) across plants cultivated in different geographical regions. Here, I used the rhizosphere and bulk soil of seven different rootstock-scion combinations (Vitis spp.) to dissect the main factors driving the microbial communities’ recruitment in ten different vineyards in Tuscany (Italy), distributed in the Pomino and Nipozzano estates of Frescobaldi company. Among the factors investigated, I focused my attention on the geographical area, soil type and rootstock-scion combination. By using high-throughput sequencing of bacterial 16S rRNA gene and fungal ITS region, I show how both bacterial and fungal communities associated with grapevine rhizosphere and bulk soils are mainly affected by the geographical area and the soil. Nonetheless, I also revealed that the rootstock-scion combination is an important driver in shaping the microbial community, explaining a higher percentage of variability in comparison with the factors rootstock and scion taken alone. Overall, the results obtained in my thesis offer a new perspective of research that aim to develop a deep understanding about the contribution of scionrootstock combinations in the microbial community ecology of the plant holobiont. Keywords: Plant-microbe interactions, edaphic microorganisms, Microbial ecology, Plant Growth Promoting Bacteria, Rootstock-scion, Grapevine.

The thesis deals with the occurrence of viral pathogens on grape - Chardonnay clones. Monitored and evaluated clones were 8, 95, 96 (foreign) on rootstocks 1103 Paulsen, SO4, Kober 5 BB and 110 Richter and VP-155/6-VP 161/6, PO-158/7 and PO-160 / 1 (Czech) on the rootstock Kober 5 BB. All plants have a controlled origin. The experiment was conducted in 2013 on the test sites in the cadastral Perná. At the beginning of vegetation were recorded values on 1 herbaceous plant -- sprouting and not-sprouting buds. During vegetation were the plants observed. From the monitored plants were harvested grapes and following parameters were checked: number and weight of the grapes, weight of berries and the stem. Furthermore, before leaf the leaves were sampled for subsequent ELISA test for viral diseases Grapevine fanleaf virus, Arabis mosaic virus, Grapevine fleck virus, Grapevine leafroll-associated virus 1 and 3, Grapevine virus A. All values were evaluated by statistical program Statistica 10.

Vancouver Island, British Columbia, is at the northern extent of natural climate zones conducive for grape growing, making vineyards susceptible to any changing weather patterns and temperature extremes. Grapevine monitoring is an important aspect of the viticulture industry, and remote sensing technologies are a powerful aid in reporting vegetation information for better vineyard management practices. However, the understanding of vine spectral responses as viewed by optical sensors has to be developed further, and was undertaken in this study. Chlorophyll pigments drive photosynthesis, a biochemical process in plants, which contributes to physiological performance and productivity, making it an appropriate leaf characteristic for detailed examination. This study aimed to develop a thorough understanding of the relationship between (i) leaf-level spectral reflectance and transmittance properties and (ii) pigment concentrations, via ground-based sampling. This was achieved through the examination of two ground campaign tools, as well as current spectral data processing techniques and workflow methods. A spectrometer and SPAD chlorophyll meter collected nondestructive measurements during leaf senescence and grape harvest, and wet chemical extraction methods determined chlorophyll content (expressed in terms of unit leaf area and leaf fresh weight). Reflectance indices,first order derivative indices, and a continuum removal approach were used to generate eighteen reflectance-based attributes. This study performed a series of chlorophyll estimation models through iterative ordinary least square regression, followed by two methods of model validation. Performance metrics indicated strong models with high explanatory power; the continuum removed depth normalized total area metric was presented as the optimal nondestructive attribute for accurate chlorophyll estimation for leaf level field campaigns (R2 = 0.93). Chlorophyll expressed in units of fresh weight yielded more consistent models than in units of leaf area. The chlorophyll meter also presented compelling results (R2 ≥ 0.78), and both sensors were determined to be appropriate for field validation campaigns for this vineyard study. / Graduate

Aquaporins (AQP) are membrane bound proteins that facilitate the movement of water and other small neutral solutes across cellular membranes. Plant aquaporins belong to a large family of highly conserved proteins called the Membrane Intrinsic Protein (MIP) superfamily. In many plant species the expression of aquaporin genes and their regulation has been linked to water stress. Grapevines respond to water stress with a variety of physiological mechanisms, including the susceptibility to xylem embolism. The formation of embolised vessels can lead to a reduction in hydraulic conductivity of the xylem. Recently, it has been hypothesised that aquaporins may contribute to the water movement required for embolism recovery of xylem vessels thus restoring the hydraulic pathway. Molecular and physiological techniques have been combined to study the putative role of plasma membrane and tonoplast membrane aquaporins in response to water stress induced xylem embolism in two cultivars of grapevine (Vitis vinifera cv. Chardonnay and Grenache). Water-stress induced cavitation was measured in the stems and petioles of pot grown grapevines of a drought tolerant (Grenache) and a drought sensitive variety (Chardonnay) by the detection of ultrasonic acoustic emissions (UAEs) over both a drying and diurnal cycle. Vulnerability curves were generated by correlating the UAEs with the leaf water potential (ψL). Varietal differences in cavitation vulnerability and hydraulic properties were observed. Grenache was more susceptible to water-stress induced xylem embolism than Chardonnay, and displayed a higher hydraulic capacity (measured by maximum hydraulic conductivity). This is most likely due to anatomical differences of the xylem vessels. Chardonnay displayed vulnerability segmentation, with cavitation occurring first in the petiole and later in the stem, before developing into “runaway” cavitation under severe water stress. Vulnerability segmentation was not observed in Grenache, with both petioles and stems equally vulnerable to the formation of xylem embolism. Under severe water stress, Grenache did not develop runaway cavitation indicating that they must have some mechanism to prevent the onset of runaway cavitation. To determine the role of aquaporins, candidate genes were identified, by screening a Vitis vinifera cv. Cabernet Sauvignon cDNA library, for aquaporin cDNAs encoding members of the Plasma membrane Intrinsic Protein (PIP) and Tonoplast Intrinsic Protein (TIP) subfamilies. The screen resulted in the identification of 11 full-length and two partial aquaporin cDNAs. Sequence analyses of these cDNAs reveal five are homologous to PIP2 aquaporins, six to PIP1 and two to the TIP aquaporins. Functional expression of the fulllength AQP cDNAs in Xenopus oocytes showed PIP2 members have significantly higher water permeability compared to PIP1 aquaporins. VvPIP2;1 showed very high water permeability which was reduced by acidic cytosolic pH, as has been reported for other members of the PIP2 family. Transcript analysis of some of these aquaporin genes provides preliminary evidence that aquaporins may contribute to differences in the hydraulic response of these two grapevine varieties to conditions of water stress. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1313316 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 2002. / Includes bibliographical references (leaves 158-180).

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-11-28T13:27:09Z No. of bitstreams: 1 Aricleia de Moraes Catarino.pdf: 1031221 bytes, checksum: 5bd0ffb507daa4b9b04ebd494fd2269c (MD5) / Made available in DSpace on 2016-11-28T13:27:09Z (GMT). No. of bitstreams: 1 Aricleia de Moraes Catarino.pdf: 1031221 bytes, checksum: 5bd0ffb507daa4b9b04ebd494fd2269c (MD5) Previous issue date: 2015-02-27 / The grapevine (Vitis spp.) belongs to the family of Vitaceae, being the botanical species V. vinifera L. and V. labrusca L. the most and widely cultivated, due to their products consumed as fresh fruits, jam, juices and wines. Despite the high economical importance, several factors may severely affect this crop, including the diseases caused by viruses. This study aimed to verify the incidence of virus present in commercial vineyards of two producing areas in Northeastern Brazil and evaluate the efficiency of some molecular methods for detecting and identifying viral species associated with grapevine. Materials showing or not symptoms were collected from grapevine genotypes in vineyards of Pernambuco, Paraiba, Bahia and Rio Grande do Sul, Brazil, and Locorotondo, Province of Bari, of the Puglia Region, Italy. The first part of the work was conducted at the Virology Laboratory of the Embrapa Uva e Vinho, RS, Brazil. For the identification of viral agents in the samples collected in Brazil, the extraction of total RNA was performed, cDNAs were obtained and tested by real time RT-PCR, using primers and probes specific for the following viruses: Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine leafroll-associated virus 2, 3 e 4 (GLRaV-2, -3 e -4), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV) and Grapevine fanleaf virus (GFLV). DNA fragments, products of the RT-qPCR, corresponding to the CP gene of each virus were eluted, linked to pGEM-T Easy vector (Promega) and used to transform bacteria. The plasmid DNA was extracted from transformed bacterial colonies, confirming the presence of the cloned fragments, which were sequenced. The grapevine material collected in Locorotondo was processed at the Istituto per la Protezione Sostenibile delle Piante (CNR-IPSP) and at the Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi “Aldo Moro”. In order to detect in multiplex test the most relevant viruses involved in the aetiology of fanleaf degeneration and the complexes of leafroll and rugose wood of grapevine, amplification techniques based on Allele Specific Primer Extension (ASPE) were tested by using the obtained cDNAs. The results showed that the techniques aggregate some advantages, such as reduction in time and relative simplicity of implementation, completely eliminating the use of toxic reagents, such as the ethidium bromide. The use of multiplex facilitates amplification of multiple targets in a single reaction, reducing the time and cost of the analyzes. / A videira (Vitis spp.) pertence à família Vitaceae, sendo as espécies botânicas V. vinifera L. e V. labrusca L. cultivadas em maior escala devido seus produtos, consumidos na forma de frutos in natura, geleias, sucos e vinhos. Apesar da grande importância econômica, vários fatores podem comprometer a produção desta cultura, incluindo as doenças causadas por vírus. O presente trabalho teve como objetivos verificar a incidência de vírus presentes em vinhedos comerciais de duas áreas produtoras do Nordeste do Brasil e avaliar a eficiência de alguns métodos moleculares para detecção e identificação de espécies virais associadas à videira. Amostras, apresentando ou não sintomas, foram coletados de genótipos de videira em propriedades situadas em Pernambuco, Paraíba, Bahia e Rio Grande do Sul, Brasil, e em Locorotondo, Província de Bari, Região da Puglia, Itália. A primeira parte do trabalho foi conduzida no Laboratório de Virologia da Embrapa Uva e Vinho, RS, Brasil. Visando a identificação dos agentes virais, nas amostras coletadas no Brasil, foram realizadas as extrações do RNA total, obtidos os cDNAs e testados por PCR em Tempo Real, empregando-se primers e sondas, específicos para os seguintes vírus: Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine leafroll-associated virus 2, 3 e 4 (GLRaV-2, -3 e -4), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV) e Grapevine fanleaf virus (GFLV). Fragmentos de DNA, produtos da RTq-PCR, correspondentes ao gene da CP de cada vírus foram eluídos, ligados ao vetor pGEM-T Easy (Promega) e utilizados na transformação de bactéria. Foi extraído o DNA plasmidial das colônias bacterianas transformadas, confirmando-se a presença dos fragmentos clonados, os quais foram sequenciados. A segunda parte, realizada com o material coletado em Locorotondo, foi processada no Istituto per la Protezione Sostenibile delle Piante (CNR-IPSP) e no Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi “Aldo Moro”. Foram utilizadas, a partir de cDNAs obtidos, técnicas de amplificação baseadas na Allele Specific Primer Extension (ASPE), visando detectar em teste multiplex os vírus mais relevantes envolvidos na etiologia da degenerescência e nos complexos do enrolamento das folhas e do lenho rugoso da videira. Os resultados obtidos mostraram que as técnicas agregam algumas vantagens, como a redução no tempo e relativa simplicidade de execução, eliminando completamente o uso de reagentes tóxicos, a exemplo do brometo de etídeo. O uso de multiplex facilita a amplificação de múltiplos alvos em uma única reação, reduzindo o tempo e o custo das análises.

A survey was conducted, from 2001 to 2004, of viruses spreading within certified grapevine material in South Africa. As far as possible, viruses were identified and detection methods established. However, unknown spherical virus-like particles were observed in samples that also contained Grapevine Leafroll Associated Virus-Type 3. The unknown spherical particles were thought to most likely be Grapevine Fleck Virus, which was previously found in South Africa. A PCR method to be used locally for the routine detection of Grapevine Fleck Virus was established and first used to determine whether any of the greenhouse and field samples with the unknown spherical viruses were infected with Grapevine Fleck Virus. During the 2001 to 2004 survey, plants with leafroll and reddening symptoms unlike classical grapevine leafroll disease were also observed. No grapevine leafroll-associated viruses could be detected in these, but the symptoms observed resembled symptoms induced by phytoplasmas in Europe. A PCR method for the routine universal detection of phytoplasmas was established and this method was used to determine if phytoplasmas were associated with the symptomatic plants found. Sequence information from PCR amplicons suggest the presence of Candidatus phytoplasma solani, found for the first time in South Africa. This important finding however requires conformation by a second laboratory. / Dissertation (MSc (Microbiology))--University of Pretoria, 2010. / Microbiology and Plant Pathology / unrestricted

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The nepoviruses are a group of nematode-transmitted plant viruses that are distributed worldwide and infect a wide range of plant species, including grapevine. Most of the nepoviruses are foreign to South Africa and to date, only Grapevine fanleaf virus (GFLV) is present. The Department of Agriculture, Forestry and Fisheries (DAFF), as the official National Plant Protection Organisation (NPPO) of South Africa, is committed to prevent the importation and spread of plant pathogens by administering the Agricultural Pests Act, 1983 (Act No. 36 of 1983). Effective measures are implemented by which the introduction of agricultural pests may be prohibited to safeguard the agricultural environment. One of the core functions of DAFF is to render a routine plant health diagnostic service for imported plants and plant products to prevent exotic pathogens from entering the country. The objective of this study was to develop a diagnostic assay for the detection of nepoviruses in grapevine. The project aimed to produce antibodies by recombinant DNA technology against bacterially expressed viral coat protein of a specific nepovirus [Tomato ringspot virus (ToRSV)] and subsequently develop a DAS-ELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) assay for the detection of the virus. The coat protein (CP) was successfully isolated from imported ToRSV-infected grapevine material. Two expression systems were utilised for expression of the ToRSV-CP, the GST gene fusion system and an Agrobacterium-mediated expression system. The GST gene fusion system was unsuccessful as insufficient soluble protein expression prevented the production of antibodies and thus the development of the DAS-ELISA assay. Tissue print immunoassay (TPIA) initially showed positive results for transient expression of the fusion protein in tobacco plants, but further confirmation proved to be inconclusive. The project also aimed to develop a real-time PCR assay for the specific detection and relative quantification of GFLV, based on a conserved region of the RNA-2 genome. A partial GFLV-RNA-2 from a South African isolate of grapevine was sequenced and used for the design of specific primers. The quantitative real-time PCR assay based on SYBR green technology proved to be sensitive in detecting levels as low as 0.11ng/reaction in infected plants, making it a highly effective diagnostic tool for the detection of GFLV. / AFRIKAANSE OPSOMMING: Die nepovirusse is 'n groep van nematode-oordraagbare plant virusse wat wêreldwyd versprei word en 'n wye verskeidenheid van plantspesies infekteer, insluitend wingerd. Die meeste van die nepovirusse is uitheems aan Suid-Afrika en tot op datum is net Wingerd netelblaar virus (GFLV) teenwoordig. Die Departement van Landbou, Bosbou en Visserye (DAFF), as die amptelike Nasionale Plant Beskermings Organisasie (NPBO) van Suid-Afrika, is daartoe verbind om die invoer en verspreiding van plantpatogene te voorkom deur administrasie van die Wet op Landbouplae, 1983 (Wet No. 36 van 1983). Doeltreffende maatreëls word geïmplementeer waardeur die invoer van landbouplae verbied word om sodoende die landbou-omgewing te beskerm. Een van die kernfunksies van DAFF is om 'n roetine plant gesondheid diagnostiese diens vir ingevoerde plante en plantprodukte te lewer om te verhoed dat eksotiese patogene die land binnedring. Die doel van hierdie studie was om 'n diagnostiese toets vir die opsporing van nepovirusse in wingerd te ontwikkel. Die projek was daarop gemik om antiliggame te vervaardig deur rekombinante DNA-tegnologie teen bakterieël-uitgedrukte virale mantelproteïen van 'n spesifieke nepovirus [Tomato ringspot virus (ToRSV)] en vervolgens ‘n DASELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) toets vir die opsporing van die virus te ontwikkel. Die mantelproteïen (CP) is met sukses geïsoleer vanaf ingevoerde ToRSV-besmette wingerdmateriaal. Twee uitdrukking stelsels is gebruik vir uitdrukking van die ToRSV-CP, die “GST gene fusion” stelsel en 'n Agrobacterium-bemiddelde uitdrukking stelsel. Die “GST gene fusion” stelsel was egter onsuksesvol aangesien onvoldoende oplosbare proteïen uitdrukking die produksie van antiliggame en dus die ontwikkeling van die DAS-ELISA toets verhoed het. “Tissue print immunoassay” (TPIA) het aanvanklik positiewe resultate getoon vir tydelike uitdrukking van die fusie proteïen in tabakplante, maar verdere bevestiging was onoortuigend. Die projek was ook daarop gemik om ‘n in-tyd polimerase ketting reaksie (PKR) toets vir die spesifieke opsporing en relatiewe kwantifisering van GFLV, gebaseer op 'n gekonserveerde volgorde van die RNA-2 genoom, te ontwikkel. 'n Gedeeltelike GFLV-RNA-2 nukleïensuurvolgorde van 'n Suid-Afrikaanse wingerd isolaat is bepaal en gebruik vir die ontwerp van spesifieke inleiers. Die kwantitatiewe in-tyd PKR toets gebaseer op SYBR groen tegnologie was sensitief genoeg om vlakke van so laag as 0.11ng/reaksie in geïnfekteerde plante op te spoor, wat dit 'n hoogs effektiewe diagnostiese hulpmiddel vir die opsporing van GFLV maak.