Cloning and partial sequencing of eggplant mottled dwarf virus isolated from potato
2010
Pourrahim, Reza, Iranian Research Institute of Plant Protection | Farzadfar,Shirin | Shahraeen, Nooh | Izadpanah, Karamatolah
Eggplant mottled dwarf virus (EMDV) is a member of the genus Nucleorhabdovirus, family Rhabdoviridae. The occurrence of EMDV on potato was reported for the first time in Iran and the virus is of great economically importance on potato crops in Iran. Despite of its economic importance, no data was available on nucleotide sequence of the virus before this study. In the present work, an isolate of EMDV (EMDV-Da) obtained from a potato field in Damavand area, was selected and its host range and as well as the partial genome sequence were determined. To this end, the virus isolate was propagated in Nicotiana rustica, physically purified by differential centrifugation and viral genomic RNA was subsequently extracted. To amplify some parts of EMDV genome, eight degenerate primers were designed based on conserved regions in different Nucleorhabdovirus species available in the GenBank. A 1045 bp fragment was obtained in the RT-PCR amplification of viral genomic RNA using a pair of primers, namely E1-F and E1-R, and its sequence was determined. Multiple sequence alignment of this sequence showed 20.1 to 59.6 nucleotide sequence identities with other seven Nucleorhabdovirus species used in this study. This sequence showed the highest and lowest identity with MMV and NCMV polymerase gene, respectively. Based on the sequence obtained for EMDV-Da, a pair of primers was designed and used in RT-PCR which successfully amplified a 520 bp DNA fragment. To our knowledge, this study reports for the first time the successful detection of EMDV in RT-PCR tests in the world, which has considerable importance for seed potato production and certification programs.
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