Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas
Autor(a) principal: | |
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Data de Publicação: | 2010 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da UEPG |
Texto Completo: | http://tede2.uepg.br/jspui/handle/prefix/2203 |
Resumo: | The grapevine (Vitis spp.) being vegetatively propagated facilitates the spread of viruses. In general, viruses are able to induce disorders in plant cells including photosynthesis and metabolic changes, causing alterations in grape quality. The purpose of Chapter I was to identify and molecularly characterize the viruses present in vector mealybugs in two establised vineyards (Rio Grande do Sul, Brazil), one of cv. Cabernet Sauvignon (Vitis vinifera), cultivated since 1995, and the of cv. Isabel (V. labrusca), cultivated since 1971. Plants were checked serologically for six viruses detected. Fragments that comprise complete or partial viral genes were amplified by RT-PCR using 17 pairs of specific primers for detection of the following viruses: Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine chrome mosaic virus (GCMV), Rupestris stem pitting-associated virus (RSPaV) and Grapevine leafroll-associated virus 1-4 (GLRaV-1 to - 4). Degenerate primers were used for detection of Closterovirus, Trichovirus and Vitivirus genera and Betaflexiviridae family. For each primer pair at least one amplicon was cloned and sequenced, identifying seven isolates of RSPaV, three isolates of GLRaV-2 and two isolates of GLRaV-3. The obtained sequences (submitted to GenBank) showed identities higher than 90% with the homologous viral isolates from the GenBank. GVA, GVB and GLRaV-3 were detected in three of five samples of mealybugs collected in Caxias do Sul (RS) and Petrolina (PE) vineyards. High genetic variability of RSPaV and unavailability of commercial antiserum have impaired the detection of this virus in grapevines. In Chapter II the objective was to produce polyclonal antiserum against the recombinant coat protein (CP) of RSPaV. The complete CP gene of this virus was amplified, cloned, sequenced and subcloned into expression vector pRSET-B, and the recombinant plasmid was used for expression in Escherichia coli cells (strain BL21:DE3). The yield of RSPaV CP, expressed per ml of culture, was 17.35 μg for rabbit immunization 2.55 mg were used. The antiserum showed a concentration of 1939 mg IgG/ml which was able to recognize the recombinant protein in Western blot and detect RSPaV in grapevine infected tissues using the indirect ELISA technique. In Chapter III physiological alterations of plant and enological quality changes of the grapes from infected grapevines GLRaV-2 and RSPaV infected were studied. For these evaluations the photosynthetic potential, total chlorophyll (a and b), total soluble sugars and starch were determined in leaves. Healthy or asymptomatic leaves showed favourable values for almost all characteristics. The saturation photosynthesis was 2.9 (C. Franc) and 2.25 (C. Sauvignon) times higher in healthy than in infected grapevines. Considering the grape quality significant differences between grapes from infected and non-infected grapevine were also observed in five from six characteristics studied. Healthy grapes showed 17.88 (cv. C. Franc) and 16.10 (cv. C. Sauvignon) oBrix values while infected showed 15.35 and 13.38, respectively. Therefore, these viruses can damage the productivity and quality production of these grape cultivars. |
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Ayub, Ricardo AntonioCPF:54603722672http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782860Z5Fajardo, Thor Vinícius MartinsCPF:57984522634http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727026U2Galvão, Carolina WeigertCPF:00534598900http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4760025Y8Zerbini, Francisco MuriloCPF:73507814668ZERBINI, Francisco M.CPF:00698287924http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4710935Y7Basso, Marcos Fernando2017-07-25T19:29:45Z2010-08-032017-07-25T19:29:45Z2010-07-06BASSO, Marcos Fernando. Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas. 2010. 133 f. Dissertação (Mestrado em Agricultura) - UNIVERSIDADE ESTADUAL DE PONTA GROSSA, Ponta Grossa, 2010.http://tede2.uepg.br/jspui/handle/prefix/2203The grapevine (Vitis spp.) being vegetatively propagated facilitates the spread of viruses. In general, viruses are able to induce disorders in plant cells including photosynthesis and metabolic changes, causing alterations in grape quality. The purpose of Chapter I was to identify and molecularly characterize the viruses present in vector mealybugs in two establised vineyards (Rio Grande do Sul, Brazil), one of cv. Cabernet Sauvignon (Vitis vinifera), cultivated since 1995, and the of cv. Isabel (V. labrusca), cultivated since 1971. Plants were checked serologically for six viruses detected. Fragments that comprise complete or partial viral genes were amplified by RT-PCR using 17 pairs of specific primers for detection of the following viruses: Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine chrome mosaic virus (GCMV), Rupestris stem pitting-associated virus (RSPaV) and Grapevine leafroll-associated virus 1-4 (GLRaV-1 to - 4). Degenerate primers were used for detection of Closterovirus, Trichovirus and Vitivirus genera and Betaflexiviridae family. For each primer pair at least one amplicon was cloned and sequenced, identifying seven isolates of RSPaV, three isolates of GLRaV-2 and two isolates of GLRaV-3. The obtained sequences (submitted to GenBank) showed identities higher than 90% with the homologous viral isolates from the GenBank. GVA, GVB and GLRaV-3 were detected in three of five samples of mealybugs collected in Caxias do Sul (RS) and Petrolina (PE) vineyards. High genetic variability of RSPaV and unavailability of commercial antiserum have impaired the detection of this virus in grapevines. In Chapter II the objective was to produce polyclonal antiserum against the recombinant coat protein (CP) of RSPaV. The complete CP gene of this virus was amplified, cloned, sequenced and subcloned into expression vector pRSET-B, and the recombinant plasmid was used for expression in Escherichia coli cells (strain BL21:DE3). The yield of RSPaV CP, expressed per ml of culture, was 17.35 μg for rabbit immunization 2.55 mg were used. The antiserum showed a concentration of 1939 mg IgG/ml which was able to recognize the recombinant protein in Western blot and detect RSPaV in grapevine infected tissues using the indirect ELISA technique. In Chapter III physiological alterations of plant and enological quality changes of the grapes from infected grapevines GLRaV-2 and RSPaV infected were studied. For these evaluations the photosynthetic potential, total chlorophyll (a and b), total soluble sugars and starch were determined in leaves. Healthy or asymptomatic leaves showed favourable values for almost all characteristics. The saturation photosynthesis was 2.9 (C. Franc) and 2.25 (C. Sauvignon) times higher in healthy than in infected grapevines. Considering the grape quality significant differences between grapes from infected and non-infected grapevine were also observed in five from six characteristics studied. Healthy grapes showed 17.88 (cv. C. Franc) and 16.10 (cv. C. Sauvignon) oBrix values while infected showed 15.35 and 13.38, respectively. Therefore, these viruses can damage the productivity and quality production of these grape cultivars.A videira (Vitis spp.), por ser propagada vegetativamente, facilita a disseminação dos vírus. O objetivo do Capítulo I foi identificar e caracterizar molecularmente os vírus presentes em dois vinhedos antigos: um da cv. Cabernet Sauvignon (CS) (Vitis vinifera) de 1995 e outro da cv. Isabel (V. labrusca) de 1971 além de detectar sorologicamente tais isolados. Foram amplificados, por RT-PCR, fragmentos que compreendem genes virais completos ou parciais, utilizando-se 17 pares de oligonucleotídeos específicos para a detecção dos seguintes vírus: rapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine leck virus (GFkV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine chrome mosaic virus (GCMV), Rupestris stem pitting-associated virus (RSPaV), Grapevine leafroll-associated virus 1 a 4 (GLRaV-1 a -4), além de oligonucleotídeos degenerados para a família Betaflexiviridae e os gêneros Closterovirus, Trichovirus e Vitivirus. Pelo menos um fragmento amplificado, para cada par de oligonucleotídeos, foi clonado e sequenciado, identificando-se sete isolados de RSPaV, três de GLRaV-2 e dois de GLRaV-3. As sequências obtidas e depositadas no GenBank apresentaram identidades superiores a 90% com os respectivos isolados virais do GenBank. Também GVA, GVB e GLRaV-3 foram detectados via RT-PCR, em três de cinco amostras de cochonilhas coletadas em vinhedos de Caxias do Sul (RS) e Petrolina (PE). A variabilidade genética do RSPaV e a indisponibilidade de antisoro comercial têm dificultado a detecção deste vírus em videiras. No Capítulo II o objetivo foi produzir anti-soro policlonal utilizando a proteína capsidial (CP) recombinante do RSPaV. O gene completo da CP deste vírus foi previamente amplificado, clonado e sequenciado e, posteriormente subclonado em vetor de expressão pRSET-B, sendo o plasmídeo recombinante utilizado para a expressão em Escherichia coli BL21:DE3. O rendimento foi de 17,35 μg de CP do RSPaV expressa por ml de cultura, sendo utilizados 2,55 mg para a imunização do coelho. O anti-soro produzido apresentou uma concentração de 1939 mg de IgG/ml, foi capaz de reconhecer a proteína recombinante em Western blot e detectar o RSPaV em tecidos infectados de videira em ELISA indireto. De forma geral, os vírus são capazes de induzir desordens na célula vegetal que incluem alterações fotossintéticas e metabólicas, refletindo na qualidade da uva. No Capítulo III estudaram-se as alterações fisiológicas e da qualidade enológica da uva produzida em videiras infectadas com os vírus GLRaV-2 e RSPaV. Foram determinados nas folhas: potencial fotossintético, clorofilas total (a e b), açúcares solúveis totais e amido. Quanto às variáveis de qualidade da uva, foram observadas diferenças significativas, em favor das uvas de plantas sadias, em 5 das 6 variáveis. Uvas sadias apresentaram 2,53 (C. Franc, CF) e 2,72 (CS) °Brix a mais do que as infectadas. Para as variáveis relacionadas às alterações fisiológicas foram observadas diferenças significativas, em favor das folhas sadias ou assintomáticas, para quase todas variáveis analisadas, sendo a fotossíntese de saturação 2,9 (CF) e 2,25 (CS) vezes superior em plantas sadias. Estes vírus podem comprometer a produtividade e a qualidade da produção destas cultivares.Made available in DSpace on 2017-07-25T19:29:45Z (GMT). No. of bitstreams: 1 Marcos Fernando Basso.pdf: 3822084 bytes, checksum: d2aa276083a740974a36a726bdce8dad (MD5) Previous issue date: 2010-07-06Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paranáapplication/pdfporUNIVERSIDADE ESTADUAL DE PONTA GROSSAPrograma de Pós-Graduação em AgronomiaUEPGBRAgriculturaVitisdiagnosevariabilidadesorologiaqualidade da uvaVitisdiagnosisvariabilityserologyquality of grapesCNPQ::CIENCIAS AGRARIAS::AGRONOMIADesenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadasinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UEPGinstname:Universidade Estadual de Ponta Grossa (UEPG)instacron:UEPGORIGINALMarcos Fernando Basso.pdfapplication/pdf3822084http://tede2.uepg.br/jspui/bitstream/prefix/2203/1/Marcos%20Fernando%20Basso.pdfd2aa276083a740974a36a726bdce8dadMD51prefix/22032017-07-25 16:29:45.211oai:tede2.uepg.br:prefix/2203Biblioteca Digital de Teses e Dissertaçõeshttps://tede2.uepg.br/jspui/PUBhttp://tede2.uepg.br/oai/requestbicen@uepg.br||mv_fidelis@yahoo.com.bropendoar:2017-07-25T19:29:45Biblioteca Digital de Teses e Dissertações da UEPG - Universidade Estadual de Ponta Grossa (UEPG)false |
dc.title.por.fl_str_mv |
Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas |
title |
Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas |
spellingShingle |
Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas Basso, Marcos Fernando Vitis diagnose variabilidade sorologia qualidade da uva Vitis diagnosis variability serology quality of grapes CNPQ::CIENCIAS AGRARIAS::AGRONOMIA |
title_short |
Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas |
title_full |
Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas |
title_fullStr |
Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas |
title_full_unstemmed |
Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas |
title_sort |
Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas |
author |
Basso, Marcos Fernando |
author_facet |
Basso, Marcos Fernando |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Ayub, Ricardo Antonio |
dc.contributor.advisor1ID.fl_str_mv |
CPF:54603722672 |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4782860Z5 |
dc.contributor.advisor-co1.fl_str_mv |
Fajardo, Thor Vinícius Martins |
dc.contributor.advisor-co1ID.fl_str_mv |
CPF:57984522634 |
dc.contributor.advisor-co1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727026U2 |
dc.contributor.advisor-co2.fl_str_mv |
Galvão, Carolina Weigert |
dc.contributor.advisor-co2ID.fl_str_mv |
CPF:00534598900 |
dc.contributor.advisor-co2Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4760025Y8 |
dc.contributor.referee1.fl_str_mv |
Zerbini, Francisco Murilo |
dc.contributor.referee1ID.fl_str_mv |
CPF:73507814668 |
dc.contributor.referee1Lattes.fl_str_mv |
ZERBINI, Francisco M. |
dc.contributor.authorID.fl_str_mv |
CPF:00698287924 |
dc.contributor.authorLattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4710935Y7 |
dc.contributor.author.fl_str_mv |
Basso, Marcos Fernando |
contributor_str_mv |
Ayub, Ricardo Antonio Fajardo, Thor Vinícius Martins Galvão, Carolina Weigert Zerbini, Francisco Murilo |
dc.subject.por.fl_str_mv |
Vitis diagnose variabilidade sorologia qualidade da uva |
topic |
Vitis diagnose variabilidade sorologia qualidade da uva Vitis diagnosis variability serology quality of grapes CNPQ::CIENCIAS AGRARIAS::AGRONOMIA |
dc.subject.eng.fl_str_mv |
Vitis diagnosis variability serology quality of grapes |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS AGRARIAS::AGRONOMIA |
description |
The grapevine (Vitis spp.) being vegetatively propagated facilitates the spread of viruses. In general, viruses are able to induce disorders in plant cells including photosynthesis and metabolic changes, causing alterations in grape quality. The purpose of Chapter I was to identify and molecularly characterize the viruses present in vector mealybugs in two establised vineyards (Rio Grande do Sul, Brazil), one of cv. Cabernet Sauvignon (Vitis vinifera), cultivated since 1995, and the of cv. Isabel (V. labrusca), cultivated since 1971. Plants were checked serologically for six viruses detected. Fragments that comprise complete or partial viral genes were amplified by RT-PCR using 17 pairs of specific primers for detection of the following viruses: Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Arabis mosaic virus (ArMV), Grapevine chrome mosaic virus (GCMV), Rupestris stem pitting-associated virus (RSPaV) and Grapevine leafroll-associated virus 1-4 (GLRaV-1 to - 4). Degenerate primers were used for detection of Closterovirus, Trichovirus and Vitivirus genera and Betaflexiviridae family. For each primer pair at least one amplicon was cloned and sequenced, identifying seven isolates of RSPaV, three isolates of GLRaV-2 and two isolates of GLRaV-3. The obtained sequences (submitted to GenBank) showed identities higher than 90% with the homologous viral isolates from the GenBank. GVA, GVB and GLRaV-3 were detected in three of five samples of mealybugs collected in Caxias do Sul (RS) and Petrolina (PE) vineyards. High genetic variability of RSPaV and unavailability of commercial antiserum have impaired the detection of this virus in grapevines. In Chapter II the objective was to produce polyclonal antiserum against the recombinant coat protein (CP) of RSPaV. The complete CP gene of this virus was amplified, cloned, sequenced and subcloned into expression vector pRSET-B, and the recombinant plasmid was used for expression in Escherichia coli cells (strain BL21:DE3). The yield of RSPaV CP, expressed per ml of culture, was 17.35 μg for rabbit immunization 2.55 mg were used. The antiserum showed a concentration of 1939 mg IgG/ml which was able to recognize the recombinant protein in Western blot and detect RSPaV in grapevine infected tissues using the indirect ELISA technique. In Chapter III physiological alterations of plant and enological quality changes of the grapes from infected grapevines GLRaV-2 and RSPaV infected were studied. For these evaluations the photosynthetic potential, total chlorophyll (a and b), total soluble sugars and starch were determined in leaves. Healthy or asymptomatic leaves showed favourable values for almost all characteristics. The saturation photosynthesis was 2.9 (C. Franc) and 2.25 (C. Sauvignon) times higher in healthy than in infected grapevines. Considering the grape quality significant differences between grapes from infected and non-infected grapevine were also observed in five from six characteristics studied. Healthy grapes showed 17.88 (cv. C. Franc) and 16.10 (cv. C. Sauvignon) oBrix values while infected showed 15.35 and 13.38, respectively. Therefore, these viruses can damage the productivity and quality production of these grape cultivars. |
publishDate |
2010 |
dc.date.available.fl_str_mv |
2010-08-03 2017-07-25T19:29:45Z |
dc.date.issued.fl_str_mv |
2010-07-06 |
dc.date.accessioned.fl_str_mv |
2017-07-25T19:29:45Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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masterThesis |
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dc.identifier.citation.fl_str_mv |
BASSO, Marcos Fernando. Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas. 2010. 133 f. Dissertação (Mestrado em Agricultura) - UNIVERSIDADE ESTADUAL DE PONTA GROSSA, Ponta Grossa, 2010. |
dc.identifier.uri.fl_str_mv |
http://tede2.uepg.br/jspui/handle/prefix/2203 |
identifier_str_mv |
BASSO, Marcos Fernando. Desenvolvimento de ferramentas sorologicas e moleculares para identificação de vírus em videiras e cochonilhas, alterações fisiológicas e na qualidade enológica da uva de videiras infectadas. 2010. 133 f. Dissertação (Mestrado em Agricultura) - UNIVERSIDADE ESTADUAL DE PONTA GROSSA, Ponta Grossa, 2010. |
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