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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., (© The Author(s) 2024. Published by Oxford University Press.)

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 . 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.

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 GVA II variant groups contain samples from both vineyards studied, suggesting that these GVA II 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.

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.

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.

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.

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.

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.

Rapid and reliable detection tools are essential for disease surveillance and outbreak management, and genomic data is essential to determining pathogen origin and monitoring of transmission pathways. Low virus copy number and poor RNA quality can present challenges for genomic sequencing of plant viruses, but this can be overcome by enrichment of target nucleic acid. A targeted whole genome sequencing (TWG-Seq) approach for the detection of cucumber green mottle mosaic virus (CGMMV) has been developed where overlapping amplicons generated using two multiplex RT-PCR assays are then sequenced using the Oxford Nanopore MinION. Near complete coding region sequences were assembled with ≥100× coverage for infected leaf tissue dilution samples with RT-qPCR cycle quantification (Cq) values from 11.8 to 38 and in seed dilution samples with Cq values 13.8 to 27. Consensus sequences assembled using this approach showed greater than 99% nucleotide similarity when compared to genomes produced using metagenomic sequencing. CGMMV could be confidently detected in historical seed isolates with degraded RNA. Whilst limited access to, and costs associated with second-generation sequencing platforms can influence diagnostic outputs, the portable Nanopore technology offers an affordable high throughput sequencing alternative when combined with TWG-Seq for low copy or degraded samples.

Obtaining complete phytoplasma genomes is difficult due to the lack of a culture system for these bacteria. To improve genome assembly, a non-ionic, low- and iso-osmotic iodixanol (Optiprep™) density gradient centrifugation method was developed to enrich for phytoplasma cells and deplete plant host tissues prior to deoxyribonucleic acid (DNA) extraction and high-throughput sequencing (HTS). After density gradient enrichment, potato infected with a ' Candidatus Phytoplasma australasia'-related strain showed a ∼14-fold increase in phytoplasma HTS reads, with a ∼1.7-fold decrease in host genomic reads compared to the DNA extracted from the same sample without density gradient centrifugation enrichment. Additionally, phytoplasma genome assemblies from libraries equalized to 5 million reads were, on average, ∼15,000 bp larger and more contiguous (N50 ∼14,800 bp larger) than assemblies from the DNA extracted from the infected potato without enrichment. The method was repeated on capsicum infected with Sweet Potato Little Leaf phytoplasma (' Ca. Phytoplasma australasia'-related strain) with a lower phytoplasma titer than the potato. In capsicum, ∼threefold more phytoplasma reads and ∼twofold less host genomic reads were obtained, with the genome assembly size and N50 values from libraries equalized to 3.4 million reads ∼137,000 and ∼4,000 bp larger, respectively, compared to the DNA extracted from infected capsicum without enrichment. Phytoplasmas from potato and capsicum were both enriched at a density of 1.049-1.058 g/ml. Finally, we present two highly contiguous ' Ca. Phytoplasma australasia' phytoplasma reference genomes sequenced from naturally infected Solanaceae hosts in Australia. Obtaining high-quality phytoplasma genomes from naturally infected hosts will improve insights into phytoplasma taxonomy, which will improve their detection and disease management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rodrigues Jardim, Tran-Nguyen, Gambley, Rodoni and Constable.)

High-throughput sequencing (HTS) of host plant small RNA (sRNA) is a popular approach for plant virus and viroid detection. The major bottlenecks for implementing this approach in routine virus screening of plants in quarantine include lack of computational resources and/or expertise in command-line environments and limited availability of curated plant virus and viroid databases. We developed: (1) virus and viroid report web-based bioinformatics workflows on Galaxy Australia called GA-VirReport and GA-VirReport-Stats for detecting viruses and viroids from host plant sRNA extracts and (2) a curated higher plant virus and viroid database (PVirDB). We implemented sRNA sequencing with unique dual indexing on a set of plants with known viruses. Sequencing data were analyzed using GA-VirReport and PVirDB to validate these resources. We detected all known viruses in this pilot study with no cross-sample contamination. We then conducted a large-scale diagnosis of 105 imported plants processed at the post-entry quarantine facility (PEQ), Australia. We detected various pathogens in 14 imported plants and discovered that de novo assembly using 21-22 nt sRNA fraction and the megablast algorithm yielded better sensitivity and specificity. This study reports the successful, large-scale implementation of HTS and a user-friendly bioinformatics workflow for virus and viroid screening of imported plants at the PEQ.

A phytoplasma was initially detected in Dypsis poivriana by nested and real-time PCR from the botanical gardens in Cairns, Queensland, Australia in 2017. Further surveys in the Cairns region identified phytoplasma infections in eight additional dying ornamental palm species ( Euterpe precatoria, Cocos nucifera , Verschaffeltia splendida, Brassiophoenix drymophloeodes , Burretiokentia hapala , Cyrtostachys renda , Reinhardtia gracilis , Carpoxylon macrospermum ), a Phoenix species, a Euterpe species and two native palms ( Archontophoenix alexandrae ). Analysis of 16S rRNA gene sequences showed that this phytoplasma is distinct as it shared less than 97.5 % similarity with all other ' Candidatus Phytoplasma' species. At 96.3 % similarity, the most closely related formally described member of the provisional ' Ca . Phytoplasma' genus was ' Ca . Phytoplasma noviguineense', a novel taxon from the island of New Guinea found in monocotyledonous plants. It was slightly more closely related (96.6-96.8 %) to four palm-infecting strains from the Americas, which belong to strain group 16SrIV and which have not been assigned to a formal ' Candidatus Phytoplasma' species taxon. Phylogenetic analysis of the 16S rRNA gene and ribosomal protein genes of the phytoplasma isolate from a dying coconut palm revealed that the phytoplasma represented a distinct lineage within the phytoplasma clade. As the nucleotide identity with other phytoplasmas is less than 97.5 % and the phylogenetic analyses show that it is distinct, a novel taxon 'Candidatus Phytoplasma dypsidis' is proposed for the phytoplasma found in Australia. Strain RID7692 (GenBank accession no. MT536195) is the reference strain. The impact and preliminary aspects of the epidemiology of the disease outbreak associated with this novel taxon are described.

In Australia, Stylosanthes little leaf (StLL) phytoplasma has been detected in Stylosanthes scabra Vogel, Arachis pintoi Krapov, Saccharum officinarum L., Carica papaya L., Medicago sativa L., and Solanum tuberosum L. The 16S rRNA gene sequence of StLL phytoplasma strains from S. scabra , C. papaya, S. officinarum and S. tuberosum were compared and share 99.93-100 % nucleotide sequence identity. Phylogenetic comparisons between the 16S rRNA genes of StLL phytoplasma and other ' Candidatus Phytoplasma' species indicate that StLL represents a distinct phytoplasma lineage. It shares its most recent known ancestry with ' Ca . Phytoplasma luffae' (16SrVIII-A), with which it has 97.17-97.25 % nucleotide identity. In silico RFLP analysis of the 16S rRNA amplicon using i PhyClassifier indicate that StLL phytoplasmas have a unique pattern (similarity coefficient below 0.85) that is most similar to that of 'Ca . Phytoplasma luffae'. The unique in silico RFLP patterns were confirmed in vitro . Nucleotide sequences of genes that are more variable than the 16S rRNA gene, namely tuf (tu-elongation factor), secA (partial translocation gene), and the partial ribosomal protein ( rp ) gene operon ( rps19-rpl22-rps3 ), produced phylogenetic trees with similar branching patterns to the 16S rRNA gene tree. Sequence comparisons between the StLL 16S rRNA spacer region confirmed previous reports of rrn interoperon sequence heterogeneity for StLL, where the spacer region of rrnB encodes a complete tRNA-Isoleucine gene and the rrnA spacer region does not. Together these results suggest that the Australian phytoplasma, StLL, is unique according to the International Organization for Mycoplasmology (IRPCM) recommendations. The novel taxon ' Ca . Phytoplasma stylosanthis' is proposed, with the most recent strain from a potato crop in Victoria, Australia, serving as the reference strain (deposited in the Victorian Plant Pathology Herbarium as VPRI 43683).

In this study, we determined the complete genome sequence of a new blunervirus isolated from tomato plants grown in an open field in Italy in the fall of 2018. Like other blunerviruses, the RNA genome of this virus is quadripartite, positive-sense, and single-stranded. Excluding the polyA tail present in each segment, the RNAs 1 and 2 are 5790 nucleotides (nt) and 3621 nt in size, respectively, and each contains a single open reading frame (ORF). The RNAs 3 and 4 are 2842 and 1924 nt long and encode five and two ORFs, respectively. BLASTp analysis of the predicted products of RNA1 and RNA2 ORF1 showed the highest sequence identity (31% and 42%) to tea plant necrotic ring blotch virus (TPNRBV), while the protein encoded by RNA 4 ORF2 had the highest sequence identity (38%) to blueberry necrotic ring blotch virus (BNRBV). These are the only two recognized members in the genus Blunervirus. When the RNA3 ORF3 and ORF5 products were compared with the blunerviruses-encoded proteins, they had the highest sequence identity (30% and 32%) to their TPNRBV-encoded homologs; however, general comparisons showed stronger matches to two different proteins from Acinetobacter baumannii. The proteins encoded by ORFs 1, 2 and 4 of RNA3 and ORF 1 of RNA4 showed no significant BLASTp hits to any known proteins in the databases. Given the limited genetic similarity of this virus to those currently available in the databases, we suggest that this is a new virus, for which we propose the name "tomato fruit blotch virus" (ToFBV). A distinct isolate of the same virus was also detected in Australia.

Grapevine rupestris vein feathering virus (GRVFV; tentative genus Marafivirus; family Tymoviridae ) was first detected from a Greek grapevine (Vitis vinifera), with asteroid mosaic-like symptoms (El Beaino et al. 2001; Ghanem-Sabanadzovic et al. 2003) and was also infected with grapevine fleck virus. GRVFV has been detected in the United States, South Africa, Canada, Spain, China, New Zealand, Brazil, Germany, Korea, Slovakia, Hungary and Pakistan (Cho et al. 2018; Mahmood et al. 2019).Transmission vectors are currently unknown. In 2018, nine grapevine samples were collected between May to July in South Australia (SA) and Western Australia (WA) (Table S1), were analysed by high-throughput sequencing (HTS) to characterise grapevine viruses in Australian vineyards. Total RNA or double stranded RNA was extracted from grapevine canes using RNeasy 96 QIAcube HT kit (Qiagen) with MacKenzie buffer (MacKenzie et al. 1997) or using CF-11 (Balijja et al. 2008). Libraries were prepared using the NEBNext® Ultra II RNA library Prep Kit (NEB) or TruSeq® Stranded mRNA Prep kit (Illumina) with Ribo-Zero®gold plant kit for ribosomal depletion (Illumina, San Diego, CA). Libraries were sequenced using Illumina Miseq (SA) or Hiseq (WA) technology with 2x300 (SA) or 2x100 (WA) paired end reads which were trimmed using Trim Galore! (0.4.0) or BBmap (38.20), respectively. De novo assembly, using the SPAdes (version 3.12.0) genome assembler with default settings, resulted in twelve near full length GRVFV genomes (6713-6737nt), eight sequences from the WA samples and four from the SA samples. WA samples 171 and 178 and SA sample BV each had two distinct GRVFV molecular variants. Variants 171-1 and 171-2 (GenBank accessions MT084811, MT084812) from sample 171 shared 83.39% nucleotide (nt) identity. Variants 178-1 and 178-2 (MT084813, MT084814) from sample 178 shared 83.54% nt identity. Variants BV6799 and BV8822 (MN974274, MN974275) from sample BV shared 82.85% nt identity. Only one GRVFV sequence was obtained from all other samples. The genome of SA isolate LC1 (MN974273) was confirmed by RT-PCR amplification and Sanger sequencing of overlapping genome regions. Tissue from the infected LC1 isolate has been deposited into the Victorian plant pathogen reference collection (VPRI accession No. 43698). When the genomes of all Australian isolates were compared, they had 78.94% to 94.37 % nt identity with each other. The SA isolates LC1, BV8822, BV6799, and SEL-L (MN974276), and the WA isolates 172 (MT084807), 179 (MT084808), 180 (MT084809), and 182 (MT084810) were most closely related to the Swiss isolate CHASS (KY513702; 82.87% to 85.46% nt identity). The WA isolates 171-1, 171-2, 178-1 and 178-2 were most closely related to the New Zealand isolate Ch8021 (MF000325; 83.21% to 93.87%). Grapevine leafroll-associated virus 1 (GLRaV-1), GLRaV-3, GLRaV-4 (strain 6 and 9), grapevine virus A, grapevine rupestris stem pitting associated virus, grapevine yellow speckle viroid 1 and hop stunt viroid were also identified in the sequencing data. This is the first report of GRVFV in Australia. All WA samples were collected during dormancy and symptoms were not observed. Sample LC1 from SA had Shiraz disease, the other SA samples were asymptomatic, and none had asteroid mosaic-like symptoms. Further research is required to determine its distribution and association with disease in Australia.

Melon necrotic spot virus (MNSV) was detected in field-grown Cucumis melo (rockmelon) and Citrullus lanatus (watermelon) plants in the Sunraysia district of New South Wales and Victoria, Australia, in 2012, 2013, and 2016, and in two watermelon seed lots tested at the Australian border in 2016. High-throughput sequencing was used to generate near full-length genomes of six isolates detected during the incursions and seed testing. Phylogenetic analysis of the genomes suggests that there have been at least two incursions of MNSV into Australia and none of the field isolates were the same as the isolates detected in seeds. The analysis indicated that one watermelon field sample (L10), the Victorian rockmelon field sample, and two seed interception samples may have European origins. The results showed that two isolates (L8 and L9) from watermelon were divergent from the type MNSV strain (MNSV-GA, D12536.2) and had 99% nucleotide identity to two MNSV isolates from human stool collected in the United States (KY124135.1, KY124136.1). These isolates also had high nucleotide pairwise identity (96%) to a partial sequence from a Spanish MNSV isolate (KT962848.1). The analysis supported the identification of three previously described MNSV genotype groups: EU-LA, Japan melon, and Japan watermelon. To account for the greater diversity of hosts and geographic regions of the MNSV isolates used in this study, it is suggested that the genotype groups EU-LA, Japan melon, and Japan watermelon be renamed to groups I, II, and III, respectively. The divergent isolates L8 and L9 from this study and the stool isolates from the United States formed a fourth genotype group, group IV. Soil collected from the site of the Victorian rockmelon MNSV outbreak was found to contain viable MNSV and the virus vector, a chytrid fungus, Olpidium bornovanus (Sahtiyanci) Karling, 18 months after the initial MNSV detection. This is a first report of O. bornovanus from soil sampled from an MNSV-contaminated site in Australia.

The near-complete genome sequence of the original Carrot virus Y (CarVY) type isolate (CarVY-Vic) collected in 1999 in Victoria, Australia, and a near-complete genome sequence from an isolate collected in 2019 from the same region (CarVY-2-22) were determined following deep sequencing. The two CarVY genome sequences shared 98% nucleotide identity., (© Crown copyright 2020.)

One hundred Prunus trees, including almond ( P. dulcis ), apricot ( P. armeniaca ), nectarine ( P. persica var. nucipersica ), peach ( P. persica ), plum ( P. domestica ), purple leaf plum ( P. cerasifera ) and sweet cherry ( P. avium ), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: Apple chlorotic leaf spot virus (ACLSV) (13/100), Apple mosaic virus (ApMV) (1/100), Cherry green ring mottle virus (CGRMV) (4/100), Cherry necrotic rusty mottle virus (CNRMV) (2/100), Cherry virus A (CVA) (14/100), Little cherry virus 2 (LChV2) (3/100), Plum bark necrosis stem pitting associated virus (PBNSPaV) (4/100), Prune dwarf virus (PDV) (3/100), Prunus necrotic ringspot virus (PNRSV) (52/100), Hop stunt viroid (HSVd) (9/100) and Peach latent mosaic viroid (PLMVd) (6/100). The results showed that PNRSV is widespread in Prunus trees in Australia. Metagenomic high-throughput sequencing (HTS) and bioinformatics analysis were used to characterise the genomes of some viruses that were detected by RT-PCR tests and Apricot latent virus (ApLV), Apricot vein clearing associated virus (AVCaV), Asian Prunus Virus 2 (APV2) and Nectarine stem pitting-associated virus (NSPaV) were also detected. This is the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV occurring in Australia. It is also the first report of ASGV infecting Prunus species in Australia, although it is known to infect other plant species including pome fruit and citrus.

Apple mosaic virus (ApMV) and prune dwarf virus (PDV) are amongst the most common viruses infecting Prunus species worldwide but their incidence and genetic diversity in Australia is not known. In a survey of 127 Prunus tree samples collected from five states in Australia, ApMV and PDV occurred in 4 (3%) and 13 (10%) of the trees respectively. High-throughput sequencing (HTS) of amplicons from partial conserved regions of RNA1, RNA2, and RNA3, encoding the methyltransferase (MT), RNA-dependent RNA polymerase (RdRp), and the coat protein (CP) genes respectively, of ApMV and PDV was used to determine the genetic diversity of the Australian isolates of each virus. Phylogenetic comparison of Australian ApMV and PDV amplicon HTS variants and full length genomes of both viruses with isolates occurring in other countries identified genetic strains of each virus occurring in Australia. A single Australian Prunus infecting ApMV genetic strain was identified as all ApMV isolates sequence variants formed a single phylogenetic group in each of RNA1, RNA2, and RNA3. Two Australian PDV genetic strains were identified based on the combination of observed phylogenetic groups in each of RNA1, RNA2, and RNA3 and one Prunus tree had both strains. The accuracy of amplicon sequence variants phylogenetic analysis based on segments of each virus RNA were confirmed by phylogenetic analysis of full length genome sequences of Australian ApMV and PDV isolates and all published ApMV and PDV genomes from other countries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The distribution of Ilarvirus species populations amongst 61 Australian Prunus trees was determined by next generation sequencing (NGS) of amplicons generated using a genus-based generic RT-PCR targeting a conserved region of the Ilarvirus RNA2 component that encodes the RNA dependent RNA polymerase (RdRp) gene. Presence of Ilarvirus sequences in each positive sample was further validated by Sanger sequencing of cloned amplicons of regions of each of RNA1, RNA2 and/or RNA3 that were generated by species specific PCRs and by metagenomic NGS. Prunus necrotic ringspot virus (PNRSV) was the most frequently detected Ilarvirus , occurring in 48 of the 61 Ilarvirus -positive trees and Prune dwarf virus (PDV) and Apple mosaic virus (ApMV) were detected in three trees and one tree, respectively. American plum line pattern virus (APLPV) was detected in three trees and represents the first report of APLPV detection in Australia. Two novel and distinct groups of Ilarvirus -like RNA2 amplicon sequences were also identified in several trees by the generic amplicon NGS approach. The high read depth from the amplicon NGS of the generic PCR products allowed the detection of distinct RNA2 RdRp sequence variant populations of PNRSV, PDV, ApMV, APLPV and the two novel Ilarvirus -like sequences. Mixed infections of ilarviruses were also detected in seven Prunus trees. Sanger sequencing of specific RNA1, RNA2, and/or RNA3 genome segments of each virus and total nucleic acid metagenomics NGS confirmed the presence of PNRSV, PDV, ApMV and APLPV detected by RNA2 generic amplicon NGS. However, the two novel groups of Ilarvirus -like RNA2 amplicon sequences detected by the generic amplicon NGS could not be associated to the presence of sequence from RNA1 or RNA3 genome segments or full Ilarvirus genomes, and their origin is unclear. This work highlights the sensitivity of genus-specific amplicon NGS in detection of virus sequences and their distinct populations in multiple samples, and the need for a standardized approach to accurately determine what constitutes an active, viable virus infection after detection by molecular based methods.

PCR amplicon next generation sequencing (NGS) analysis offers a broadly applicable and targeted approach to detect populations of both high- or low-frequency virus variants in one or more plant samples. In this study, amplicon NGS was used to explore the diversity of the tripartite genome virus, Prunus necrotic ringspot virus (PNRSV) from 53 PNRSV-infected trees using amplicons from conserved gene regions of each of PNRSV RNA1, RNA2 and RNA3. Sequencing of the amplicons from 53 PNRSV-infected trees revealed differing levels of polymorphism across the three different components of the PNRSV genome with a total number of 5040, 2083 and 5486 sequence variants observed for RNA1, RNA2 and RNA3 respectively. The RNA2 had the lowest diversity of sequences compared to RNA1 and RNA3, reflecting the lack of flexibility tolerated by the replicase gene that is encoded by this RNA component. Distinct PNRSV phylo-groups, consisting of closely related clusters of sequence variants, were observed in each of PNRSV RNA1, RNA2 and RNA3. Most plant samples had a single phylo-group for each RNA component. Haplotype network analysis showed that smaller clusters of PNRSV sequence variants were genetically connected to the largest sequence variant cluster within a phylo-group of each RNA component. Some plant samples had sequence variants occurring in multiple PNRSV phylo-groups in at least one of each RNA and these phylo-groups formed distinct clades that represent PNRSV genetic strains. Variants within the same phylo-group of each Prunus plant sample had ≥97% similarity and phylo-groups within a Prunus plant sample and between samples had less ≤97% similarity. Based on the analysis of diversity, a definition of a PNRSV genetic strain was proposed. The proposed definition was applied to determine the number of PNRSV genetic strains in each of the plant samples and the complexity in defining genetic strains in multipartite genome viruses was explored.

Current atmospheric CO2 levels are about 400 μmol mol(-1) and are predicted to rise to 650 μmol mol(-1) later this century. Although the positive and negative impacts of CO2 on plants are well documented, little is known about interactions with pests and diseases. If disease severity increases under future environmental conditions, then it becomes imperative to understand the impacts of pathogens on crop production in order to minimize crop losses and maximize food production. Barley yellow dwarf virus (BYDV) adversely affects the yield and quality of economically important crops including wheat, barley and oats. It is transmitted by numerous aphid species and causes a serious disease of cereal crops worldwide. This study examined the effects of ambient (aCO2 ; 400 μmol mol(-1) ) and elevated CO2 (eCO2 ; 650 μmol mol(-1) ) on noninfected and BYDV-infected wheat. Using a RT-qPCR technique, we measured virus titre from aCO2 and eCO2 treatments. BYDV titre increased significantly by 36.8% in leaves of wheat grown under eCO2 conditions compared to aCO2 . Plant growth parameters including height, tiller number, leaf area and biomass were generally higher in plants exposed to higher CO2 levels but increased growth did not explain the increase in BYDV titre in these plants. High virus titre in plants has been shown to have a significant negative effect on plant yield and causes earlier and more pronounced symptom expression increasing the probability of virus spread by insects. The combination of these factors could negatively impact food production in Australia and worldwide under future climate conditions. This is the first quantitative evidence that BYDV titre increases in plants grown under elevated CO2 levels., (© 2015 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.)

Barley yellow dwarf virus-PAV (BYDV-PAV) is associated with yellow dwarf disease, one of the most economically important diseases of cereals worldwide. In this study, the impact of current and future predicted temperatures for the Wimmera wheat growing district in Victoria, Australia on the titre of BYDV-PAV in wheat was investigated. Ten-day old wheat (Triticum aestivum, cv. Yitpi) seedlings were inoculated with BYDV-PAV and grown at ambient (5.0-16.1°C, night-day) or elevated (10.0-21.1°C, night-day) temperature treatments, simulating the current Wimmera average and future daily temperature cycles, respectively, during the wheat-growing season. Whole above-ground plant samples were collected from each temperature treatment at 0 (day of inoculation), 3, 6, 9, 12, 15, 18, 21 and 24 days after inoculation and the titre of BYDV-PAV was measured in each sample using a specific one-step multiplex normalised reverse transcription quantitative PCR (RT-qPCR) assay. Physical measurements, including plant height, dry weight and tiller number, were also taken at each sampling point. The titre of BYDV-PAV was significantly greater in plants grown in the elevated temperature treatment than in plants grown in the ambient treatment on days 6, 9 and 12. Plants grown at elevated temperature were significantly bigger and symptoms associated with BYDV-PAV were visible earlier than in plants grown at ambient temperature. These results may have important implications for the epidemiology of yellow dwarf disease under future climates in Australia., (Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.)

Pepper chat fruit viroid (PCFVd), a species of Pospiviroid, was first discovered in a capsicum crop in the Netherlands in 2006 (4) and was then reported only in Thailand (2) and Canada. The mechanism of international spread was not known, but movement with traded seed was suspected. PCFVd is transmissible through capsicum seed (4) and very probably through tomato seed, like other pospiviroids. The viroid causes disease in capsicum and tomato and experiments by others indicate a capacity to cause disease in potato. It poses a biosecurity threat to crops internationally. PCFVd was intercepted by the Australian Government Department of Agriculture, Fisheries, and Forestry (DAFF) in five shipments of tomato seed (Solanum lycopersicum) exported from Israel and Thailand in September and October 2012. Batches of up to 20,000 seeds were sampled from each seed lot in a shipment and total nucleic acids were extracted from sub-samples, each of about 400 seeds, following a method similar to Hoshino et al. (1). PCFVd was initially detected when reverse transcription PCR using the generic pospiviroid primers Pospi1-FW and Pospi1-RE (3) produced amplicons of 189 bp, which were then sequenced. The PCFVd specific primers AP FW1 and AP RE2 (4) were used to amplify the remainder of the viroid genome, which was directly sequenced. Overlapping sequences were aligned to produce complete sequences of 349 bases, one from seed from Thailand and two from seed from Israel (GenBank: KC762952, KC762953, KC762954). Searches of the GenBank nucleotide non-redundant database indicated close matches with sequences from PCFVd isolates from tomato in Thailand (2); alignments generated by BLAST showed the sequences differed from those from Thailand at only 2 to 18 nucleotide positions, equating to 95 to 99% identity. PCFVd sequences from seed from Thailand were almost identical (>99%) to the sequences from seed from Israel. Many sub-samples were negative, indicating that the number of contaminated seeds was very small in some shipments. The positive sub-samples as a proportion of the total number of sub-samples tested from the five shipments was 1/1, 1/5, 1/1, 12/50, and 7/50. Tomato and capsicum seed are produced in many countries and often traded through second countries. The infected tomato seed shipments intercepted by DAFF were destroyed or re-exported following Australian regulations. Other countries were informed through the International Plant Protection Convention. This pest viroid has not been intercepted by Australian authorities before and has not been detected in recent Australian survey work (data not shown). References: (1) S. Hoshino et al. Res. Bull. Plant Prot. Japan 42:75, 2006. (2) K. Reanwarakorn et al. New Dis. Rep. 24:6, 2011 (3) J. Th. J. Verhoeven et al. EJPP 110:823, 2004. (4) J. Th. J. Verhoeven et al. Virus Res. 144:209, 2009.