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1 Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada
2 Department of Plant Agriculture, University of Guelph, 50 Stone Road East, Guelph, ON N1G 2W1, Canada
Correspondence
Baozhong Meng
bmeng{at}uoguelph.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers of the sequences determined in this work are DQ278605 [GenBank] –DQ278650 [GenBank] .
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Perhaps as a result of the prolonged history of cultivation and the more recent practice of grafting in viticulture, grapevines are known to be host to a great number of viruses. At present, 55 distinct RNA viruses belonging to seven families and 20 genera have been identified in grapevines (Martelli, 2003). It is likely that some of the viruses have co-existed with grapevines since ancient times, whilst others might have been introduced more recently from other plant hosts.
Grapevine Rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus (Martelli & Jelkmann, 1998) within the newly erected family Flexiviridae (Adams et al., 2004). GRSPaV is closely associated with the disease Rupestris stem pitting (RSP) (Goheen, 1988), a component of the rugose wood disease complex, which are among the most widespread and devastating disease syndromes affecting grapevines (Martelli, 1993). GRSPaV is perhaps the most prevalent virus of grapevines and is commonly detected in cultivated grapevines (Meng & Gonsalves, 2003, and references therein). Nonetheless, the aetiological role of GRSPaV in RSP has not been established.
The genomes of four isolates of GRSPaV have been sequenced completely. Two nearly identical isolates of GRSPaV were sequenced independently in two laboratories (Meng et al., 1998; Zhang et al., 1998). Recently, the genome sequences of two additional GRSPaV isolates were determined (Meng et al., 2005). The first isolate, GRSPaV-SG1, was detected in the majority of the ‘St George’ plants that had been used as the standard indicator for RSP, whilst the other, GRSPaV-BS, was obtained from ‘Bertille Seyve 5563’, a French–American hybrid wine grape. The nucleotide sequence identities among these two new isolates and the first sequenced isolate GRSPaV-1 are 87.3 % between GRSPaV-1 and GRSPaV-SG1, 84.3 % between GRSPaV-1 and GRSPaV-BS and 83.9 % between GRSPaV-SG1 and GRSPaV-BS (Meng et al., 2005). Based on data obtained from a biological indexing experiment, the authors demonstrated that infection with GRSPaV-SG1 was asymptomatic, since it did not induce RSP symptoms on graft-inoculated ‘St George’ plants (Meng et al., 2005).
Previous work has suggested that GRSPaV is genetically diverse, consisting of numerous sequence variants (Meng et al., 1999b; Rowhani et al., 2000; Casati et al., 2003; Santos et al., 2003; Terlizzi & Credi, 2003). The initial discovery of genetic diversity of GRSPaV was based on identification of viral clones with different sequences and on the detection of mixtures of viral variants from three grapevine varieties (Meng et al., 1999b). This finding was confirmed by several recent reports. For example, Rowhani et al. (2000) reported the existence of three ‘strains' of GRSPaV. Santos et al. (2003) identified four groups of GRSPaV variants by using a pair of broad-spectrum primers. Similarly, Casati et al. (2003) detected three tentative groups of sequence variants from isolates obtained from Italy and California. Using single-strand conformation polymorphism of cDNA clones and sequence comparison, Terlizzi & Credi (2003) also identified three groups of GRSPaV variants.
Most of the studies on the genetic diversity of GRSPaV conducted so far have involved only the genomic regions encoding the whole or part of the capsid protein (CP) and the isolates assayed were from Eurasian grapevine varieties belonging to the species Vitis vinifera. Since many of the sequence data were reported as meeting abstracts, they were not required to be deposited into the public database and are thus unavailable. As a result, a general picture of the genetic variability of GRSPaV could not be obtained.
The objectives of this investigation were: (i) to compare the efficacy of two pairs of primers targeting different genomic regions for assessing genetic diversity of GRSPaV; (ii) to compare the population structures of GRSPaV from grapevine scion varieties (including V. vinifera and interspecific hybrids) with those from rootstock varieties; and (iii) to obtain an overall picture of the genetic diversity and population structure of GRSPaV. We demonstrate the existence of at least four sequence variant groups of GRSPaV. We also reveal that the population structures of GRSPaV in scion varieties differ drastically from those in rootstock varieties.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. In total, 24 isolates were assayed. Each of the isolates was derived from a single plant and only one isolate was analysed for each of the following varieties: five V. vinifera varieties (‘Pagadebit 2’, ‘Canino 9’, ‘Trebbiano 12’, ‘Merlot’ and ‘Pinot Noir’); five interspecific hybrid varieties (‘Seyve Villard 3160’, ‘Colobel 257’, ‘Ravat 34’, ‘Seyval’ and ‘Niagara’); and four rootstock varieties [‘Grande Glabre’ (Vitis riparia), ‘Kober 5BB’, ‘Paulsen 1103’ and ‘Millardet et de Grasset (Millardet) 101-14’]. Furthermore, 10 isolates each from an individual plant of Vitis rupestris ‘St George’ collected from Sidney, British Columbia, and Geneva, New York, were also assayed. ‘Pagadebit 2’, ‘Canino 9’ and ‘Trebbiano 12’ were from the Foundation Vineyard of the Dipartimento di Colture Arboree, University of Bologna, Italy, and were kindly provided by Dr R. Credi. ‘Merlot’ was from the Casa Larga Vineyard at Fairport, New York, while ‘Pinot Noir’ was from J. Doolittle's vineyard at Trumansburg, New York. ‘Seyve Villard 3160’, ‘Colobel 257’, ‘Ravat 34’, ‘Seyval’ and ‘Grande Glabre’ were kindly provided by P. Forsline of the US Department of Agriculture-Plant Genetic Resources Unit at Geneva, New York. The remaining samples were from the Grapevine Breeding Collection Vineyard of the University of Guelph at Vineland, Ontario, Canada. All but two of the isolates were collected from the germplasm collections where they were grown on their own roots. The exceptions were ‘Pinot Noir’ and ‘Merlot’, which were collected from commercial vineyards in New York. However, there was no information about the rootstock varieties used for these two isolates.
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. Viral genomic ssRNAs were isolated from ‘Niagara’, ‘Kober 5BB’, ‘Paulsen 1103’ and ‘Millardet 101-14’ using leaves collected in early August following the method of Steward & Nassuth (2001).
RT-PCR.
Resulting ssRNAs or dsRNAs were reverse transcribed into cDNAs at 42 °C with Moloney murine leukemia virus reverse transcriptase (Promega) following the manufacturer's instructions. First-strand cDNAs were amplified via PCR using Taq DNA polymerase (New England Biolabs). Primers used were RSP13 and RSP14, which amplify a region within the helicase domain of ORF1 (nt 4373–4711) resulting in a DNA fragment of 339 bp (Meng et al., 1999a) and RSP21 and RSP22, which target the central region of the CP gene (nt 7917–8357) resulting in a DNA fragment of 441 bp (Meng et al., 2003).
Cloning, sequencing and sequence analysis.
RT-PCR products were cloned into pGEM-T (Promega) and the products of ligation reactions were used to transform JM109 cells (Promega). Recombinant clones containing inserts of the expected size were quick-screened using PCR, purified using the Miniprep kit (Qiagen) and sequenced on an ABI 373 sequencer (Applied Biosystems). The sequences of cDNA clones derived from different viral isolates were aligned using CLUSTAL_X (Thompson et al., 1997). Phylogenetic trees were constructed using the Bayesian method (MrBayes version 3.01; Ronquist & Huelsenbeck, 2003). The parameters for the evolutionary model were determined by the maximum-likelihood ratio test (Goldman, 1993) and calculated using MODELTEST version 3.06 (Posada & Crandall, 1998). Four Markov chains were used and the dataset was run for 4x106 generations to allow for adequate time of convergence. The default ‘prior’ settings were used. Trees were sampled every 100 generations. We designated the first 30 000 sample trees as ‘burn in’ and used the last 10 000 sample trees to estimate the consensus tree and the Bayesian posterior probabilities. Two separate runs, which included a total of four independent tree searches, were conducted and the resulting trees were compared and pooled.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). To determine whether mixed infection with GRSPaV is a general phenomenon for scion varieties, we initiated this study to analyse further the genetic diversity and population structure of 10 isolates, each from a different grapevine scion variety. Five of the isolates were from V. vinifera varieties (‘Pagadebit 2’, ‘Trebbiano 12’, ‘Canino 9’, ‘Merlot’ and ‘Pinot Noir’) and the other five were from interspecific hybrids (‘Seyve Villard 3160’, ‘Colobel 257’, ‘Ravat 34’, ‘Seyval’ and ‘Niagara’) (Table 1
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The viral populations within these 10 isolates were dissected through cloning and sequencing of RT-PCR products amplified with primers RSP13 and RSP14. This primer pair targets the helicase-encoding region of ORF1 (Fig. 1a) and was designed based on the consensus sequence of multiple viral variants (Meng et al., 1999a). Between four and 12 clones were sequenced for each isolate. In total, the nucleotide sequences of 69 clones were determined. Fig. 1(b) presents one of the final phylogenetic trees generated from the Bayesian analysis with the Bayesian posterior probability marked on the tree. Due to the large number of clones that were compared relative to the small number of informative sites for each of the clones, some of the branches had posterior probability values below 50 %. However, four major groups of sequence variants could be discerned (Fig. 1b). To confirm the validity of the clustering, we conducted a phylogenetic analysis using the neighbour-joining method, which resulted in an identical clustering pattern (data not shown). Furthermore, a similar clustering pattern could clearly be seen in the phylogenetic tree of clones derived from the CP region (Fig. 1c). Group I contained the reference isolate GRSPaV-1 and 17 cDNA clones derived from five isolates: ‘Seyve Villard 3160’, ‘Seyval’, ‘Ravat 34’, ‘Colobel 257’ and ‘Pagadebit 2’. Viral variants within this group had sequence identities of 91.4–99.4 %. Group II contained the reference isolate GRSPaV-SG1 and 29 cDNA clones originating from eight isolates: ‘Trebbiano 12’, ‘Merlot’, ‘Pagadebit 2’, ‘Canino 9’, ‘Pinot Noir’, ‘Ravat 34’, ‘Seyve Villard 3160’ and ‘Seyval’ (Table 1). The viral variants within this group were 86.7–99.1 % identical in nucleotide sequence. Group III contained the reference isolate GRSPaV-BS and 14 clones derived from four isolates: ‘Merlot’, ‘Trebbiano 12’, ‘Seyval’ and ‘Ravat 34’. The viral variants within this group had nucleotide sequence identities of 91.4–99.1 %. Finally, group IV was the smallest and contained nine clones from ‘Pinot Noir’, ‘Merlot’ and ‘Niagara’ (Fig. 1b). The variants within this group were 92.9–97.6 % identical in nucleotide sequence.
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). For example, the isolates ‘Ravat 34’ and ‘Seyval’ each contained viral variants from groups I, II and III. Among the 12 clones derived from ‘Seyval’, seven belonged to group I, one belonged to group II, and four belonged to group III, with sequence identities ranging from 87.3 to 98.5 % among themselves. The composition of variants in isolate ‘Ravat 34’ was slightly different; among the 10 clones sequenced, two belonged to group I, seven belonged to group II, while the other belonged to group III. The clones from ‘Ravat 34’ had 87.9–93.5 % sequence identities among themselves. The remaining seven isolates each contained two distinct types of viral variant. However, the types of sequence variants detected in these isolates varied. For example, among the eight clones from the isolate ‘Pinot Noir’, seven belonged to group II and one belonged to group IV, with nucleotide sequence identities ranging from 76.7 to 99.7 %. Isolates ‘Pagadebit 2’, ‘Seyve Villard 3160’ and ‘Colobel 257’ each contained viral variants from groups I and II (Table 1).
To verify that RSP13 and RSP14 were effective in covering the spectrum of sequence variants of GRSPaV, primers RSP21 and RSP22 were used in RT-PCR to obtain cDNA clones from seven of the above 10 isolates. This primer pair targets the central region of the CP gene (Fig. 1a) and was designed based on the consensus sequence of three viral variants (Meng et al., 2003). Again, between four and 12 clones were sequenced for each of the isolates and a total of 44 clones was sequenced (Table 1). To link our data with those from others, we also included sequences of clones representing each of the four groups recently identified by Nolasco et al. (2006) (sequences were kindly provided by Dr Nolasco prior to their release in the GenBank). The phylogenetic relationships of these clones are presented in Fig. 1(c). Similar to data obtained using primers RSP13 and RSP14, four groups of viral variants were again identified (Fig. 1c). Group I contained the reference isolate GRSPaV-1 and 22 clones from ‘Colobel 257’, ‘Niagara’, ‘Seyve Villard 3160’, ‘Ravat 34’ and ‘Pagadebit 2’. Clone E105-G representing the group 2b of Nolasco et al. (2006) fell within this group. Group II contained the reference isolate GRSPaV-SG1, as well as 15 clones from ‘Ravat 34’, ‘Trebbiano 12’, ‘Pagadebit 2’ and ‘Merlot’. Clone M31-35 representing the group 2a of Nolasco et al. (2006) belonged to this group. Group III contained the reference isolate GRSPaV-BS and five clones from ‘Niagara’, as well as clone E105-P representative of the group 3 of Nolasco et al. (2006). Lastly, group IV contained three clones from ‘Merlot’ and several representative clones from the group 1 of Nolasco et al. (2006) (Fig. 1c).
After compiling the data obtained, four groups of viral variants were identified by using the two pairs of primers targeting two different regions of the GRSPaV genome. Based on the presence of the reference isolates, we concluded that the first three groups of viral variants identified by both primer pairs were the same. However, the relationship of group IV identified using primers RSP21/RSP22 compared with that identified using primers RSP13/RSP14 could not be determined at the present time due to the lack of a completely sequenced reference isolate.
Primers RSP13 and RSP14 seemed to be equally effective (in the case of ‘Pagadebit 2’) or more effective (in the case of ‘Trebbiano 12’, ‘Seyve Villard 3160’, ‘Colobel 257’ and ‘Ravat 34’) than primers RSP21 and RSP22, as the former primer pair detected more sequence variants (Table 1). In the case of ‘Merlot’, two types of sequence variants were detected by using each pair of the primers. However, only one type of sequence variant was detected in common for both primer pairs. For example, primers RSP13 and RSP14 detected viral variants belonging to groups III and IV, whilst RSP21 and RSP22 detected viral variants belonging to groups II and IV (Table 1). For ‘Niagara’, although two types of sequence variant were detected with both primer pairs, those detected with RSP13/RSP14 belonged to groups II and IV, whilst those detected by primers RSP21/RSP22 belonged to groups I and III (Table 1).
Genetic analysis of viral isolates from rootstock varieties: evidence for single infections
To examine the genetic diversity and population structure of GRSPaV in rootstock varieties, we assayed 14 isolates derived from five grapevine rootstocks. A single isolate was obtained from each of the following rootstock varieties: ‘Grande Glabre’ (V. riparia), ‘Kober 5BB’, ‘Paulsen 1103’ and ‘Millardet 101-14’. The other 10 isolates were obtained from 10 individual plants of V. rupestris ‘St George’ that were maintained as indicator selections at Sidney, British Columbia, Canada, or at Geneva, New York. All of these rootstocks were grown on their own roots. Based on the finding that primers RSP13/RSP14 were more effective than primers RSP21/RSP22, only the former primers were used to analyse the genetic diversity of isolates from rootstocks.
In sharp contrast with the isolates from scion varieties, each of these rootstock varieties was infected with only a single type of sequence variant, either group I or group II (Table 1 and Fig. 1b). No viral variants belonging to groups III or IV were detected in any of these rootstock varieties. Furthermore, the type of viral variant detected in a particular rootstock seemed to correlate with the genotype of the rootstock. For example, the eight clones obtained from ‘Grande Glabre’ were 100 % identical to each other and belonged to group I. It is interesting to note that ‘Grande Glabre’ is a selection of wild V. riparia and has been used as a parent for breeding several rootstock varieties. Similarly, the six clones derived from ‘Kober 5BB’ were 99.4–100 % identical to one another and were 97.9 % identical to GRSPaV-1. Interestingly, ‘Kober 5BB’ is a selection of seedlings derived from a cross between Vitis berlandieri (the female parent) and V. riparia (the male parent). Moreover, the four clones derived from ‘Millardet 101-14’ were 100 % identical to one another and were 98.8 % identical to GRSPaV-1.
On the other hand, the seven clones derived from ‘Paulsen 1103’ were 98.2–100 % identical amongst themselves and were 97.9 % identical to GRSPaV-SG1; all belonged to group II. ‘Paulsen 1103’ is a selection of seedlings derived from a cross between V. berlandieri (the female parent) and V. rupestris (the male parent). For the 10 isolates from ‘St George’ plants, all 52 cDNA clones fell into group II, although slight variation was observed in the composition of sequence variants among these isolates. For example, seven isolates contained GRSPaV-SG1, one isolate contained GRSPaV-SG2, one isolate contained both GRSPaV-SG1 and -SG2, and the last contained GRSPaV-SG1, -SG2 and -SG3. The nucleotide sequences of GRSPaV-SG1, -SG2 and -SG3 differed by 3.5–9.8 % (Meng et al., 2005).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Like most crop plants, grapevines were always grown on their own roots until the 1860s when Phylloxera, a devastating root-attacking insect, was accidentally introduced from North America into the vineyards in Western Europe, resulting in the mass death of grapevines. To combat this problem, Phylloxera-resistant Vitis species that are grown in the wild in North America were introduced into Europe to be used as rootstocks (Pongracz, 1983; Howell, 1987; Reisch & Pratt, 1996). Since then, rootstocks have been used increasingly in viticulture. Today, the vast majority of grapevines throughout the world are grown on rootstocks. As a result, grapevines are known to be infected by a large number of viruses. GRSPaV is perhaps one of the most prevalent viruses that infect grapevines (Meng & Gonsalves, 2003).
Despite the recent efforts towards understanding the genetic diversity of GRSPaV (Meng et al., 1999b; Rowhani et al., 2000; Casati et al., 2003; Santos et al., 2003; Terlizzi & Credi, 2003), an extensive analysis and an overall picture of the genetic diversity and population structure of GRSPaV are still lacking. After this study was completed, Nolasco et al. (2006) reported on the genetic variability of GRSPaV isolates collected from Portugal. The present study involved extensive analyses of the genetic diversity and population structure of GRSPaV, since it was based entirely on direct analysis of nucleotide sequences derived from GRSPaV-infected grapevines belonging to three Vitis species and various interspecific hybrids. Furthermore, the isolates assayed included a number of commonly used scion and rootstock varieties. Several important findings were made. First, in agreement with the report of Nolasco et al. (2006), we identified four distinct groups of GRSPaV variants. Second, we demonstrated that the scion varieties assayed were mostly infected with a mixture of distinct viral variants (Table 2). Third, we revealed, for the first time, that the viral population in GRSPaV-infected rootstocks, or at least the ones assayed here, consists of a homogeneous population of identical or nearly identical variants from the same lineage (Fig. 1b; Table 2). Last, we revealed specific associations between two of the variant groups with two of the North American Vitis species as well as their progeny, shedding light on the possible origin and evolution of GRSPaV.
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should be designated ‘GRSPaV-1’ lineage; those from our group II and group 2a of Nolasco et al. (2006) should be designated ‘GRSPaV-SG1’ lineage; the viral variants from our group III and group 3 of Nolasco et al. (2006) should be designated ‘GRSPaV-BS’ lineage; whilst those from our group IV based on primers RSP21/RSP22 and those from group 1 of Nolasco et al. (2006) should be designated ‘GRSPaV-VS’ lineage. The last lineage is so named in recognition of detection of similar viral sequences from wild Vitis sylvestris by Nolasco and associates. We recommend that sequences obtained in the future also be treated in a similar fashion.
As shown in Table 2, sequence variants of the GRSPaV-SG1 lineage were the most prevalent, as they were detected in all scion varieties assayed, as well as in two rootstock varieties. Sequence variants of the GRSPaV-1 lineage were the second most prevalent and were detected in six of the scion varieties and three rootstock varieties. Sequence variants of the GRSPaV-BS lineage were less common and were detected in five of the scion varieties. Sequence variants of the GRSPaV-VS lineage seemed to be the least prevalent, as they were detected only in three of the scion varieties. Moreover, the latter two lineages were not detected in any of the rootstock varieties assayed.
Interestingly, very different patterns of population structure were identified for isolates derived from scion varieties versus those from rootstock varieties. All of the isolates from rootstock varieties contained a homogeneous population of a single type of viral variant of either the GRSPaV-1 or the GRSPaV-SG1 lineage. In contrast, nine of the 10 isolates from scion varieties comprised sequence variants that belonged to two, three and possibly even four lineages. The isolates derived from two of the French–American hybrid scion varieties, ‘Ravat 34’ and ‘Seyval’, clearly comprised variants of three lineages. The interspecific hybrid scion variety ‘Niagara’ seemed to be infected with variants from all four lineages. However, each of the two pairs of primers detected only two groups of variants. Further research is needed to determine whether the isolate derived from ‘Niagara’ indeed comprises four distinct sequence variants. Mixed infections with divergent GRSPaV variants have been reported recently by others (Rowhani et al., 2000; Casati et al., 2003; Nolasco et al., 2006).
What is the explanation of the phenomenon that scion varieties are infected mostly with mixtures of divergent sequence variants, whilst rootstock varieties are infected with a homogeneous population of a single variant? Based on the seemingly specific association between the type of sequence variants and the genotypes of the host plant, we propose the following scenario. GRSPaV-1 may have co-existed with V. riparia for a long time. Likewise, GRSPaV-SG1 may have been in co-existence with V. rupestris since ancient times. As V. riparia and V. rupestris grapevines or their hybrids have been used commonly as rootstocks on to which different scion varieties are grown, viruses carried in these rootstocks could have been transmitted into the scion varieties through grafting. The same scion variety may have been grafted on to different rootstocks at different times and in different geographical regions. As a result, different viral variants accumulated in the scion varieties, leading to the mixed infection of many of the commonly used scion varieties with multiple sequence variants of the virus. This more recent viticultural practice of grafting, coupled with the asymptomatic infection of GRSPaV and frequent regional and international exchange of grapevine propagation materials, may have played a major role in the commonly observed mixed infection of scion varieties.
It is worth noting that the genetic variability detected may encompass mutations resulting from the polymerases used in RT-PCR. In general, RT-PCR produces less than 0.5 % errors (Teycheney et al., 2005). Nonetheless, such low levels of errors should not have much impact on the validity of our conclusions. Nor can the mixed infections be explained by accumulation of point mutations within the same plant over time. Despite the fact that viral RNA-dependent RNA polymerases possess a higher mutation rate due to their lack of proofreading capacity, an RNA virus may not change much with time unless it adapts to a new host. As stated by Gibbs et al. (1999), ‘a potential to vary need not result in variability, as the latter depends on the balance between mutation and selection’. Relative genetic stability has been demonstrated for other plant RNA viruses. For example, isolates of Turnip yellow mosaic virus from Europe and Australia differ by only 3–5 % in nucleotide sequence, despite their isolation for at least 13 000 years (Gibbs et al., 1999). Thus, it is more likely that the mixed infection of scion varieties with diverged variants of GRSPaV has resulted from separate introduction of distinct sequence variants into the same plants.
Grapevines are host to a large number of taxonomically distinct viruses. Several recent studies have demonstrated that mixed infections of grapevine scion varieties with divergent variants of several other viruses, including Grapevine fanleaf virus (Vigne et al., 2004), Grapevine leafroll-associated viruses 1 and 3 (Little et al., 2001; Turturo et al., 2005), Grapevine virus A (Goszczynski & Jooste, 2003) and Grapevine virus B (Shi et al., 2004). Mixed infections have also been reported for citrus with Citrus tristeza virus (Ayllón et al., 2001; Rubio et al., 2001) and for apple with Apple chlorotic leaf spot virus (Candresse et al., 1995). It will be interesting to examine whether rootstocks also play a role in these situations.
It remains an open question as to how the wild Vitis species became infected with different variants of GRSPaV in the first place. It is possible that the ancestor of GRSPaV was with Vitis from the start. As different Vitis species diverged, the virus also co-evolved and diverged. As a result, GRSPaV-1 adapted to V. riparia, whilst GRSPaV-SG1 adapted to V. rupestris and it is only within the past one and a half centuries that these two viral variants were brought into V. vinifera varieties through grafting. Alternatively, both viral variants may have existed in V. vinifera varieties and been transmitted horizontally to V. riparia and V. rupestris rootstocks by insect vectors or by humans. Testing of wild-grown species of Vitis that are in geographical separation from V. vinifera will probably provide clues that are important in answering this question.
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ACKNOWLEDGEMENTS |
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Received 15 September 2005;
accepted 7 February 2006.
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