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.