Received: 16 February 2017 / Accepted: 5 June 2017
© Springer Science+Business Media, LLC 2017
Waite Diagnostics, School of Agriculture, Food and Wine,
The University of Adelaide, Adelaide 5005, Australia
A total of 575 commercial grapevine (Vitis vinifera) samples from Australia have been tested for a newly emerging virus, Grapevine Pinot gris virus (Trichovirus,
Betaflexiviridae) during 2015–2017. Nine samples from two states tested positive. Six of these were from New South Wales and 3 from South Australia in a total of
eight varieties. All these varieties were imported from Europe within the last 4–19 years. A fragment of 431 nucleotides on the coat protein gene of Grapevine Pinot
gris virus was targeted for virus identification by RT-PCR. The virus specificity of each positive sample was confirmed by sequencing followed by the BLASTn analysis
which showed an identity of up to 99.3% to the virus sequences in the NCBI database. The phylogenetic tree as well as pairwise sequence identity showed that, although
the virus sequence in each variety was unique, they all grouped with the isolates from Europe and well away from the South Korean and several Chinese isolates. This is the first report of the occurrence of Grapevine Pinot gris virus in Australia.
Grapevine Pinot gris virus, Trichovirus phylogenetic tree, Single-tube RT-PCR, Sequence demarcation tool
Vitis Vinifera, an exotic crop to Australia, arrived with the first fleet of white European settlers in 1788 . At the same time, viruses, which are mostly limited to the phloem tissue, arrived with infected cuttings. In Australia, the gene-pool of exotic viruses in major crops such as grapevine is under strict control by adopting biosecurity measures at the borders. While up to 65 viruses infect the grapevine , none of the Australian native viruses has ever been detected in this host (N. Habili, unpublished).
Here, we report the entry of Grapevine Pinot gris virus (GPGV: Trichovirus, Betaflexiviridae) into Australia which has recently been discovered in Italy  using Next Gene Sequencing (NGS). The infected plants are either symptomless or show the symptoms of leaf mottling and deformation.
GPGV has been reported from Europe, South Korea, China, Canada and the United States . In the Bordeaux region of France, GPGV has been found in Vitis vinifera
cv. Merlot which was grafted onto the Gravesac rootstock . In Canada, GPGV has been detected in cultivars Syrah, Cabernet Franc, Riesling and Vidal Blanc as well as in a French-American Vitis hybrid . The virus has now been found in commercial vineyards at Napa Valley in cultivars Chardonnay, Cabernet Sauvignon and Cabernet Franc . It was also detected in the Foundation block of UC Davis (USA) in one plant of six of cv. Touriga Nacional which was imported from Portugal in 1981 . No symptoms were observed on this plant and no positive samples were found in the neighbouring vines, suggesting the lack of natural spread. The eriophyid mite, Colomerus vitis can transmit GPGV experimentally .
In the present survey, a 431-nt segment of the coat protein (CP) gene of GPGV has been targeted in singletube RT-PCR  using specific primers CP-F 50-ATAGCAGTTGAAGGGACCT-30 and CP-R 50- AAGCCGTGATAGCATTAGT-30 . We used the total nucleic acid (NA) extraction protocol of MacKenzie et al.  which was modified by the absorption of NA to a silica matrix as previously described . All the nucleic acid extracts were checked for their quality and adjusted to a similar
concentration before use. Bioline Tetro reverse transcriptase (Cat No. BIO-65050) and Bioline MyTaq SH DNA polymerase (Cat. No. BIO-21112) were added to a cocktail comprising 1:1 (v/v) mixture of the buffer for each enzyme supplied by the manufacturer (Bioline, England). BLASTn, Mega 7 and Sequence Demarcation Tool (SDT)  were
used for sequence analysis.
|Variety||Grower’s code||Accession no.||Location||Sample collection date||Year imported||% identityn|
|Tempranillo||T2||KY243032||Wentworth, NSWb||Nov. 2016||2006||99.0|
|Tempranillo||T5||KY243033||Wentworth, NSW||Nov. 2016||2006||99.3|
|Fiano||Fi12||KY243034||Wentworth, NSW||Nov. 2016||1998||98.1|
|Riesling||Ri16||KY243035||Wentworth, NSW||Nov. 2016||2006||99.0|
|Graciano||Gr35||KY243036||Wentworth, NSW||Nov. 2016||2006||99.3|
|Moscato Giallo||Mos38||KY243037||Wentworth, NSW||Nov. 2016||1998||98.8|
|Cabernet Sauvignon||1124 (Ca1)||KY415994||South Australia||Dec. 2016||2011||99.0|
|Cabemet Sauvignon||1125 (Ca2)||KY415994||South Australia||Dec. 2016||2011||99.0|
|Malbec||595||Nsc||South Australia||Dec. 2016||2011||Ns|
|a The % identity was calculated using 431 nt of the CP of the GPGV isolate Riesling 25-3 (Ac. No. KX522755) from Germany as a reference
b NSW New South Wales
c Not sequenced
Table 1 List of varieties tested positive for GPGV in Australia
Since June 2015, a total of 575 grapevine samples from various grape growing states in Australia have been tested. In November 2016, 19 commercial samples from New
South Wales (NSW) and in December 2016, 13 from South Australia (SA) were tested for virus (Table 1). The mother vines of these samples have been independently imported from Europe to Australia within the last 4–19 years. Six of the 19 samples from NSW were positive which belonged to the following varieties: Fiano, Graciano, Muscat, Riesling and Tempranillo (two clones). Of the 13 samples from SA, 3 samples from cv Malbec and Cabernet Sauvignon (two clones) tested positive as indicated by the presence of the 431 bp segment in the PCR gel. Each amplicon of the expected size was directly sequenced in duplicate at both orientations by Sanger sequencing (AGRF, Adelaide). The BLASTn analysis confirmed that the 431 bp sequence from each infected variety showed an identity of up to 99.3% with the GPGV CP gene in the database (Table 1). The 431 bp sequence of eight GPGV isolates was deposited in the NCBI database and compared with the available sequences using Mega 7 (Fig. 1).
Trans to Springer to view full of Figure Pic.
The phylogenetic tree indicated that, although the analysed sequence variant was unique to each variety, they showed up to 99.3% identity and grouped with the clade of
the European isolates well away from the Korean and Chinese isolates. This is confirmed by the amino acid sequence analysis from the same CP segment (not shown) as well as by pairwise sequence identity matrix using SDT . In this colour-coded pairwise sequence alignment programme the identities decreased from 100 to 93% while
the colour changed from dark red to dark blue (Fig. 2). The Australian isolates generally gave a red colour with European counterparts, indicating a higher pairwise sequence identity than those from China and South Korea which positioned in the blue zone (Fig. 2). In terms of sequence identity, SDT shows minor differences between two isolates more clearly than the phylogeny tree. For example, in Fig. 2, the virus isolate, Cab 2 from Australia is similar to the Mer isolate from France as confirmed by the data in the tree (Fig. 1). The major difference is their pairwise identity with the Slovakian isolate, SK13 (see KF134125_SK13 on x-axis in Fig. 2). In fact, the Australian Cab 2 was imported from France by a private importer (N. Habili, unpublished). These pairwise sequence footprints can be used to track the origin of the virus detected in imported cuttings.
It is important to note that 10 progeny plants vegetatively propagated from two mother plants of each clone of cv. Cabernet Sauvignon were infected while the neighbouring progeny from non-infected varieties tested negative, indicating the apparent lack of natural transmission of the virus as confirmed by the other workers .
Since GPGV is a new discovery arising from NGS, the association of the attributed symptoms  has not been confirmed and remains to be evaluated by biological indexing of the generated infectious cDNA clones of the viral RNA. When cuttings from symptomatic samples from the field were planted in a greenhouse, no symptoms were observed (Reynard, personal communication). The epidemiology of GPGV is further complicated following the detection of GPGV in two symptomatic herbaceous hosts in a vineyard in Italy . The threat of the GPGV invasion can be largely reduced by testing all recently imported mother plants and destroying virus positive vines and their progeny.
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