Detecção de vírus da videira por RT-PCR em tempo real e por extensão de primers alelo-específicos e caracterização molecular de isolados do Nordeste Brasileiro

Abstract

The grapevine (Vitis spp.) belongs to the family of Vitaceae, being the botanical species V. vinifera L. and V. labrusca L. the most and widely cultivated, due to their products consumed as fresh fruits, jam, juices and wines. Despite the high economical importance, several factors may severely affect this crop, including the diseases caused by viruses. This study aimed to verify the incidence of virus present in commercial vineyards of two producing areas in Northeastern Brazil and evaluate the efficiency of some molecular methods for detecting and identifying viral species associated with grapevine. Materials showing or not symptoms were collected from grapevine genotypes in vineyards of Pernambuco, Paraiba, Bahia and Rio Grande do Sul, Brazil, and Locorotondo, Province of Bari, of the Puglia Region, Italy. The first part of the work was conducted at the Virology Laboratory of the Embrapa Uva e Vinho, RS, Brazil. For the identification of viral agents in the samples collected in Brazil, the extraction of total RNA was performed, cDNAs were obtained and tested by real time RT-PCR, using primers and probes specific for the following viruses: Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine leafroll-associated virus 2, 3 e 4 (GLRaV-2, -3 e -4), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV) and Grapevine fanleaf virus (GFLV). DNA fragments, products of the RT-qPCR, corresponding to the CP gene of each virus were eluted, linked to pGEM-T Easy vector (Promega) and used to transform bacteria. The plasmid DNA was extracted from transformed bacterial colonies, confirming the presence of the cloned fragments, which were sequenced. The grapevine material collected in Locorotondo was processed at the Istituto per la Protezione Sostenibile delle Piante (CNR-IPSP) and at the Dipartimento di Scienze del Suolo, della Pianta e degli Alimenti, Università degli Studi “Aldo Moro”. In order to detect in multiplex test the most relevant viruses involved in the aetiology of fanleaf degeneration and the complexes of leafroll and rugose wood of grapevine, amplification techniques based on Allele Specific Primer Extension (ASPE) were tested by using the obtained cDNAs. The results showed that the techniques aggregate some advantages, such as reduction in time and relative simplicity of implementation, completely eliminating the use of toxic reagents, such as the ethidium bromide. The use of multiplex facilitates amplification of multiple targets in a single reaction, reducing the time and cost of the analyzes.