Genomic variation in pepper vein yellows viruses in Australia, including a new putative variant, PeVYV-10.

Since the first identification and full sequence of the polerovirus pepper vein yellows virus in Australia in 2016, virus surveys of crops and weeds have sporadically identified PeVYV in different hosts and locations. Genomic comparisons of 14 PeVYV-like isolates using RT-PCR products spanning the 3' end of the RdRp region (ORF 2), the intergenic region, ORF 3a, ORF 4, and ORF 3 (1388 nt) showed that four of the PeVYV isolates might be a new variant or PeVYV-like virus. From six PeVYV-positive plants, eight PeVYV-like sequences were obtained by high-throughput sequencing, as two hosts, 5352 and 5634, contained two slightly different PeVYV-like isolates. Three of the PeVYV-like isolates were most closely related to PeVYV-6 and PeVYV-5, and two isolates were closely related to PeVYV-9 and PeVYV-2. The other three isolates shared only 69-74% nucleotide sequence identity across the whole genome with any of the other PeVYVs, despite sharing 73-98%, 87-91%, and 84-87% amino acid sequence identity in ORF 3a, ORF 3, and the RdRp (ORF 2), respectively, suggesting that this virus is a new PeVYV-like virus, which we have tentatively called PeVYV-10. This is also the first report of a PeVYV-like virus infecting garlic.

The observation of taxonomic boundaries for the 16SrII and 16SrXXV phytoplasmas using genome-based delimitation.

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.

Comparative genomics and genomic diversity of Pseudomonas syringae clade 2b-a in Australia.

BACKGROUND: A zucchini disease outbreak with unusual symptoms associated with Pseudomonas syringae clade 2b was identified in Bundaberg, Australia during autumn 2016. To investigate the genetic diversity of the 11 Australian isolates obtained from the outbreak, the genomes were compared to the publicly available P. syringae strains in phylogroup 2. RESULTS: Average nucleotide identity refined the P. syringae clade 2b-a into four clusters (Cluster A, B, C1 and C2), an expansion from the previously identified A, B and C. Australian isolates were in Cluster A, C1 and C2. Genomic analyses highlighted several key factors that may contribute to the virulence of these isolates. Six orthologous groups, including three virulence factors, were associated with P. syringae phylogroup 2 cucurbit-infecting strains. A region of genome plasticity analysis identified a type VI secretion system pathway in clade 2b-a strains which could also contribute to virulence. Pathogenicity assays on isolates KL004-k1, KFR003-1 and 77-4C, as representative isolates of Cluster A, C1 and C2, respectively, determined that all three isolates can infect pumpkin, squash, watermelon and zucchini var. Eva with different levels of disease severity. Subsequently, type III effectors were investigated and four type III effectors (avrRpt2, hopZ5, hopC1 and hopH1) were associated with host range. The hopZ effector family was also predicted to be associated with disease severity. CONCLUSIONS: This study refined the taxonomy of the P. syringae clade 2b-a, supported the association between effector profile and pathogenicity in cucurbits established in a previous study and provides new insight into important genomic features of these strains. This study also provided a detailed and comprehensive resource for future genomic and functional studies of these strains.

Characterization of Pseudomonas syringae Isolated from Systemic Infection of Zucchini in Australia.

Zucchini plants with symptoms including twisted petioles, necrotic leaves, crown rot, and internal fruit rot were found in Bundaberg, Australia, at a commercial field for the first time during late autumn 2016, resulting in direct yield losses of 70 to 80%. Three Pseudomonas syringae strains isolated from symptomatic leaf (KL004-k1), crown (77-4C), and fruit (KFR003-1) were characterized and their pathogenicity evaluated on pumpkin, rockmelon, squash, and zucchini. Biochemical assays showed typical results for P. syringae. The three isolates differed, however, in that two produced fluorescent pigment (KFR003-1 and 77-4C) whereas the third, KL004-k1, was nonfluorescent. Multilocus sequence analysis classified the isolates to phylogroup 2b. The single-nucleotide polymorphism analysis of core genome from the Australian and closely related international isolates of P. syringae showed two separate clusters. The Australian isolates were clustered based on fluorescent phenotype. Pathogenicity tests demonstrated that all three isolates moved systemically within the inoculated plants and induced necrotic leaf symptoms in zucchini plants. Their identities were confirmed with specific PCR assays for P. syringae and phylogroup 2. Pathogenicity experiments also showed that the Eva variety of zucchini was more susceptible than the Rosa variety for all three isolates. Isolate KL004-k1 was more virulent than 77-4C on pumpkin, rockmelon, squash, and zucchini. This study expands the knowledge of P. syringae isolates that infect cucurbits and provides useful information for growers about the relative susceptibility of a range of cucurbit species.

'Candidatus Phytoplasma stylosanthis', a novel taxon with a diverse host range in Australia, characterised using multilocus sequence analysis of 16S rRNA, secA, tuf, and rp genes.

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 iPhyClassifier 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).

A metagenomic investigation of phytoplasma diversity in Australian vegetable growing regions.

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.

The Establishment and Spread of a Newly Introduced Begomovirus in a Dry Tropical Environment Using Tomato Yellow Leaf Curl Virus as a Case Study.

Early detection of tomato yellow leaf curl virus (TYLCV) in a previously unaffected tomato production district in Australia allowed its spread to be evaluated spatially and temporally. The population dynamics of the TYLCV vector, Bemisia argentifolii (silverleaf whitefly, SLW), were also evaluated. The district is a dry tropical environment with a clear break to commercial production during the summer wet season. The incidence of TYLCV within crops and its prevalence through the district was influenced by weather, location, vector movements, and the use of Ty-1 virus-resistant hybrids. Rainfall had an important influence, with late summer and early autumn rain suppressing the levels of SLW and, by contrast, a dry summer supporting faster population growth. The use of Ty-1 hybrids appears to have reduced the incidence of TYLCV in this district. There was limited use of Ty-1 hybrids during 2013, and by season end, crops had moderate levels of SLW and high virus incidence. The 2015 and early 2016 season had high SLW populations, but TYLCV incidence was lower than in 2013, possibly due to the widespread adoption of the Ty-1 hybrids reducing virus spread. This study provides valuable epidemiology data for future incursions of begomoviruses, and other viruses spread by SLW.

Development of a Novel Tissue Blot Hybridization Chain Reaction for the Identification of Plant Viruses.

Assays for the high throughput screening of crops for virus monitoring need to be quick, easy, and low cost. One method involves using tissue blot immunoassays (TBIA), where plant stems are blotted onto nitrocellulose membrane and screened with available antibodies against a range of viruses. TBIAs are inexpensive but limited by antibody availability and specificity. To circumvent the antibody limitations, we developed the tissue blot hybridization chain reaction (TB-HCR). As with TBIA, plant stems are blotted onto a nitrocellulose membrane, however, TB-HCR involves using nucleic acid probes instead of antibodies. We demonstrated for the first time that TB-HCR can be used for plant viruses by designing and testing probes against species from several virus genera including begomovirus, polerovirus, luteovirus, cucumovirus, and alfamovirus. We also explored different hairpin reporter methods such as biotin/streptavidin-AP and the Alexa Fluor-488 Fluorophore. TB-HCR has applications for low-cost diagnostics for large sample numbers, rapid diagnostic deployment for new viruses, and can be performed as a preliminary triage assay prior to downstream applications.

Iodixanol density gradients as an effective phytoplasma enrichment approach to improve genome sequencing.

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.

Detection and Distribution of Viruses Infecting Garlic Crops in Australia.

The distribution of viruses in eastern Australian field garlic was evaluated. Detection assays were developed that involved generic RT-PCR for viruses in the Allexivirus, Carlavirus and Potyvirus genera followed by virus-specific colorimetric dot-blot hybridization. Assays targeted the potyviruses (onion yellow dwarf virus (OYDV), shallot yellow stripe virus (SYSV), and leek yellow stripe virus (LYSV)), the carlaviruses (garlic common latent virus (GCLV) and shallot latent virus (SLV)), and the allexiviruses (garlic viruses A, B, C, X (GarVA, -B, -C, -X) and shallot virus X (ShVX)). Virus incidence in crops was consistently high, with most plants infected with at least one virus from each genus. OYDV, LYSV, SLV, and GCLV were commonly detected. Three of the four allexiviruses were in all districts surveyed but varied in incidence, whereas ShVX and SYSV were not detected. There was no association between virus species complement and bulb size, indicating size is not a good predictor of the virus status of planting material. The variation of virus incidence across different Australian growing districts and in different cultivars implies multiple introductions of viruses rather than spread within the country. The genetic diversity observed within coat protein sequences of some virus species also supports multiple separate introductions.

A Rapid and Cost-Effective Identification of Invertebrate Pests at the Borders Using MinION Sequencing of DNA Barcodes.

The rapid and accurate identification of invertebrate pests detected at the border is a challenging task. Current diagnostic methods used at the borders are mainly based on time consuming visual and microscopic examinations. Here, we demonstrate a rapid in-house workflow for DNA extraction, PCR amplification of the barcode region of the mitochondrial cytochrome oxidase subunit I (COI) gene and Oxford Nanopore Technologies (ONT) MinION sequencing of amplified products multiplexed after barcoding on ONT Flongle flow cells. A side-by-side comparison was conducted of DNA barcode sequencing-based identification and morphological identification of both large (>0.5 mm in length) and small (<0.5 mm in length) invertebrate specimens intercepted at the Australian border. DNA barcode sequencing results supported the morphological identification in most cases and enabled immature stages of invertebrates and their eggs to be identified more confidently. Results also showed that sequencing the COI barcode region using the ONT rapid sequencing principle is a cost-effective and field-adaptable approach for the rapid and accurate identification of invertebrate pests. Overall, the results suggest that MinION sequencing of DNA barcodes offers a complementary tool to the existing morphological diagnostic approaches and provides rapid, accurate, reliable and defendable evidence for identifying invertebrate pests at the border.

Genome Characterization of Two Carrot Virus Y Isolates from 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.