Development of TaqMan RT-qPCR for the detection of regulated citrus viruses and viroids in Aotearoa New Zealand.

The major citrus species include several economically important fruits, such as orange, mandarin, lemon, limes, grapefruit and pomelos. Since the 1980 s, total production and consumption of citrus has grown strongly with the current annual worldwide production at over 105 million tonnes. New Zealand's citrus exports, for instance, had an estimated worth of NZ$ 11.6 million (approx. US$ 7 million) in 2020. Citrus plants are prone to viral diseases, which can lead to substantial economic losses. In New Zealand, the citrus Import Health Standard (IHS) has identified 22 viruses and viroids that are subject to regulation and requires citrus nursery stock to be free of these pathogens. As such, there is a need for reliable, sensitive, and rapid detection methods to screen for these viruses and viroids during post entry quarantine. In this study, we developed TaqMan RT-qPCR assays for the detection of nine of these regulated viruses and viroids, namely citrus leaf rugose virus (CiLRV), citrus leprosis virus C (CiLV-C), citrus leprosis virus C2 (CiLV-C2), citrus leprosis virus N (CiLV-N), citrus psorosis virus (CPsV), citrus yellow mosaic virus (CYMV), citrus bent leaf viroid (CBLVd), citrus viroid V (CVd-V), and citrus viroid VI (CVd-VI). These assays have been validated and found to be highly sensitive, specific, and reliable. The implementation of these assays will facilitate the safe importation of citrus nursery stock, thus safeguarding the country's horticultural and economic interests.

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 Streptocarpus flower break virus in Streptocarpus hybrids in Aotearoa New Zealand.

Streptocarpus (Cape primrose, family Gesneriaceae) is a genus of plants native to Southern Africa commonly grown indoors for their foliage and trumpet-shaped flowers. In Aoteroa New Zealand (NZ) to date, no viruses have been reported to infect plants of the Gesneriaceae (Veerakone et al. 2015). In September 2022, a plant of Streptocarpus hybrid exhibiting necrotic rings was observed in a hobbyist's greenhouse in Auckland, NZ. High-Throughput Sequencing (HTS) using MinIONTM (Oxford Nanopore Technologies), was applied as a first screen (Liefting et al. 2021). Phylogenetic analysis was performed using Geneious Prime 2021 (Biomatters Ltd, NZ). A BLASTn search with 622,847 obtained reads resulted in 3,260 and 4,340 matches to the sequences of Streptocarpus flower break tobamovirus (SFBV) and Impatiens necrotic spot orthotospovirus (INSV), respectively. A near-complete (98.5%) genome sequence of SFBV was obtained (GenBank accession No. OQ970154), which shared 99.52% nucleotide identity to a SFBV type isolate from Germany (GenBank accession No. NC_008365). A phylogenetic tree was also generated (e-Xtra). To confirm the presence of both viruses, leaf tissue was rub inoculated onto herbaceous indicator plants as described by Tang et al. (2013). Chenopodium amaranticolor and C. quinoa plants developed local lesions while Nicotiana occidentalis plants showed local necrosis followed by systemic leaf puckering by 14 days post inoculation (dpi). Nicotiana benthamiana and N. clevelandii plants showed systemic chlorosis but N. tabacum plants did not exhibit any symptoms by 28 dpi. Samples from indicators and Streptocarpus were tested by RT-PCR (SFBV) or RT-qPCR (INSV), using in-house designed primers: SFBV-forward (5'-GTCATCAGCCGGAGAGGTTC-3'), SFBV-reverse (5'-AGGGCGAGTCTCTTCCTCTG-3'), INSV-forward (5'-CAATCAGAGGGTGACTTGGAA-3'), INSV-reverse (5'-GACTTTCCGAAGACTTGATGC-3') and INSV-probe (5'-CCATTGTCCTTTATCATTCCAACAAG-3'). RT-PCR products (across MP and CP regions) of the expected size (357 bp) were amplified from the Streptocarpus sample and symptomatic indicators. All amplicons were sequenced in both directions and found to be identical to the obtained HTS sequence. The presence of INSV was confirmed in all Streptocarpus and inoculated indicators except N. tabacum by INSV-specific RT-qPCR. A further 77 Streptocarpus plants were collected from a greenhouse in Auckland that holds a collection of multiple Streptocarpus cultivars from across NZ and overseas. Twenty-five plants, either displaying flower colour-break (only one plant) or asymptomatic (24 plants), tested positive for SFBV by RT-PCR. All amplicons were sequenced and found to be identical. SFBV was first described from naturally infected Streptocarpus plants in 1995 in the Netherlands (Verhoeven et al. 1995), and then in Germany (Heinze et al. 2006) and the United States (Pappu & Druffel 2007). While INSV has been found in NZ in several plant genera (Veerakone et al., 2015), to our knowledge, this is the first report of SFBV in NZ. SFBV was thought to be associated with colour breaking of Streptocarpus flowers (Verhoeven et al. 1995) but the virus was detected in asymptomatic Streptocarpus plants in this study and in California (Pappu & Druffel 2007). Given SFBV-infected plants were purchased from several sources, and leaf cuttings for propagation are shared among hobbyists, SFBV is likely to have spread throughout NZ. How this will affect production is unclear at this stage.

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.

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.

Analysis of the genome of grapevine red blotch virus and related grabloviruses indicates diversification prior to the arrival of Vitis vinifera in North America.

In this study 163 complete whole-genome sequences of the emerging pathogen grapevine red blotch virus (GRBV; genus Grablovirus, family Geminiviridae) were used to reconstruct phylogenies using Bayesian analyses on time-tipped (heterochronous) data. Using different combinations of priors, Bayes factors identified heterochronous datasets (3×200 million chains) generated from strict clock and exponential tree priors as being the most robust. Substitution rates of 3.2×10-5 subsitutions per site per year (95% HPD 4.3-2.1×10-5) across the whole of the GRBV genome were estimated, suggesting ancestral GRBV diverged from ancestral wild Vitis latent virus 1 around 9 000 years ago, well before the first documented arrival of Vitis vinifera in North America. Whole-genome analysis of GRBV isolates in a single infected field-grown grapevine across 12 years identified 12 single nucleotide polymorphisms none of which were fixed substitutions: an observation not discordant with the in silico estimate. The substitution rate estimated here is lower than those estimated for other geminiviruses and is the first for a woody-host-infecting geminivirus.

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.

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.

Searching for the Mechanism that Mediates Mefenoxam-Acquired Resistance in Phytophthora infestans and How It Is Regulated.

Phytophthora infestans, the causal agent of late blight disease of potatoes, is mainly controlled by the use of fungicides. Isolates that are resistant to commonly used fungicides have been reported. Also, several studies show that originally mefenoxam-sensitive isolates acquire resistance to this fungicide when exposed to sublethal concentrations. This phenomenon, termed "mefenoxam-acquired resistance," has been observed in different Phytophthora species and seems to be unique to mefenoxam. In this study, we aimed to elucidate the molecular mechanism mediating this type of resistance as well as a possible regulatory process behind it. A combination of computational analyses and experimental approaches was used to identify differentially expressed genes with a potential association to the phenomenon. These genes were classified into seven functional groups. Most of them seem to be associated with a pleiotropic drug resistance (PDR) phenotype, typically involved in the expulsion of diverse metabolites, drugs, or other substances out of the cell. Despite the importance of RNA Polymerase I for the constitutive resistance of P. infestans to mefenoxam, our results indicate no clear interaction between this protein and the acquisition of mefenoxam resistance. Several small non-coding RNAs were found to be differentially expressed and specifically related to genes mediating the PDR phenotype, thus suggesting a possible regulatory process. We propose a model of the molecular mechanisms acting within the cell when P. infestans acquires resistance to mefenoxam after exposed to sublethal concentrations of the fungicide. This study provides important insights into P. infestans' cellular and regulatory functionalities.

ICTV Virus Taxonomy Profile: Secoviridae 2022.

Members of the family Secoviridae are non-enveloped plant viruses with mono- or bipartite linear positive-sense ssRNA genomes with a combined genome of 9 to 13.7 kb and icosahedral particles 25-30 nm in diameter. They are related to picornaviruses and are members of the order Picornavirales. Genera in the family are distinguished by the host range, vector, genomic features and phylogeny of the member viruses. Most members infect dicotyledonous plants, and many cause serious disease epidemics. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Secoviridae, which is available at ictv.global/report/secoviridae.

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.

Grapevine Asteroid Mosaic-Associated Virus is Resident and Prevalent in Wild, Noncultivated Grapevine of New York State.

In North America, uncultivated, free-living grapevines (Vitis spp.) frequently grow alongside their cultivated counterparts, thus increasing the potential for exchange of microbiota. For this study, we used high-throughput sequencing (HTS) of small RNAs to survey for virus populations in free-living grapevines of the Finger Lakes region of New York State. Of 32 grapevines analyzed, 23 were free-living vines, while the remaining 9 were commercially grown Vitis vinifera plants from the same region. In total, 18 (78.3%) of the free-living grapevines tested were positive for grapevine asteroid mosaic-associated virus (GAMaV) infection by HTS, with detection confirmed by seminested reverse-transcription PCR and sequencing of nine isolates. Phylogenetic analyses of an ungapped alignment of the New York GAMaV sequences (length: 2,334 nucleotides) with the five known full-length or close to full-length global sequences showed that the New York isolates were broadly grouped. Of the nine cultivated plants, eight were infected with both hop stunt viroid and grapevine yellow speckle viroid 1, three were singly infected with grapevine leafroll-associated virus 3, and one harbored GAMaV. This limited survey of free-living grapevines, one of the first to use HTS, has highlighted the high incidence of a virus associated with disease in commercial V. vinifera.

Proposed revision of the family Secoviridae taxonomy to create three subgenera, "Satsumavirus", "Stramovirus" and "Cholivirus", in the genus Sadwavirus.

We present a taxonomic proposal for revision of the family Secoviridae, a taxon of plant viruses in the order Picornavirales. We propose the reorganization of the genus Sadwavirus to create three new subgenera and to update the classification of five existing species. The proposed subgenera are "Satsumavirus" (one species: Satsuma dwarf virus), "Stramovirus" (two species: Strawberry mottle virus and Black raspberry necrosis virus) and "Cholivirus" (two species: Chocolate lily virus A and Dioscorea mosaic associated virus).

Engineering partial resistance to cucumber mosaic virus in tobacco using intrabodies specific for the viral polymerase.

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants.

A rapid, sensitive and inexpensive method for detection of grapevine red blotch virus without tissue extraction using loop-mediated isothermal amplification.

Grapevine red blotch virus (GRBV) is an emerging virus of significant viticultural importance throughout North America. Here, we report the development of a simple protocol for point-of-use detection of GRBV. Extraction of nucleic acids is not required; instead, the whole intact plant can simply be pricked with a sterile pipette tip, which is then incubated in sterile distilled water to provide the sample template in a loop-mediated isothermal amplification (LAMP) reaction. This method is 10,000 times more sensitive than conventional PCR, costs under a dollar per sample, and can be completed from sampling to readout in just over half an hour.

Evidence for the splicing of grablovirus transcripts reveals a putative novel open reading frame.

Grapevine red blotch virus (GRBV) is type member of the newly identified genus Grablovirus. It possesses a single-stranded circular DNA genome of around 3200 nucleotides encoding three open reading frames (ORFs) in both the virion sense, the V1 (CP), V2 and V3, and complementary sense, C1 (RepA), C2 and C3. As shown for members of the genus Mastrevirus, the C1 and C2 ORFs are predicted to fuse through splicing to form a replication-associated protein (Rep). Data obtained using high-throughput sequencing (RNA-Seq) of three RNA-enriched populations, extracted from GRBV-infected grapevine (Vitis vinifera), confirmed the presence of the predicted C1-C2 intron (nts 2288-2450), but in addition identified a larger virion-sense intron (nts 251-589) spanning the V2 ORF. Evidence for both introns in a number of isolates was supported by bioinformatic analysis of publicly available datasets (n=20). These observations were further supported by RT-PCR analyses in both GRBV-infected grapevine and transient expression assays where GRBV genome segments were agro-inoculated onto Nicotiana benthamiana. The donor site of the virion-sense intron is located within two small ORFs, V0 and V02, while the acceptor site is two-thirds along the V2 ORF. Splicing at these positions is predicted to delete the N terminus of the encoded V2 protein. Comparative analyses of full-length GRBV sequences and the related tentative grabloviruses Prunus geminivirus A and wild Vitis virus 1 support the existence of both introns and V0. The probable regulatory role of these introns in the GRBV infection cycle is discussed.

The Distribution and Detection of Grapevine red blotch virus in its Host Depend on Time of Sampling and Tissue Type.

Grapevine red blotch virus (GRBV) is the causal agent of grapevine red blotch, an emerging disease that affects cultivated grapevine such as Vitis vinifera. The ability to detect viruses in grapevine is often hindered by low virus titers compounded by a variable distribution in the plant and seasonal variations. In order to examine these two variables in relation to GRBV, we developed a quantitative polymerase chain reaction (qPCR) method that incorporates both internal and external references to enhance assay robustness. In greenhouse-grown vines infected with GRBV, qPCR identified highest virus titers in the petioles of fully expanded leaves and significantly reduced levels of virus in the shoot extremities. In vineyard-grown vines infected with GRBV, the virus titer in July and October 2016 followed a pattern similar to that found for the greenhouse-grown plants but, most strikingly, close to half (44%) of the samples analyzed in June 2015 tested negative for infection. The technique presented and results obtained highlight the variability of virus distribution in its host and provide a useful guide for selecting the best tissues for optimal GRBV diagnosis.

Prevalence and Genetic Diversity of Grabloviruses in Free-Living Vitis spp.

The distribution and diversity of grapevine red blotch virus (GRBV) and wild Vitis virus 1 (WVV1) (genus Grablovirus; family Geminiviridae) were determined in free-living Vitis spp. in northern California and New York from 2013 to 2017. Grabloviruses were detected by polymerase chain reaction in 28% (57 of 203) of samples from California but in none of the 163 samples from New York. The incidence of GRBV in free-living vines was significantly higher in samples from California counties with high compared with low grape production (χ2 = 83.09; P < 0.001), and in samples near (<5 km) to compared with far (>5 km) from vineyards (χ2 = 57.58; P < 0.001). These results suggested a directional spread of GRBV inoculum predominantly from vineyards to free-living Vitis spp. WVV1 incidence was also significantly higher in areas with higher grape production acreage (χ2 = 16.02; P < 0.001). However, in contrast to GRBV, no differential distribution of WVV1 incidence was observed with regard to distance from vineyards (χ2 = 0.88; P = 0.3513). Two distinct phylogenetic clades were identified for both GRBV and WVV1 isolates from free-living Vitis spp., although the nucleotide sequence variability of the genomic diversity fragment was higher for WWV1 (94.3 to 99.8% sequence identity within clade 1 isolates and 90.1 to 100% within clade 2 isolates) than GRBV (98.3% between clade 1 isolates and 96.9 to 100% within clade 2 isolates). Additionally, evidence for intraspecific recombination events was found in WVV1 isolates and confirmed in GRBV isolates. The prevalence of grabloviruses in California free-living vines highlights the need for vigilance regarding potential grablovirus inoculum sources in order to protect new vineyard plantings and foundation stock vineyards in California.