Identification of a Tobamovirus Infecting New Guinea Impatiens

Abstract

A putative tobamovirus was isolated from infected New Guinea Impatiens showing leaf symptoms. Virus particles were propagated in Nicotiana benthamiana and purified from infected plants, and the viral RNA was extracted. The ligation-anchored/PCR technique (LA/PCR), based on the ligation of the viral RNA to an EcoRI-linearised plasmid (Bluescript), was used to amplify the 3’end of the viral genome, using a degenerate primer designed from published tobamovirus sequences and the universal reverse primer on the plasmid. The LA/PCR-generated product, corresponding to part of the viral 3’ non-coding region (3’NC), was cloned and sequenced. From this sequence information, we designed a specific primer, to be used in a RT/PCR reaction together with another degenerate primer, located upstream of the movement protein gene. The resulting PCR product, covering the putative movement protein gene, coat protein gene (CP), and part of the 3’NC, was cloned and sequenced.