Grapevine (Vitis spp.) propagation material is profusely exchanged across geographic and regulatory boundaries. Trading germplasm increases the diversity of cultivars and rootstocks with enhanced viticultural attributes but also risks the dissemination of pests and detrimental pathogens such as viruses in new grape production areas. Therefore, regulations are established to facilitate the safe trade of Vitis propagation material with desirable traits. Regrettably regulations are sometimes inadequate. Consequently, the accessibility of desired germplasm for growers might be unduly delayed, providing opportunities to circumvent regulations by illegally introducing germplasm of interest, amidst risking epidemics of viruses of concern, including quarantined agents, and jeopardizing the health of vineyards. To address some of the regulatory limitations, scientists from around the world recently defined phantom agents in fruit crops, including grapevines, and provided a compelling case for their exclusion from regulatory oversight. Simultaneously, a group of virologists realized the need to complement the list of phantom agents in grapevines by considering additional viruses, virus-like diseases and viroids that should not be subject to regulatory oversight. Here, we present a list of nine viruses, 14 virus-like diseases, nine viroids, and 129 presumed viruses of the grapevine, that are not phantom agents but should be excluded from regulation or should not be regulated. Our list is anticipated to assist policy makers adopt regulations that expedite the safe exchange of Vitis germplasm across regulatory boundaries while reducing incentives for illicit introductions.

Grapevine red blotch virus (GRBV; species Grablovirus vitis, genus Grablovirus, family Geminiviridae) poses a significant threat to viticulture worldwide, impacting grapevine health and wine quality. Here, we report the first detection and tracing of GRBV in Australia and provide a summary of a subsequent survey to determine the extent of GRBV distribution in Western Australia. Additionally, the study introduces a tiled amplicon sequencing method, which, when combined with long-read nanopore sequencing, enables rapid GRBV genome sequencing. Our analysis suggests a single introduction of GRBV from North America to Australia through the state of Victoria and subsequently to Western Australia. Finally, this study provides insight into the epidemiology of GRBV, based on strain variation and distribution, which is a crucial step in supporting an emergency biosecurity response and implementing effective control measures to safeguard the sustainability of the wine industry in Australia.

The first detection of grapevine red blotch virus (syn. GRBV; species Grablovirus vitis, genus Grablovirus, family Geminiviridae) in Australia was initially reported in several grapevine varieties in Western Australia in 2022, but its impact and spread in the country is still currently unknown. In this study, GRBV was detected in 14 locally selected and imported wine, table and dried grapes varieties from a historical germplasm collection in Victoria (n = 12) and in germplasms in South Australia (n = 2), using a combination of a nested endpoint polymerase chain reaction (PCR) assay and tiled amplicon genome sequencing. Phylogenetic and median-joining network analyses indicated that there may have been at least three separate introductions of GRBV into the historical germplasm collection. The phylogenetic, sequence identity, median-joining network, and single nucleotide polymorphism analyses indicate a close relationship between the South Australian, Western Australian and most Victorian GRBV isolates, which appear to have emerged from an introduction of infected Vitis vinifera cv. Perle de Csaba in the 1960s. Spatial analyses and the known history of the 14 infected Victorian and South Australian varieties, also provide evidence of GRBV spread via vegetative propagation of Perle de Csaba from the historical collection to South Australia and Western Australia. The analyses also indicated that a very slow and inefficient spread of GRBV between vines in the same vineyard has also possibly occurred via an unknown vector. Seasonal testing of selected varieties from the Victorian collection suggests winter as the optimal time for GRBV testing under environmental conditions in north-west Victoria.

Xylella fastidiosa is a plant-pathogenic bacterium that poses a serious threat to the production of economically important plant species including grapes, almonds, olives and a broad range of amenity plants, causing significant economic losses worldwide. While multiple molecular detection assays have been developed for X. fastidiosa, there is a lack of molecular tools available for detection and differentiation of the closely related pear pathogen, Xylella taiwanensis. In this study, we present a novel conventional PCR assay with primers that can amplify both Xylella species. The amplified product could be sequenced and used for discrimination between the two species and the subspecies within the fastidiosa species. This PCR assay was designed using a genome-informed approach to target the ComEC/Rec2 gene of both Xylella species, ensuring a higher specificity than other previously developed PCR assays. A test performance study across five national plant diagnostic laboratories in Australia and New Zealand demonstrated this assay's high sensitivity and specificity to all known species and subspecies within the Xylella genus. This PCR assay can be used for Xylella identification at the species and subspecies level and is compatible with Sanger sequencing and nanopore sequencing for rapid turnaround time. The newly developed conventional PCR assay presented here offers rapid detection and accurate identification of both Xylella species from plant, insect vector or bacterial samples, enabling timely implementation of biosecurity measures or disease management responses.

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.

This collaborative work by over 180 researchers from 40+ countries addresses the challenges posed by “phantom agents”—putative pathogenic agents named in literature without supporting data on their existence. Those agents remain on regulatory lists, creating barriers in trade and plant certification. Historically identified based solely on symptoms, these agents lack isolates or sequence data, making reliable detection or risk assessment impossible. After reviewing over 120 such agents across 10 key plant genera, we recommend their removal from regulatory lists and call for revised standards aligned with modern diagnostics. This effort seeks to streamline germplasm exchange, benefiting global agriculture by removing the constraints imposed by phantoms.

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (n=46) or during collection efforts between January 2015 and June 2022 (n=149). The sampled hosts were classified as crop (n=155), weed (n=24), ornamental (n=7), native plant (n=6), and insect (n=3) species. Most samples came from Queensland (n=78), followed by Western Australia (n=46), the Northern Territory (n=32), New South Wales (n=17), and Victoria (n=10). Of the 195 draft phytoplasma genomes, 178 met our genome criteria for comparison using an average nucleotide identity approach. Ten distinct phytoplasma species were identified and could be classified within the 16SrII, 16SrXII (PCR only), 16SrXXV, and 16SrXXXVIII phytoplasma groups, which have all previously been recorded in Australia. The most commonly detected phytoplasma taxa in this study were species and subspecies classified within the 16SrII group (n=153), followed by strains within the 16SrXXXVIII group (‘Ca. Phytoplasma stylosanthis’; n=6). Several geographic- and host-range expansions were reported, as well as mixed phytoplasma infections of 16SrII taxa and ‘Ca. Phytoplasma stylosanthis’. Additionally, six previously unrecorded 16SrII taxa were identified, including five putative subspecies of ‘Ca. Phytoplasma australasiaticum’ and a new putative 16SrII species. PCR and sequencing of the 16S rRNA gene was a suitable triage tool for preliminary phytoplasma detection. Metagenomic sequencing, however, allowed for higher-resolution identification of the phytoplasmas, including mixed infections, than was afforded by only direct Sanger sequencing of the 16S rRNA gene. Since the metagenomic approach theoretically obtains sequences of all organisms in a sample, this approach was useful to confirm the host family, genus, and/or species. In addition to improving our understanding of the phytoplasma species that affect crop production in Australia, the study also significantly expands the genomic sequence data available in public sequence repositories to contribute to phytoplasma molecular epidemiology studies, revision of taxonomy, and improved diagnostics.

Shiraz disease (SD) is one of the most destructive viral diseases of grapevines in Australia and is known to cause significant economic loss to local growers. Grapevine virus A (GVA) was reported to be the key pathogen associated with this disease. This study aimed to better understand the diversity of GVA variants both within and between individual SD and grapevine leafroll disease (LRD) affected grapevines located at vineyards in South Australia. Amplicon high throughput sequencing (Amplicon-HTS) combined with median-joining networks (MJNs) was used to analyze the variability in specific gene regions of GVA variants. Several GVAII variant groups contain samples from both vineyards studied, suggesting that these GVAII variants were from a common origin. Variant groups analyzed by MJNs using the overall data set denote that there may be a possible relationship between variant groups of GVA and the geographical location of the grapevines.

Gene- and genome- based approaches were used to determine whether Vigna little leaf (ViLL) phytoplasma, which occurs in northern Australia, is a distinct ' Candidatus Phytoplasma' species. The ViLL 16S rRNA gene sequences exhibited the highest known similarity to species in the 16SrXXIX- A and 16SrIX- D subgroups, namely ' Candidatus Phytoplasma omanense' (98.03- 98.10%) and ' Candidatus Phytoplasma phoenicium' (96.87-97.20%), respectively. A total of 48 single- copy orthologue genes were identified to be shared among the two draft ViLL phytoplasma genomes, 30 publicly available phytoplasma genomes, and one Acholeplasma laidlawii genome as the outgroup taxon. Phylogenomic assessments using the 48 shared single- copy orthologue genes supported that ViLL and 'Ca. Ca . Phytoplasma phoenicium' were closely related yet distinct species. The 16S rRNA gene sequence analysis and phylogenomic assessment indicate that ViLL phytoplasmas are a distinct taxon. As such, a novel species, ' Candidatus Phytoplasma vignae', is proposed. Strain BAWM- 336 (genome accession number JAUZLI000000000) detected in Momordica charantia (bitter melon) serves as the reference strain of this species, with infected plant material deposited in the Victorian Plant Pathology Herbarium (VPRI) as VPRI 44369.

The Australian Biosecurity Genomic Database (ABGD) is a curated collection of reference viral genome sequences based on the Australian National Notifiable Disease List of Terrestrial Animals. It was created to facilitate the screening of high-throughput sequencing (HTS) data for the potential presence of viruses associated with notifiable disease. The database includes a single verified sequence (the exemplar species sequence, where relevant) for each of the 60 virus species across 21 viral families that are associated with or cause these notifiable diseases, as recognized by the World Organisation for Animal Health. The open-source ABGD on GitHub provides usage guidance documents and is intended to support building a culture in Australian HTS communities that promotes the use of quality-assured, standardized, and verified databases for Australia's national biosecurity interests. Future expansion of the database will include the addition of more strains or subtypes for highly variable viruses, viruses causing diseases of aquatic animals, and genomes of other types of pathogens associated with notifiable diseases, such as bacteria. Database URL: https://github.com/ausbiopathgenDB/AustralianBiosecurityGenomicDatabase.

Within the 16SrII phytoplasma group, subgroups A-X have been classified based on restriction fragment length polymorphism of their 16S rRNA gene, and two species have been described, namely 'Candidatus Phytoplasma aurantifolia' and 'Ca. Phytoplasma australasia'. Strains of 16SrII phytoplasmas are detected across a broad geographic range within Africa, Asia, Australia, Europe and North and South America. Historically, all members of the 16SrII group share ≥97.5 % nucleotide sequence identity of their 16S rRNA gene. In this study, we used whole genome sequences to identify the species boundaries within the 16SrII group. Whole genome analyses were done using 42 phytoplasma strains classified into seven 16SrII subgroups, five 16SrII taxa without official 16Sr subgroup classifications, and one 16SrXXV-A phytoplasma strain used as an outgroup taxon. Based on phylogenomic analyses as well as whole genome average nucleotide and average amino acid identity (ANI and AAI), eight distinct 16SrII taxa equivalent to species were identified, six of which are novel descriptions. Strains within the same species had ANI and AAI values of >97 %, and shared ≥80 % of their genomic segments based on the ANI analysis. Species also had distinct biological and/or ecological features. A 16SrII subgroup often represented a distinct species, e.g., the 16SrII-B subgroup members. Members classified within the 16SrII-A, 16SrII-D, and 16SrII-V subgroups as well as strains classified as sweet potato little leaf phytoplasmas fulfilled criteria to be included as members of a single species, but with subspecies-level relationships with each other. The 16SrXXV-A taxon was also described as a novel phytoplasma species and, based on criteria used for other bacterial families, provided evidence that it could be classified as a distinct genus from the 16SrII phytoplasmas. As more phytoplasma genome sequences become available, the classification system of these bacteria can be further refined at the genus, species, and subspecies taxonomic ranks.

Five grapevine viruses, including grapevine geminivirus A (GGVA), grapevine Syrah virus 1 (GSyV-1), grapevine Cabernet Sauvignon reovirus (GCSV), grapevine virus F (GVF) and grapevine Red Globe virus (GRGV) have been detected in four South Australian vineyards using metagenomic high throughput sequencing for the first time in Australia. Most of them were present in asymptomatic grapevines and some were present in grapevines with leafroll disease that were infected by several other viruses, including grapevine leafroll-associated viruses. Therefore, the association of GGVA, GSyV-1, GCSV, GVF and GRGV with disease in Australia is unknown.

Shiraz disease (SD) is an economically important virus-associated disease that can significantly reduce yield in sensitive grapevine varieties and has so far only been reported in South Africa and Australia. In this study, RT-PCR and metagenomic high-throughput sequencing was used to study the virome of symptomatic and asymptomatic grapevines within vineyards affected by SD and located in South Australia. Results showed that grapevine virus A (GVA) phylogroup II variants were strongly associated with SD symptoms in Shiraz grapevines that also had mixed infections of viruses including combinations of grapevine leafroll-associated virus 3 (GLRaV-3) and grapevine leafroll-associated virus 4 strains 5, 6 and 9 (GLRaV-4/5, GLRaV-4/6, GLRaV-4/9). GVA phylogroup III variants, on the other hand, were present in both symptomatic and asymptomatic grapevines, suggesting no or decreased virulence of these strains. Similarly, only GVA phylogroup I variants were found in heritage Shiraz grapevines affected by mild leafroll disease, along with GLRaV-1, suggesting this phylogroup may not be associated with SD.

Seed lots of tomato and capsicum (Solanum lycopersicon and Capsicum annuum, respectively) are required to be free of quarantine pests before their entry to Australia is permitted. Testing of samples from 118 larger seed lots in the period 2019-2021 revealed that 31 (26.3%) carried one or more of four Tobamovirus species, including tomato mottle mosaic virus (ToMMV), which is a quarantine pest for Australia. Testing of samples from a further 659 smaller seed lots showed that 123 (18.7%) carried a total of five Tobamovirus species, including ToMMV and tomato brown rugose fruit virus (ToBRFV), which is also a quarantine pest for Australia. Estimated prevalence of contamination by tobamoviruses ranged from 0.388% to 0.004% in contaminated larger seed lots. Analyses of these data allow us to estimate probabilities of detection of contamination under different regulatory settings.

High-quality complete genomes of five Xylella fastidiosa strains were assembled by combining Nanopore and Illumina sequencing data. Among these, International Collection of Micro-organisms from Plants (ICMP) 8731, ICMP 8742 and ICMP 8745 belong to subspecies fastidiosa while ICMP 8739 and ICMP 8740 were determined as subspecies multiplex. The strains were further classified into sequence types.

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.

The detection of cucumber green mottle mosaic (CGMMV) in the Northern Territory (NT), Australia, in 2014 led to the introduction of strict quarantine measures for the importation of cucurbit seeds by the Australian federal government. Further detections in Queensland, Western Australia (WA), New South Wales and South Australia occurred in the period 2015–2020. To explore the diversity of the current Australian CGMMV population, 35 new coding sequence complete genomes for CGMMV isolates from Australian incursions and surveys were prepared for this study. In conjunction with published genomes from the NT and WA, sequence, phylogenetic, and genetic variation and variant analyses were performed, and the data were compared with those for international CGMMV isolates. Based on these analyses, it can be inferred that the Australian CGMMV population resulted from a single virus source via multiple introductions.

Grapevine leafroll disease affects the health status of grapevines worldwide. Most studies in Australia have focused on grapevine leafroll-associated viruses 1 and 3, while little attention has been given to other leafroll virus types, in particular, grapevine leafroll-associated virus 2 (GLRaV-2). A chronological record of the temporal occurrence of GLRaV-2 in Australia since 2001 is reported. From a total of 11,257 samples, 313 tested positive, with an overall incidence of 2.7%. This virus has been detected in 18 grapevine varieties and Vitis rootstocks in different regions of Australia. Most varieties were symptomless on their own roots, while Chardonnay showed a decline in virus-sensitive rootstocks. An isolate of GLRaV-2, on own-rooted Vitis vinifera cv. Grenache, clone SA137, was associated with severe leafroll symptoms after veraison with abnormal leaf necrosis. The metagenomic sequencing results of the virus in two plants of this variety confirmed the presence of GLRaV-2, as well as two inert viruses, grapevine rupestris stem pitting-associated virus (GRSPaV) and grapevine rupestris vein feathering virus (GRVFV). No other leafroll-associated viruses were detected. Among the viroids, hop stunt viroid and grapevine yellow speckle viroid 1 were detected. Of the six phylogenetic groups identified in GLRaV-2, we report the presence of four groups in Australia. Three of these groups were detected in two plants of cv. Grenache, without finding any recombination event. The hypersensitive reaction of certain American hybrid rootstocks to GLRaV-2 is discussed. Due to the association of GLRaV-2 with graft incompatibility and vine decline, the risk from this virus in regions where hybrid Vitis rootstocks are used cannot be overlooked.

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.

Cucumber green mottle mosaic virus (CGMMV) is a Tobamovirus of economic importance affecting cucurbit crops and Asian cucurbit vegetables. Non-host crops of CGMMV, including capsicum (Capsicum annum), sweetcorn (Zea mays), and okra (Abelmoschus esculentus), were tested for their susceptibility to the virus, with field and glasshouse trials undertaken. After 12 weeks post-sowing, the crops were tested for the presence of CGMMV, and in all cases, no CGMMV was detected. Commonly found within the growing regions of cucurbits and melons worldwide are weeds, such as black nightshade (Solanum nigrum), wild gooseberry (Physalis minima), pigweed (Portulaca oleracea), and Amaranth species. Several weeds/grasses were tested for their ability to become infected with CGMMV by inoculating weeds directly with CGMMV and routinely testing over a period of eight weeks. Amaranthus viridis was found to be susceptible, with 50% of the weeds becoming infected with CGMMV. To further analyse this, six Amaranth samples were used as inoculum on four watermelon seedlings per sample and tested after eight weeks. CGMMV was detected in three of six watermelon bulk samples, indicating that A. viridis is a potential host/reservoir for CGMMV. Further research into the relationship between CGMMV and weed hosts is required. This research also highlights the importance of proper weed management to effectively manage CGMMV.