The Molecular Identification Of Pathogenic Viruses Of Grapevince Leafroll Disease In China

Grapevine leafroll disease (GLD) is one of the most destructive virus diseases of grapevines, has frequently found in grape producing areas all around the world. The disease is caused by complex viruses. Until now, the identified Grapevine leafroll-associated viruses(GLRaVs) have up to eleven species and name as GLRaV-1 to GLRaV-11 based on their identification order. All These viruses are belonged to family Closteroviridae in different genus. GLRaV-1, 3, 4, 5, 6, 8 and 9 are members of the genus Ampelovirus, GLRaV-2 is the member of the genus Closterovirus and and other GLRaVs have not designed to any genus. Among these GLRaVs, only GLRaV-1, GLRaV-2 and GLRaV-3 have insect vectors.In this study, five GLRaVs in grapevine samples showing leafroll symptoms collected from Zhengzhou grapevine germplasm resources of the nursery collection were detected by enzyme-linked immunosorbent assay (ELISA). Totally twenty eight grapevine varieties were tested. Results showed that GLRaV-1 was most frequently occurred virus and its detection rate was up to 35.7%. The detection rates of GLRaV-3, GLRaV-6 and GLRaV-7 were 25%, 14.3% and 17.9%, respectively. GLRaV-2 had a relatively lower rate of 7.1%.Double strained RNA (dsRNA) was extracted from the phloem tissues of dormant canes of 85 grapevine samples showing leafroll symptoms was used as the template for RT-PCR. The primer sets LR7HSP70L and LR7HSP70D designed based on the conserved sequence of HSP70 gene in Closteroviridae were used for the amplifying of HSP70 gene. Fragments with a size about 590 bp were amplified from 14 samples. All these PCR products were cloned and sequenced. The obtained sequences were analyzed using the BLASTn (NCBI) program. Results showed that the obtained HSP70 fragments had very high similarities with those from GLRaV-1, GLRaV-3 and GLRaV-7, respectively. The sizes of amplified fragments of GLRaV-1 and GLRaV-3 were 602 bp, and amplified fragment size of GLRaV-7 was 590 bp.Clones with the amplified fragments were randomly selected for PCR-SSCP analysis. Results showed that clones from sample 'BAIYA' produced four kinds of SSCP bands and clones from the sample 'A FANG' produced two kinds of SSCP bands. Clones showing different SSCP types were selected and sequenced. Results also showed that the PCR product from 'BAIYA' were a mixture of HSP70 fragments from GLRaV-l,GLRaV-3 and GLRaV-7, and the PCR product from sample ' A FANG ' were a mixture of HSP70 fragments from GLRaV-3 and GLRaV-7. These results further comfirmed the complex infection of GLRaVs in grapevines.Sequence analysis showed that the HSP70 gene from GLRaV-1 had a high sequence divergence with 82.6% to 91.0% identities at the nucleotide (nt) level and 85.0% to 96.0% identities at the amino acid (aa) level. Also, the HSP70 gene from GLRaV-3 had 82.6%-91.0% identities at nt level and 85.0% -96.0% identities was at aa level, and the HSP70 gene from GLRaV-7 had 94.1%-100% identities at nt level and 91.8%-100% at aa level.