Caracterização biológica e molecular de isolados de vírus pertencentes ao gênero Tobamovirus provenientes de Capsicum annuum L.)
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Data
2003-01
Autores
Cezar, Márcia Aparecida [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O objetivo deste trabalho foi caracterizar isolados de tobamovírus coletados em campos de produção comercial de pimentão e pimenta nas regiões de Lins, Sorocaba e Salto, Estado de São Paulo. A coleção de isolados foi submetida a testes sorológicos utilizando-se antissoros policlonais para o ToMV e PMMoV e em seguida à purificação biológica por meio de lesões monolesionais em Nicotiana glutinosa, onde algumas das lesões foram transferidas para Nicotiana cleevelandi, hospedeira multiplicadora de tobamovírus. Os isolados foram mantidos em plantas de Nicotiana cleevelandi por meio de sucessivas passagens mecânicas. Os isolados foram inoculados em uma série diferencial de Capsicum spp: C. annuum cv. ECW (L+L+); C. annuum cv. Magda (L+L+); C. annuum cv. Tisana (L1L1); C. frutescens cv. Tabasco (L2L2); C. chinense PI 159236 (L3 L3); C. chacoense PI 260429 (L4L4). Foram inoculados três plantas de cada genótipo diferencial no estádio de primeira folha verdadeira e estas avaliadas durante 21 dias. Realizou-se a identificação molecular destes isolados utilizando-se oligonucleotídeos universais e específicos para as espécies de TMV, ToMV e PMMoV em reação de RT-PCR de uma só etapa, utilizando-se a transcriptase reversa AMV e a Taq DNA polimerase. A especificidade dos oligonucleotídeos foi inicialmente avaliada por meio de comparação da seqüência nucleotídica dos oligonucleotídeos com a do genoma de espécies de tobamovírus disponíveis no Genbank.O teste biológico confirmou a presença de tobamovírus nas amostras coletadas a partir de pimentão e pimenta, entretanto não permitiu diferenciar as espécies de vírus presentes. De acordo com os resultados obtidos através da inoculação dos isolados em genótipos diferenciais, observou-se que os isolados Pe-01, Pe-03, Pe-05 e Pe-12... .
The objective of this work was to characterize virus isolates belonging to the Tobamovirus genus collected from commercial sweet and hot peppers fields surrounding the cities of Lins, Sorocaba and Salto, São Paulo State. The collection of isolates was subjected to serological detection using ToMV and PMMoV antisserums, followed by single local lesion passages in Nicotiana glutinosa. Some of the local lesion isolates were selected and inoculated in Nicotiana cleevelandi plants, a propagative host of tobamoviruses. The isolates were maintained in Nicotiana cleevelandi plants through successive sap-inoculations. These isolates were further inoculated on the differential genotypes of Capsicum spp: C. annuum cv. ECW (L+L+); C. annuum cv. Magda (L+L+); C. annuum cv. Tisana (L1L1); C. frutescens cv. Tabasco (L2L2); C. chinense PI 159236 (L3 L3); C. chacoense PI 260429 (L4L4). Three plants of each differential genotypes were inoculated at the first leaf stage, and evaluated during 21 days. The same isolates were submitted to the molecular identification using degenerated and specific primers for the TMV, ToMV and PMMoV species, in a one step RTPCR protocol, using the AMV reverse transcriptase and the Taq DNA polymerase. The specificity of the primers where first evaluated by a nucleotide sequence alignment of the primers with the sequence of the genome of tobamovirus disposable in the Genbank. The biological test has confirmed the presence of tobamovirus in the sweet pepper and hot pepper samples, but could not differentiate the species of virus. Using the differential genotypes of Capsicum spp, the isolates Pe-01, Pe-03, Pe-05 andPe-12 could be classified as belonging to the P1-2 pathotype. The isolate Pe-08 belongs to the P1 and Pe-02, Pe-04, Pe-06, Pe-07, Pe-09, Pe-10, Pe-11 and Pe-13 as P0 pathotype. By the RT-PCR the isolates Pe-06, Pe-07, Pe-09, Pe-10... (Complete abstract, click electronic address below).
The objective of this work was to characterize virus isolates belonging to the Tobamovirus genus collected from commercial sweet and hot peppers fields surrounding the cities of Lins, Sorocaba and Salto, São Paulo State. The collection of isolates was subjected to serological detection using ToMV and PMMoV antisserums, followed by single local lesion passages in Nicotiana glutinosa. Some of the local lesion isolates were selected and inoculated in Nicotiana cleevelandi plants, a propagative host of tobamoviruses. The isolates were maintained in Nicotiana cleevelandi plants through successive sap-inoculations. These isolates were further inoculated on the differential genotypes of Capsicum spp: C. annuum cv. ECW (L+L+); C. annuum cv. Magda (L+L+); C. annuum cv. Tisana (L1L1); C. frutescens cv. Tabasco (L2L2); C. chinense PI 159236 (L3 L3); C. chacoense PI 260429 (L4L4). Three plants of each differential genotypes were inoculated at the first leaf stage, and evaluated during 21 days. The same isolates were submitted to the molecular identification using degenerated and specific primers for the TMV, ToMV and PMMoV species, in a one step RTPCR protocol, using the AMV reverse transcriptase and the Taq DNA polymerase. The specificity of the primers where first evaluated by a nucleotide sequence alignment of the primers with the sequence of the genome of tobamovirus disposable in the Genbank. The biological test has confirmed the presence of tobamovirus in the sweet pepper and hot pepper samples, but could not differentiate the species of virus. Using the differential genotypes of Capsicum spp, the isolates Pe-01, Pe-03, Pe-05 andPe-12 could be classified as belonging to the P1-2 pathotype. The isolate Pe-08 belongs to the P1 and Pe-02, Pe-04, Pe-06, Pe-07, Pe-09, Pe-10, Pe-11 and Pe-13 as P0 pathotype. By the RT-PCR the isolates Pe-06, Pe-07, Pe-09, Pe-10... (Complete abstract, click electronic address below).
Descrição
Palavras-chave
Pimentão, Virus, Virus - Identificação
Como citar
CEZAR, Márcia Aparecida. Caracterização biológica e molecular de isolados de vírus pertencentes ao gênero Tobamovirus provenientes de Capsicum annuum L.). 2003. ix, 42 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Ciências Agronômicas, 2003.