Grapevine virus A variants of group II associated with Shiraz disease in South Africa are present in plants affected by Australian Shiraz disease, and have also been detected in the USA

Authors


E-mail: GoszczynskiD@arc.agric.za

Abstract

Primers were designed for RT-nested PCR amplification of the highly variable 293-nt fragment from the 5′ terminal part of the Grapevine virus A (GVA) replicase gene, specific to South African variants of molecular groups I and II. This new technique, along with RT-PCR for simultaneous amplification of variants of groups I, II and III, as well as cloning of amplicons, single-strand conformation polymorphism (SSCP) analysis of clones and sequencing, were used to investigate the populations of variants infecting 16 local Shiraz grapevines with different Shiraz disease (SD) status. The techniques were also used to study variants in GVA-infected grapevines from Australia and the USA. The Australian grapevines included seven plants of cvs Shiraz and Merlot affected by Australian Shiraz disease (AuSD), and one plant of cv. Crimson Seedless with unknown AuSD status. Grapevines from the USA included plants of cvs Chardonnay, Thompson Seedless and an unknown cultivar. The results confirmed the association of certain genetic variants of group II with SD and showed the common presence of these variants in AuSD-affected grapevines from Australia. Interestingly, a variant of this group was also detected in grapevine cv. Chardonnay from the USA, although the disease has not yet been reported from that country. The study also supports an earlier observation that members of group II, closely related to variant GTR1-2, are not associated with the disease. The variants were found only in SD-free grapevines. Results show that variants of the most divergent group III, which are common in South Africa, are also present in Australia and the USA. These variants are not associated with SD, but frequently occur in mixed infections with members of group II in plants affected by this disease in South Africa.

Introduction

Shiraz disease (SD) is of great concern to the South African grapevine industry because it is highly destructive to grapevines of noble cultivars, such as Shiraz and Merlot, and is spreading naturally in vineyards. SD-affected plants are easy to identify in vineyards as the canes do not mature. Instead, they remain green for an extended period through the season and are rubbery in texture. Also, the leaves are shed much later than in uninfected grapevines (Goussard & Bakker, 2006). Once infected plants show symptoms of SD, they never recover, and die within 3–5 years. Shiraz disease is latent in most cultivars and rootstocks, but it can be transmitted easily from these to SD-susceptible grapevines by grafting on infected rootstocks or by topworking of grapevines. The importance of this destructive disease is emphasized by the fact that the Shiraz (syn. Syrah) cultivar is planted widely worldwide, especially in Australia, France and the USA. A disease similar to SD was reported from Australia, and has been temporarily named Australian Shiraz disease (AuSD) (Habili et al., 2003; Habili & Randles, 2004). Although infected vines in Australia do not usually die, their yield is greatly reduced.

Shiraz disease was first reported in 1985 from South Africa (Corbett & Wiid, 1985). Since then, its suspected viral aetiology has remained a mystery. In 2003, it was shown that SD is transmitted by the mealybug Planococcus ficus, and then it was found that a vitivirus, Grapevine virus A (GVA), was closely associated with the disease (Goszczynski & Jooste, 2003a). Results suggest that GVA is also associated with AuSD (Habili et al., 2003; Habili & Randles, 2004). Studies have revealed extensive genetic heterogeneity of this virus in South African vineyards (Goszczynski & Jooste, 2003b). Three divergent molecular groups of the virus (I–III) were identified. Results showed that the variants of molecular group II were closely associated with SD, and variants of molecular group III were present in GVA-infected, SD-susceptible grapevines that did not express symptoms of the disease (Goszczynski, 2007). It was further revealed that, although the majority of variants of molecular group II were associated with strong symptoms of the disease, a variant of this group, GTR1-2, was present in a consistently SD-free Shiraz plant. The genome of this putative nonpathogenic to Shiraz variant of GVA is clearly different from variants associated with SD in open reading frames 1 (ORF1), 2 and 3, encoding replicase, a protein of unknown function and the movement protein, respectively, as well as in the 5′ untranslated region (5′UTR) and 3′UTR. An especially high divergence was found at the 5′ terminal of ORF1, between the methyltransferase and AlkB domains (Goszczynski et al., 2008). In addition, a 119-nt recombinant ORF2 GVA fragment was discovered in the variant GVA P163-M5 of molecular group II, and this clearly induced more severe symptoms in the alternative herbaceous host, Nicotiana benthamiana, than other variants of this group (Goszczynski et al., 2008). This variant was isolated from grapevine cv. Cinsaut Blanc used by the grapevine industry as a reliable positive control source of SD in woody indexing. In summary, all the research data accumulated to date suggest that certain genetic variants of GVA of group II are involved in the aetiology of Shiraz disease.

The aims of this study were to (i) develop a new RT-nested PCR technique for sensitive detection of GVA variants of group II, (ii) confirm the association of certain genetic variants of molecular group II with Shiraz disease, and (iii) investigate the presence of these variants in grapevines affected by Australian Shiraz disease.

Materials and methods

Cane samples of GVA-infected cv. Shiraz grapevines, comprising 12 plants affected by SD and 12 plants free of visible symptoms of the disease, were collected from Groenhoff vineyard in the Western Cape, South Africa, in 2000. DsRNA was isolated from these grapevines immediately after the plants were collected, and stored at −20°C. All local grapevines, those affected as well as those not affected by the disease, also tested positive for an ampelovirus, Grapevine leafroll-associated virus 3 (GLRaV-3) (D. E. Goszczynski, Plant Protection Research Institute, Pretoria, unpublished data) The overseas GVA-infected grapevines used in this study comprised seven AuSD-affected plants of cvs Shiraz and Merlot and one plant of cv. Crimson Seedless from Australia and three plants from the USA. Plants from the USA included grapevine cvs Chardonnay, Thompson Seedless and an unknown cultivar, indicated in this study as 2USA, 6USA and 8USA, respectively.

Detailed virus status records available for seven GVA-infected Australian grapevines are shown in Table 1. These grapevines were tested for a wide range of viruses, as described by Habili & Randles (2002). According to this data, five grapevines were also infected with the ampeloviruses, Grapevine leafroll-associated virus 1 (GLRaV-1) and/or Grapevine leafroll-associated virus 9 (GLRaV-9). Only two samples were ampelovirus-free (Table 1). The three GVA-positive grapevines from the USA were also infected with GLRaV-3 (plant 2USA) and GLRaV-4 (plants 6USA and 8USA) (Dr J. Monis, Eurofins STA Laboratories, California, USA, personal communication).

Table 1.   Results of detailed virus status analysis of selected Australian grapevinesa used in this study
  1. 0: negative; +: positive; nt: not tested.

  2. aAll grapevines were infected with AuSD except plant 10Au (Crimson Seedless), for which the disease status was not known.

  3. bLR1–9: Grapevine leafroll-associated virus 1–9 ; GRSPaV: Grapevine rupestris stem pitting-associated virus; GVA: Grapevine virus A; GFkV: Grapevine fleck virus (variants A and B); GFLV: Grapevine fanleaf virus; ArMV: Arabis mosaic virus; Phyto: phytoplasmas.

Grapevine codeCultivarLR1bLR2LR3LR4LR5LR9GRSPaVGVAGFkV-AGFkV-BGFLVArMVPhyto
3AuSD+Merlot0000ntnt+++0000
4AuSD+Shiraz+000ntnt++0000nt
5AuSD+Shiraz+0000+++0000nt
6AuSD+Merlot+00000+++0000
7AuSD+Shiraz00000+++0000nt
10AuCrimson Seedless0nt0ntnt+nt+ntntntntnt
11AuSD+Shiraz000000++0000nt

Isolation of dsRNA from South African grapevines, RT-PCR amplifications, purification of PCR amplicons from agarose, cloning, SSCP analysis of clones, sequencing and sequence analysis were all carried out as described by Goszczynski (2007). In the case of overseas grapevines, total RNA was purified using a QIAGEN RNeasy Mini kit. One microlitre of this RNA was used in 10 μL reverse transcription (RT) in the laboratory. Two sets of GVA-specific primers were primarily used in RT-PCR amplifications in this study: V1F, V2R, V3F and V4R (this work), and MP2F, MP1R (Goszczynski, 2007). The first set of primers was used in RT-nested PCR. The primer pair V1F/V2R (5′-TGCTTGGAGAGATTTATCAGGGC-3′/5′-GCCTTATCCCAACCCAG-3′) was used for RT and the first round of PCR, and the primer pair V3F/V4R (5′-TTCAGATTCATGGAGAG-3′/5′-CCACCGGTGTAGCTGTA-3′) was used for the second round of PCR. Reverse transcription (RT) was performed at 42°C for 1 h. Thermal cycling parameters in the first round of PCR were as follows: one cycle of 94°C for 4 min; 35 cycles of 94°C for 30 s, 65°C for 30 s, 72°C for 1 min; and a final elongation at 72°C for 5 min. Thermal cycling parameters in the second round of PCR were exactly the same as those described for the first round of PCR, except that the annealing temperature of the primers was 56°C. The primer pair MP2F/MP1R (5′-TCTGAACAAGGCCCTGCA-3′/5′-AGATTCTTGCCATGGGGCAT-3′) (Goszczynski, 2007) was used in RT-PCR. Thermal cycling parameters were as described above for RT and the second round of nested PCR. The final amplicons were about 293 and 254 bp, corresponding to the replicase (ORF1, V3F-V4R) and movement protein genes (ORF3, MP2F-MP1R) of the virus, respectively.

SSCP analysis of cloned sequences were carried out in 15% acrylamide/bis-acrylamide (29·2/0·8), 0·75-mm gels in 0·5× TBE buffer at 5°C for 1 h, using Mini-protean II dual slab cell (Bio-Rad) (Goszczynski, 2007).

Nucleotide and amino acid sequence alignments, and analyses of the homology of the sequences (percentage identity) were carried out using the dnaman version 5·2·9 (Lynnon Biosoft, 1994–2001, Quebec) software package. Percentages of similarity of amino acid sequences (% of positives) were determined using the Basic Local Alignment Search Tool (blast) of the National Centre for Biotechnology Information (NCBI), Bethesda, USA. Phylogenetic analyses were conducted using mega version 4 (Tamura et al., 2007). Phylogenetic trees were constructed with the neighbour-joining method (Saitou & Nei, 1987) using evolutionary distances calculated according to the method of Kimura (1980). A bootstrap analysis of the data, based on 1000 permutations, was used to assess the statistical confidence of the topologies of phylogenetic trees.

Included in the analyses were sequences from the following isolates of GVA: Is151, GTG11-1, BMO32-1, KWVMO4-1, P163-M5, GTR1-2, GTR1-1 and P163-1, which were deposited in the GenBank/EMBL database with accession numbers X75433, DQ855084, DQ855087, DQ855083, DQ855082, DQ855086, DQ787959 and DQ855088, respectively. The sequence data of Grapevine virus B (GVB) and Grapevine virus E (GVE), with accession numbers X75448 and AF057136, were also used in the study.

Results

Designing primers specific to the variable part of the replicase gene, and their evaluation in RT-nested PCR

Several forward and reverse oligonucleotide primers, flanking the highly variable 5′ terminal part of the replicase gene, were designed manually from the alignments of sequences of variants representing all three molecular groups (I–III) of GVA variants identified in South Africa (Goszczynski et al., 2008) (alignments not shown). The initial aim was to design primers for the specific and sensitive RT-nested PCR amplification of variants of group II. The dsRNAs extracted from N. benthamiana infected with various members of groups I (GTG11-1), II (BMO32-1, KWVMO4-1, GTR1SD-1, P163-M5 and GTR1-2) and III (P163-1) were used to evaluate the quality of the primers. The primer pairs V1F/V2R and V3F/V4R were selected. Results revealed that, in addition to members of group II, a variant of group I was also amplified using these primers. Only the P163-1 variant of group III was not detected (results not shown). Thus, results suggest that the technique is specific to GVA variants of groups II and I. The virus amplifications were strongly positive even when the annealing temperature in the first round of RT-nested PCR was increased to 65°C. This high annealing temperature was used in all later applications of the technique. The variant-specificity of the primers was confirmed by cloning the amplicons and sequencing (results not shown). The amplicons of about 293 bp were complementary to the GVA sequence located between 1659 and 1951 nt in the virus genome. As reported earlier, the virus is most variable in this region (Goszczynski et al., 2008). Results of the analysis indicate that this variability existed not only between variants of different groups, but also among members of the same group. Variants of group II associated with strong symptoms of SD shared, respectively, 73·7–89·4% and 78·6–91·8% nucleotide and amino acid identity in this genomic region. However, the amino acid sequence similarity of these variants, 90–98%, made them a uniform group, clearly divergent from the putative nonpathogenic variant of group II, GTR1-2 (Introduction), sharing 81–85% amino acid similarity (Table 2). The variants of the group II associated with SD were highly divergent in this region from variants of groups I and III, sharing only 62–72% amino acid similarity (Table 2).

Table 2.   Molecular divergence within group II of Grapevine virus A (GVA) variants associated with Shiraz disease (SD) and between these and other variants, including the putative nonpathogenic variant GTR1-2 of this group, in the extensively variable fragment of the 5′ terminal of the replicase genea
  1. aThe 293-nt fragment of ORF1 corresponding to amplicon of variants of groups I and II obtained in RT-nested PCR based on primers pairs V1F/V2R and V3F/V4R.

  2. b’Amino acid similarity’ refers to positive substitutions determined using NCBI BLAST.

  3. cGTR1SD-1, BMO32-1, KWVMO4-1 and P163-M5.

  4. dGTG11-1 (group I), P163-1 and GTR1-1 (group III).

GVA variantsNucleotide identity (%)Amino acid identity (%)Amino acid similarity (%)b
Associated with SDc73·7–89·478·6–91·890–98
GTR1-270·0–73·067·3–71·481–85
Otherd57·0–64·549·0–58·262–72

GVA variants RT-nested PCR amplified from local and overseas grapevines using the primer pair V1F/V2R and V3F/V4R

Using the RT-nested PCR technique, strong amplifications of GVA were obtained for all 12 SD-affected South African Shiraz plants used in this study. Unexpectedly, clearly positive amplifications were also obtained for most of the SD-free plants (not shown). The amplifications were also strongly positive for six out of eight RNA extracts of AuSD-affected plants from Australia, and all three GVA-positive grapevines from the USA. Two AuSD-affected plants, 4AuSD+ and 5AuSD+, were consistently negative using this technique (Table 3).

Table 3.   Summary of Grapevine virus A (GVA) variants of molecular groups I, II and III, which were identified in this study in local and overseas grapevines with various Shiraz disease (SD, AuSD) status
  1. nt: samples not tested; –: samples very weakly or not amplified in RT-PCR.

  2. aAccording to disease symptoms at the time of sampling.

  3. bSee Figures 1a, 2b and 3b.

  4. cVariants closely related to putative nonpathogenic variant GVA GTR1-2 (see Fig. 1a).

  5. dVariants which cluster in group III in Figure. 1a, but also show visible divergence from variants of this group.

  6. eGrapevines of cultivars not susceptible to Shiraz disease.

Plant disease statusaPlant codePrimers used in RT-PCR and GVA variants identified after sequencing of PCR ampliconsbGVA variants status of plants
V1F/V2R + V3F/V4RMP2F/MP1R
SD+GT4IIntII
GT6IIntII
GT9I, IIntI, II
GT14I, IIntI, II
GT15IIntII
GTG102010SD+III, II, IIII, II, III
SD−GTG1IIcntIIc
GTG2I, IIcI, IIII, IIc, III
GTG3IIIIII
GTG4IIII, IIII, II, III
GTG7I, IIc, IIIdntI, IIc, IIId
GTG8IIcntIIc
GTG9IntI
GTG102000SD−IIIIII
GTG102010SD−II, IIII, III
GTR4II, IIII, III
GTR5I, IIcntI, IIc
AuSD+3AuSDIIIIII
4AuSDIIII
5AuSDII, IIIII, III
6AuSDIIII, IIIII, III
7AuSDIIII
11AuSDIIII
12AuSDIIII
Unknowne10AuIIIdIIId
2USAIIII, IIIII, III
6USAII
8USAII

Six and 10 selected PCR products of amplifications of GVA from South African Shiraz grapevines affected and not affected by SD, respectively, and all products of amplifications of GVA from Australia and the USA were cloned and sequenced. Phylogenetic analysis of the sequence data showed that variants of group II associated with SD were present in all six SD-affected South African grapevines (Table 3; Fig. 1a), confirming the earlier results reported by Goszczynski (2007). Of the six GVA-positive Australian grapevines, variants of group II associated with SD were detected easily in five plants, all of them affected by AuSD (Table 3; Fig. 1a). Surprisingly, the analysis revealed that a variant of group II closely related to variants associated with SD was amplified from one sample, 2USA (grapevine cv. Chardonnay), from the USA. In the remaining two USA grapevines, 6USA and 8 USA, only variants of group I were found (Table 3; Fig. 1a).

Figure 1.

 (a) Phylogenetic tree depicting Grapevine virus A (GVA) variants of three molecular groups (I–III), which were identified in local (SD−, SD+), Australian (highlighted) and USA grapevines with various Shiraz disease (SD, AuSD) status (Tables 1 & 3) using RT-nested PCR based on primers V1F/V2R, V3F/V3R, cloning, SSCP analysis of clones and sequencing. Reference GVA variants are boxed. Arrows indicate sequences of variants of molecular group II associated with SD, which were amplified from South African SD-negative plants GTG3 and GTG4, and a cv. Chardonnay grapevine from the USA (DG36.2USA). Broken lines indicate two putative pathogenic groups of group II variants. In the analysis, sequence data for Grapevine virus B (GVB) and Grapevine virus E (GVE) were also used as outgroups. (b) SSCP analysis of clones of GVA sequences which were RT-nested PCR amplified from four South African SD-negative Shiraz grapevines (GTG1, GTG7, GTG8, GTG9). Selected clones indicated by numbers were sequenced. Boxed numbers indicate sequences closely related to putative nonpathogenic variant GTR1-2 of molecular group II. Other sequences clustered in groups I and III [see (a)]. ‘?’ indicates a sequence with unknown identity.

The variants of group II associated with SD were also found in two SD-negative South African grapevines, GTG3 and GTG4 (Table 3; Fig. 1a). At the time of sampling, the grapevines did not express any visible symptoms of SD. The variants were not detected in the remaining eight Shiraz plants free of this disease. These grapevines were infected with either GVA variants of group I, or variants of group II closely related to GTR1-2, the putative nonpathogenic variant (Table 3; Fig. 1a). The results of SSCP analysis of the population of variants of SD-free Shiraz plants GTG1, GTG7, GTG8 and GTG9 are shown in Figure 1b. The results strongly suggest that the SD-negative grapevines GTG1 and GTG8 were dominated by variants closely related to variant GTR1-2, while grapevine GTG7 was infected with a mixture of variants of group I and those related to group III (see below), and variants closely related to GTR1-2 (Fig. 1a). In plant GTG9, only variants of group I were found. The fact that GTR1-2-related variants were only detected in SD-negative grapevines supports the hypothesis that these variants of group II are not associated with Shiraz disease (Goszczynski & Jooste, 2003a; Goszczynski, 2007; Goszczynski et al., 2008).

Unexpectedly, the sequence data revealed positive amplifications of two variant sequences which clustered with variants of group III (Fig. 1a). One of these variants was present in SD-free grapevine GTG7.SD– from South Africa, and another, represented here by clone DG30.10Au in a cv. Crimson Seedless grapevine from Australia (Fig. 1a). Detectable phylogenetic divergence of these variants from other variants of group III (Fig. 1a) suggests that they may represent a new, yet unknown, group of variants of GVA. Results of a preliminary investigation suggest that this putative new and group III-related GVA variant was the only, or the clearly dominant, variant in the Australian Crimson Seedless grapevine. The amplification of the DG30·10Au sequence from this Australian grapevine, which is related to variants of group III, raised the question whether variants of group III, which are common in South Africa (Goszczynski & Jooste, 2003b; Goszczynski, 2007), are also present in overseas vineyards. The variants were not detected in Italy (Murolo et al., 2008).

GVA variants RT-PCR amplified from local and overseas grapevines using primers MP2F/MP1R

According to earlier results (Goszczynski, 2007), RT-PCR based on primers MP2F/MP1R specifically amplifies the ORF3 fragment of variants of groups I, II and III identified in South Africa. An additional advantage of the primers is that denatured amplicons of group II variants migrate in 15% polyacrylamide gels as clearly ‘diffused’ bands. The phenomenon has been observed so far only for members of group II (Goszczynski, 2007), and can be used for rapid identification of these variants. Surprisingly, strongly positive RT-PCR amplifications, using MP2F/MP1R primers, were obtained only for four out of seven GVA-positive AuSD-affected plants from Australia (results not shown). The remaining three AuSD-affected plants, 7AuSD+, 11AuSD+ and 12AuSD+, which, according to previous experiments (Table 3) were infected with variants of group II, and a plant, 10Au, infected with a variant phylogenetically related to members of group III (Fig. 1a), were weakly positive. In the case of the three USA samples, all of them were positively amplified but strong amplification was obtained only for one sample, 2USA (grapevine cv. Chardonnay) (results not shown). DsRNAs extracted from four SD-negative plants of South African Shiraz (GTG2, GTG3, GTG4 and GTR4) were also used in the amplifications. In this case, the primary targets were SD-negative plants GTG3 and GTG4, in which only variants of group II associated with SD were found earlier (Table 3; Fig. 1a). Other dsRNAs, purified from SD-negative plants GTG2 and GTR4, were used as a control to monitor amplifications of the variants of various groups by the technique. GVA was strongly amplified from all these local grapevines (not shown).

The products of all strongly positive amplicons of GVA from overseas and local grapevines were cloned. As expected, SSCP analysis of clones and the sequence data revealed that AuSD-affected Australian plants were infected with variants of group II that clustered with variants associated with SD in South Africa (Fig. 2b). SSCP results suggested that plants 3AuSD+ and 4AuSD+ were dominated by these variants (Fig. 2a). Variants of group II were also found in the grapevine 2USA from the USA. Variants of group III were readily detected in plants 5AuSD+, 6AuSD+ and 2USA. Results suggested that the population of these variants was dominant in grapevine 2USA (Fig. 2a). Surprisingly, this time, only variants of group II associated with SD were found again in SD-negative South African plant GTG3 (Table 3; Fig. 2). This suggested that these were the only, or the clearly dominant, variants in this plant. Variants of group III were easily detected in another SD-negative plant, GTG4, in which only the group II variants associated with SD were found previously (Table 3; Fig. 2). The other SD-negative plants used in this analysis, GTG2 and GTR4, were both only infected with a mixture of variants of groups I and III (Table 3). Interestingly, group II variants associated with SD, which were present in SD-free Shiraz plants GTG3 and GTG4, had no ‘diffused’ SSCP patterns. The diffused pattern was also hardly visible for these variants present in plant 2USA, contrary to the clearly diffused patterns observed for the group II variants from all AuSD-affected plants (Fig. 2a). Because of the limited number of samples investigated in this work, it is not clear how common this feature is and what it means. Previous results indicated that the ‘diffused’ form of the DNA strand can be transformed to the ‘not-diffused’ form by only one nt change (Goszczynski, 2007).

Figure 2.

 (a) Selected results of SSCP analysis of clones of Grapevine virus A (GVA) variants RT-PCR amplified from various overseas and local grapevines using primers MP2F/MP1R. GVA-infected grapevines cv. Shiraz affected by Australian Shiraz disease (AuSD+) from Australia and not exhibiting Shiraz disease symptoms (SD−) from South Africa, plus a cv. Chardonnay grapevine from the USA (2USA) were studied. Clones indicated by numbers were sequenced. Circled numbers indicate variants of group II which cluster with members of this group associated with Shiraz disease. Other variants identified were members of groups I and III. ‘pp’ indicates a sequence encoding a plant protein. (b) Phylogenetic analysis of sequence data obtained. Sequences of group II amplified from SD-negative Shiraz GTG3 are indicated by a vertical line, and those of groups II and III amplified from the other SD-negative grapevine, GTG4, by diamonds. Variants amplified from a USA grapevine are shaded. Reference GVA variants are boxed. Sequence data for Grapevine virus B (GVB) and Grapevine virus E (GVE) were also used.

GVA variants identified in SD-free and SD-affected grapevines propagated from SD-free mother plant

In 2000, cuttings of SD-negative Shiraz, GTG102000SD− were collected, and a dsRNA extract was prepared. Some of these cuttings were successfully rooted and potted. Two plants became established and were used in this study. The plants did not show symptoms of SD for 10 years. In the summer of 2010, one of them exhibited mild symptoms of this disease. DsRNAs were purified from these two grapevines. DsRNA from these, and from the original mother plant purified in 2000, were used in amplifications using primers V1F/V2R and V3F/V4R and MP2F/MP1R, reported in this paper. The virus was positively amplified using the first set of primers, V1F/V2R and V3F/V4R, and only from dsRNAs purified from plants GTG102010SD− and GTG102010SD+. In this case, cloning of the amplicons, SSCP analysis of clones and sequencing revealed homogenous populations of variants of group I (represented by clone 49.GTG102010SD−) in the SD-negative plant, and group II associated with SD (represented by clone 51.GTG102010SD+) in the SD-affected plant (Fig. 1a). Amplifications using the second set of primers, MP2F/MP1R, specific to variants of groups I, II and III, were successful for all dsRNAs, including those isolated from the mother plant GTG102000SD−. This time the SSCP analysis and sequencing of clones revealed variable populations of variants (Fig. 3a, b). Only variants of group III were found in the mother plant, GTG102000SD−. The population of these variants could be divided into two subpopulations (Fig. 3b). Identical variants of both subpopulations were found in grapevines GTG102010SD− and GTG102010SD+, confirming the common origin of these three plants. In addition, variants of group I were detected in plants GTG102010SD− and GTG102010SD+. The variants of this group were not detected in the mother plant GTG102000SD−. Variants of group II associated with Shiraz disease were found only in SD-affected plant GTG2010SD+ (Fig. 3a, b). SSCP patterns of 64 clones, and sequence data of selected clones, suggested that only six clones were sequences of the group II variants associated with SD. This suggests that the variants were minor in plant GTG102010SD+. As mentioned above, this plant exhibited mild symptoms of the disease. The results suggest that variants of group III were dominant in this plant (Fig. 3a).

Figure 3.

 (a) Results of SSCP analysis of clones of sequences RT-PCR amplified from Shiraz disease (SD)-free grapevine cv. Shiraz, GTG102000SD−, and two plants propagated from this grapevine, GTG102010SD− and GTG102010SD+, with different SD status. Primer pair MP2F/MP1R was used in amplifications. Clones indicated by numbers were sequenced. Circled numbers indicate clones of group II variants associated with SD. Others are clones of variants of groups I and III. ‘pp’ indicates sequences encoding plant proteins. (b) Phylogenetic analysis of obtained sequence data. Reference variants are boxed. Sequence data for Grapevine virus B (GVB) and Grapevine virus E (GVE) were also used.

Discussion

The results presented in this study and summarized in Table 3 confirm an association of certain genetic variants of group II with South African Shiraz disease reported earlier by Goszczynski (2007). Using an RT-nested PCR technique based on a set of primers V1F/V2R and V3F/V4R, specific to the variable part of the replicase gene, as well as cloning of amplicons, SSCP analysis of clones and sequencing, group II variants associated with Shiraz disease were found readily in all Shiraz grapevines affected by the disease. These variants were not detected in eight out of 10 Shiraz plants free of this disease. The exceptions were two SD-negative plants, GTG3 and GTG4, in which these group II variants were also present. The plants were symptomless at the time of sampling. It is possible that the disease in these plants may have been latent at an early stage of infection, and that the group II variants associated with the disease were at low titre, dominated by GVA variants not associated with SD. This possibility seems probable for plant GTG4, in which variants of group III were also readily detected using primers MP2F/MP1R (Fig. 2a). However, it cannot explain the absence of the disease in the plant GTG3. SSCP analysis of cloned GVA sequences amplified from this plant suggested that the variant of group II associated with SD was the only variant, or was clearly dominant in this plant (Fig. 2a), and that the plant should express symptoms of the disease. As the exact aetiology of SD is not yet known, it is possible that another pathogen, along with GVA variants of group II, is necessary to induce this disease. Also, it cannot be ruled out that the variant of group II associated with SD present in the SD-free GTG3 plant was a recombinant, or a mutant that was not able to induce the disease.

The suggested association of GVA variants of group II with Shiraz disease is supported by the presence of these variants in plants affected by AuSD in Australia. The variants were detected in all seven samples of RNA extracted from AuSD-affected plants. It is important to note here that the result combines the results of the application of two sets of primers V1F/V2R and V3F/V4R and MP2F/MP1R in the amplifications of GVA from Australian grapevines. The primers, targeting ORF1 and ORF3 of GVA, respectively, were designed using the sequence data of South African variants of the virus and, although all local variants of group II were efficiently amplified by both sets of primers, none of them detected all the Australian variants of this group. This also seems to apply to overseas variants of groups I and III (Table 3).

The association of group II variants with SD is also supported by the results of GVA variants analysis of two Shiraz grapevines, GTG102010SD− and GTG102010SD+, with different SD status, which were propagated from an SD-negative mother plant, GTG102000SD−. The variants of group II associated with SD were found only in the SD-affected plant. In SD-negative plants, including the mother plant, only variants of group I and/or III were detected (Table 3). The absence of variants of group II in the mother plant suggests that the variant of this group present in the SD-affected plant was recently transmitted from another grapevine plant. For 10 years the GTG10 grapevines were kept as SD-free in the collection of SD-positive grapevines. The disease was recorded in only one of them in 2010. Similar results of GVA variants analysis were reported earlier for grapevine cv. Shiraz, GTR1SD, by Goszczynski (2007).

The results of this study clearly support the suggestion by Goszczynski (2007) that GVA variants of group II closely related to variant GTR1-2 are not associated with Shiraz disease. These variants were detected only in grapevines free of this disease (Table 3; Fig. 1). It is interesting to note that GTR1-2-related variants were never detected along with members of group II associated with SD.

The variants of group III, usually found mixed with the group II variants in SD-affected plants in South Africa (Goszczynski, 2007), were also easily detected in AuSD-affected Shiraz grapevines in Australia (Fig. 2). Although these variants alone are not associated with SD (Goszczynski, 2007), their common presence in mixed infections with group II variants associated with SD in South Africa is interesting. The detection of these variants in a grapevine from the USA suggests that they are present in vineyards worldwide, although group III variants were not found in a study in Italy (Murolo et al., 2008).

The results of this study, and those reported earlier (Goszczynski, 2007; Goszczynski et al., 2008), suggest that certain GVA variants of group II are associated with both SD and AuSD. Observations also suggest that a further pathogen may be necessary to induce SD in South Africa. All local GVA-infected grapevines used in this study were also infected with an ampelovirus GLRaV-3, which is associated with leafroll (LR) disease. Recently, GVA variants of group II associated with SD were found in a few grapevines which did not exhibit SD symptoms (D. E. Goszczynski, unpublished data). The plants did not exhibit symptoms of LR disease either, which suggested that the plants were not infected with GLRaV-3. The identification of these GVA variants was based on a short fragment of the GVA genome, and these particular variants might be different in other parts of the genome. However, there is another possible hypothesis. The variants of group II associated with SD are always easily detected and are the dominant variant populations in plants exhibiting strong cane symptoms of the disease (D. E. Goszczynski, unpublished data). This suggests that the variants are present at a high titre in affected plants. To reach a high titre in grapevine, the virus must defend itself from the plant’s defence system. The published data suggest that GVA has a relatively weak ability to suppress gene silencing (Zhou et al., 2006) compared to members of the Closteroviridae family (Chiba et al., 2006; Dolja et al., 2006). According to Dolja et al., (2006), these large and slowly replicating viruses have developed a multicomponent and multilevel counter-defence system. Effective suppression of the grapevine’s antiviral defence system by closteroviruses might be the reason that GVA in plants affected by Shiraz disease in South Africa always occurs together with GLRaV-3, a member of this family (D. E. Goszczynski, unpublished data). The virus is very common in local vineyards. This hypothesis is interesting and will be investigated. The AuSD may follow a different strategy, as it does not always kill affected grapevines and is not associated with GLRaV-3 (Table 1). However, it is intriguing that, according to detailed virus status records available for six AuSD-affected grapevines, four of these GVA-infected plants were also positive for ampeloviruses GLRaV-1 and/or GLRaV-9 (Table 1). It would be interesting to know whether AuSD symptoms in the affected plants were stronger when these ampeloviruses were present. Unfortunately, the data were not available.

The detection of a variant of group II, closely related to variants associated with SD in grapevine cv. Chardonnay from the USA is very interesting. The disease has not yet been reported from this country. In addition to GVA of group II, this grapevine was also infected with GLRaV-3. Thus, as is the case in South Africa, there appear to be all the viral components required to induce the disease. Although Chardonnay is not susceptible to SD, the disease can be transmitted easily to SD-susceptible grapevines by grafting and by the common vineyard pest, the mealybug P. ficus.

The accumulated data on the association of variants of GVA with SD and AuSD presented here and reported earlier (Goszczynski, 2007; Goszczynski et al., 2008) may elucidate the aetiology of these diseases during future experiments to transmit the virus to grapevines. At present, the virus can be transmitted mechanically relatively easily from grapevines to N. benthamiana, but attempts to transmit it back to grapevines (fulfillment of Koch’s third postulate) remain unsuccessful. Recently, clear progress was made using an infectious DNA clone of the virus (Muruganantham et al., 2008).

In summary, the present study (i) includes a new RT-nested PCR technique for sensitive detection of GVA variants of group I and II, (ii) firmly confirms the association of certain genetic variants of molecular group II with Shiraz disease, (iii) reveals the common presence of these variants in grapevines affected by Australian Shiraz disease, (iv) strongly suggests that GVA GTR1-2 related variants of molecular group II are not pathogenic to Shiraz (the variants were detected only in SD-negative plants), and (v) suggests that SD may be present in the USA because the variant of group II associated with SD in SA was detected in a grapevine from USA vineyards.

Acknowledgements

This research was partially supported by Winetech, South Africa. We thank Dr J. Monis (Eurofins STA labs in California) for providing the USA GVA isolates used in this study.

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