Identification and detection of Potato virus Y strains by molecular methods in tobacco fields of North Iran
Journal Title: International Research Journal of Applied and Basic Sciences - Year 2012, Vol 3, Issue 7
Abstract
Tobacco viral diseases are very important because of their impact on the quality and quantity of crops. PVY is one of the most important tobacco viruses and has a worldwide distribution. This virus is classified in the genus Potyvirus, family Potyviridae with a singlestranded positive-sense genomic RNA. In order to identify the spatial distribution and phylogenetic analysis of PVY isolates, a total of 182 samples were collected in Summer 2010 from different tobacco fields from Minoodasht, Dozain, Aliabad, Fazelabad and Ghorogh areas in Golestan province. PVY infection in collected suspicious plants was investigated using polyclonal antiserum in DAS-ELISA test. Of the all collected samples 90 samples were proved to be infected with PVY. To identify the symptoms of each collected isolate, sap of positive samples in DAS-ELISA was inoculated on indicator plants such as Nicotiana tabacum cv. Samsun (necrotic spots on the leaf and mosaic), N. whiteburley (mosaic), N. rustica (vein necrotic), N. glutinosa (chlorotic spots and mosaic), Chenopodium amaranticolor (chlorotic spots on the leaf), Physalis floridana (vein clearing and chlorotic spots) and Datura metel (vein clearing). Viral RNA was extracted using RNX-Plus kit. The extracted RNA was reverse transcribed using oligo dT primer to create the respective cDNA, and was amplified in RT-PCR using a PVY-specific primer pair designed on CP region. After verification on 1% agarose gel in electrophoresis, the expected band of ~1000 bp size was amplified in infected samples. PCR products were directly sequenced and the sequences of about 850 nucleotides were aligned with other 30 sequences in the GenBank. Multiple alignment and phylogenetic analyses were performed by ClustalX and BLAST programs in BioEdit software and phylogenetic dendrogram was produced using neighbor-joining method. The results showed that all PVY sequences can be placed into 4 clades. Iranian isolates fall into two distinct clades, Aliabad and Fazelabad isolates were grouped with isolates from Italia, Spain and Bushehr, while Minoodasht isolate was grouped with isolates from Netherlands, United Kingdom and Taiwan. Genetic distances between and within groups confirmed the phylogenetic analysis results.
Authors and Affiliations
Fateme zinati fakhrabad*| Former Graduate student, Gorgan University of Agriculture Sciences and Natural Resources,zinati_fateme@yahoo.com, Asadollah ahmadikhah| Assistant Professor, Department of Agriculture Biotecnology. Gorgon University of Agricultural Sciences and Natural Resources, saeed nasrollahnejad| Associate professor, Department of Plant Protection. Gorgon University of Agricultural Sciences and Natural Resources.
Keywords
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