Caracterização de um isolado de Yam mild mosaic virus (YMMV) obtido de Dioscorea trifida, sequenciamento do genoma e produção de antissoro policlonal

Abstract

The yam (Dioscorea spp.) has an important economic and social role in the Northeast of Brazil, providing food with high value to the human diet. It is also a source of income to poor family, employing large number of manpower. Among the phytosanitary problems of this crop, the fungal and nematode diseases are the most important ones. The virus diseases also have high importance, especially for causing cultivar degeneracy, with reduction of plant vigor and consequent qualitative and quantitative production losses. Three viruses stand out in Brazil with occurrence on main cultivars used in Northeast, Yam mosaic virus (YMV) and Yam mild mosaic virus (YMMV), belonging to the genus Potyvirus, family Potyviridae and one virus of the genus Badnavirus, family Caulimoviridae. Recently, two more viruses from the genus Begomovirus and Curtovirus genus, family Geminiviridae were detected. In the present work, initially, the detection and characterization of an YMMV isolate obtained from D. trifida were undertaken by electronic microscope analysis including visualization of cytoplasmatic inclusions typical of the genus Potyvirus. In addition, molecular studies with specific primers for YMMV in RT–PCR, followed by RT–PCR with degenerate primers to viruses of the Potyvirus genus, cloning the product corresponding to the coat protein and phylogenetic analysis including sequences available at the GenBank were done. Sequencing of the complete genome of a YMMV isolate and a detailed study on the nucleotide sequence of this virus by cDNA amplification and cloning in plasmids pGEM-T Easy and the pCR 4 Topo were also realized, and the data deposited in GenBank. The diagnosis of plant virus diseases can be realized by serological and molecular techniques. In yam crop, molecular tools normally are used due to difficulties in acquiring specific antisera, making more expense the identification of viral agents. Polyclonal antiserum was produced in rabbit for YMMV, using the coat protein of an isolate of YMMV expressed in vitro using Escherichia coli system.