A method of delivering short synthetic double-stranded RNA (dsRNA) to stimulate RNA interference (RNAi)-mediated control for grapevine Pinot gris virus (GPGV) in grapevines was developed and evaluated in this study. The dsRNA molecule targeting the RNA-dependent RNA polymerase (RdRp) gene of the GPGV genome was designed and produced by a twostep polymerase chain reaction (PCR) approach followed by in vitro transcription of the amplicon. A significant decrease in virus titre was observed seven days after dipping shoot tips of GPGV-infected tissue culture (TC) plantlets into a solution of GPGV-RdRp-dsRNA followed by re-introduction to TC. The effect was more pronounced in shoot tips dipped in the GPGV-RdRp-dsRNA solution for 24 hours than in tips dipped for two hours. This study represents the first successful demonstration of dsRNA-mediated control in TC plantlets for GPGV and offers a promising avenue to provide virus-free material to nurseries, contributing to the overall health and sustainability of the viticulture industry.

Grapevine Pinot gris virus (GPGV; genus Trichovirus in the family Betaflexiviridae ) was detected in Australia in 2016, but its impact on the production of nursery material and fruit in Australia is still currently unknown. This study investigated the prevalence and genetic diversity of GPGV in Australia. GPGV was detected by reverse transcription-polymerase chain reaction (RT-PCR) in a range of rootstock, table and wine grape varieties from New South Wales, South Australia, and Victoria, with 473/2171 (21.8%) samples found to be infected. Genomes of 32 Australian GPGV isolates were sequenced and many of the isolates shared high nucleotide homology. Phylogenetic and haplotype analyses demonstrated that there were four distinct clades amongst the 32 Australian GPGV isolates and that there were likely to have been at least five separate introductions of the virus into Australia. Recombination and haplotype analysis indicate the emergence of new GPGV strains after introduction into Australia. When compared with 168 overseas GPGV isolates, the analyses suggest that the most likely origin of Australian GPGV isolates is from Europe. There was no correlation between specific GPGV genotypes and symptoms such as leaf mottling, leaf deformation, and shoot stunting, which were observed in some vineyards, and the virus was frequently found in symptomless grapevines.

Grapevine rubodvirus 2 (syn. Grapevine Muscat rose virus; GMRV, genus Rubodvirus, family Phenuiviridae) is a negative-strand RNA virus with a tri-segmented genome (Diaz-Lara et al., 2019). It has only been described in a symptomless grapevine (Vitis vinifera cv. Muscat Rose) from the USDA National Clonal Germplasm Repository, which originated from Argentina (Diaz-Lara et al., 2019). In December 2021, shoots with mottling and deformation symptoms on leaves were collected from a grapevine cv. Malbec, at a vineyard in the Sunraysia region of Victoria, Australia. Total RNA was extracted from 0.3 g leaf tissue (MacKenzie et al., 1997), libraries were prepared using a TruSeq® Stranded Total RNA Library Prep Plant with Ribo-Zero Plant kit (Illumina, USA) and high throughput sequencing was done using a NovaSeq platform (Illumina, USA) with 2 × 150 bp paired-end reads. Sequence reads were trimmed using Trim Galore! Version 0.6.5 and assembled into contigs using SPADES Version 3.13.0 (Bankevich et al., 2012). BLASTn analysis identified a total of 46 viral contigs, of which 36 contigs aligned most closely with three RNA genome segments of GMRV, one contig matched with Grapevine Pinot gris virus (GPGV) and nine contigs matched with Grapevine rupestris stem pitting-associated virus. Reference mapping using Bowtie2 Version 2.3.4.2 (Langmead & Salzberg, 2012) illustrated 100% completeness of the Australian GMRV genomic regions and RNA1, RNA2 and RNA3 had 97.6%, 99.3% and 98.6% nucleotide identity, respectively, to the three published GMRV RNA segments (GenBank Accession Nos. MK728654.1, MK728655.1 and MK728656.1). The presence of RNA 1 and RNA 3 in the sample was confirmed using two published GMRV-specific endpoint RT-PCR assays (Diaz-Lara et al., 2019). A primer pair (GMRV-MP-Forward 5′-TGGGTGGCTATCATCACTGT-3′; GMRV-MP-Reverse 5′-CATCTCTTGCACCTTTCGGC-3′) was developed in this study to amplify a 620 bp nucleotide region of the movement protein gene (MP) and confirmed the presence of RNA2. Direct bi-directional Sanger sequencing of amplicons also confirmed the detection of RNA1, RNA 2 and RNA3 by RT-PCR. The Australian GMRV isolate was also detected using RT-PCR and Sanger sequencing in a previously uninfected Cabernet Franc grapevine that was grafted with the infected Malbec material, further confirming its presence in the sample and graft transmissibility. Additional samples were collected from six Malbec grapevines at a second vineyard in Sunraysia, which had no visible symptoms and were grown from Malbec clones imported from Argentina. Amplicons of the expected size were observed in one sample after testing with each of the three RT-PCR assays. To our knowledge, this is the first report of GMRV in Australia and the sequences for RNA1, RNA2 and RNA3 are available in GenBank (OQ418650, OQ418651 and OQ418652, respectively). There are no known vectors of GMRV but the virus is transmitted through vegetative propagation. Therefore, use of planting material in which the virus hasn't been detected is advisable to minimise risk. The correlation between the Australian GMRV isolate and symptoms in diseased Malbec requires further investigation because of the presence of other viruses, particularly GPGV, that might also be associated with the symptoms observed. However, detection of both GMRV and GPGV in the asymptomatic cv. Malbec sample suggests that neither virus are the sole cause of the disease. Further surveillance is required to determine the distribution of GMRV and its association with disease in Australia. This research was funded by La Trobe University through their provision of a La Trobe Full Fee Research Scholarship and La Trobe University Graduate Research Scholarship, and a PhD research scholarship by Wine Australia. We would like to acknowledge Agriculture Victoria Research as this research was conducted using the facilities of Agriculture Victoria.