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.

Complete nucleotide sequence of sweetbriar rose curly-top associated virus, a tentative member of the genus Waikavirus.

A novel virus, tentatively named "sweetbriar rose curly-top associated virus" (SRCTaV), was identified in sweetbriar rose (Rosa rubiginosa) using high-throughput sequencing. The complete genome sequence of SRCTaV was determined and characterized. Phylogenetic analysis revealed that SRCTaV is closely related to members of the genus Waikavirus.

First report of Grapevine Algerian latent virus in carnation in New Zealand.

Carnation (Dianthus caryophyllus) is a popular ornamental plant widely used as a cut flower and in landscaping. In New Zealand, several viruses are known to infect plants of the genus Dianthus: arabis mosaic virus, carnation etched ring virus (CERV), carnation latent virus, carnation mottle virus, carnation necrotic fleck virus, carnation ringspot virus, carnation vein mottle virus and cucumber mosaic virus (Veerakone et al. 2015). In October 2020, a carnation sample with leaf chlorotic spots and distortion from a home garden in Auckland, New Zealand was submitted to the Plant Health and Environment Laboratory (PHEL) for virus testing. Leaf tissue of the sample was mechanically inoculated onto a range of herbaceous species using the method described in Tang et al. (2013). Chenopodium amaranticolor and C. quinoa plants developed local necrotic pinpoint spots while Nicotiana benthamiana, N. clevelandii, and N. occidentalis plants exhibited systemic leaf mosaic symptoms 7 days post-inoculation. The carnation plant and all five symptomatic indicator species tested positive for tombusviruses using an in-house designed generic RT-qPCR (available on request). Direct sequencing of the ~140 bp PCR product revealed the presence of grapevine Algerian latent virus (GALV). To further characterise the detected sequence, forward (5'-GTAGCGATGTATTGGGATAAGGA-3') and reverse (5'-TGCCGACACCCCGAAAGGT-3') primers were designed based on an alignment of the conserved region in the coat protein (CP) of 19 GALV isolates deposited in GenBank. Products of the expected size of 406 bp were amplified from all infected plants and their sequences found to be identical (GenBank accession No. OM891837). BLAST searches showed that the CP region of the sequence shared 97.0% (nucleotide) and 97.8% (amino acid) identity to the type isolate of GALV (GenBank accession no. NC_011535). GALV was first reported in Italy from a symptomless Algerian grapevine (Vitis vinifera) (Gallitelli et al., 1989), and is the only report of GALV in Vitis in the world. Since then, GALV has been reported in Germany, the Netherlands and Japan in several ornamental plant species including Alstroemeria sp. (Tomitaka et al., 2016), Gypsophila paniculata, Limonium sinuatum (Koenig et al., 2004, Fujinaga et al., 2009) and Solanum mammosum (Ohki et al., 2006). These infected ornamental host plants were reported to show various types of foliar symptoms, including chlorotic leaf spots. The GALV-infected carnation plant in this study was tested by PCR for all viruses that are known to infect D. caryophyllus in New Zealand, and CERV was identified. It is therefore unclear if the observed symptoms were induced by either GALV or CERV, or if they were the results of a synergistic interaction between GALV and CERV. Samples from a further 11 plants, comprised of nine symptomatic Dianthus spp. and two asymptomatic Alstroemeria spp. were collected from the same address and tested individually using the GALV-specific RT-PCR. GALV (along with CERV) was detected from all Dianthus plants while the Alstroemeria samples were negative. To our knowledge, this is the first report of GALV in New Zealand, and the first report in the host Dianthus in the world. Given the GALV-infected carnation plants were purchased from a local garden centre between 2007-2020, and plants from this garden centre have been widely distributed over this period of time to various customers, the virus is very likely to have spread throughout the country.

New Virus Diagnostic Approaches to Ensuring the Ongoing Plant Biosecurity of Aotearoa New Zealand.

To protect New Zealand's unique ecosystems and primary industries, imported plant materials must be constantly monitored at the border for high-threat pathogens. Techniques adopted for this purpose must be robust, accurate, rapid, and sufficiently agile to respond to new and emerging threats. Polymerase chain reaction (PCR), especially real-time PCR, remains an essential diagnostic tool but it is now being complemented by high-throughput sequencing using both Oxford Nanopore and Illumina technologies, allowing unbiased screening of whole populations. The demand for and value of Point-of-Use (PoU) technologies, which allow for in situ screening, are also increasing. Isothermal PoU molecular diagnostics based on recombinase polymerase amplification (RPA) and loop-mediated amplification (LAMP) do not require expensive equipment and can reach PCR-comparable levels of sensitivity. Recent advances in PoU technologies offer opportunities for increased specificity, accuracy, and sensitivities which makes them suitable for wider utilization by frontline or border staff. National and international activities and initiatives are adopted to improve both the plant virus biosecurity infrastructure and the integration, development, and harmonization of new virus diagnostic technologies.

International Trade and Local Effects of Viral and Bacterial Diseases in Ornamental Plants.

Since the 1950s, there have been major changes in the scope, value, and organization of the ornamental plant industry. With fewer individual producers and a strong trend toward consolidation and globalization, increasing quantities of diverse plant genera and species are being shipped internationally. Many more ornamentals are propagated vegetatively instead of by seed, further contributing to disease spread. These factors have led to global movement of pathogens to countries where they were not formerly known. The emergence of some previously undescribed pathogens has been facilitated by high-throughput sequencing, but biological studies are often lacking, so their roles in economic diseases are not yet known. Case studies of diseases in selected ornamentals discuss the factors involved in their spread, control measures to reduce their economic impact, and some potential effects on agronomic crops. Advances in diagnostic techniques are discussed, and parallels are drawn to the international movement of human diseases.

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.

Sensitive detection of Tomato ringspot virus by real-time TaqMan RT-PCR targeting the highly conserved 3'-UTR region.

A real-time TaqMan RT-PCR assay was developed for the rapid and sensitive detection of Tomato ringspot virus (ToRSV), an important plant virus which infects a wide range of fruit and ornamental crops. Primers and a probe were designed based on the highly conserved 3'-untranslated region (UTR) sequences of ToRSV, to amplify a 182bp fragment of this region of RNA-1 and RNA-2. The assay was demonstrated to reliably amplify all ToRSV isolates tested. The detection limit was estimated to be about 12 copies of the ToRSV target region. No amplification was observed from the RNA of other nepoviruses or healthy host species. A comparison with a published conventional RT-PCR and a SYBR-based qRT-PCR indicated that both of the published assays lacked reliability and sensitivity, as neither were able to amplify all ToRSV isolates tested, and both were approximately 1000 times less sensitive than the novel TaqMan real-time assay. This TaqMan real-time assay was tested using four different reagent kits and was shown to be robust and stable, with no significant differences in sensitivity between kits. It is expected that the implementation of this TaqMan real-time RT-PCR assay will facilitate efficient phytosanitary certification of nursery stock requiring testing for ToRSV by regulatory agencies, and will also have wider uses for the general detection of ToRSV in a range of hosts.

Detection of Tomato black ring virus by real-time one-step RT-PCR.

A TaqMan-based real-time one-step RT-PCR assay was developed for the rapid detection of Tomato black ring virus (TBRV), a significant plant pathogen which infects a wide range of economically important crops. Primers and a probe were designed against existing genomic sequences to amplify a 72 bp fragment from RNA-2. The assay amplified all isolates of TBRV tested, but no amplification was observed from the RNA of other nepovirus species or healthy host plants. The detection limit of the assay was estimated to be around nine copies of the TBRV target region in total RNA. A comparison with conventional RT-PCR and ELISA, indicated that ELISA, the current standard test method, lacked specificity and reacted to all nepovirus species tested, while conventional RT-PCR was approximately ten-fold less sensitive than the real-time RT-PCR assay. Finally, the real-time RT-PCR assay was tested using five different RT-PCR reagent kits and was found to be robust and reliable, with no significant differences in sensitivity being found. The development of this rapid assay should aid in quarantine and post-border surveys for regulatory agencies.