Lavender Harbors More Viruses than Previously Thought: First Report of Raspberry Ringspot Virus and Phlox Virus M in Lavandula × intermedia.

Plants of the genus Lavandula are thought to be rarely infected by viruses. To date, only alfalfa mosaic virus, cucumber mosaic virus, tobacco mosaic virus, and tomato spotted wilt virus have been reported in this host. In this study, we identified for the first time raspberry ringspot virus (RpRSV) and phlox virus M (PhlVM) in lavender using herbaceous indexing, enzyme-linked immunosorbent assay, and high-throughput sequencing. Nearly complete genome sequences for both viruses were determined. Phylogenetic and serological characterizations suggest that the obtained RpRSV isolate is a raspberry strain. A preliminary survey of 166 samples indicated RpRSV was spread only in the lavender cultivar 'Grosso', while PhlVM was detected in multiple lavender cultivars. Although RpRSV raspberry strain may have spread throughout Auckland and nearby areas in New Zealand, it is very likely restricted to the genus Lavandula or even to the cultivar 'Grosso' due to the absence or limited occurrence of the nematode vector. Interestingly, all infected lavender plants, regardless of their infection status (by RpRSV, PhlVM, or both) were asymptomatic. RpRSV is an important virus that infects horticultural crops including grapevine, cherry, berry fruits, and rose. It remains on the list of regulated pests in New Zealand. RpRSV testing is mandatory for imported Fragaria, Prunus, Ribes, Rosa, Rubus, and Vitis nursery stock and seeds for sowing, while this is not required for Lavandula importation. Our study revealed that lavender could play a role not only as a reservoir but also as an uncontrolled import pathway of viruses that pose a threat to New Zealand's primary industries.

Detection, Characterization, and Distribution of the First Case of Pepino Mosaic Virus in Aotearoa New Zealand.

Tomato (Solanum lycopersicum L., family Solanaceae) represents one of the most economically valuable horticultural crops worldwide. Tomato production is affected by numerous emerging plant viruses. We identified, for the first time in New Zealand (NZ), Pepino mosaic virus (PepMV) in greenhouse grown tomato crops using a combination of methods from electron microscopy and herbaceous indexing to RT-qPCR and high-throughput sequencing. Phylogenetic and genomic analysis of a near-complete PepMV genome determined that the detected strain belonged to the mild form of the CH2 lineage of the virus. Subsequently, a delimiting survey of PepMV was conducted, and PepMV was detected at four additional locations. PCR-derived sequences obtained from samples collected from different greenhouses and from herbaceous indicator plants were identical to the original sequence. Since PepMV has never been reported in NZ before, seed pathways are speculated to be the most likely source of entry into the country.

First report of Fig virus B in Ficus carica in New Zealand.

Fig (Ficus carica) has been cultivated since ancient times, and is now grown worldwide, both for its fruit and as an ornamental plant. Several viruses and viroids are associated with Fig mosaic disease (FMD), a disease complex occurring worldwide (Preising et al. 2021). Fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig mild mottle-associated virus (FMMaV), and fig badnavirus 1 (FBV-1) are known to infect fig in New Zealand (Minafra et al. 2012; Veerakone et al. 2015). In December 2020, leaf samples from a fig tree growing on the roadside at St Heliers, Auckland, showing dieback with foliar chlorotic mosaic symptoms, was received for virus testing. Total nucleic acid was extracted from the symptomatic leaves using a KingFisher™ mL Purification System (Thermofisher Scientific, Waltham, MA) with an InviMag Plant DNA Mini Kit (Invitek Molecular GmbH, Germany) and subjected to high-throughput sequencing on an Oxford Nanopore Technologies MinION device using the method described in Liefting et al. 2021. All sequence analysis was performed using Geneious Prime 2021.1.1 (https://www.geneious.com). A total of 355,858 reads that passed quality check were subjected to BLASTn search against the NCBI nt database as described in Liefting et al. 2021. The following viruses produced hits: FMV, FBV-1, FMMaV and a fig closterovirus. The presence of FMV, FBV-1 and FMMaV were confirmed by species specific RT-PCRs. To identify the closterovirus, reads were mapped to closteroviruses reported in fig including the recently identified tentative species fig virus A (FiVA; GenBank accession no MN817232) and fig virus B (FiVB; GenBank accession no. MN817233). Five viral contigs ranging from 939 to 2,340 nucleotides (nt) were obtained from mapping to FiVB. Subsequently, a 6.4 kb sequence (GenBank accession no. OQ968551) from the 3' region of the NZ isolate was amplified by overlapping RT-PCR using primers designed from the contig sequences. The sequence shared 79.5% nucleotide (nt) identity with FiVB The original sample and a further 25 symptomatic and 10 asymptomatic fig samples, collected from the Auckland area between 2016 and 2021, were tested using FiVB specific RT-PCR and Sanger sequencing using primers FiVB-F1 (5'-GAGGGAGAGATGTAGATGC-3') and FiVB-R2 (5'-TGTCGTCGATATCGTTGTGT-3'), designed to amplify a 725 nt fragment in the 70 kDa heat shock protein (HSP70) ORF. Products of the expected size were amplified from the original sample and three symptomatic samples and their sequences found to be identical. BLAST searches showed that the sequence (GenBank accession no. ON553403) shared 82.7% nt and 87.3% amino acid (aa) identity to an isolate of FiVB (GenBank accession no MN817233). These additional positive samples were collected from a small home nursery where the plants were propagated from cuttings and have been distributed locally, suggesting the virus is very likely to have a limited spread throughout the Auckland area. All three FiVB infected samples were also positive for FMV. However, the association of FiVB with FMD symptoms is unknown. FiVB was first identified from a latex sample exuded from a fig tree collected from Japan (Park et al. 2021) and is the only report of FiVB in the world to date. Although an identical sequence from Argentina, named fig closterovirus 1, was submitted to GenBank, the origin of this isolate is not known. To our knowledge, this is the first report of FiVB in New Zealand.

Complete genome sequence of a novel potyvirus infecting Miscanthus sinensis (silver grass).

Here, we describe the full-length genome sequence of a novel potyvirus, tentatively named "Miscanthus sinensis mosaic virus" (MsiMV), isolated from Miscanthus sinensis (silver grass) held in a post-entry quarantine facility after being imported into Western Australia, Australia. The MsiMV genome is 9604 nucleotides (nt) in length, encoding a 3071-amino-acid (aa) polyprotein with conserved sequence motifs. The MsiMV genome is most closely related to that of sorghum mosaic virus (SrMV), with 74% nt and 78.5% aa sequence identity to the SrMV polyprotein region. Phylogenetic analysis based on the polyprotein grouped MsiMV with SrMV, sugarcane mosaic virus (SCMV), and maize dwarf mosaic virus (MDMV). This is the first report of a novel monopartite ssRNA virus in Miscanthus sinensis related to members of the genus Potyvirus in the family Potyviridae.

Partial biological and molecular characterization of a novel citrivirus from Nandina domestica.

We report the complete genome sequence of a novel virus isolated from Nandina domestica 'Firepower' in Auckland, New Zealand. It was mechanically transmitted to Nicotiana species, although all of these infections were symptomless. The complete genome of the new virus is 8892 nucleotides (nt) long, excluding the 3' poly(A) tail, contains three open reading frames (ORF), and is most closely related to citrus leaf blotch virus (CLBV) Actinidia isolate (CLBV-Act; 72% nt sequence identity), a member of the genus Citrivirus. Replicase and coat proteins, encoded by genome ORFs 1 and 3 respectively, shared 81-83% and 76-79% amino acid (aa) sequence identity, respectively, with CLBV-Act. Computer-based analysis suggests that this novel virus is the result of recombination between CLBV-Act and an unknown virus, highlighting the importance of this phenomenon for betaflexivirus evolution.

Characterization of the bacterial communities of psyllids associated with Rutaceae in Bhutan by high throughput sequencing.

BACKGROUND: Several plant-pathogenic bacteria are transmitted by insect vector species that often also act as hosts. In this interface, these bacteria encounter plant endophytic, insect endosymbiotic and other microbes. Here, we used high throughput sequencing to examine the bacterial communities of five different psyllids associated with citrus and related plants of Rutaceae in Bhutan: Diaphorina citri, Diaphorina communis, Cornopsylla rotundiconis, Cacopsylla heterogena and an unidentified Cacopsylla sp. RESULTS: The microbiomes of the psyllids largely comprised their obligate P-endosymbiont 'Candidatus Carsonella ruddii', and one or two S-endosymbionts that are fixed and specific to each lineage. In addition, all contained Wolbachia strains; the Bhutanese accessions of D. citri were dominated by a Wolbachia strain first found in American isolates of D. citri, while D. communis accessions were dominated by the Wolbachia strain, wDi, first detected in D. citri from China. The S-endosymbionts from the five psyllids grouped with those from other psyllid taxa; all D. citri and D. communis individuals contained sequences matching 'Candidatus Profftella armatura' that has previously only been reported from other Diaphorina species, and the remaining psyllid species contained OTUs related to unclassified Enterobacteriaceae. The plant pathogenic 'Candidatus Liberibacter asiaticus' was found in D. citri but not in D. communis. Furthermore, an unidentified 'Candidatus Liberibacter sp.' occurred at low abundance in both Co. rotundiconis and the unidentified Cacopsylla sp. sampled from Zanthoxylum sp.; the status of this new liberibacter as a plant pathogen and its potential plant hosts are currently unknown. The bacterial communities of Co. rotundiconis also contained a range of OTUs with similarities to bacteria previously found in samples taken from various environmental sources. CONCLUSIONS: The bacterial microbiota detected in these Bhutanese psyllids support the trends that have been seen in previous studies: psyllids have microbiomes largely comprising their obligate P-endosymbiont and one or two S-endosymbionts. In addition, the association with plant pathogens has been demonstrated, with the detection of liberibacters in a known host, D. citri, and identification of a putative new species of liberibacter in Co. rotundiconis and Cacopsylla sp.

The complete nucleotide sequence and genome organisation of a novel member of the family Betaflexiviridae from Actinidia chinensis.

We report the complete genome sequence of a novel virus, tentatively named "actinidia seed-borne latent virus" (ASbLV), isolated from Actinidia chinensis in Auckland, New Zealand. The complete genome of ASbLV is 8,192 nucleotides long, excluding the 3' poly(A) tail, contains four open reading frames, and is most closely related to Caucasus prunus virus (56% nucleotide sequence identity), a member of the genus Prunevirus. Based on the demarcation criteria of the family Betaflexiviridae, ASbLV is a new member of the genus Prunevirus.

An internet-based bioinformatics toolkit for plant biosecurity diagnosis and surveillance of viruses and viroids.

BACKGROUND: Detection and preventing entry of exotic viruses and viroids at the border is critical for protecting plant industries trade worldwide. Existing post entry quarantine screening protocols rely on time-consuming biological indicators and/or molecular assays that require knowledge of infecting viral pathogens. Plants have developed the ability to recognise and respond to viral infections through Dicer-like enzymes that cleave viral sequences into specific small RNA products. Many studies reported the use of a broad range of small RNAs encompassing the product sizes of several Dicer enzymes involved in distinct biological pathways. Here we optimise the assembly of viral sequences by using specific small RNA subsets. RESULTS: We sequenced the small RNA fractions of 21 plants held at quarantine glasshouse facilities in Australia and New Zealand. Benchmarking of several de novo assembler tools yielded SPAdes using a kmer of 19 to produce the best assembly outcomes. We also found that de novo assembly using 21-25 nt small RNAs can result in chimeric assemblies of viral sequences and plant host sequences. Such non-specific assemblies can be resolved by using 21-22 nt or 24 nt small RNAs subsets. Among the 21 selected samples, we identified contigs with sequence similarity to 18 viruses and 3 viroids in 13 samples. Most of the viruses were assembled using only 21-22 nt long virus-derived siRNAs (viRNAs), except for one Citrus endogenous pararetrovirus that was more efficiently assembled using 24 nt long viRNAs. All three viroids found in this study were fully assembled using either 21-22 nt or 24 nt viRNAs. Optimised analysis workflows were customised within the Yabi web-based analytical environment. We present a fully automated viral surveillance and diagnosis web-based bioinformatics toolkit that provides a flexible, user-friendly, robust and scalable interface for the discovery and diagnosis of viral pathogens. CONCLUSIONS: We have implemented an automated viral surveillance and diagnosis (VSD) bioinformatics toolkit that produces improved viruses and viroid sequence assemblies. The VSD toolkit provides several optimised and reusable workflows applicable to distinct viral pathogens. We envisage that this resource will facilitate the surveillance and diagnosis viral pathogens in plants, insects and invertebrates.

Opium poppy mosaic virus, a new umbravirus isolated from Papaver somniferum in New Zealand.

A novel virus, tentatively named "opium poppy mosaic virus" (OPMV), was isolated from Papaver somniferum (opium poppy) with leaf mosaic and mottling symptoms in Auckland, New Zealand, in 2006. The virus was mechanically transmitted to herbaceous plants of several species, in which it induced local and/or systemic symptoms. No virus particles were observed by electron microscopy in the diseased P. somniferum or any of the symptomatic herbaceous plants. The complete genomic sequence of 4230 nucleotides contains four open reading frames (ORF) and is most closely related (59.3 %) to tobacco bushy top virus, a member of the genus Umbravirus. These data suggest that OPMV is a new umbravirus.

High-Throughput Sequencing Methods for the Detection of Two Strawberry Viruses in Post-Entry Quarantine.

High-throughput sequencing (HTS) technologies may be a useful tool for testing imported plant germplasm for multiple pathogens present in a sample, offering strain-generic detection not offered by most PCR-based assays. Metatranscriptomics (RNAseq) and tiled amplicon PCR (TA-PCR) were tested as HTS-based techniques to detect viruses present in low titres. Strawberry mottle virus (SMoV), an RNA virus, and strawberry vein banding virus (SVBV), a DNA virus, were selected for comparison of RNAseq and TA-PCR with quantitative PCR assays. RNAseq of plant ribosomal RNA-depleted samples of low viral titre was used to obtain datasets from 3 M to 120 M paired-end (PE) reads. RNAseq demonstrated PCR-like sensitivity, able to detect as few as 10 viral copies/µL when 60 million (M) PE reads were generated. The custom TA-PCR primer panels designed for each virus were successfully used to recover most of the reference genomes for each virus. Single- and multiple-target TA-PCR allowed the detection of viruses in samples with around 10 viral copies/µL with a minimum continuous sequence length recovery of 500 bp. The limit of detection of the HTS-based protocols described here is comparable to that of quantitative PCR assays. This work lays the groundwork for an increased flexibility in HTS detection of plant viruses.

Comparison of the complete genome sequence of two closely related isolates of 'Candidatus Phytoplasma australiense' reveals genome plasticity.

BACKGROUND: 'Candidatus Phytoplasma australiense' is associated with at least nine diseases in Australia and New Zealand. The impact of this phytoplasma is considerable, both economically and environmentally. The genome of a NZ isolate was sequenced in an effort to understand its pathogenicity and ecology. Comparison with a closely related Australian isolate enabled us to examine mechanisms of genomic rearrangement. RESULTS: The complete genome sequence of a strawberry lethal yellows (SLY) isolate of 'Candidatus Phytoplasma australiense' was determined. It is a circular genome of 959,779 base pairs with 1126 predicted open reading frames. Despite being 80 kbp larger than another 'Ca. Phytoplasma australiense' isolate PAa, the variation between housekeeping genes was generally less than 1% at a nucleotide level. The difference in size between the two isolates was largely due to the number and size of potential mobile units (PMUs), which contributed to some changes in gene order. Comparison of the genomes of the two isolates revealed that the highly conserved 5' UTR of a putative DNA-directed RNA polymerase seems to be associated with insertion and rearrangement events. Two types of PMUs have been identified on the basis of the order of three to four conserved genes, with both PMUs appearing to have been present in the last common ancestor of 'Ca. Phytoplasma asteris' and 'Ca. Phytoplasma australiense'. Comparison with other phytoplasma genomes showed that modification methylases were, in general, species-specific. A putative methylase (xorIIM) found in 'Ca. Phytoplasma australiense' appeared to have no analogue in any other firmicute, and we believe has been introduced by way of lateral gene transfer. A putative retrostransposon (ltrA) analogous to that found in OY-M was present in both isolates, although all examples in PAa appear to be fragments. Comparative analysis identified highly conserved 5' and 3' UTR regions of ltrA, which may indicate how the gene is excised and inserted. CONCLUSIONS: Comparison of two assembled 'Ca. Phytoplasma australiense' genomes has shown they possess a high level of plasticity. This comparative analysis has yielded clues as to how rearrangements occur, and the identification of sets of genes that appear to be associated with these events.

Development and Validation of a Bioinformatic Workflow for the Rapid Detection of Viruses in Biosecurity.

The field of biosecurity has greatly benefited from the widespread adoption of high-throughput sequencing technologies, for its ability to deeply query plant and animal samples for pathogens for which no tests exist. However, the bioinformatics analysis tools designed for rapid analysis of these sequencing datasets are not developed with this application in mind, limiting the ability of diagnosticians to standardise their workflows using published tool kits. We sought to assess previously published bioinformatic tools for their ability to identify plant- and animal-infecting viruses while distinguishing from the host genetic material. We discovered that many of the current generation of virus-detection pipelines are not adequate for this task, being outperformed by more generic classification tools. We created synthetic MinION and HiSeq libraries simulating plant and animal infections of economically important viruses and assessed a series of tools for their suitability for rapid and accurate detection of infection, and further tested the top performing tools against the VIROMOCK Challenge dataset to ensure that our findings were reproducible when compared with international standards. Our work demonstrated that several methods provide sensitive and specific detection of agriculturally important viruses in a timely manner and provides a key piece of ground truthing for method development in this space.

Application of Oxford Nanopore Technology to Plant Virus Detection.

The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT's feasibility as a valuable component to the diagnostician's toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.

A New 'Candidatus Liberibacter' Species Associated with Diseases of Solanaceous Crops.

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with 'Candidatus Liberibacter' species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named 'Candidatus Liberibacter solanacearum'. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

Phylogenetic Analysis of "Candidatus Phytoplasma australiense" Reveals Distinct Populations in New Zealand.

ABSTRACT The phytoplasma "Candidatus Phytoplasma australiense" has been reported from New Zealand and Australia, where it has been associated with a range of host plants, especially since the 1970s. Partial tuf gene sequences of 36 New Zealand (NZ) isolates from four different host genera revealed nine different variants, which clustered into two distinct groups without any obvious correlation with host or geographic region. Phylogenetic analysis of these sequences, together with those available from Australian isolates, revealed three distinct clades: one found solely in Australia, one found solely in NZ, and a third with representatives from both countries. These divisions are consistent with differences observed in the 16-23S rRNA internal transcribed spacer region; therefore, we conclude that they represent three distinct subgroups: tuf 1, tuf 2, and tuf 3. We estimated a time of divergence for the three clades based on a synonymous substitution rate calculated by comparing the complete tuf gene sequence from the Loofah witches'-broom phytoplasma and "Candidatus Phytoplasma australiense". Using a calibration date of 110 million years, the estimated time to a common ancestor for all clades (6 to 9 million years ago) suggests divergence during the Miocene, well after the geological separation of NZ and Australia.

Development of a duplex one-step RT-qPCR assay for the simultaneous detection of Apple scar skin viroid and plant RNA internal control.

Apple scar skin viroid (ASSVd) is an important quarantine pathogen for international movement of pome germplasm as it can cause significant damage to pip fruit. A one-step real-time RT-PCR assay was developed for the rapid and sensitive detection of ASSVd. The assay was able to detect a wide range of ASSVd isolates and was highly specific compared to a published conventional RT-PCR. The detection limit of the new assay was estimated to be about 100 copies of the ASSVd target. The assay can be run as a duplex with the nad5 internal control primers and probe to simultaneously check the PCR competency of the samples therefore reducing the risk of false negatives. It is expected that this real-time RT-PCR assay will facilitate efficient testing for ASSVd by regulatory services, and will also have a wider use for the general detection of ASSVd in a range of pip fruit.

Cosmid cloning and sample sequencing of the genome of the uncultivable mollicute, Western X-disease phytoplasma, using DNA purified by pulsed-field gel electrophoresis.

Western X-disease (WX) phytoplasma is an uncultivable, intracellular pathogen of plants and insects with an AT-rich genome of 670 kb. As part of the genome sequencing project of WX phytoplasma, we have cloned approximately 50% of its genome into the pcosRW2 cosmid vector using DNA purified from pulsed-field gels. One of the cosmid clones with an insert of 24.6 kb was sequenced, which along with the cosmid end sequences, represents 60 kb of unique sequence from the WX phytoplasma genome. The putative genes identified in this sequence represent a wide variety of functions and many had not been previously identified in a phytoplasma.

Phytoplasma plasmid DNA extraction.

Phytoplasma plasmids have generally been detected from DNA extracted from plants and insects using methods designed for the purification of total phytoplasma DNA. Methods include extraction from tissues that are high in phytoplasma titre, such as the phloem of plants, with the use of CsCl-bisbenzimide gradients that exploit the low G+C content of phytoplasma DNA. Many of the methods employed for phytoplasma purification have been described elsewhere in this book. Here we describe in detail two methods that are specifically aimed at isolating plasmid DNA.

Genome wide sequence analysis grants unbiased definition of species boundaries in "Candidatus Phytoplasma".

The phytoplasmas are currently named using the Candidatus category, as the inability to grow them in vitro prevented (i) the performance of tests, such as DNA-DNA hybridization, that are regarded as necessary to establish species boundaries, and (ii) the deposition of type strains in culture collections. The recent accession to complete or nearly complete genome sequence information disclosed the opportunity to apply to the uncultivable phytoplasmas the same taxonomic approaches used for other bacteria. In this work, the genomes of 14 strains, belonging to the 16SrI, 16SrIII, 16SrV and 16SrX groups, including the species "Ca. P. asteris", "Ca. P. mali", "Ca. P. pyri", "Ca. P. pruni", and "Ca. P. australiense" were analyzed along with Acholeplasma laidlawi, to determine their taxonomic relatedness. Average nucleotide index (ANIm), tetranucleotide signature frequency correlation index (Tetra), and multilocus sequence analysis of 107 shared genes using both phylogenetic inference of concatenated (DNA and amino acid) sequences and consensus networks, were carried out. The results were in large agreement with the previously established 16S rDNA based classification schemes. Moreover, the taxonomic relationships within the 16SrI, 16SrIII and 16SrX groups, that represent clusters of strains whose relatedness could not be determined by 16SrDNA analysis, could be comparatively evaluated with non-subjective criteria. "Ca. P. mali" and "Ca. P. pyri" were found to meet the genome characteristics for the retention into two different, yet strictly related species; representatives of subgroups 16SrI-A and 16SrI-B were also found to meet the standards used in other bacteria to distinguish separate species; the genomes of the strains belonging to 16SrIII were found more closely related, suggesting that their subdivision into Candidatus species should be approached with caution.