Exogenous application of double-stranded RNA to reduce grapevine Pinot gris virus titre in in vitro grown Vitis vinifera

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

Generic Amplicon Deep Sequencing to Determine Ilarvirus Species Diversity in Australian Prunus

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

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

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

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

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

Optimization of a Loop Mediated Isothermal Amplification (LAMP) Assay for In-Field Detection of Dichelobacter nodosus With aprV2 (VDN LAMP) in Victorian Sheep Flocks

Dichelobacter nodosus is the primary etiological agent of footrot in sheep and has a variety of virulence factors. Of these, AprV2, an extracellular protease, has been shown to be capable of causing severe or “virulent” disease symptoms under the right conditions. Due to this, a loop-mediated isothermal amplification (LAMP) assay for the detection of aprV2-positive D. nodosus (VDN LAMP) was developed and evaluated for field use. A sample of 19 sheep flocks (309 sheep) in Victoria, Australia, were tested to determine the optimum conditions for in-field VDN LAMP assay use and sampling, for detecting aprV2-positive D. nodosus infected sheep. VDN LAMP performance was compared to a validated rtPCR that detects aprV2 and the benign strain counterpart, aprB2, using biologically duplicate samples to determine sensitivity and specificity. Flocks were sampled either in winter-spring (moist) or early summer (dry) conditions and had a range of clinical expressions of the disease ovine footrot. Variables considered for optimizing field performance were: sample collection method, sample preparation, clinical expression of disease, and nature of the feet when sampled (moist vs. dry, clean vs. soiled). The test was found to perform best when sheep were sampled with moist, clean feet, using a dry swab with the sample prepared in alkaline polyethylene glycol, pH 13.0, as the collection buffer. A sensitivity of 89% and specificity of 97% was seen when used in-field under these conditions, when compared to aprV2 detection by rtPCR, with “very good” agreement to rtPCR results. This study shows the VDN LAMP test is easy to use in-field to identify the presence of aprV2-positive D. nodosus in sheep flocks

‘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)

Genetic diversity and recombination between turnip yellows virus strains in Australia

Disease outbreaks caused by turnip yellows virus (TuYV), a member of the genus Polerovirus, family Luteoviridae, regularly occur in canola and pulse crops throughout Australia. To understand the genetic diversity of TuYV for resistance breeding and management, genome sequences of 28 TuYV isolates from different hosts and locations were determined using high-throughput sequencing (HTS). We aimed to identify the parts of the genome that were most variable and clarify the taxonomy of viruses related to TuYV. Poleroviruses contain seven open reading frames (ORFs): ORF 0–2, 3a, and 3–5. Phylogenetic analysis based on the genome sequences, including isolates of TuYV and brassica yellows virus (BrYV) from the GenBank database, showed that most genetic variation among isolates occurred in ORF 5, followed by ORF 0 and ORF 3a. Phylogenetic analysis of ORF 5 revealed three TuYV groups; P5 group 1 and group 3 shared 45–49% amino acid sequence identity, and group 2 is a recombinant between the other two. Phylogenomic analysis of the concatenated ORFs showed that TuYV is paraphyletic with respect to BrYV, and together these taxa form a well-supported monophyletic group. Our results support the hypothesis that TuYV and BrYV belong to the same species and that the phylogenetic topologies of ORF 0, 3a and 5 are incongruent and may not be informative for species demarcation. A number of beet western yellow virus (BWYV)- and TuYV-associated RNAs (aRNA) were also identified by HTS for the first time in Australia