Diversity of viroids infecting grapevines in South Africa

doi:10.21203/rs.3.rs-1742186/v1

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Diversity of viroids infecting grapevines in South Africa

https://doi.org/10.21203/rs.3.rs-1742186/v1

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published 06 Feb, 2023

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Seven viroid species have been reported to infect grapevine namely, hop stunt viroid (HSVd), grapevine yellow speckle viroid 1 (GYSVd-1), grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Japanese grapevine viroid (JGVd), grapevine latent viroid (GLVd), and citrus exocortis viroid (CEVd). In this study, RNA sequence (RNA-Seq) data, from 229 Vitis accessions from the field maintained South African Vitis germplasm collection vineyard, were analysed to determine the diversity of the viroids present. Five of the seven known grapevine infecting viroids were very commonly found, with 214 of the 229 samples containing at least one viroid species. HSVd, GYSVd-1, GYSVd-2, AGVd, and JGVd were identified in the RNA-Seq data of the samples and confirmed with RT-PCR detection and Sanger sequencing. The HSVd sequences indicated the presence of two variants, with one showing multiple nucleotide insertions. AGVd and GYSVd-2 did not display significant sequence diversity, confirming past international studies. GYSVd-1 occurred as four major variants worldwide and representatives of all four were identified in this vineyard. This is first report on the diversity of viroids infecting grapevine in South Africa and the first report of JGVd outside of Japan. Further studies are needed to fully assess the population and to identify potentially new viroid species.

Viroids

diversity

RT-PCR

Next Generation Sequencing

Grapevine

Viroids are RNA molecules which are small (245–400 nucleotides), single stranded, circular, non-encapsidated and non-protein-coding. They possess high levels of self-complimentary sequences which result in the formation of secondary structures. Viroids are classified into two families based on structural features and functionality: the Pospiviroidae and the Avsunviroidae (1–4). Viroids of the Pospiviroidae family contain a central conserved region (CCR) within a rod-like secondary structure (2,5) and replicate in the infected host cell’s nucleus (6). In contrast, viroids of the Avsunviroidae family do not possess a CCR, adopt a secondary branch-like structure, contain hammerhead ribozymes, and replicate in the host cells plastids (2, 7–9).

Seven species of viroids are known to infect grapevines, namely hop stunt viroid (HSVd) (10), citrus exocortis viroid (CEVd) (11), Australian grapevine viroid (AGVd) (12), grapevine yellow speckle viroid 1 (GYSVd-1) (13), grapevine yellow speckle viroid 2 (GYSVd-2) (14), grapevine latent viroid (GLVd) (15), and Japanese Grapevine Viroid (JGVd) (16). A hammerhead viroid-like RNA (GHVd) was recently detected in grapevines in California, Italy, France and Greece, however its true viroid nature has yet to be determined (17–20). GYSVd-1 and GYSVd-2 are the causal agents of yellow speckle disease which is characterised by yellow spots on the grapevine leaves (14). These two viroids also exhibit a synergism with grapevine fanleaf virus which results in severe vein banding in grapevine (21). The remaining viroid species do not result in disease in grapevines (22), though CEVd and HSVd do cause disease in other host plants. It is thought that these latently infected plants may be inoculum sources for other susceptible hosts, such as HSVd moving into hops from HSVd infected grapevines, resulting in outbreaks of hop stunt disease in Japan (23).

GYSVd-1 and GYSVd-2 both have a global distribution and are found in nearly all grape growing regions (22) though GYSVd-1 has a greater level of diversity when compared to GYSVd-2 (24, 25). GYSVd-1 possesses a heterogenous population which is divided into four types based on variation in nucleic acid sequences and symptom inducing ability (26). The variation in symptom inducing ability may be due to the sequence variation in the pathogenicity domain of the viroid genome (26, 27). Types 1, 2 and 3 have been reported from Australia, with only type 1 and 3 inducing disease, while type 4 has been reported from Iran with both symptomatic and asymptomatic variants occurring in the population (22, 27, 28). AGVd is one of the least studied of the grapevine viroids, however population studies suggests that it occurs as a series of variants clustering into three possible groups based on geographical origin (29, 30). CEVd occurs as a range of variants falling in two groups with no association to host or geographical origin (31). Population studies on HSVd have demonstrated a series of variants which often cluster together based on host origin with grapevine isolates clustering together (23, 31). Phylogenetic analyses have suggested that HSVd orginated in grapevine and ‘jumped’ into hops resulting in Hop Stunt disease (23). Sano et al. (23) has also suggested that HSVd in hops is in the process of becoming adapated to this new host and is therefore virulent in hops. GLVd was first reported from symptomless grapevines in China (15) and Italy (32). JGVd was first reported from symptomless grapevines in Japan (16).

No vectors are known for the grapevine viroids, and dissemination occurs through mechanical inoculation and distribution of infected propagation material (33). The first report of grapevine infecting viroids in South Africa was in 2017 in the Western Cape grape growing region (34, 35). The South African grapevine industry is largely based on planting material derived from nuclear vines which have been subjected to heat therapy and meristem tip culture. This method of virus elimination has been shown to be ineffective at removing viroids (36). As no testing is done for viroids in the South African Certification Scheme of grapevines, it is not known which viroid species are present locally, and the genetic diversity of these in South African vineyards. This study has provided some insight into the presence and diversity of the grapevine infecting viroids in the South African Vitis germplasm collection, Nietvoorbij, Stellenbosch.

During December 2019, petioles of all replicates of the 229 Vitis species, cultivars, and interspecific crosses, were collected from the Vitis Germplasm collection Block D2 at the ARC-Nietvoorbij, Stellenbosch, South Africa. Each cultivar accession is made up of between one and five replicate vines, which were sampled and pooled as a single sample. Total RNA was isolated from these pooled samples using the method of White et al., (37). RNA quality control and quantification was performed using an Implen N60 spectrophotometer (Implen, Munich, Germany). A total of 300ng from each sample was used to prepare RNAtag-seq libraries (38), with 32 samples per library and subjected to Illumina RiboZero (plant leaf) ribo-depletion (Illumina, San Diego, CA, USA). The resulting libraries were sequenced using an Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) at the Agricultural Research Council, Biotechnology Platform, Pretoria, South Africa. Sequencing data was demultiplexed using the Je suite (39). Sequence data was trimmed, filtered, and assembled using CLC Genomics Workbench (CLC) (Qiagen Bioinformatics, Aarhus Denmark). Assembled contigs of between 200 and 400 bp in size were extracted and subjected to BLAST analysis (E-value cut off: 1e-5; query coverage: ≥ 80%) against the NCBI GenBank RefSeq of viruses and viroids using the CLC BLASTn function. Contigs were then extracted subjected to BLASTn analysis at NCBI Genbank to confirm identity.

In order to capture viroid diversity potentially overlooked by the analysis of 200-400bp contigs, reads were first mapped to the Vitis vinifera genome (GCF_000003745.3) using CLC Workbench mapping tool (Qiagen Bioinformatics, Aarhus Denmark) with default parameters (match score-1, mismatch cost-2, length fraction-0.5, similarity fraction-0.8) (40). Unmapped reads were then mapped to reference sequences of all known viroid (www.ncbi.nlm.nih.gov/genome/viruses) species using the CLC Workbench reference mapping tool (Qiagen Bioinformatics, Aarhus Denmark) using the following parameters, match score-1, mismatch cost-2, length fraction-0.9, similarity fraction-0.9. Consensus sequences were extracted from CLC and aligned with the reference sequences, using CLUSTALW 1.6 in MEGA7.0 (41).

In cases where only a partial viroid genome sequence was obtained, these isolates were analysed again using reference mapping using representative datasets of each viroid species. These representative datasets were constructed as follows; all existing full-length genome sequences of the five viroids infecting grapevine were downloaded from NCBI Genbank and aligned using CLUSTALW 1.6 in MEGA7.0 (41). Only a single representative was selected amongst sources with near identical nucleotides (98–99% similarity) to serve as reference sequences in further analysis.

Phylogenetic relationships were determined from the aligned sequences using the Maximum Likelihood method (1000 bootstrap replications, General Time Reversible model) implemented in MEGA 7.0. Percentage identity scores were determined using the Sequence Demarcation Tool (SDTv1.2) (42). Representative samples were selected based on sequence alignments and phylogenetic analysis and analysed to determine the predicted secondary structures of minimal free energy were determined using the web-based RNA secondary structure prediction tool RNAstructure (29, 42).

Samples containing divergent viroid strains as determined by referencing mapping, de novo assembly, sequence alignments, and phylogenetic analyses were sampled again. Leaf and petiole tissue was sampled from up to 5 vines per cultivar, total RNA was extracted using a modified CTAB extraction method and pooled (37). One step RT-PCR (43) was performed using the primers listed in Table 1. Amplification was done using the following program: one cycle of reverse transcription for 45 min at 37 ˚C, 2 min at 50 ˚C, 5 min at 94 ˚C, and 35 cycles of 30s at 94˚C, 30s at each primer annealing temperature, 30 s at 72 ˚C, followed by a terminal extension of 5 min at 72 ˚C. The final elongation step was conducted for 5 min at 72°C. PCR products were analysed by electrophoresis on a 3% agarose gel. PCR products were submitted for bidirectional Sanger sequencing at the Central Analytical Facility at Stellenbosch University.

Table 1

Primers used for RT-PCR of five viroid species.
Species	Primer Sequence	Band size	Annealing Temp	Reference
HSVd	AS-5’-GCCCCGGGGCTCCTTTCTCAGGTAAG-3’ S-5’-CGCCCGGGGCAACTCTTCTCAGAATCC-3’	296	56˚C	(44)
GYSVd-1	AS-5’-GCGGGGGTTCCGGGGATTGC-3. S-5’-TAAGAGGTCTCCGGATCTTCTTGC-3’	367	55˚C	(26)
GYSVd-2	P1-5’-ACTAGTACTTTCTTCTATCTCCCGAAGC-3’ P2-5’-ACTAGTCCGAGGACCTTTTCTAGCGCTC-3	370	60˚C	(45)
AGVd	AS-5’-GTCGACGACGAGTCGCCAGGTGAG-3’ S-5’-GTCGACGAAGGGTCCTCAGCAGAG-3’	370	64˚C	(46)
JGVd	JG-R2–5’-AGCCTCTCTCTGTCCATCGG-3’ JG-F2–5’-GAGGCGCTTTTTCTTTTCTCCTA-3’	290	55˚C	(16)
GHVd	Vir-F2-5'-GTGTGGTGCTCCTGACGAGTCCA-3' Vir-R2-5'-CTAGCCTAGGAGCGAATCTGCCA-3'	320	60˚C	(17)

Using both reference mapping and BLASTn analysis, viroids were identified in 214 of the 229 (93.4%) accessions analysed. HSVd was the most abundant viroid, present in 170 samples (70.4%), followed by GYSVd-1, present in 106 samples (46.3%), then GYSVd-2, present in 74 samples (32.3%), AGVd, present in 19 samples (8.3%), and JGVd, in one sample (0.29%). Viroid sources were named after their location in the germplasm collection and the host cultivar. Samples 06 − 04 Bourboulenc, 05 − 02 Azal Branco, 11 − 02 Dauphine, and 14 − 07 Gobernador Benegas were infected with the four main viroid species. Full sequences of Grapevine Hammerhead Viroid-like RNA were identified in 33 of the 229 cultivars (14.4%) (Supplementary Data Table 2). Figure 1 shows the number of vines with various combinations of mixed infections of GYSVD-1, GYSVd-2, HSVd, and AGVd.

Sequence comparisons revealed that the majority of HSVd isolates obtained in this study were most similar to HSVd isolate PGH-2 (GenBank accession KR909028) with high levels of sequence identity (98–100%). However, 34 samples proved to be the exception, and appeared to have multiple insertions towards the end of the CCR, with these isolates appearing most similar to HSVd isolate SDLY-23 (GenBank accession KY270463) (Fig. 2). Samples 11–15 Donzillinho do Galego and 46 − 13 St Laurant are most similar to sequence HSVd VL-IS2 (GenBank accession MF774866) (94 to 95% similarity). The presence of both sequence variants was confirmed using RT-PCR. Five isolates which are most like isolate PGH-2 were analysed using RNAstructure and changes in the Terminal Left (TL) domain were observed. Two nucleotide insertions are present in the TL domain of sample 06–09 Blauer Kadarka which may have resulted in this structural change. Sample 24 − 14 Optenhorst has five single nucleotide polymorphisms (SNPs) in the TL domain which resulted in changes in the structure. Sample 35 − 09 Viura has two insertions, one in the TL domain and one in the CCR, these two insertions have resulted in changes to the TL structure but not the CCR structure. Isolate 11 − 03 David possesses the same insertions as sample 06–09 Blauer Kadarka and there is no apparent difference between the two structures. No other variations were observed in the CCR, pathogenicity (P) and variable (V) domains (Fig. 3a). Four of the 34 samples which were observed to have multiple insertions at the end of CCR were analysed using RNAstructure, these samples all possess modifications to the CCR (Fig. 3b). Sample 11–15 Donzillinho de Galego has six SNPs, two deletions and 16 insertions, ten of which occur in the CCR. This has resulted in structure changes to both the CCR and P domain of this isolate. Samples 20 − 04 Mourvedre and 04–08 Anglianico possess ten insertions in the CCR which result in structure changes of the CCR. Sample 22 − 15 Mourisco de Semente has two SNPs in the P domain and 12 insertions, 10 in the CCR and two in the V domain, the 10 insertions have resulted in the structure changes in the CCR which has not occurred in the other domains.

Sequence analysis revealed that each of the four types of GYSVd-1 is present in the D2 vineyard. This can be observed in the phylogenetic analyses (Fig. 4), as well as sequence comparisons between the four types of GYSVd-1 with isolates sharing similarities ranging between 89–99%. The presence of GYSVd-1 was confirmed using RT-PCR. Mixed infections of the four GYSVD-1 types were confirmed through reference mapping and BLASTn analysis using reference sequences for the four types of GYSVd-1. Three isolates of GYSVd-1 type 3, and two isolates of GYSVd-1 type 1 were analysed using RNAstructure. No changes in nucleotide bases were observed in CCR and very limited variation observed in the V and P domains. Sample 05 − 02 Azal Branco represents the most of common secondary structure present in the GYSVd-1 type 3 isolates. Minor changes in the V and P domain between 05 − 02 Azal Branco and samples 47 − 01 Deckrot and 05–08 Baroque were observed. Three SNPs were observed in the V domain of sample 05–08 Baroque which may contribute to the structural change also observed in the TL domain. Sample 47 − 01 Deckrot possesses six insertions and three SNPs which are in the TL domain, resulting in structural modifications in the TL region and throughout the rest of the genome (Fig. 5a). Samples 03–12 Albanello and 12 − 02 Fernao Pires, which represent GYSVd-1 type 1 differ significantly from 05 − 02 Azal Branco, however, both samples possess similar secondary structures.

Sequence comparisons reveal that the isolates of GYSVd-2 are most similar to four references sequences, GYSVd-2 isolate 1–5 (GenBank accession DQ377126), GYSVd-2 isolate Maragheh (GenBank accession JN008867), GYSVd-2 isolate clone 1 (GenBank accession MG780425) and GYSVd-2 isolate Sup4 (GenBank accession LR735996), ranging from 98–100% similarity in nucleotide identity. The overall similarity in nucleotide identity between the isolates obtained in this study range from 95–100% (Fig. 6). Infection of GYSVd-2 was confirmed using RT-PCR. Sequence comparisons revealed that the GYSVd-2 strains amplified were most like reference isolate GYSVd-2 clone 1 (MG780425). Sample 08 − 01 Chenin Blanc has shown to have at least two variants of GVSVd-2 based on sequence comparisons between the sequences obtained from the NGS data and the sequences obtained from RT-PCR. Seven isolates were analysed using RNAstructure and changes were observed in the P domain of sample 08 − 01 Chenin Blanc due to the presence of four SNPs and changes in TL domain of sample 12 − 02 Fernao pires due to the presence of five SNPs in that region (Fig. 5b).

Sequence comparisons reveal that the majority of the AGVd isolates are most like references sequence AGVd isolate WM35-VII (GenBank accession MH521286) (98.4–100%), while two isolates are most similar to AGVd isolate 6061 (GenBank accession KF007271) (99%) and AGVd isolate Xatam (KF876037) (99%). The similarity between these isolates and the AGVd reference genome (GenBank accession NC003553) range from 96-99.5%. The overall similarity between the isolates obtained in this study range from 95.1–99.5%. All AGVd isolates obtained in this study cluster together apart from 09 − 03 Cinsault Gris (Fig. 7). Five isolates were analysed using RNAstructure. Sample 08–08 Chasselas Blanc possess three SNPs and one deletion which resulted in structural change to the TL domain. Sample 18 − 01 Lindley possesses five SNP’s spread across the genome, and these have resulted in no structure changes. Sample 26 − 14 Pontak possesses three SNPs spread across the genome, without structural changes (Fig. 5c). RT-PCR confirmation confirmed the presence of AGVd in samples 05 − 02 Azal Branco and 09 − 03 Cinsault Gris and comparison of these sequences to the sequences obtained from the NGS data reveal no significant changes.

From the 229 accessions in the South African Vitis germplasm collection, Nietvoorbij, Stellenbosch, 370 viroid sequences of five of the six known grapevine infecting viroid species, as well as 33 sequences of GHVd were obtained. This study is the first in-depth study on the diversity of grapevine infecting viroids in South Africa, and the first report of GHVd and JGVd in South Africa.

Differences between nucleotide sequences of isolates obtained for HSVd suggest that it follows a quasispecies model (31, 44). There appears to be an insertion in the HSVd CCR domain which can be observed in other isolates from Genbank. These unique HSVd isolates can be divided into two groups, one similar to HSVd isolate SDLY-23, which originates from strawberry, and the other with similarity to HSVd isolate PGH-2 from grapevine. Several Vitis accessions may contain a mixed infection of different strains of HSVd and the observed difference may be a result of recombination between strains (2, 28). The HSVd sequences, which share the most similarity to HSVd isolate PGH-2, have several SNPs distributed across the genomes, which have led to changes in the secondary structure of the molecule. The HSVd sequences which are most like HSVd isolate SDLY-23 have many insertions present just after the CCR in the P domain which has resulted in major structural changes. Changes in secondary structure may reflect in other properties such as host interaction and disease expression (26, 27, 47).

GYSVd-1 occurs in a naturally heterogenous population and can be separated into four types (22, 28, 47). Results of this study indicate that GYSVd-1 type 1, GYSVd-1 type 3, and GYSVd-1 type 4 are present in the South African Vitis germplasm collection, with type three being the more abundant of the two. Results also indicate that some vines may contain mixed infections, such as sample 05 − 02 Azal Branco containing type 1 and type 3. All three types have been implicated in diseases (26, 28, 45, 46), however the nucleotide identity difference between the three is relatively low, ranging from 89–91%. These differences may contribute to the variability and transient expression of the yellow speckle-vein banding symptoms (26, 48). The occurrence of mixed infections is important for diagnostic testing as only certain types can cause disease symptoms. Therefore, cultivars which have tested positive for GYSVd-1 may need to be tested using type specific RT-PCR. Yellow speckle disease is more prominent during the height of the summer, and therefore surveys for disease expression and sampling for leaf and petiole tissue should occur during this time. Differences between nucleotide sequences of GYSVd-1 types 1, 3 and 4 isolates obtained in this study as well as phylogenetic analyses support the suggestion that GYSVd-1 follows a quasispecies model (28). The GYSVd-1 sequences identified here have SNPs distributed throughout the genomes. This has resulted in modifications to the secondary structure of the viroids, and this may influence disease expression (8). Changes in secondary structure may reflect in other properties such as host interaction (48). Collectively, these GSYVd-1 isolates were obtained from a variety of Vitis hosts including different table and wine grape species. The lack of significant variation between isolates suggests that they are not under diversifying selection (25).

GYSVd-2 has been shown to be less genetically diverse than GYSVd-1 (45) but also forms a quasispecies (8, 28, 49). GYSVd-2 isolates obtained in this study showed limited genetic variation with sequence identities between isolates ranging from 97–100%. Several samples contain SNPs however there was a single dominant sequence present throughout most of the samples. Those isolates containing nucleotide changes may experience a change in secondary structure. The rod-shaped secondary structure of the viroid has been shown to serve as functional motifs which interact with cellular factors of the host to accomplish various aspects of replication and disease induction (48). These GSYVd-2 viroids were obtained from a variety of hosts including different table and wine grape species and cultivars, and lack of variation between isolates suggests that they are not under diversifying selection (45). Only GYSVd-1 and − 2 result in disease in grapevine (33), symptoms were not surveyed during this study due to the presence of grapevine leaf roll-associated viruses and vitiviruses in the majority of the 229 accessions.

AGVd variants can be distinguished from one another by geographic origin (8, 28, 49). Several isolates obtained in this study possess a number of SNPs. These isolates containing nucleotide changes experience a change in secondary structure when compared to the reference NC_003553. These changes in secondary structure may reflect in other properties such as host interaction (18, 30). Sample 09 − 03 Cinsault Gris is distinguished from the other isolates, this may be because the sequence is shorter than the others. A similar effect can be seen with KY114494, which is 329bp in length and groups outside of the AGVd sequences (29). Phylogenetic analyses and nucleotide sequence identity show that AGvd does not possess a high degree of genetic variability (30). These AGVd isolates were obtained from a variety of hosts including different table and wine grape cultivars and lack of variation between isolates suggests that they are not under diversifying selection (33, 47).

JGVd is a tentative new viroid species of 367bp detected in symptomless grapevines in Japan (16) and was identified in sample 44 − 02 Katta Khurgan and confirmed using RT-PCR. JGVd is thought to have originated from sequence recombination between multiple viroid sequences, similar to how AGVd is thought to have originated (16, 50). GHVd is a poorly characterized circular, single stranded RNA molecule discovered through the bioinformatic analysis of small interfering RNAs of grapevine in Italy (19). It is 375 nucleotides in length and shares many structural characteristics of viroids or of some small circular satellite RNA molecules. GHVd has been detected in Italy, France (17), and Greece (20). GHVd was detected and confirmed using RT-PCR for the first time in South Africa during this study but further studies are needed to understand the exact nature of this molecule.

This study has provided the first insight into the diversity of grapevine infecting viroids in South Africa. Given the high level of stringency applied during this study, further studies are needed to determine if any new viroid species are potentially present in South African vineyards. With the increasing temperatures across the winelands, especially over the summer period, more expression of yellow speckle disease may be observed (33, 47). It may therefore to be prudent to consider adding GYSVd-1 and GYSVd-2 to the list of viral agents targeted for elimination in the industry.

Author contributions SWM, and GP contributed to the study design. DR and GP contributed towards sample collection and processing. SWM, DR, JTB, and GP, wrote the manuscript.

Funding This work has received no specific grant from any funding body.

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Conflict of interest The authors declare that they have no conflict of interest.

Ethical standards The manuscripts constitutes original research and has not been previously published. All authors are in agreement for manuscript submission, which complies with the ethical standards of the journal.

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Supplementary data table 2: Results of NGS analysis, using both Reference mapping and nBLAST, of block D2 indicating positive viroid infection.

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Diversity of viroids infecting grapevines in South Africa

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