Development of Molecular Detection Methodologies and Characterization of a New Virus Associated with Rootstock Stem Lesion

A Closterovirus was isolated from a Vitis vinifera grapevine cv. Redglobe table grape causing incompatibility and stem lesion symptoms on certain rootstocks (e.g., Kober 5BB and 5C, 1103P and 3309C). The virus associated with the disease was named Grapevine rootstock stem lesion associated virus (GRSLaV). The full length of the viral genome has been sequenced which was contained 14583 nucleotides. The genome organization was included 9 open reading frames (ORF) and very similar to Grapevine leafroll associated virus 2 (GLRaV-2) and confirmed to be belonged to Closterovrus genus of plant viruses along with GLRaV-2 and Citrus tristeza virus. Members of this genus are proven or suspected to be transmitted in nature by aphids. A nucleotide sequence comparison was made between GLRaV-2 and GRSLaV and found 72-80%homology between the sequences. Different amount of homologies were found between different ORFs, for example, 72%homology between ORF1a, 80%between ORF1b and 77%between ORF6 (a gene which is responsible for viral coat protein). The relationship of GRSLaV was also compared with other viruses causing leafroll disease in grapevine in a phylogenetic map and confirmed our previous data. In this map, GRSLaV was shown to be closely allied to GLRaV-2 and more closely related to CTV than any other GLRaVs (GLRaV-1, -3, -4, -5, and -7) which are members of the genus Closterovirus. Viral coat protein was synthesized in an artificial media and used to immunize a rabbit and produce specific antibody. This antibody was not reacting very well in ELISA, but the Western Blot (an immunoassay test) experiment showed that GRSLaV was serologically related to GLRaV-2. A pair of PCR primers were designed for use in PCR detection of the virus in grapevine. A limited field survey was conducted and over 100 samples were tested. The results indicated that the virus is not widely distributed in commercial vineyards. It is noteworthy that the virus was detected by RT-PCR assay in four samples collected from poorly performing wine grapevines in commercial vineyards.