Análise molecular em alho consumo nacional e importado para detecção da presença de Allexivírus

Abstract

Garlic is a monocotyledon belonging to the family Aliaceae, originating in Central Asia. In Brazil, it was introduced by the Portuguese, at the time of discovery, being widely used in cooking until the present day. Botanically, it is an agrarian herbaceous, with approximately 50 cm of height, propagated by bulbilho, which allows the maintenance of the agronomic characteristics. This method of propagation allows an efficient dissemination of pathogens, mainly viruses, which are one of the main groups of pathogens of this culture, which can cause a reduction of productive potential between 20 and 80%. Garlic is a natural host of viral species belonging to the genera Potyvirus, Carlavirus and Allexivirus. The latter was recently established as a member of the Flexiviridae family and groups Garlic mite-borne filamentous virus (GarMbFV), Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C Garlic virus D (GarV-D), Garlic virus E (GarV-E), Garlic virus X (GarV-X) and Shallot virus X (ShV-X). In Brazil, imported garlic has been used for planting, which constitutes a risk of introduction of exotic viral species in the country. Currently, the detection of allexivirus in bulbs can be performed by serological and / or molecular tests, and planting is necessary to obtain foliar tissue with high concentration of viral particles, which requires, on average, 30 days. Serological tests are efficient, however, because of the difficulty in producing high quality antisera, are currently less indicated than molecular methods. Aiming to reduce the time for analysis of detection of allexivirus in garlic, a methodological adjustment for nucleic acid extraction was sought directly from the leaf primordia of bulbilhos. Samples of garlic, from Rio Grande do Sul and imported from Argentina were analyzed using foliar primordia obtained by dissecting the bulbiles to extract total RNA using the Trizol reagent. Amplifications of the target genomic fragment were performed via RT-PCR. A band at the time corresponding to 500 bp was observed on agarose gel in the two samples studied. PCR products were transferred to membranes for Southern blot testing, for which a cold probe capable of detecting Allexivirus species was used, from which testing the presence of allexivirus was confirmed. Another allexivirus detection analysis was carried out on samples of garlic consumption imported from Argentina, China and Spain, following the same methodology, being verified a band corresponding to 500 bp in samples from Argentina and China. Southern Blot test, in which a specific cold probe was used to detect GarV-C, confirmed viral infection by the aforementioned species. According to the results obtained, it was found that the extraction of total RNA directly from leaf primordia, combined with the use of molecular techniques, presents itself as a fast and efficient method for detection of allexivirus. It was also verified that most of the analyzed samples are infected with allexivirus, evidencing a sanitary pattern that contraindicates the use of this type of material as seed, with the risk of introduction of exotic viral species.