The effect of exposure to sub-lethal chlorine stress on Salmonella enterica serotypes Typhimurium and Heidelberg was examined in this study. Both serotypes demonstrated an acquired tolerance to chlorine with the adapted cells growing in concentrations above the MIC. Chlorine induced a morphological change to the rugose variant in Salmonella. The biofilm formation of the adapted and control cells was tested on food-contact surfaces at room temperature and 37°C. The chlorineapted rugose formed stronger biofilms (P < 0.05) when compared to smooth (adapted and control) on both surfaces tested and at both temperatures. The possibility of crossaptation to antibiotics and low pH was evaluated. Adapted rugose showed reduced susceptibility against some of the antibiotics tested. Chlorine does not aid in the survival of Salmonella enterica at low pH. Chlorine stress can select for tolerant Salmonella cells that attach strongly to food-contact surfaces and after some time may become less susceptible to antimicrobials.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-28T10:50:51Z No. of bitstreams: 1 texto completo.pdf: 293374 bytes, checksum: 3cac7991cb8c86d500d4d51179490aa4 (MD5) / Made available in DSpace on 2017-04-28T10:50:51Z (GMT). No. of bitstreams: 1 texto completo.pdf: 293374 bytes, checksum: 3cac7991cb8c86d500d4d51179490aa4 (MD5) Previous issue date: 2004-02-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Plantas de feijão-vagem do cultivar Novirex apresentando sintomas de mosaico e enrolamento de vagens, sem deformação foliar evidente, foram coletadas em 2002 no município de Cordisburgo, MG. Estudos anteriores de microscopia eletrônica, eletroforese da proteína e do RNA viral e sorologia identificaram o isolado como o comovírus Bean rugose mosaic virus (BRMV). Neste trabalho deu-se prosseguimento à caracterização do isolado, por meio de análises em laboratório e casa de vegetação envolvendo produção e avaliação de anti- soro policlonal, determinação da gama de hospedeiros, estudo da transmissão do vírus por besouros crisomelídeos e avaliação de perdas em feijoeiro como resultado de infecção isolada ou em conjunto com o potyvírus Bean common mosaic virus (BCMV). O procedimento adotado para purificação possibilitou a obtenção de vírus purificado em rendimento satisfatório para a obtenção de anti-soro. A titulação dos anti-soros obtidos foi realizada por ELISA indireto, obtendo-se reações positivas com a diluição máxima testada (1:70.000), e nenhuma reação com extrato de planta sadia. Das 22 espécies vegetais testadas, Chenopodium quinoa apresentou inicialmente lesões locais cloróticas e posteriormente infecção sistêmica com mosaico e distorção foliar. Nos cultivares de feijão e soja observou-se sintomas de mosaico e bolhosidade, em conformidade com os resultados esperados para o BRMV. O isolado de BRMV foi transmitido pelo besouro crisomelídeo Cerotoma arcuata a uma taxa de 33,3%. Este isolado foi inoculado em plantas de feijoeiro ‘Ouro Negro’ e de feijão-vagem ‘Novirex’, levando a uma redução do peso de vagens por planta de 44,6% e 65,7%, respectivamente. O Bean common mosaic virus (BCMV) ocasionou porcentagens de redução de peso de vagens menores em ‘Novirex’ (13,1%) em comparação a ‘Ouro Negro’ (50,4%). Quando o BRMV foi inoculado inicialmente, seguido do BCMV, verificou-se redução do peso de vagens por planta de até 69,3% para ‘Novirex’ e de 91,5% para ‘Ouro Negro’. Não se observou diferença significativa em peso ou número de vagens por planta quando estas foram inoculadas seqüencialmente com o BCMV seguido do BRMV. / Bean plants of the cultivar Novirex, showing an atypical pod curling symptom without mosaic or leaf distortion, were collected in 2002 at Cordisburgo, MG. Previous studies involving electron microscopy and electrophoretic analysis of viral protein and RNA identified the isolate as the comovirus Bean rugose mosaic virus (BRMV). The present work continued the characterization of the isolate, and included its purification and production of a polyclonal antiserum, determination of a partial host range, vector transmission studies, and estimates of yield losses in beans due to single or mixed infection with the potyvirus Bean common mosaic virus (BCMV). The protocol adopted for virus purification led to purified preparations with high yield, and the antisera obtained after rabbit immunization reacted with the maximum dilution tested (1:70.000) in indirect ELISA, without any reactions with sap from healthy plants. Out of the 22 plant species tested as hosts, Chenopodium quinoa reacted with chlorotic local lesions which evolved to mosaic and leaf distortion in non-inoculated leaves. Bean and soybean cultivars reacted with mosaic of varied intensities, as expected for BRMV. The isolate was transmitted by Ceratoma arcuata to 33,3% of the inoculated plants. Upon inoculation onto ‘Ouro Negro’ and ‘Novirex’ beans, the total weight of pods per plant was reduced by 44,6% and 65,7%, respectively. Single infection by BCMV led to a smaller reduction of pod weight in ‘Novirex’ (13,1%) compared to ‘Ouro Negro’ (50,4%). When BRMV was inoculated first, followed by BCMV, total pod weight was reduced by up to 69,3% in ‘Novirex’ and 91,5% in ‘Ouro Negro’. No statistically significant differences were observed in total weight or number of pods per plant after inoculation with BCMV followed by BRMV. / Dissertação importada do Alexandria

Many soybean [Glycine max (L.) Merrill] cultivars have narrow leaflet shape but it is not known if all of these lines derive this trait from the ln gene or another locus. This project was conducted to determine the inheritance of the narrow leaflet trait in several soybean genotypes and wild [Glycine soja Sieb. et Zucc.] accessions, and also to determine the allelism of the genes for this trait in the selected lines. The parents, F1, F2 and F2:3 generations were grown at Kentland Research Farm near Blacksburg, VA or in the greenhouse. The F2 and F2:3 generations (where available) were observed for segregation in leaflet shape. The populations were scored as having either broad or narrow leaflets using visual classification and leaf measurements when necessary. 'Camp' was crossed with broad leaflet parent 'Essex' to study the inheritance of the narrow leaflet trait in Camp. Observation of the F2 and F2:3 generations lead to the conclusion that a single recessive gene controls leaflet shape in Camp. Narrow leaf parents 'SRF 400' and Camp were crossed with lines having the ln gene (T41, S56, and D64-4731). None of the crosses among Camp, T41, SRF 400, S56 and D64-4731 segregated for leaflet shape in the F2 generation leading to the conclusion that they all have the ln allele at the same locus controlling lanceolate leaflet shape. T313, a line containing a gene for narrow rugose leaflets (lnr), was crossed with Camp to study allelism between the lnr and ln genes. Segregation for leaflet shape was observed in the F2 and F2:3 generations allowing the conclusion that the lnr gene controlling the narrow rugose leaflet trait in T313 is at a locus independent from the ln gene. A deficiency of narrow rugose plants was observed in all of the populations with T313 as a parent, and was theorized as being caused by selection against lnr gametes. After adjustment for the lnr deficiency, the F2 data appeared to fit a 9 broad : 3 narrow : 4 narrow rugose ratio. Three G. soja lines were crossed to broad and narrow leaflet parents and the F2 generations were examined to determine the inheritance of the very narrow leaf phenotype. The results indicate that there are one or two recessive genes controlling narrow leaflet shape in the G. soja accessions, which are not allelic to the ln gene. Since these populations were not advanced to the F3 generation, definite conclusions cannot be drawn about the genetics of the very narrow leaf phenotype. / Master of Science

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-11-28T13:39:39Z No. of bitstreams: 1 Monique Bezerra Nascimento.pdf: 1822147 bytes, checksum: 8f2d6b8a88935d10d34decf26f6730b3 (MD5) / Made available in DSpace on 2016-11-28T13:39:39Z (GMT). No. of bitstreams: 1 Monique Bezerra Nascimento.pdf: 1822147 bytes, checksum: 8f2d6b8a88935d10d34decf26f6730b3 (MD5) Previous issue date: 2015-02-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The grapevine (Vitis spp.) virus diseases affect severely the production, the quality of the grape and reduce the life of the vineyards. In Brazil, among them, the complexes of rugose wood caused by Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine virus D (GVD) and the leafroll, caused by Grapevine rupestris stem pitting-associated virus (GRSPaV) and Grapevine leafroll-associated virus -1 to -4, -4 strain 5 (GLRaV-1 to -4, -4 strain 5) are very important. The first part of the present work had the objectives of evaluating the effects of these viruses on agronomic traits, such as, fresh weight of the bunch, °Brix, weight of pruned branches, stem diameters of rootstock and canopy, in symptomatic and asymptomatic plants in Niagara Rosada and Merlot vineyards, and to survey commercial nurseries for GRSPaV, GVA, GVB, Grapevine fleck virus (GFkV) and GLRaV-3. All plants were indexed by real-time RT-PCR. Asymptomatic infected grapevines, however, were affected negatively by the viral infection, but to a lesser intensity in comparison with the symptomatic ones. The nurseries presented a high incidence of GRSPaV and GVA, being characterized as sources of inoculum for vineyards formed with these materials. The second part of the work aimed the molecular characterization of isolates of the following viruses: Prunus necrotic ringspot virus (PNRSV), agent of rose mosaic (Rosa sp.), obtained from rose; GRSPaV, GVA and GLRaV-2, from symptomatic and asymptomatic grapevine plants. Despite the economic importance of PNRSV for stone fruit trees in Brazil, only peach isolates have been molecular characterized in Brazil, it was observed high percentages of identity, among the isolates GRSPaV, GVA and GLRaV-2, which made it difficult to explain the variation in symptom expression or their absence, based on the sequence differences. In this study, it was conducted, for the first time, the molecular characterization of the complete genes to the coat protein (CP) and movement protein (MP) of a Brazilian PNRSV isolate obtained from rose. The obtained results demonstrated the high importance of using virus-free propagative materials to achieve the highest agronomic potential of the grapevine cultivars and to reduce losses caused by these pathogens. / As viroses da videira (Vitis spp.) afetam severamente a produção, a qualidade da uva e diminuem a vida útil dos vinhedos. No Brasil, destacam-se, o complexo do lenho rugoso, causados por Grapevine virus A (GVA), Grapevine virus B (GVB) e Grapevine virus D (GVD), e o enrolamento das folhas, causado por Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine leafroll-associated virus -1 ao -4, -4 estirpe 5 (GLRaV-1 ao -4, -4 estirpe 5). A primeira parte do presente trabalho teve como objetivos avaliar os efeitos destes vírus sobre as variáveis agronômicas relacionadas ao vigor da planta, tais como, peso fresco do cacho, °Brix, peso de ramos podados, diâmetros do tronco do porta-enxerto e da copa, em plantas sintomáticas e assintomáticas em vinhedos das cultivares Niágara Rosada e Merlot, além do levantamento de vírus em matrizeiros comerciais para GRSPaV, GVA, GVB, Grapevine fleck virus (GFkV) e GLRaV-3. Todas as plantas foram indexadas por RT-PCR em tempo real. Videiras assintomáticas, porém infectadas, foram afetadas negativamente pela infecção viral, porém em menor intensidade, quando comparadas com as sintomáticas. Os matrizeiros testados apresentaram alta incidência de GRSPaV e GVA, ficando caracterizados como fontes de inóculo destes patógenos para vinhedos formados com esses materiais. Na segunda parte do trabalho, visou-se a caracterização molecular de isolados dos seguintes vírus: Prunus necrotic ringspot virus (PNRSV), agente do mosaico da roseira (Rosa sp.), obtido da roseira; GRSPaV, GVA e GLRaV-2, de videiras sintomáticas e assintomáticas. Apesar da importância econômica do PNRSV para as fruteiras de caroço no Brasil, apenas isolados brasileiros, obtidos de pessegueiro, tinham sido caracterizados molecularmente. Dentro de cada espécie, os isolados de GRSPaV, GVA e GLRaV-2 demonstraram altas porcentagens de identidade, de modo que, as diferenças entre eles, dificilmente, explicariam as variações na expressão e ausência de sintomas. Neste estudo, foi realizada pela primeira vez a caracterização molecular dos genes completos da capa proteica (CP) e da proteína de movimento (MP) de um isolado brasileiro do PNRSV, obtido em roseira no Brasil. Os resultados obtidos demonstraram a utilização de material propagativo livre de vírus como opção para atingir o melhor potencial agronômico das cultivares de videira e reduzir perdas ocasionadas por estes patógenos.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-07-19T13:40:35Z No. of bitstreams: 1 texto completo.pdf: 4542523 bytes, checksum: c68041abd5ef22ab9466db888a2ebc85 (MD5) / Made available in DSpace on 2017-07-19T13:40:35Z (GMT). No. of bitstreams: 1 texto completo.pdf: 4542523 bytes, checksum: c68041abd5ef22ab9466db888a2ebc85 (MD5) Previous issue date: 2001-11-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Um complexo viral causando mosaico dourado e deformação (rugosidade) foliar em tomateiros na região do Triângulo Mineiro, MG, Brasil, foi obtido por meio de moscas brancas virulíferas, e denominado TGV-Ub1. Os vírus presentes neste complexo viral foram transmitidos via extrato vegetal tamponado (EVT) a partir de folhas de tomateiro para plantas de Nicotiana benthamiana. A análise via PCR utilizando oligonucleotídeos universais para o gênero Begomovirus indicou a amplificação de dois fragmentos distintos para o componente A e para o componente B, sugerindo que o complexo viral TGV-Ub1 é composto por dois vírus distintos. De fato, dois geminivírus foram purificados biologicamente a partir do complexo viral TGV-Ub1, e causaram em tomateiro sintomas de mosaico dourado suave, sem rugosidade. O complexo viral TGV--Ub1 infectou apenas plantas da família Solanaceae, incluindo várias espécies de Nicotiana e Solanum tuberosum, e excluindo S. melongena, S. gilo, Capsicum annuum e C. frutescens. Obtiveram-se 87 clones com cerca de 2.600 nucleotídeos a partir de folhas de tomateiro infectado pelo complexo viral TGV-Ub1. A comparação das seqüências completas de nucleotídeos dos clones pUb1-49 (DNA-A), pUb1-62 e pUb1-81 (ambos DNA-B) indicou que esses componentes pertencem a novos begomovírus. Com base em análise filogenética, esses componentes foram classificados como pertencente a um begomovírus do hemisfério ocidental. Os clones pUb1-49 e -81 possuem regiões comuns idênticas. Este vírus foi denominado Tomato rugose mosaic virus (TRMV). O clone pUb1-62 possui região comum distinta do TRMV e de todos os demais geminivírus descritos. O DNA-A cognato de pUb1-62 não foi encontrado. Clones contendo 1,5 cópia dos componentes genômicos clonados em pUb1-49, -62 e -81 foram construídos. A análise da infectividade desses clones em Santa Clara e em N. benthamiana, por meio de bombardeamento de partículas, demonstrou que a combinação dos clones correspondentes ao genoma do TRMV causou sintomas sistêmicos nos dois hospedeiros, semelhantes àqueles causados pelos isolados puros obtidos neste trabalho. A combinação dos clones pUb1-49 e -62 não resultou em infecção sistêmica, indicando que esses dois componentes não formam pseudo-recombinantes viáveis. O TRMV foi transmitido via EVT a partir de folhas de N. benthamiana para N. benthamiana, e, por enxertia para S. tuberosum e Datura stramonium. Amostras de tomateiro apresentando sintomas semelhantes a mosaico dourado foram coletadas em três municípios da região do Triângulo Mineiro. Análise via PCR indicou que 148 das 170 amostras coletadas estavam infectadas por begomovírus. Das amostras infectadas, apenas uma hibridizou a 68°C com sonda para o componente A do TRMV, 109 hibridizaram a 58°C com esta sonda e 39 não hibridizaram com esta sonda em nenhuma das duas temperaturas, indicando que a maioria das amostras continha geminivírus geneticamente relacionado ao TRMV. Os componentes TRMV-A e TRMV-B foram detectados, respectivamente, em plantas de Nicandra physaloides (joá de capote) e Phaseolus vulgaris (feijão comum) coletadas em lavouras de tomateiro. Análise genética baseada em PCR-RFLP de fragmentos amplificados a partir das amostras coletadas indicou a existência de um alto grau de diversidade genética de begomovírus em tomateiros da região do Triângulo Mineiro. / A viral complex causing golden mosaic and leaf distortion (rugosity) in tomato plants in the Triângulo Mineiro region, Minas Gerais, Brasil, was obtained from viruliferous whiteflies, and named TGV-Ub1. This viral complex was sap-transmitted from tomato leaves to Nicotiana benthamiana plants. PCR amplification using universal primers for the genus Begomovirus yielded two distinct fragments for the A and B components of the viral genome. This suggested that the TGV-Ub1 complex was composed of two distinct viruses. Indeed, two geminiviruses were biologically purified from the TGV-Ub1 complex, and shown to cause symptoms of mild golden mosaic with no rugosity on tomato plants. The TGV-Ub1 complex infected only plants in the Solanaceae, including several Nicotiana species and Solanum tuberosum, but excluding S. melongena, S. gilo, Capsicum annuum and C. frutescens. Eighty-seven clones corresponding to full-length (~2.600 nucleotides) viral genomes were obtained from tomato leaves infected with TGV-Ub1. Comparisons of the complete nucleotide sequences of clones pUb1-49 (DNA-A), pUb1-62 and pUb1-81 (both DNA-B) indicated that they comprise new begomoviruses. By phylogenetic analysis, these components were identified as belonging to a begomovirus from the western hemisphere. Clones pUb1-49 and -81 showed identical common regions. This virus was named Tomato rugose mosaic virus (TRMV). Clone pUb1-62 has a distinct common region from TRMV and all other known geminiviruses. A cognate DNA-A for pUb1-62 was not found. Clones containing 1.5 copies of the genomic components cloned in pUb1-49, -62 and -81 were constructed. Infectivity analysis of these clones in Santa Clara and N. benthamiana plants using particle bombardment demonstrated that the combination of clones corresponding to the TRMV genome resulted in systemic symptoms in both hosts. These symptoms were analogous to those observed when using the pure isolates obtained in this study. The combination of pUb1-49 and -62 did not result in systemic infection, indicating that these two components do not form viable pseudo-recombinants. TRMV was sap-transmitted from N. benthamiana leaves to N. benthamiana plants, and by grafting to S. tuberosum and Datura stramonium. Samples from tomato plants showing symptoms similar to golden mosaic were collected in three different municipal districts in the region of Triângulo Mineiro. PCR analysis indicated that 148 out of 170 collected samples were infected with a begomovirus. From these infected samples, only one hybridized at 68°C to a specific probe for the DNA-A of TRMV. However, 109 samples hybridized at 58°C to the same probe. Thirty- nine samples did not hybridize to this probe at either temperature. These results indicated that most of the samples contained geminiviruses genetically related to TRMV. TRMV-A and TRMV-B were detected, respectively, in plants of Nicandra physaloides and Phaseolus vulgaris growing naturally nearby tomato fields. A genetic analysis based on PCR-RFLP from fragments obtained from collected samples demonstrated the existence of a high degree of genetic diversity of begomoviruses in tomato plants cultivated in the region of Triângulo Mineiro.

No description available.

The effect of sub-lethal chlorine stress on various strains/serotypes of Salmonella on biofilm formation and thermal resistance was studied. The effect of oxidative stress (induced by 150 ppm of chlorine in TSB) on Salmonella biofilm formation on polystyrene and stainless steel surfaces at three temperatures (4°C, 30°C, and room temperature) in nutrient rich (full strength TSB) and nutrient limited conditions (1/10th TSB) was evaluated. On polystyrene surface, chlorine stressed S. Heidelberg (strain ID 72), S. Newport (strain ID 107) and S. Typhimurium (ATCC 14028) formed stronger (P < 0.05) biofilms at 30°C. On stainless steel, the chlorine stressed S. Heidelberg (ATCC 8326) and S. Enteritidis (ATCC 4931) at room temperature formed stronger (P < 0.05) biofilms as compared to the non-stressed control cells. The thermal resistance of short-term (1h) and long-term (27d) chlorine stressed Salmonella Heidelberg and S. Typhimurium were compared with the non-stressed controls at three different temperatures (55°C, 58°C and 61°C) and two growth phases (logarithmic and stationary). The short-term stressed log phase cells (both serotypes) were found to be more sensitive (P< 0.05) to thermal inactivation in TSB. Upon long-term sub-lethal chlorine exposure, Salmonella developed a rugose morphotype on tryptic soy agar at 37°C. The rugose morphotype provided significant thermal protection (P< 0.05) against heat stress as compared to smooth morphotype. In chicken broth, at 55°C, short-term chlorine stressed stationary phase S. Typhimurium displayed a higher D55 value compared to non-stressed cells. The findings from this research reveal that some Salmonella strains have the potential to form stronger biofilms and exhibit higher thermal tolerance upon exposure to sub-lethal chlorine concentration.

Mathematical theory of partial differential equations (PDEs) is a pretty old classical area with wide range of applications to almost every branch of science and engineering. With the advanced development of functional analysis and operator theory in the last century, it became a topic of analysis. The theory of homogenization of partial differential equations is a relatively new area of research which helps to understand the multi-scale phenomena which has tremendous applications in a variety of physical and engineering models, like in composite materials, porous media, thin structures, rapidly oscillating boundaries and so on. Hence, it has emerged as one of the most interesting and useful subject to study for the last few decades both as a theoretical and applied topic. In this thesis, we study asymptotic analysis (homogenization) of second-order partial differential equations posed on an oscillating domain. We consider a two dimensional oscillating domain (comb shape type) consisting of a fixed bottom region and an oscillatory (rugose) upper region. We introduce optimal control problems for the Laplace equation. There are mainly two types of optimal control problems; namely distributed control and boundary control. For distributed control problems in the oscillating domain, one can apply control on the oscillating part or on the fixed part and similarly for boundary control problem (control on the oscillating boundary or on the fixed part the boundary). We consider all the four cases, namely distributed and boundary controls both on the oscillating part and away from the oscillating part. The present thesis consists of 8 chapters. In Chapter 1, a brief introduction to homogenization and optimal control is given with relevant references. In Chapter 2, we introduce the oscillatory domain and define the basic unfolding operators which will be used throughout the thesis. Summary of the thesis is given in Chapter 3 and future plan in Chapter 8. Our main contribution is contained in Chapters 4-7. In chapters 4 and 5, we study the asymptotic analysis of optimal control problems namely distributed and boundary controls, respectively, where the controls act away from the oscillating part of the domain. We consider both L2 cost functional as well as Dirichlet (gradient type) cost functional. We derive homogenized problem and introduce the limit optimal control problems with appropriate cost functional. Finally, we show convergence of the optimal solution, optimal state and associate adjoint solution. Also convergence of cost-functional. In Chapter 6, we consider the periodic controls on the oscillatory part together with Neumann condition on the oscillating boundary. One of the main contributions is the characterization of the optimal control using unfolding operator. This characterization is new and also will be used to study the limiting analysis of the optimality system. Chapter 7 deals with the boundary optimal control problem, where the control is applied through Neumann boundary condition on the oscillating boundary with a suitable scaling parameter. To characterize the optimal control, we introduce boundary unfolding operators which we consider as a novel approach. This characterization is used in the limiting analysis. In the limit, we obtain two limit problems according to the scaling parameters. In one of the limit optimal control problem, we observe that it contains three controls namely; a distributed control, a boundary control and an interface control.

Thesis (MSc (Genetics))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The first aim of this study was to reliably and rapidly detect Grapevine rupestris stem pittingassociated virus (GRSPaV) in grapevine. This was achieved by screening 94 grapevines using crude plant extracts in both quantitative and conventional reverse transcription polymerase chain reaction (RT-PCR). The second aim was to establish a technique capable of differentiating GRSPaV sequence variants. The application of this technique is for the largescale screening of diseased vines to associate sequence variants of GRSPaV with disease symptoms. Nested quantitative polymerase chain reaction and high resolution melting assays (qPCR-HRM) were developed for three regions of the GRSPaV genome (coat protein, RNAdependant RNA-polymerase and triple gene block movement protein). The qPCR-HRM technique using the high saturation dye, EvaGreen™, and the Rotor-Gene™ 6000 analyzer was validated with a panel of sixteen sequence-characterized viral isolates. Diluted RT-PCR products and cloned cDNA gave the most consistent amplification plots and dissociation profiles. RT-PCR products generated from total RNA extracts were used as template for qPCR-HRM assays and for direct sequencing of sixteen samples in the three aforementioned regions. The average amplification efficiency for qPCR was 1.52±0.04. Auto-calling of userdefine genotypes was performed at a confidence interval of 70%. Phylogenetic analysis of the three regions of the GRSPaV genome was performed with published GenBank sequences to confirm the HRM data. The dominant sequence variants found in the South African sample set radiated with Group II, reference full-length variant GRSPaV-SG1. GRSPaV-infected samples can in future be subjected to qPCR-HRM assays developed during this study. This can be performed to establish similarity to known genotypes and therefore phylogenetic groups. Mixed infection of sequence variants and quasi-species were a common occurrence. The assay will be useful in establishing correlation of specific genotypes to different phenotypical expression of viral disease. This could provide insight into the etiology of diseases associated with GRSPaV. / AFRIKAANSE OPSOMMING: Die eerste doel van hierdie studie was om die virus wat met Rupestris-stamverpitting (Grapevine rupestris stem pitting-associated virus of “GRSPaV”) in wingerd verbind is, vinnig en betroubaar op te spoor. Dit is bereik deur 94 wingerdstokke vir die teenwoordigheid van die virus te toets met beide kwantitatiewe en konvensionele trutranskripsie polimerase kettingreaksies (RT - PCR) vanaf ongesuiwerde plant-ekstraksies. Die tweede doel was die daarstelling van ’n tegniek om onderskeid te tref tussen variante van GRSPaV met verskillende nukleotiedvolgordes. Hierdie tegniek kan op groot skaal gebruik word om ge-affekteerde wingerdstokke te toets om sodoende siektesimptome met spesifieke variante van GRSPaV te verbind. Ge-neste kwantitatiewe polimerase-kettingreaksies (qPCR) en hoë-resolusie smelt-analises (HRM) is ontwikkel vir drie streke van die GRSPaV-genoom (mantelproteïen, RNS-afhanklike RNS-polimerase en trippelgeenblok bewegingsproteïen). Die tegniek van qPCR-HRM met die hoë-versadingingskleurstof EvaGreen™ en die Rotor- Gene™ 6000 ontleder se geldigheid is bevestig deur vergelyking met ’n paneel van sestien virus-isolate waarvan die volgorde reeds bepaal is. Verdunde RT-PCR-produkte en gekloneerde DNS het die mees konsekwente amplifikasie-uitstipping en dissosiasieprofiele opgelewer. RT-PCR-produkte wat vanuit totale RNS-ekstrakte verkry is, is as templaat vir qPCR-HRM-analises gebruik. Dieselfde produkte is ook gebruik, om die volgorde van sestien monsters in drie streke direk te bepaal. Die gemiddelde amplifikasiedoeltreffendheid van die qPCR was 1.52±0.04. Gebruiker-gedefinieerde genotipes is deur middel van outooproeping teen ’n vertroue-interval van 70% uitgevoer. Filogenetiese analises vir drie streke van die GRSPaV-genoom is uitgevoer met gepubliseerde GenBank-volgordes om die HRMdata te bevestig. Die dominante volgorde-variante in die stel Suid-Afrikaanse monsters het ooreengestem met Groep II, vollengte-verwysingsvariant GRSPaV-SG1. Monsters wat met GRSPaV besmet is kan in die toekoms onderwerp word aan die qPCR-HRM-analises wat in hierdie studie ontwikkel is. Dit kan uitgevoer word om ooreenkomste met bekende genotipes te bepaal, en dus ook met filogenetiese groepe. Die besmetting van plante met meer as een volgorde-variant het algemeen voorgekom. Die kwasi-spesies populasie-struktuur van die virus het ook gedurig na vore gekom. Die toets sal nuttig wees in die bepaling van korrelasies tussen spesifieke genotipes en verskillende fenotipiese voorkomste van virussiektes. Dit kan insig verleen in die etiologie van siektes wat met GRSPaV verbind word.

No description available.