Molecular variants of Grapevine rupestris stem pitting-associated virus infecting grapevines (Vitis spp.) in Brazil/Variantes moleculares de Grapevine rupestris stem pitting-associated virus infectando videiras (Vitis spp.) no Brasil.
Nine genotypes were sampled (one sample/ genotype) in three grapevine collections in Rio Grande do Sul, Sao Paulo and Pernambuco States, Brazil. The evaluated V. vinifera plants were symptomatic, exhibiting foliar symptoms related to viruses and other genotypes were asymptomatic (Table 1). The GRSPaV CP isolates were characterized in enriched dsRNAs extracted from 30g of bark scrapings per sample using CF11 cellulose. Sequencing data was generated from a complementary DNA library that was constructed by Macrogen (Korea) or Eurofins (USA). The Illumina HiSeq2000 platform was used to generate the pairedend reads. CLC Genomics Workbench software was used for quality trimming and de novo contig assembly from the reads. All contigs were analyzed using NCBI's Blast program (http://www.ncbi.nlm.nih.gov/ blast) against the viral RefSeq databases and also all contigs identified as GRSPaV were individually analyzed using Blastn against the GenBank database (FAJARDO et al., 2017).
Multiple sequence alignments of nucleotides (nt) and deduced amino acids (daa) were performed using ClustalX 2.1 and a pairwise nucleotide sequence identity matrix was generated using Sequence Demarcation Tool version 1.2 (SDT v1.2) (MUHIRE et al., 2014). The matrix generation of nt and daa identities were also performed using BioEdit v.7.2.5 software. The 15 sequences obtained in this research were aligned with 12 representative isolates of GRSPaV from the GenBank, which accession numbers of the nt sequences of the GRSPaV CPs used for phylogenetic analysis are presented in Table 1. Phylogenetic relationships were determined from the aligned sequences by using the neighbor-joining (NJ) method using Kimura 2-parameter with gamma distribution (G) and 2,000 bootstrap replications implemented in Molecular Evolutionary Genetics Analysis (MEGA 7.0.21) software package.
Twenty rooted rootstocks of grapevine cv. 110R (V. berlandieri x V. rupestris), not inoculated, exhibiting severe vein necrosis on leaves were sampled for total RNA extractions using the RNeasy Plant Mini kit (Qiagen) from 100mg of petioles of symptomatic leaves, grinding plant tissue in liquid nitrogen. Primer pairs used to amplify the partial CP of GRSPaV for one-step RT-PCR were 48V/49C (LIMA et al., 2006). The RT-PCR in a single step was carried out using One Step RT-PCR kit (Qiagen) and reactions were performed according to the manufacturer with 4gL of total RNA (ca. 400ng). The RT-PCR reactions and amplification cycling, amplified DNA analysis, elution of DNA fragments, cloning, purification of recombinant plasmids from Escherichia coli and nucleotide comparisons were performed as described by BASSO et al. (2010). The automatic nucleotide sequencing was performed with two clones.
Fifteen complete sequences of the GRSPaV CP gene (780 nt and 259 daa length) were obtained by NGS from nine different grapevine genotypes including wild and commercial cultivars of wine and table grapevines (Table 1). Fifteen CP sequences for Brazilian isolates of GRSPaV were deposited in GenBank under accession numbers KT008367 through KT008381 (Table 1). Multiple alignments between these sequences showed identities ranging from 82% to 99% (Figure 1A) and, 93% to 100%, nt and daa; respectively, suggesting high variability among the CPs of Brazilian isolates.
The CP-based phylogenetic relationships of GRSPaV isolates from three Brazilian states were compared among themselves and with reference sequences from other grape growing regions. This analysis, using the NJ method, included a total of 15 CP sequences of local isolates of GRSPaV and 12 reference isolates (sequence variants) retrieved from the GenBank. Results showed segregation of 13 Brazilian GRSPaV CP sequences into three major lineages represented by the GRSPaV-BS, -SG1 and RSPaV-1 isolates (Table 1, Figure 1B). Only two studied isolates (VVIT and VVCG2) did not cluster in the previously defined groups (lineages) (Figure 1B); although, these isolates showed high nucleotide identities of CP gene with reference isolates of GRSPaV, 90% with BS and 90% with 3138-07, respectively (Table 1). Each one of these lineages was assigned to a reference isolate to maintain a standardized nomenclature of GRSPaV sequence variant groups in analogy with a previous report by NOLASCO et al. (2006) and MENG et al. (2006). There have already been reports on the occurrence of sequence variants of GRSPaV in vineyards worldwide (LIMA et al., 2006).
GRSPaV is implicated in the rupestris stem pitting disease of the rugose wood complex responsible for graft incompatibility, delayed bud burst, severe decline, stem pitting, and even death of vines. This virus has been demonstrated to comprise a family of molecular variants, and a specific relationship between some of the viral variant groups and distinct Vitis species appears to exist. It seems that the RSPaV-1 and GRSPaV-SG1 lineages are more prevalent, whereas the GRSPaV-BS and GRSPaV-VS (SY) lineages are less common (MENG & GONSALVES, 2007). These authors also mentioned that viral variants that are distinct from these four variant groups (lineages) may exist in nature, and that additional distinct variants may be discovered later on.
From grapevines cv. 110R, exhibiting vein necrosis, it was possible to amplify a DNA fragment (330bp) (GenBank KY321511) with 100% nt identity between this and the CF210 (EF690384) Brazilian isolate and 99% with PG isolate (Table 1). Some information about biological properties associated with molecular variants of GRSPaV is available such as decline and incompatibility problems associated with GRSPaV-SY (AY368590) (LIMA et al., 2006).
Reliable and comprehensive detection and phylogenetic analysis of molecular variants of GRSPaV infecting grapevines were achieved after applying NGS and conventional RT-PCR. This result contributes to the improvement of the viral diagnosis, since it expands the knowledge on the genetic variability of GRSPaV in Brazil at the level of sequence variants. It is necessary to extend this study to sample productive Brazilian vineyards aiming to determine the incidence of representative variants in Brazilian grape growing areas. This study revealed that genetic variability of GRSPaV is also present in Brazilian grapevine genotypes and the information should be taken into consideration in symptomatological assessments and biological and molecular indexing.
Received 12.20.16 Approved 06.06.17 Returned by the author 09.18.17
To Empresa Brasileira de Pesquisa Agropecuaria (Embrapa) for the financial support to this research (Project 02.13.14.002) and Fundacao de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS) for the student scholarship. To Tatsuya Nagata (UnB, Brazil) and Maher Al Rwahnih (FPS, UC Davis, USA) for their help with bioinformatic analysis of NGS.
BASSO, M.F. et al. Molecular detection and identification of virus associated with symptomatic and symptomless grapevines. Ciencia Rural, v.40, p.2249-2255, 2010. Available from: <http://dx.doi. org/10.1590/S0103-84782010001100001>. Accessed: June 26, 2017.
BOUYAHIA, H. et al. Grapevine rupestris stempitting-associated virus is linked with grapevine vein necrosis. Vitis, v.44, p.133-137, 2005. Available from: <http://ojs.openagrar.de/index.php/VITIS/ article/view/4322>. Accessed: June 26, 2017.
FAJARDO, T.V.M. et al. High-throughput sequencing applied for the identification of viruses infecting grapevines in Brazil and genetic variability analysis. Tropical Plant Pathology, 2017. Available from: <http://link.springer.com/article/10.1007/s40858-017-0142 8>. Accessed: June 26, 2017. doi: 10.1007/s40858-017-0142-8.
HU, G.-J. et al. Molecular characterizations of two grapevine rupestris stem pitting-associated virus isolates from China. Archives of Virology, v.160, p.2641-2645, 2015. Available from: <http://link.springer.com/article/10.1007/s00705-015-2544-6>. Accessed: June 26, 2017.
LIMA, M.F. et al. Molecular analysis of a California strain of Rupestris stem pitting-associated virus isolated from declining Syrah grapevines. Archives of Virology, v.151, p.1889-1894, 2006. Available from: <http://link.springer.com/article/10.1007/ s00705-006-0742-y>. Accessed: Jun. 26, 2017.
MENG, B. et al. Genetic diversity analyses of grapevine Rupestris stem pitting-associated virus reveal distinct population structures in scion versus rootstock varieties. Journal of General Virology, v.87, p.1725-1733, 2006. Available from: <http:// jgv.microbiologyresearch.org/content/journal/jgv/10.1099/ vir.0.81533-0>. Accessed: June 26, 2017.
MENG, B., GONSALVES, D. Grapevine rupestris stempitting-associated virus: A decade of research and future perspectives. Plant Viruses, v. 1, n.1, p.52-62, 2007. Available from: <http://www.globalsciencebooks.info/Online/GSBOnline/ images/0706/PV_1(1)/PV_1(1)52-62o.pdf>. Accessed: June 26, 2017.
MUHIRE, B.M. et al. SDT: A virus classification tool based on pairwise sequence alignment and identity calculation. PLOS ONE, v.9, e108277, 2014. Available from: <http://journals. plos.org/plosone/article?id=10.1371/journal.pone.0108277>. Accessed: June 26, 2017.
NOLASCO, G. et al. Rupestris stem pitting associated virus isolates are composed by mixtures of genomic variants which share a highly conserved coat protein. Archives of Virology, v.151, p.83-96, 2006. Available from: <http://link.springer.com/ article/10.1007/s00705-005-0611-0>. Accessed: June 26, 2017.
TERLIZZI et al. Detection of multiple sequence variants of Grapevine rupestris stem pitting-associated virus using primers targeting the polymerase domain and partial genome sequencing of a novel variant. Annals of Applied Biology, v.159, p.478-490, 2011. Avalilable from: <http://dx.doi.or g/10.1111/j. 1744-7348.2011.00512.x>. Accessed: Sep. 18, 2017.
Thor Vinicius Martins Fajardo (1) *, Alexis Cardama Kin (2), Osmar Nickel (1)
(1) Empresa Brasileira de Pesquisa Agropecuaria (Embrapa Uva e Vinho), CP 130, 95701-008, Bento Goncalves, RS, Brasil. E-mail: thor.fajardo@embrapa. br. * Corresponding author.
(2) Universidade Estadual do Rio Grande do Sul (UERGS), Bento Goncalves, RS, Brasil.
Caption: Figure 1--A. Pairwise nucleotide sequence identity matrix of 15 GRSPaV isolates from Brazil and 12 representative isolates from the GenBank, generated using SDT software. B. Phylogenetic relationship among GRSPaV isolates based on the multiple alignment of the complete coat protein gene nucleotide sequences of 15 isolates from Brazil and other 12 isolates and sequence variants retrieved from the GenBank. The tree was constructed by the neighborjoining method using Kimura 2-parameter with gamma distribution (G) and 2,000 bootstrap replications implemented using MEGA 7.0.21 software package. Names of GRSPaV isolates and origins were included according to the GenBank. Reference variants for each lineage are marked with an asterisk and specific clusters (groups) are indicated as defined by NOLASCO et al. (2006) or MENG et al. (2006). Bar: number of nucleotide substitutions per site.
Table 1--Information about sequences of GRSPaV included in study and available in GenBank. Country Genotypes Isolate RS-Brazil V. vinifera cv. Cabernet Sauvignon CS-BR RS-Brazil V. vinifera cv. Cabernet Sauvignon VVCS2 RS-Brazil V. flexuosa VF1 RS-Brazil V. flexuosa VF2 RS-Brazil V. labrusca cv. Isabel VLIS1 RS-Brazil V. labrusca cv. Isabel VLIS2 SP-Brazil V. labrusca cv. Isabel VLIS3 SP-Brazil V. labrusca cv. Isabel ISA-BR SP-Brazil V. gigas VG PE-Brazil V. vinifera cv. Syrah VVSY1 PE-Brazil V. vinifera cv. Syrah VVSY2 PE-Brazil V. vinifera cv. Tempranillo TEMP-BR RS-Brazil V. vinifera cv. Italia VVIT RS-Brazil V. vinifera cv. CG90450 VVCG1 RS-Brazil V. vinifera cv. CG90450 VVCG2 RS-Brazil V. berlandieri x V. rupestris cv. 110R VN-BR USA V. riparia RSPaV-1 Italy V.vinifera cv. Moscato Giallo GRSPaV-MG USA V.vinifera cv. Syrah GRSPaV-SY Canada Vitis sp. cv. Bertille Seyve 5563 GRSPaV-BS USA Vitis vinifera cv. Pinot Noir GRSPaV-PN USA grapevine GRSPaV USA V. rupestris cv. St. George GRSPaV-SG1 Canada V. vinifera 3138-07 Italy V. vinifera cv. Pinot Gris PG USA V. riparia cv. Grande Glabre GRSPaV-GG Portugal V. sylvestris Vs279-2 USA V. vinifera GRSPaV-WA Country GenBank Nucleotide Variant accessions identity with lineage sequence variant RS-Brazil KT008367 BS (96%) BS RS-Brazil KT008368 3138-07 (96%) SG1 RS-Brazil KT008369 BS (97%) BS RS-Brazil KT008370 RSPaV-1 (99%) RSPaV-1 RS-Brazil KT008371 BS (95%) BS RS-Brazil KT008372 BS (92%) BS SP-Brazil KT008373 BS (99%) BS SP-Brazil KT008374 RSPaV-1 (99%) RSPaV-1 SP-Brazil KT008375 RSPaV-1 (99%) RSPaV-1 PE-Brazil KT008376 BS (95%) BS PE-Brazil KT008377 PG (92%) RSPaV-1 PE-Brazil KT008378 3138-07 (96%) SG1 RS-Brazil KT008379 BS (90%) ? RS-Brazil KT008380 BS (96%) BS RS-Brazil KT008381 3138-07 (90%) ? RS-Brazil KY321511 ** PG (99%) --- USA NC_001948 -- RSPaV-1 Italy FR691076 -- SG1 USA AY368590 -- SY Canada AY881627 -- BS USA AY368172 -- PN USA AF026278 -- RSPaV-1 USA AY881626 -- SG1 Canada JX559646 -- SG1 Italy HE591388 -- RSPaV-1 USA JQ922417 -- RSPaV-1 Portugal AY927684 -- SY USA KC427107 -- SG1 * The Brazilian States of Rio Grande do Sul (RS), Sao Paulo (SP) and Pernambuco (PE), ** partial CP gene and "?" not defined.