Identificação e purificação de isolados de Cucumber mosaic virus e triagem de genótipos de pimentão para resistência
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Data
2001-12
Autores
Frangioni, Desiré Spada dos Santos [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
No período de 1998 à 2001, 17 amostras de pimentão ‘Magali-R’ (Capsicum annuum L.) com sintomas de mosaico sistêmico, nanismo, deformação foliar e dos frutos, causando severos danos e perdas econômicas, foram coletadas nas regiões produtoras de Paulínia, Lins, Jacareí, Guaiçara e Lucianópolis, municípios do Estado de São Paulo. Essas amostras foram caracterizadas através de círculo de hospedeiras, DAS-ELISA com o emprego de anticorpo monoclonal contra o CMV-I e CMV-II. Um isolado de cada região foi caracterizado molecularmente, quanto ao subgrupo através da RT-PCR e RFLP com a endonuclease de restrição MspI. As amostras foram inoculadas em plantas indicadoras que reagiram com sintomas típicos do Cucumber mosaic virus (CMV), entre estas, Nicotiana glutinosa demonstrando mosaico sistêmico e ausência de necrose, indicou que os isolados do CMV, em estudo, pertenciam ao subgrupo I. N. glutinosa foi utilizada para manter os isolados, para os testes de ELISA, extração de RNA total e como multiplicadora do vírus para a purificação. Todos os isolados testados no DAS-ELISA reagiram positivamente ao anticorpo monoclonal específico para o CMV-I e negativamente para o CMV-II. Os isolados 1, 3, 4, 15 e 17 foram amplificados com sucesso através da RT-PCR, e produziram um fragmento de 486 pb, tamanho esperado para o subgrupo I. Fragmento que ao ser digerido pela MspI, resultou em duas bandas de 150 e 336 pb, padrão esperado para o CMV-I. O método utilizado na purificação foi eficiente, resultando em uma preparação do CMV purificado, cujo rendimento foi de 40 mg/kg de tecido infectado, permitindo a imunização de galinhas e a produção de um antissoro policlonal específico à partir das gemas dos ovos. O antissoro apresentou alto título em ELISA indireto. A triagem de genótipos de pimentão para a resistência ao CMV... .
During the years 1998 to 2001, 17 samples of sweet peppers ‘Magali- R’ (Capsicum annuum L.) showing systemic mosaic, stunt, leaf and fruit distortion, causing severe damage and economic losses, were collected from Paulínia, Lins, Jacareí, Guaiçara and Lucianópolis, regions of São Paulo State. These samples were characterized by host range and DAS-ELISA with monoclonal antibodies against CMV I and CMV II. One isolate from each region was also characterized by RT-PCR and RFLP with restriction enzyme MspI. The plants host range revelead the presence of the Cucumber mosaic virus (CMV) in all samples. The symptom reaction in Nicotiana glutinosa was sistemic mosaic without necrosis, indicating that all CMV isolates belongs to subgroup I. The CMV isolates were also propagated and mantained in this host for the ELISA test and total RNA extration. All isolates tested by DAS-ELISA reacted positively with CMV I specific monoclonal antibodies and negatively with CMV II. The isolates 1, 3, 4, 15 e 17 were successfully amplified by the RTPCR and produced a fragment of 486 pb, the expected size for CMV subgroup I. These fragments when digested by MspI produced two bands of 150 and 336 pb, restriction pattern expected for the CMV subgroup I. Purified CMV yielding was 40mg/kg of infected leaves. The method used was efficient to produce a good purified preparation utilized to immunize chickens that produced specific antiserum with high titer. Screening of sweet peppers resistance to CMV showed immunity of AF-97A and tolerance of AF-188, AF-1178, AF-98 A, AF-99 A and AF-136 A. The hybrid progenies Lamuyo, Reinger, Mayata, Mônica, Magali, Mikalor, Esterel, Melody and “All Big” cultivar showed immune plants. These results are promising to sweet pepper breeding for resistance to CMV.
During the years 1998 to 2001, 17 samples of sweet peppers ‘Magali- R’ (Capsicum annuum L.) showing systemic mosaic, stunt, leaf and fruit distortion, causing severe damage and economic losses, were collected from Paulínia, Lins, Jacareí, Guaiçara and Lucianópolis, regions of São Paulo State. These samples were characterized by host range and DAS-ELISA with monoclonal antibodies against CMV I and CMV II. One isolate from each region was also characterized by RT-PCR and RFLP with restriction enzyme MspI. The plants host range revelead the presence of the Cucumber mosaic virus (CMV) in all samples. The symptom reaction in Nicotiana glutinosa was sistemic mosaic without necrosis, indicating that all CMV isolates belongs to subgroup I. The CMV isolates were also propagated and mantained in this host for the ELISA test and total RNA extration. All isolates tested by DAS-ELISA reacted positively with CMV I specific monoclonal antibodies and negatively with CMV II. The isolates 1, 3, 4, 15 e 17 were successfully amplified by the RTPCR and produced a fragment of 486 pb, the expected size for CMV subgroup I. These fragments when digested by MspI produced two bands of 150 and 336 pb, restriction pattern expected for the CMV subgroup I. Purified CMV yielding was 40mg/kg of infected leaves. The method used was efficient to produce a good purified preparation utilized to immunize chickens that produced specific antiserum with high titer. Screening of sweet peppers resistance to CMV showed immunity of AF-97A and tolerance of AF-188, AF-1178, AF-98 A, AF-99 A and AF-136 A. The hybrid progenies Lamuyo, Reinger, Mayata, Mônica, Magali, Mikalor, Esterel, Melody and “All Big” cultivar showed immune plants. These results are promising to sweet pepper breeding for resistance to CMV.
Descrição
Palavras-chave
Virus do mosaico do pepino, Virus - Identificação, Virus - Purificação, Pimentão - Resistência a doenças e pragas
Como citar
FRANGIONI, Desiré Spada dos Santos. Identificação e purificação de isolados de Cucumber mosaic virus e triagem de genótipos de pimentão para resistência. 2001. x, 98 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Ciências Agronômicas, 2001.