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ARS Home » Pacific West Area » Davis, California » Crops Pathology and Genetics Research » Research » Publications at this Location » Publication #262722
Title: Biological, molecular, and serological studies of a novel strain of grapevine leafroll associated virus 2

Author
item ALKOWNI, RAED - An-Najah National University
item ZHANG, YUN-PING - University Of California
item ROWHANI, ADIB - University Of California
item UYEMOTO, JERRY - US Department Of Agriculture (USDA)
item MINAFRA, ANGELANTONIO - University Of Bari

Submitted to: Virus Genes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2011
Publication Date: 4/13/2011
Citation: Alkowni, R., Zhang, Y., Rowhani, A., Uyemoto, J.K., Minafra, A. 2011. Biological, molecular, and serological studies of a novel strain of grapevine leafroll associated virus 2. Virus Genes. DOI 10.1007/s11262-011-0607-7.

Interpretive Summary: A heretofore unknown virus causing graft-incompatibility on selected rootstocks (i.e. 3309C, 5BB, 5C, 1616C, and 1103P) was isolated and molecularly characterized. It proved to be related to Grapevine leafroll associated virus type 2 (GLRaV-2) and due to its novel biological properties, molecular variation, and discovery in Red Globe grapevine was designated GLRaV-2RG. GLRaV-2RG is symptomless in graft-inoculated grape plants of Red Globe, Cabernet Sauvignon, Cabernet franc, Rupestris St. George, LN33, etc and presumably all grape varieties and whereby, infected sources were unknowingly collected and propagated into nursery stocks for field planting. Primers were developed to molecularly detect the virus and cane collections made from only negative tested sources.

Technical Abstract: In California, a novel closterovirus was detected in ‘Redglobe’ grapevine, associated with graft incompatibility and given a trivial name “Grapevine rootstock stem lesion associated virus (GRSLaV)”. The virus was lethal on test plants growing on rootstocks: 1616C, 5BB, 5C, 3309C and 1103 P, whereas latent infections occurred on another 13 scion-rootstock combinations.. In contrast, GLRaV-2 type (type strain) produced only typical leafroll symptoms. Double-stranded RNA was extracted from infected Redglobe grapevines, cloned, sequenced and determined a genome length of 16,527 nucleotides. Computer-assisted analysis of sequenced open-reading-frames (ORFs) revealed a genome organization typical of monopartite viruses in the genus Closterovirus with nine ORFs, encoding putatively the followings: 327 kDa ORF1a, 53 kDa ORF1b (RdRp; translated via ribosomal frameshift), 6 kDa ORF2 (p6), 65 kDa ORF3 (HSP70h); 62 kDa ORF4 (HSP90); 25 kDa ORF5 (CPm); 22 kDa ORF6 (CP); 19 kDa ORF 7 and 24 kDa ORF8. Phylogenetic comparisons revealed closest similarity (range 71 to 79% homology, among all ORFs) with GLRaV-2 type. Also the 3’-UTR of GRSLaV consisted of 223 nucleotides with an extended oligo(A) tract similar to that of GLRaV-2 type, Beet yellow stunt virus and Beet yellows virus. Recombinant GRSLaV coat protein was expressed in E. coli, purified and immunized in a rabbit to produce polyclonal antiserum. Serological data matched the molecular data, whereby exposed plant tissue extracts of grapevines infected by both viruses (GRSLaV and GLRaV-2) reacted positively with homologous and hererologous viral antisera but not with healthy grapevine extracts in ELISA and Western blot tests. Based on the comparative sequence data and shared antigens, GRSLaV was now considered a strain of GLRaV-2 and redesignated as Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG). Primers specific for GLRaV-2RG were developed, which did not amplify GLRaV-2 type strain. When both sets of specific primers were used in assays of different grapevine collections, the incidence of the respective viruses varied considerably e.g. 1.7% and 13.5%, respectively for GLRaV-2RG and GLRaV-2 type.