Identification And Molecular Characteristics Of Viruses From Grapevines Caused Leafroll And Ringspot

Grapevine virus disease is one of the important factors affecting the yield and quality of grape. There are many kinds of grapevine virus diseases reported in the world, among them, grapevine leafroll disease(GLD) and grapevine infectious degeneration disease are common in China, and can cause serious economic loss. The typical symptom of GLD is leafroll, which can be caused by several grapevine leafroll viruses(GLRa Vs). For the fanleaf degradation disease, its symptoms behave various and complex. Ring spot, as a typical symptom of the fanleaf degradation disease, is not yet unknown that what viruses are associated with it. So, the study will identify which viruses are associated with the leafroll and ringspot symptom on grapevines, and also will analyze the molecular characteristics of several relative viruses.1.The detection result of GLRa V-1, 4, 7 in grapevine samples. The detection rates of GLRa Vs are very low, especially Grapevine leafroll-associated virus-1, 4 and 7(GLRa V-1, 4 and 7). In order to improve the detection sensitivity and determine the prevelance and occurrence of GLRa V-1, 4 and 7 in China, more than 100 grapevine samples were collected from different provinces and autonomous regions, and were tested for the existence of GLRa V-1,-4 and-7 by RT-PCR(reverse transcription-polymerase chain reaction) and RT- nested PCR using the primers, designed against the CP and HSP70 gene of every virus. The results showed that nested PCR had higher sensitivity and can detect more virus isolates for every virus than conventional PCR method. The detection rate of GLRa V-1,-4 and-7 was 27.3 %( 36/132), 43.2 %( 57/132) and 23.4 %( 44/188), respectively.2.The analysis of genetic diversity of Chinese GLRav-1、4 and 7 isolates. The total 43 CP and 21 HSP70 gene sequences of GLRa V-1 isolates were obtained in this study. Phylogenetic analysis of global CP and HSP70 gene sequences showed that all variants belonged to eight and seven groups, respectively, and Chinese isolates were divided into five and four groups. For CP gene sequence variants, group 4 was a new group, which only comprised Chinese isolates. The results also showed that natural selection, rather than random processes, led to the evolution of variants belonging to CP gene sequence variants in group 2 and group 8. Furthermore, three new recombination events were identified in the GLRa V-1 CP gene population. The total 22 CP and 19 HSP70 gene sequences of GLRa V-4 isolates were obtained in this study. Phylogenetic analysis results showed that GLRa V-4 variants other than GLRa V-4 strain 5、6、9、Pr and Car can be divided into two groups. One group includes Chinese isolates and other isolates from several countries, and named group1. Another group includes Japanese GLRa V-4 isolate Ru1 and Ru2, and named group 2. The recombination analysis result of GLRa V-4 HSP70 gene sequence showed that the sequence LN-Ths4 clone3 and HB-JF clone4 are recombinant.For GLRa V-7, the 45 CP and 16 HSP70 gene sequences were obtained in this study. The results showed that the obtained CP gene sequences showed 89.12%~100%/88.94~100% at the nucleotide/amino acid level, HSP70 gene sequences showed 85.33%~100%/88.97~100% at the nucleotide/amino acid level. Phylogenetic analysis results showed that CP gene sequences can be divided into five groups, and HSP70 gene sequences can be divided into four groups. Group 3 and 4 in the CP and HSP70 are identified as two new groups, which only include Chinese isolates. In addition, a recombinant LN-ZF clone9 was identified in this study.3.Small RNA sequencing of grapevine samples showed ring spot and the genome determination of the relative viruses. Small RNA sequencing was carried out in ‘Ayaneskeal’ and ‘Beta’ grapevines showed ring spot, and one asymptomatic ‘Beta’ sample as a control. The result showed that a new geminivirus was found in the ‘Ayaneskeal’ grapevine, and two new Grapevine berry inner necrosis virus(GINV) variants were found in the ‘Ayaneskeal’ grapevine and ‘Beta’ grapevine, respectively. The genome of the new geminivirus was amplified and determined by PCR, RCA and standard Sanger sequencing, and the result showed that the complete genome of the geminivirus is 2905 bp. The genome of the obtained geminivirus includes the highly conserved nucleotide sequence ‘TAATATTAC’, which is specific in geminivirus. The predicted result of ORFs showed that the geminivirus genome include seven ORFs, six ORFs had 23%~67% homology with the previously reported geminivirus sequences. So, the present study suggested that the geminivirus may be a new geminivirus first reported in grapevine, and seemingly behave latent in some grapevine clutivars, thus provisionally named Grapevine latent geminivirus(GLGV).Two complete genomes of GINV variants were determined from the ‘Ayaneskeal’ and ‘Beta’ grapevines behaving ring spot. The Chinese GINV isolates LN_ANSJ_RS(KU971246) and LN_BETA_RS(KU234316) had 71.02%/76.75% and 71.29%/77.15%, and had 78.91%/86.67% and 80.27%/89.74% homology at nucleotide/amino acid level of RP gene with the Japanese isolate(D88448), respectively. The RP gene and CP gene of the Chinese isolate LN_ANSJ_RS and LN_BETA_RS had 74.87%/82.09% and 83.16%/94.36% homology at nucleotide/amino acid level, respectively. Phylogenetic analysis of RP, MP and CP between the GINV isolates and other Trichovirus members showed all phylogenetic trees had the similar results that LN_ANSJ_RS, LN_BETA_RS and Japanese isolate(D88448) were divided into a large branch, but the three isolate forms a independent branch, respectively. To our best knowledge, this is the first report of GINV in China. The results also indicated that GINV may be closely related with the ring spot symptom.4.Detection and genetic diversity of grapevine berry inner necrosis virus(GINV).The One hundred and ninety-five grapevine samples, collected from 15 provinces and autonomous regions, were used for the detection of GINV. PCR and nested PCR were carried out using different primer sets, which were designed against CP and HSP70 gene of GINV. The detection results showed that 70 in 195 samples(35.9%) tested positive for GINV. Seventeen grapevines belonging to 13 different grapevine cultivars behaved ringspot symptom, and in which 16 tested positive for GINV. Phylogenetic analysis of CP and MP gene showed that all GINV variants can be divided into three groups. Group1 and Group2 consist of Chinese GINV isolates, and Group3 only includes Japanese isolates(D88448) and a Chinese isolate LN-Beta2.