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Article

Occurrence of Grapevine Leafroll-Associated Virus-3 (GLRaV-3), Complete Nucleotide Sequence and Cultivar Susceptibility to a GLRaV-3 Isolate from Shaanxi Province of China

by
Xinyi Hao
1,2,
Bolei Jiao
1,
Yunlei Wang
1,
Boxing Shang
1 and
Yan Xu
1,*
1
State Key Laboratory of Crop Stress Biology in Arid Region, College of Horticulture, Northwest A&F University, Xianyang 712100, China
2
School of Food & Wine, Ningxia University, Yinchuan 750021, China
*
Author to whom correspondence should be addressed.
Horticulturae 2022, 8(1), 73; https://doi.org/10.3390/horticulturae8010073
Submission received: 18 November 2021 / Revised: 28 December 2021 / Accepted: 31 December 2021 / Published: 14 January 2022
(This article belongs to the Special Issue Stress Biology of Horticultural Plants)
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Abstract

:
Grapevine (Vitis spp.) is globally one of the most economically important fruit crops. China is the largest grapevine-growing country of the world and Shaanxi province is one of the major grapevine-growing provinces in the country. A survey of GLRaV-3 found it widespread, with 57–100% infection frequencies, in both wine and table grapevine cultivars of three grapevine-growing regions of Shaanxi province. The virus infection frequencies varied with cultivars and regions. In order to obtain the full genomic length of a new GLRaV-3 isolate, GLRaV-3-Sau (accession number MK988555), was sequenced. This isolate has a genome of 18026 nucleotides, and 14 open reading frames (ORFs). The full-genome of the isolate GLRaV-3-Sau shared 85.88% nucleotide identity to GLRaV-3-LN, another isolate found in China. Coat protein (CP) genes of GLRaV-3 isolates were identical (99%) to the Vitis vinifera isolate (accession number HQ185608.1) from the USA. Immunohistochemistry for virus localization found that distribution patterns were similar in red-berried cultivar ‘Cabernet Sauvignon’ and white-berried cultivar ‘Chardonnay’, and GLRaV-3 is restricted in phloem tissue of vascular bundles. Virus transmission by micrografting found virus transmission efficiency was higher in ‘Chardonnay’ and ‘Thompson Seedless’ than in ‘Hunan-1’, indicating that ‘Hunan-1’ was less sensitive to GLRaV-3. As far as we know, these are the most comprehensive comparisons on the genome and CP genes of GLRaV-3 worldwide and the first to have found that the grapevine ‘Hunan-1’ is less susceptible to GLRaV-3.

1. Introduction

Grapevine (Vitis spp.) is globally one of the most economically important fruits. Grapevine leafroll disease (GLD) is one of the most serious virus diseases infecting grapevine and widely occurs in many grapevine-growing countries [1,2,3]. GLD severely affects plant growth, reduces photosynthesis [4,5,6], and alters berry composition [7,8,9], eventually resulting in reduced yield and quality of berries [2,10].
Grapevine leafroll-associate virus-3 (GLRaV-3), a type member of the genus Ampelovirus in the family Cloteroviridae, is the major causal agent of GLD [2,3]. GLRaV-3 is a phloem-limited virus [11], transmitted by grafting [12] and vectors such as mealy bugs, soft scale and scale insects [2,13,14]. To date, full length genomes are available for at least 59 isolates of GLRaV-3 from the major grapevine-growing regions of the world [15,16,17,18,19,20,21,22,23].
However, the data have been limited in China [23,24]. China is one of the major grapevine-growing country globally, accounting for 11.5% and 16.7% of the acreage and yield of the world in 2019, respectively [25]. Shaanxi province is one of the major grapevine-growing regions for both wine and table grapevines in China. The prevalence of GLRaV-3 was 100% in Shaanxi province, and the result showed that GLRaV-3 is the predominant virus associated with GLD in China [26]. Up to now, there was one full-length genome sequence of GLRaV-3 from north China [24], but no data from northwest China.
In the present study, a survey of GLRaV-3 was conducted in three major grapevine-growing regions of Shaanxi province of China. The first complete genome of the isolate, GLRaV-3-Sau from northwest China, was sequenced and the evolutionary relationship coupled with genetic diversity among 59 fully sequenced genomes was analyzed, and the virus distribution pattern in red-berried cultivar ‘Cabernet Sauvignon’ and white-berried cultivar ‘Chardonnay’ was compared by virus localization. The susceptibility to GLRaV-3 in ‘Chardonnay’, ‘Thompson Seedless’ and ‘Hunan-1’ was tested via the virus transmission and relative level of GLRaV-3.

2. Materials and Methods

2.1. Plant Materials

A survey of GLRaV-3 was conducted in three commercial vineyards of three major grapevine-growing regions in Shaanxi, including Xianyang (107°38′ E, 34°11′ N, altitude 452 m), Xi’an (108°49′ E, 33°85′ N, altitude 415 m), and Weinan (109°22′ E, 34°47′ N, altitude 396 m) during the growing season (May–June 2018). All grapevines surveyed were grafted-propagated and were 4 years old. Thirty samples (fully opened leaves) with leafroll-like symptoms were collected from each of the 7 grapevine cultivars, including 3 wine (‘Cabernet Sauvignon’, ‘Chardonay’, ‘Riesling’) and 4 table grapevine (‘Hutai’, ‘Red Globe’, ‘Shine Muscat’, ‘Summer Black’), in the three regions, and used for GLRaV-3 detection by a reverse transcription polymerase chain reaction (RT-PCR), as described below. The samples were stored at −80 °C until usage.

2.2. Virus Detection by RT-PCR

GLRaV-3 was detected by RT-PCR as described by [27], using primers referenced in [28]. A total of 0.5 g of grape leaf were used for RNA extraction. Synthesis of cDNA was performed from 30 μg of total RNA, and the 546 bp bands were obtained by PCR.

2.3. Sequencing of Full Genome of GLRaV-3-Sau

The wine grape, ‘Cabernet Sauvignon’, infected with GLRaV-3 from Shaanxi, was utilized for sequencing of the full genome of GLRaV-3. Total RNA was extracted, as described above for the detection of the virus by RT-PCR. The expected target bands were amplified by PCR. The 5′ terminal fragments were confirmed by the 5′ Rapid Amplification of cDNA Ends Kit (version 2.0, Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instruction, and the 3′ terminal fragments were confirmed, as described by [29]. The sequence of the ISAB-BR isolate from grapevine (KX701860.1) was used as a reference to design the specific primers. The PCR reaction (12.5 μL 2xTaq PCR PreMix, 2 μL DNA, 1 μL forward primer, 1 μL reverse primer, 8.5 μL ddH2O) followed the programs according to [29]. According to the Star Prep Gel Extraction Kit (GenStar, Beijing, China) manufacturer’s instructions to purify the PCR products, the purified cDNA bands were cloned into pCombia 2300 vector, which was used to transform Escherichia coli strain top10. The full-length genome of GLRaV-3-Sau was assembled by 28 overlapping RT-PCRs, conducted by DNAMAN (LynnonBiosoft, San Ramon, CA, USA). Phylogenetic analyses were performed by the Molecular Evolutionary Genetics Analysis tool. Phylogenetic trees of complete genomes were established through the neighbor-joining (NJ) method, with 1000 replicates bootstrapping, while the phylogenetic trees of coat protein (CP) sequences were constructed with the maximum likelihood method by MEGA software (MEGA v.4.2).

2.4. Micrografting and Virus Localization

Stock shoots in vitro (10 cm in length with 5 healthy, developed leaves) were used for micrografting. Scion and rootstock preparation followed the protocols outlined in [12]. Micrografts were cultured on 1/2 MS medium and placed on the same culture conditions as described, for 3 weeks. The cultures were maintained at 24 °C under a 16 h photoperiod with a light intensity of 50 μm/(m2·s), provided by cool-white fluorescent tubes.
GLRaV-3 was localized in micrografts after 3 weeks of micrografting. The virus immunolocalization was described by [12]. The sections were treated with PBS containing 4% BSA for 30 min, followed by overnight incubation at 4 °C, with rabbit polyclonal antibodies coat protein to GLRaV-3. After washing three times, the sections were covered with mouse anti-rabbit monoclonal antibodies at room temperature for 30 min. After rinsing three times with PBS again, samples were stained with Fuchsin substrate solution. The sections were observed via a light microscope.

2.5. Analyses of Relative Levels of GLRaV-3 mRNA

The relative levels of GLRaV-3 mRNA were analyzed using qRT-PCR in grapevine leaves from these rootstocks (Figure 1B) after 2, 3, 4, and 5 weeks of micrografting. The total RNA of leaf samples was extracted and cDNA was synthesized, according to [30]. The primers described by [31] were employed for quantification of GLRaV-3, and the 18S ribosomal RNA of grapevine was used as the reference gene [32]. qPCRs were carried out using an IQ5 machine (BIORAD, USA), using Reagent kits (RR820A, Takara, Japan) and the reaction conditions described by [30]. Expression levels of GLRaV-3 mRNA were standardized according to the reference gene, using the 2−∆∆Cq method [33].

2.6. Experiment Design and Data Analysis

The micrografting experiments were arranged in a two-factor design (grafting combination and time), and repeated three times. Five grapevine leaves were collected from each of the three independent experiments in three major grapevine-growing regions in Shaanxi. Five biological replicates were implemented in the analyses of relative levels of GLRaV-3 mRNA and full-length genome of GLRaV-3 in the experiments.

3. Results

3.1. Occurrence of GLRaV-3

The 30 samples of each grapevine cultivars surveyed, including 3 wine and 4 table grapevines grown in the three major grapevine-growing regions in Shaanxi province of China and infected with GLRaV-3, with high infection frequencies (Table 1).

3.2. Full Genome of GLRaV-3

The complete genome of the GLRaV-3-Sau isolate was sequenced and consisted of 18026 nucleotides. The isolate, GLRaV-3-Sau, was accessioned in GenBank under the accession number, MK988555. The 5′UTRs and 3′UTRs of the isolate, GLRaV-3-Sau, reported in this study, contain 255 nt and 280 nt, respectively. Phylogenetic analysis of the full-length genome indicated that GLRaV-3-Sau shared 85.9% nucleotide identity with GLRaV-3-LN, another Chinese isolate found in grapevine plants grown in Beijing, while it shared 64.85–99.06% with other isolates, including 139, GH11, and ISAB-BR (Figure 2).
The GLRaV-3-Sau isolate’s genomic RNA was found to have 14 ORFs. ORF1 of the GLRaV-3-Sau isolate contains ORF1a and ORF1b (Figure 3).
Phylogenetic analysis of the full-length CP sequence showed that GLRaV-3 isolates were segmented into six groups. As shown in Figure 4, the CP gene of GLRaV3-Sau belongs to Group 2 and shared high CP identity (99%) to the Vitis vinifera cv. merlot isolate (accession number HQ185608.1) from the USA (Figure 4).

3.3. Distribution Pattern of GLRaV-3 in Micrografts

Virus-specific antibodies were used to locate GLRaV-3 by recognizing the CP antigen. After Fuchsin staining, infected cells (Figure 5B) produced a purple-color reaction, which is not present in healthy cells (Figure 5A,C). In the shoots infected with the virus, GLRav-3 was detected only in the phloem tissue (Figure 5D). The distribution pattern was similar in red-berried cultivar ‘Cabernet Sauvignon’ and white-berried cultivar ‘Chardonnay’, and GLRaV-3 is restricted to the phloem tissue of vascular bundles (Figure 5E,F).

3.4. Analysis of Susceptibility to GLRaV-3

The grapevine cultivars, ‘Chardonnay’, ‘Thompson Seedless’ and ‘Hunan-1’, were used to study the susceptibility to GLRaV-3. The GLRaV-3 infected ‘Cabernet Sauvignon’ as the scions were grafted on virus-free ‘Chardonnay’, ‘Thompson Seedless’ and ‘Hunan-1’. The virus transmission percentages are listed in Table 2.
Quantitative analysis of the infected rootstocks showed that the effects of grafting combination, time, and their interaction were significant at p = 0.01, 0.05, and 0.01, respectively. After 2–5 weeks of grafting, the relative level of GLRaV-3 in ‘Hunan-1’ was lower than that in ‘Chardonnay’ and ‘Thompson Seedless’. After 5 weeks of grafting, there was no significant difference in GLRaV-3 between ‘Chardonnay’ and ‘Thompson Seedless’, but it was significantly higher than ‘Hunan-1’. The results showed that ‘Hunan-1’ is less susceptible to GLRaV-3 (Figure 6).

4. Discussion

Shaanxi province represents a major region for grapevine production in northwest China. It was previously reported that 47% of samples collected from the three grapevine-growing regions in central China were infected with GLRaV-3, analyzed by RT-PCR [23]. Another study reported 100% infection frequency of GLRaV-3 in more than 300 grapevine samples collected from the main grape-growing regions in China [24]. All these data indicated that GLRaV-3 widely occurred in grapevines with high infection frequencies in China.
In this study, the full-length genome of the GLRaV-3-Sau isolate was sequenced; this isolate has 18026 nucleotides (nt), which shows high nucleotide identity to the isolate GLRaV-3-LN (18,563 nt) reported from China [24]. Up to now, complete genomes of 59 GLRaV-3 isolates have been reported in the world (Supplement Table S1). Most of them contain long 5′UTRs and short conserved 3′UTRs. The length of 5′UTRs ranged from 848 nt to 158 nt, and that of 3′UTRs ranged from 362 nt to 14 nt. The 5′UTRs and 3′UTRs of the isolate, GLRaV-3-Sau, are similar to the GLRaV-3 isolate ISAB-BR from Brazil (accession number KX756669).
Interestingly, the genomic RNA of GLRaV-3-Sau isolate was found to have 14 ORFs. Most of the GLRaV-3 isolates have 13 ORFs [2,3,15,16,24], while isolates CA-1, CA-2, and NY-1 were reported to have 12 ORFs [13]. ORF1 of the isolate GLRaV-3-Sau contains ORF1a and ORF1b. ORF1a is divided into OFR1a1 and OFR1a2, and the initiation codon of OFR1a1 is from 256 nt to 3432 nt, and that of OFR1a2 is from 3767 nt to 6973 nt, which encode for methyltransferase (MET) and helicase (HEL) domains. ORF1a1 and ORF1a2 encode for methyltransferase, superfamily RNA helicase, and RNA dependent RNA polymerase domains [2,34,35,36], and function in RNA accumulation, virus infection, and systemic transmission of BYV [37,38] and GLRaV-2, with the capability of RNA demethylation [39,40]. ORF2 encodes a 6kDa protein and ORF 3-7 contain conserved replication genes [2]. ORF6 encodes for coat protein. The functions of ORF8-12 are not clear, and are proposed to be involved in the inhibition of the host RNA interference defense, viral long-distance transport, and some unknown functions [41].
The analysis of the phylogenetic diversity of GLRaV-3 isolates indicated viral properties, including evolutionary relationships, host range, and resistance breaking; they are located in different regions along the genome, and in many cases comprise more than one functional region [42,43]. Further studies on the complete genome sequence analysis are needed to clarify the biological characteristics of GLRaV-3.
Immunolocalization has been widely used for virus localization [12,44,45,46]. This technique was used for virus localization in in vitro shoots of grapevine ‘Cabernet Sauvignon’ and ‘Chardonnay’, whereby it was observed that GLRaV-3 was present only in the phloem tissue in grapevine ‘Cabernet Sauvignon’ and ‘Chardonnay’. Our results confirmed that GLRaV-3 is a phloem-limited virus [2,47], and the distribution pattern of GLRaV-3 is similar in grapevine ‘Cabernet Sauvignon’ and ‘Chardonnay’.
Micrografting and qRT-PCR were used for studying and screening for susceptibility to GLRaV-3. The first report on the successful use of micrografting was to test for grapevine fanleaf virus susceptibility [48]. A previous micrografting study with GLRaV-1 and GVA found that ‘George’ and ‘AXR1’ were less susceptible to infection by GLRaV-1 and GVA than ‘Freedom’ and ‘101-14’ when used as rootstocks, suggesting that the reduced susceptibility may be a result of their Vitis. rupestris parentage [49]. Our results showed ‘Hunan-1’ had reduced susceptibility to GLRaV-3. The grapevine cultivars possessed were found out for tolerance of virus, providing an important clue for grapevine breeding.

5. Conclusions

GLRaV-3 is widespread, with high infection frequencies, in the three major grapevine-growing regions in Shaanxi province of China. The new isolate, GLRaV-3-Sau, discovered from ‘Cabernet Suavigon’, has 14 ORFs, rather than 13 ORFs reported from most of the other known GLRaV-3 isolates. The distribution pattern of GLRav-3 was similar in red-berried cultivar ‘Cabernet Sauvignon’ and white-berried cultivar ‘Chardonnay’, and GLRaV-3 is restricted to phloem tissue of vascular bundles. The grapevine cultivar ‘Hunan-1’ had reduced susceptibility to GLRaV-3, which is the first report of grapevine with reduced susceptibility to GLRaV-3.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae8010073/s1, Table S1. Fifty-nine complete genomes of GLRaV-3 worldwide [15,16,17,18,19,21,24,31,50,51,52,53,54].

Author Contributions

All authors contributed to the study conception and design. The experiments were conceived and designed by X.H. and Y.X. The experiments were performed by B.J., B.S. and Y.W. The paper was written by X.H., B.J. and Y.X. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Ningxia Hui Autonomous Region Key Research and Development Program (2021BEF02015), Ningxia Hui Autonomous Region Flexible Introduction Team Project (2020RXTDLX08), Shaanxi Province Agriculture Research Systems for the Grape Industry and Shaanxi Province Key Research and Development Program (2018ZDXMNY053-1).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The full-length genome sequence of GLRaV-3-Sau was deposited in GenBank under the accession number, MK988555, in NCBI.

Acknowledgments

The authors thank Xiu-Ming Zhang and Kun Zhou for their help on the data analysis. The authors declare that they have no conflicts of interest.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Figure 1. Micrograft development of GLRaV-3-infected in vitro scions of ‘Cabernet Sauvignon’ grapevine grafted onto healthy ‘Chardonnay’, ‘Thompson seedless’ and ‘Hunan-1’ in vitro plants as the rootstock. (A) Scion. (B) Rootstock. (C) Micrograft. Bars = 0.5 cm.
Figure 1. Micrograft development of GLRaV-3-infected in vitro scions of ‘Cabernet Sauvignon’ grapevine grafted onto healthy ‘Chardonnay’, ‘Thompson seedless’ and ‘Hunan-1’ in vitro plants as the rootstock. (A) Scion. (B) Rootstock. (C) Micrograft. Bars = 0.5 cm.
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Figure 2. Unrooted phylogenetic tree derived from the nucleotide sequences of grapevine leafroll-associated virus 3 from grape (GLRaV3-Sau, pink dot) and other available isolates in GenBank. The phylogenetic tree was constructed by the neighbor-joining (NJ) method with 500 bootstrap replicates, and bootstrap support is indicated at branch points. The scale bar represents the number of nucleotide replacements per site. The black, purple, red, and green boxes indicate ‘ISAB-BR’, ‘139’, ‘LN‘, ‘GH-11’, respectively.
Figure 2. Unrooted phylogenetic tree derived from the nucleotide sequences of grapevine leafroll-associated virus 3 from grape (GLRaV3-Sau, pink dot) and other available isolates in GenBank. The phylogenetic tree was constructed by the neighbor-joining (NJ) method with 500 bootstrap replicates, and bootstrap support is indicated at branch points. The scale bar represents the number of nucleotide replacements per site. The black, purple, red, and green boxes indicate ‘ISAB-BR’, ‘139’, ‘LN‘, ‘GH-11’, respectively.
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Figure 3. Schematic diagram of the GLRaV-3 genome to scale. Lines above the genome map indicate the positions of the ORFs and their corresponding numbers. In the genome map, boxes indicate positions of genes, with gene products and domains indicated. UTR, untranslated region; MET, methyltransferase; HEL, helicase; POL, RNA dependent RNA polymerase; Hsp70h, heat shock protein 70 homolog; Hsp90h, heat shock protein 90 homolog; CP, coat protein; CPm, minor coat protein.
Figure 3. Schematic diagram of the GLRaV-3 genome to scale. Lines above the genome map indicate the positions of the ORFs and their corresponding numbers. In the genome map, boxes indicate positions of genes, with gene products and domains indicated. UTR, untranslated region; MET, methyltransferase; HEL, helicase; POL, RNA dependent RNA polymerase; Hsp70h, heat shock protein 70 homolog; Hsp90h, heat shock protein 90 homolog; CP, coat protein; CPm, minor coat protein.
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Figure 4. Unrooted phylogenetic tree, generated with CP nucleotide sequence of the isolate GLRaV3-Sau and other GLRaV-3 isolates available in GenBank. The red square denotes ‘GLRaV3-Sau’.
Figure 4. Unrooted phylogenetic tree, generated with CP nucleotide sequence of the isolate GLRaV3-Sau and other GLRaV-3 isolates available in GenBank. The red square denotes ‘GLRaV3-Sau’.
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Figure 5. Histological observation and virus location in micrograft. (A,C), Negative control; (B,D), Positive control; the plantlets of GLRaV-3 infected ‘Cabernet Sauvignon’ grafted on healthy ‘Chardonnay’ Bar = 0.5 mm (A,B,E,F), 5 µm (C,D).
Figure 5. Histological observation and virus location in micrograft. (A,C), Negative control; (B,D), Positive control; the plantlets of GLRaV-3 infected ‘Cabernet Sauvignon’ grafted on healthy ‘Chardonnay’ Bar = 0.5 mm (A,B,E,F), 5 µm (C,D).
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Figure 6. Relative level of GLRaV-3 of rootstocks from the scions of ‘Cabernet Sauvignon’ infected GLRaV-3 at different weeks after micrografting. C, Ch, TS, and HN = ‘Cabernet Sauvignon’, ‘Chardonnay’, ‘Thompson Seedless’ and HN-1, respectively. +V = virus-infected scion. –V = virus-free rootstock. Data presented as means ± SE; The different letters are significantly different at p < 0.05.
Figure 6. Relative level of GLRaV-3 of rootstocks from the scions of ‘Cabernet Sauvignon’ infected GLRaV-3 at different weeks after micrografting. C, Ch, TS, and HN = ‘Cabernet Sauvignon’, ‘Chardonnay’, ‘Thompson Seedless’ and HN-1, respectively. +V = virus-infected scion. –V = virus-free rootstock. Data presented as means ± SE; The different letters are significantly different at p < 0.05.
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Table
Table 1. Infection frequencies (%) of GLRaV-3, analyzed by RT-PCR in the three major grapevine-growing regions in Shaanxi province of China.
Table 1. Infection frequencies (%) of GLRaV-3, analyzed by RT-PCR in the three major grapevine-growing regions in Shaanxi province of China.
Type and Cultivars of GrapevineGrapevine-Growing RegionMean
Wine GrapeXianyangXi’anWeinan
‘Cabernet Sauvignon’90.086.7100.092.2
‘Chardonay’86.783.393.387.8
‘Riesling’63.356.790.070.0
Table grape
‘Hutai’80.073.366.773.3
‘Red Globe’76.760.080.072.2
‘Shine Muscat’83.383.393.386.6
‘Summer Black’70.066.786.774.5
Mean78.672.987.1
For cultivars, the highest (92.2%) and lowest (70.0%) mean infection frequencies were found in ‘Cabernet Sauvignon’ and ‘Riesling’, respectively. GLRaV-3 infection frequencies also varied with the regions, with the highest (86.7%) and lowest (73.3%) frequencies found in Weinan and Xi’an, respectively (Table 1). This indicated that GLRaV-3 was widespread in Shaanxi province.
Table
Table 2. GLRaV-3 infected percentages (%) of different graft combinations at different weeks.
Table 2. GLRaV-3 infected percentages (%) of different graft combinations at different weeks.
Graft CombinationsWeeks after Micrografting
2345
C(+V)/Ch(−V)70100100100
C(+V)/TS(−V)60100100100
C(+V)/HN(−V)204060100
C, Ch, TS, and HN = ‘Cabernet Sauvignon’, ‘Chardonnay’, ‘Thompson Seedless’ and HN-1, respectively. +V = virus-infected scion. −V = virus-free rootstock.
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Hao, X.; Jiao, B.; Wang, Y.; Shang, B.; Xu, Y. Occurrence of Grapevine Leafroll-Associated Virus-3 (GLRaV-3), Complete Nucleotide Sequence and Cultivar Susceptibility to a GLRaV-3 Isolate from Shaanxi Province of China. Horticulturae 2022, 8, 73. https://doi.org/10.3390/horticulturae8010073

AMA Style

Hao X, Jiao B, Wang Y, Shang B, Xu Y. Occurrence of Grapevine Leafroll-Associated Virus-3 (GLRaV-3), Complete Nucleotide Sequence and Cultivar Susceptibility to a GLRaV-3 Isolate from Shaanxi Province of China. Horticulturae. 2022; 8(1):73. https://doi.org/10.3390/horticulturae8010073

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Hao, Xinyi, Bolei Jiao, Yunlei Wang, Boxing Shang, and Yan Xu. 2022. "Occurrence of Grapevine Leafroll-Associated Virus-3 (GLRaV-3), Complete Nucleotide Sequence and Cultivar Susceptibility to a GLRaV-3 Isolate from Shaanxi Province of China" Horticulturae 8, no. 1: 73. https://doi.org/10.3390/horticulturae8010073

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