Grapevine virus diseases affect the yield and quality of grape (Vitis vinifera) [1]. However, with development of grape facility cultivation, especially the seedings were dispatching frequently among different areas, virus diseases have more serious. At present, sixty-five species of viruses were reported to infect grapevine, and some of them can cause serious economic losses worldwide [2]. So far, eleven virus species have been reported in China [3]. The diagnostic methods of the virus mainly including biological assay, serum-testing, molecular biology and election microscopy. Meanwhile, one of the primary detection method of small RNA-seq combined with RT-PCR technique was applied, while the primers were designed according to the species-specific virus sequences.
In September 2017, the leaves with symptoms spotting and crinkle were found in Shandong province from the grapevine (Cabernet Gernischt), which one of the most common wine grape varieties in China. To detect the potential viruses in these samples, total RNA was extracted by TRIzol reagent and small RNA-seq was performed on the field samples. We acquired 23,725,796 clean reads and conducted MEGABLAST using assembled contigs against the viral reference database from NCBI and identified 42 contigs associated with the following seven viral genomes: Grapevine virus A (GVA) (2 contigs), Tomato leaf curl mali virus (ToLCMLV) (1 contigs), Tomato pseudo-curly top virus (TPCTV) (1 contigs), Turnip curly top virus (TCTV) (1 contigs), Prune dwarf virus (PDV) (11 contigs), Prunus necrotic ringspot virus (PNRSV) (22 contigs), and Plum bark necrosis and stem pitting-associated virus (PBNSPaV) (4 contigs)(Table 1).
Table 1
Analysis of the data of virus-derived sRNAs in grapevine
Gene bank NO. | Virus | Transcript counts | Total length | homogeneity analysis |
NC_004364.1 | PNRSV-RNA 3 | 9 | 766 | 98%~100% |
NC_008038.1 | PDV | 9 | 596 | 95%~100% |
NC_004363.1 | PNRSV- RNA 2 | 7 | 474 | 98%~100% |
NC_004362.1 | PNRSV- RNA 1 | 6 | 453 | 86%~100% |
NC_009992.1 | PBNSPaV | 4 | 224 | 92%~100% |
NC_031340.1 | GVA | 2 | 150 | 100% |
NC_003825.1 | TPCTV | 1 | 79 | 100% |
NC_005348.1 | ToLCMLV | 1 | 79 | 100% |
NC_014324.1 | TCTV | 1 | 71 | 97% |
NC_008037.1 | PDV- RNA 2 | 1 | 54 | 100% |
NC_008039.1 | PDV- RNA 1 | 1 | 54 | 100% |
To confirm the presence of the seven viruses in these grapevines, seven pairs of specific primers were utilized to detect the symptomatic samples by RT-PCR and DNA sequencing (Table 2), which showed that amplified products have the expected sequence size of GVA and PBNSPaV in the samples. And then another pair of primers (PBN-13558-F and PBN-14116-R) (Table 2) was designed to amplify a ~ 550 bp fragment of PBNSPaV. The amplified 550 nt PCR products were cloned and sequenced using the universal primers M13F and M13R. BLAST analysis showed that the 550 bp sequences have 98% identities with PBNSPaV (KC590346.1), which was reported to infect Prunus salicina in France [4]. This ~ 550 bp sequence of PBNSPaV from grapevine in this study was submitted to GenBank with the accession number MH371356. Phylogenetic tree was constructed using MEGA 5.1 software packages and it was suggested that PBNSPaV (MH371356) have the nearest relationship with the isolate from France Australia (LC523035) (Fig. 1). PBNSPaV is a viral species that belongs to the genus Ampelovirus, family Closteroviridae and has been shown to be worldwide distributed, affecting a broad range of Prunus species [5–7]. To our knowledge, this is the first report of PBNSPaV infecting grapevine in China. More attention will be paid on the early detection of PBNSPaV, on performance and quality of the grape.
Table 2
No. | Virus | Primer | Sequence |
1 | GVA | GVA-(400–420)-F | 5-tggatctgtgagaagggaacg-3 |
GVA-(977–1000)-R | 5-cgtatacggagtcaattggaacgt-3 |
2 | TPCTV | TPTV-(400–419)-F | 5-gtttcgcggagcgaaatccg-3 |
TPTV-(880–900)-R | 5-ttcttcatcgccggacatcca-3 |
3 | TCTV | TCTV-CP-F | 5-ctcccggttacgatttggcc-3 |
TCTV-CP-R | 5-ccaccaggatcattatcaagaataatcc-3 |
5 | PDV | PD-CP-135-F | 5-TTCCGAGTGGATGCTTCACG-3 |
PD-CP-564-R | 5-CATCGAGTGTTGGAGGTACTGAG-3 |
6 | PNRSV | PN- R2-184-F | 5-TGTTTGAAGCGTACATGGTGAC-3 |
PN-R2-1096-R | 5-GCTTCGCGGAAAGTCGGTAC-3 |
7 | PBNSPaV | PBN-9508-F | 5-agttttgttttcactgcatgtag-3 |
PBN-10170-R | 5-caacctgaaacgagtggaac-3 |
PBN-13568-F | 5-GGATTAGGTGAGGTGTGGTTGAC-3 |
PBN-14139-R | 5-GTGCATTGCCGATTCCCGGAC-3 |