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.

TASPERT: Target-Specific Reverse Transcript Pools to Improve HTS Plant Virus Diagnostics.

High-throughput sequencing (HTS) is becoming the new norm of diagnostics in plant quarantine settings. HTS can be used to detect, in theory, all pathogens present in any given sample. The technique's success depends on various factors, including methods for sample management/preparation and suitable bioinformatic analysis. The Limit of Detection (LoD) of HTS for plant diagnostic tests can be higher than that of PCR, increasing the risk of false negatives in the case of low titer of the target pathogen. Several solutions have been suggested, particularly for RNA viruses, including rRNA depletion of the host, dsRNA, and siRNA extractions, which increase the relative pathogen titer in a metagenomic sample. However, these solutions are costly and time-consuming. Here we present a faster and cost-effective alternative method with lower HTS-LoD similar to or lower than PCR. The technique is called TArget-SPecific Reverse Transcript (TASPERT) pool. It relies on pathogen-specific reverse primers, targeting all RNA viruses of interest, pooled and used in double-stranded cDNA synthesis. These reverse primers enrich the sample for only pathogens of interest. Evidence on how TASPERT is significantly superior to oligodT, random 6-mer, and 20-mer in generating metagenomic libraries containing the pathogen of interest is presented in this proof of concept.

Studies on the Occurrence of Viruses in Planting Material of Grapevines in Southwestern Germany.

Viral diseases in viticulture lead to annual losses in the quantity and quality of grape production. Since no direct control measures are available in practice, preventive measures are taken to keep the vines healthy. These include, for example, the testing of propagation material for viruses such as Arabis mosaic virus (ArMV), Grapevine fanleaf virus (GFLV) or Grapevine leafroll-associated virus 1 (GLRaV-1) and 3 (GLRaV-3). As long-term investigations have shown, GLRaV-1 (2.1%) occurs most frequently in southwestern German wine-growing regions, whereas GLRaV-3 (<0.1%) is almost never found. However, tests conducted over 12 years indicate that there is no general decline in virus-infected planting material. Thus, it can be assumed that a spread of the viruses via corresponding vectors still takes place unhindered. Beyond the examinations regulated within the German Wine Growing Ordinance, one-time tests were carried out on Grapevine Pinot gris virus (GPGV). This analysis showed that GPGV was found in 17.2% of the samples.

Discovery of Four Novel Viruses Associated with Flower Yellowing Disease of Green Sichuan Pepper (Zanthoxylum Armatum) by Virome Analysis.

An emerging virus-like flower yellowing disease (FYD) of green Sichuan pepper (Zanthoxylum armatum v. novemfolius) has been recently reported. Four new RNA viruses were discovered in the FYD-affected plant by the virome analysis using high-throughput sequencing of transcriptome and small RNAs. The complete genomes were determined, and based on the sequence and phylogenetic analysis, they are considered to be new members of the genera Nepovirus (Secoviridae), Idaeovirus (unassigned), Enamovirus (Luteoviridae), and Nucleorhabdovirus (Rhabdoviridae), respectively. Therefore, the tentative names corresponding to these viruses are green Sichuan pepper-nepovirus (GSPNeV), -idaeovirus (GSPIV), -enamovirus (GSPEV), and -nucleorhabdovirus (GSPNuV). The viral population analysis showed that GSPNeV and GSPIV were dominant in the virome. The small RNA profiles of these viruses are in accordance with the typical virus-plant interaction model for Arabidopsis thaliana. Rapid and sensitive RT-PCR assays were developed for viral detection, and used to access the geographical distributions. The results revealed a correlation between GSPNeV and the FYD. The viruses pose potential threats to the normal production of green Sichuan pepper in the affected areas due to their natural transmission and wide spread in fields. Collectively, our results provide useful information regarding taxonomy, transmission and pathogenicity of the viruses as well as management of the FYD.

Genomic detection and molecular characterization of two distinct isolates of cycas necrotic stunt virus from Paeonia suffruticosa and Daphne odora.

Complete genome sequences of two cycas necrotic stunt virus (CNSV) isolates from Paeonia suffruticosa and Daphne odora were determined. Phylogenetic trees and pairwise comparisons using complete RNA1- and RNA2-encoded polyproteins showed that the two CNSV isolates are divergent (83.19%-89.42% in polyprotein 1 and 73.61%-85.78% in polyprotein 2). A comparative analysis based on taxonomic criteria for the species demarcation of nepoviruses confirmed that they are not new species but distinct variants. This is the first report of the complete genome sequences of CNSV detected in P. suffruticosa and D. odora, and the first report of CNSV infecting P. suffruticosa.

Detection and quantification of four viruses in Prunus pollen: Implications for biosecurity.

Pollen transmitted viruses require accurate detection and identification to minimize the risk of spread through the global import and export of pollen. Therefore in this study we developed RT-qPCR assays for the detection of Cherry leaf roll virus (CLRV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry virus A (CVA), four viruses that infect pollen of Prunus species. Assays were designed against alignments of extant sequences, optimized, and specificity was tested against known positive, negative, and non-target controls. An examination of assay sensitivity showed that detection of virus at concentrations as low as 101 copies was possible, although 102 copies was more consistent. Furthermore, comparison against extant assays showed that in both pollen and plant samples, the newly developed RT-qPCR assays were more sensitive and could detect a greater range of isolates than extant endpoint RT-PCR and ELISA assays. Use of updated assays will improve biosecurity protocols as well as the study of viruses infecting pollen.

Phylogeography of the soil-borne vector nematode Xiphinema index highly suggests Eastern origin and dissemination with domesticated grapevine.

The soil-borne nematode Xiphinema index is closely linked to its main host, the grapevine, and presents a major threat to vineyards worldwide due to its ability to transmit Grapevine fanleaf virus (GFLV). The phylogeography of X. index has been studied using mitochondrial and microsatellite markers in samples from most regions of its worldwide distribution to reveal its genetic diversity. We first used the mitochondrial marker CytB and illustrated the low intraspecific divergence of this mainly meiotic parthenogenetic species. To generate a higher polymorphism level, we then concatenated the sequences of CytB and three mitochondrial markers, ATP6, CO1 and ND4, to obtain a 3044-bp fragment. We differentiated two clades, which each contained two well-supported subclades. Samples from the eastern Mediterranean and the Near and Middle East were grouped into three of these subclades, whereas the samples from the western Mediterranean, Europe and the Americas all belonged to the fourth subclade. The highest polymorphism level was found in the samples of one of the Middle and Near East subclades, strongly suggesting that this region contained the native area of the nematode. An east-to-west nematode dissemination hypothesis appeared to match the routes of the domesticated grapevine during Antiquity, presumably mainly dispersed by the Greeks and the Romans. Surprisingly, the samples of the western subclade comprised only two highly similar mitochondrial haplotypes. The first haplotype, from southern Iberian Peninsula, Bordeaux and Provence vineyards, exhibited a high microsatellite polymorphism level that suggests introductions dating from Antiquity. The second haplotype contained a highly predominant microsatellite genotype widespread in distant western countries that may be a consequence of the massive grapevine replanting following the 19th-century phylloxera crisis. Finally, our study enabled us to draw a first scaffold of X. index diversity at the global scale.

Caraway yellows virus, a novel nepovirus from Carum carvi.

A novel nepovirus was identified and characterised from caraway, and tentatively named caraway yellows virus (CawYV). Tubular structures with isomeric virus particles typical for nepoviruses were observed in infected tissues by electron microscopy. The whole genome of CawYV was identified by high throughput sequencing (HTS). It consists of two segments with 8026 nt for RNA1 and 6405 nt for RNA2, excluding the poly(A) tails. CawYV-RNA1 shared closest nt identity to peach rosette mosaic virus (PRMV) with 63%, while RNA2 shared 41.5% with blueberry latent spherical virus (BLSV). The amino acid sequences of the CawYV protease-polymerase (Pro-Pol) and capsid protein (CP) regions share the highest identities with those of the subgroup C nepoviruses. The Pro-Pol region shared highest aa identity with PRMV (80.1%), while the CP region shared 39.6% to soybean latent spherical virus. Phylogenetic analysis of the CawYV-Pro-Pol and -CP aa sequences provided additional evidence of their association with nepoviruses subgroup C. Based on particle morphology, genomic organization and phylogenetic analyses, we propose CawYV as a novel species within the genus Nepovirus subgroup C.

High Risk Blueberry Viruses by Region in North America; Implications for Certification, Nurseries, and Fruit Production.

There is limited information on the distribution of blueberry viruses in the U.S. or around the world other than where the viruses were first discovered and characterized. A survey for blueberry viruses was carried out in the U.S. in 2015⁻2017. Most blueberry viruses have been characterized to the point that sensitive diagnostic assays have been developed. These assays are based on ELISA or variations of PCR, which were employed here to determine the presence of blueberry viruses in major blueberry production and nursery areas of the U.S. The viruses included in this study were: blueberry fruit drop (BFDaV), blueberry latent (BlLV), blueberry leaf mottle (BLMoV), blueberry mosaic (BlMaV), blueberry red ringspot (BRRV), blueberry scorch (BlScV), blueberry shock (BlShV), blueberry shoestring (BlSSV), blueberry virus A (BVA), peach rosette mosaic (PRMV), tobacco ringspot (TRSV), and tomato ringspot (ToRSV). In the Pacific Northwest BlShV was the most widespread virus, with BlScV and ToRSV detected in a limited number of fields in Oregon and Washington, but BlScV was widespread in British Columbia. In the upper midwest, the nematode-borne (ToRSV, TRSV), aphid-transmitted (BlSSV and BVA) and pollen-borne (BLMoV) viruses were most widespread. In the northeast, TRSV, ToRSV, and BlScV, were detected most frequently. In the southeast, BRRV and BNRBV were the most widespread viruses. BlLV, a cryptic virus with no known symptoms or effect on plant growth or yield was present in all regions. There are other viruses present at low levels in each of the areas, but with the lower incidence they pose minimal threat to nursery systems or fruit production. These results indicate that there are hotspots for individual virus groups that normally coincide with the presence of the vectors. The information presented highlights the high risk viruses for nursery and fruit production each pose a different challenge for control.

Molecular characterization and complete genome of a novel nepovirus from red clover.

During high throughput sequencing (HTS) of leaves from a symptomatic red clover plant, a new RNA virus, tentatively named red clover nepovirus A (RCNVA), was discovered. The complete genomic sequence was determined and characterized. Particularly noteworthy was that RCNVA shares high sequence identities in RNA1 with a group of phylogenetically related nepoviruses while homologies in the RNA2 segments are markedly lower. Based on the genomic organization and phylogenetic attributes, RCNVA should be classified as a novel virus of the genus Nepovirus (subfamily Comovirinae, family Secoviridae, order Picornavirales).

Genome sequence and detection of peach rosette mosaic virus.

Peach rosette mosaic disease was first described in the 1940s affecting peach and plum. It was later determined that peach rosette mosaic virus (PRMV) is the causal agent of the disease. PRMV, a member of the genus Nepovirus, infects several perennial crops including stone fruit, grape and blueberry as well as several weed species found in orchards around the world. The molecular characterization of the virus is limited to partial genome sequences making it difficult to develop reliable and sensitive molecular detection tests; the reason that detection is routinely performed using ELISA with antibodies risen against a single virus isolate. Given the potential economic impact of the virus and the modes of transmission which, in addition to nematodes, include seed we studied PRMV in more depth using a modified dsRNA extraction protocol to obtain the virus genome. We determined the full nucleotide sequence and developed a protocol that detects conserved regions present in RNA 1 and RNA 2, making it an excellent alternative to the detection protocols used today.

Rapid detection of fifteen known soybean viruses by dot-immunobinding assay.

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time.

Next generation sequencing and molecular analysis of artichoke Italian latent virus.

Next-generation sequencing (NGS) allowed the assembly of the complete RNA-1 and RNA-2 sequences of a grapevine isolate of artichoke Italian latent virus (AILV). RNA-1 and RNA-2 are 7,338 and 4,630 nucleotides in length excluding the 3' terminal poly(A) tail, and encode two putative polyproteins of 255.8 kDa (p1) and 149.6 kDa (p2), respectively. All conserved motifs and predicted cleavage sites, typical for nepovirus polyproteins, were found in p1 and p2. AILV p1 and p2 share high amino acid identity with their homologues in beet ringspot virus (p1, 81% and p2, 71%), tomato black ring virus (p1, 79% and p2, 63%), grapevine Anatolian ringspot virus (p1, 65% and p2, 63%), and grapevine chrome mosaic virus (p1, 60% and p2, 54%), and to a lesser extent with other grapevine nepoviruses of subgroup A and C. Phylogenetic and sequence analyses, all confirmed the strict relationship of AILV with members classified in subgroup B of genus Nepovirus.

Molecular characterization of a new soybean-infecting member of the genus Nepovirus identified by high-throughput sequencing.

The complete nucleotide sequence of a new soybean-infecting member of the genus Nepovirus (provisionally named "soybean latent spherical virus" [SLSV]) was identified by high-throughput sequencing of RNAs from soybean leaf samples from North Dakota, USA. The sequences of RNAs 1 (8,190 nt) and 2 (5,788 nt) were completed by rapid amplification of cDNA ends. Each contained a single long open reading frame and a 3' nontranslated region of greater than 1,500 nt. The predicted amino acid sequences of the two ORFs were most closely related to nepoviruses in subgroup C. Full-length cDNAs of RNAs 1 and 2 were cloned and used to inoculate soybean plants, which did not display obvious symptoms. These results suggest that SLSV represents a new species in the genus Nepovirus.

Complete nucleotide sequence of a new variant of grapevine fanleaf virus from northeastern China.

The complete RNA1 and RNA2 sequences of a new grapevine fanleaf virus isolate (GFLV-SDHN) from northeastern China were determined. The two RNAs are 7,367 and 3,788 nucleotides (nt) in length, respectively, excluding the poly(A) tails. Compared to other GFLV isolates, GFLV-SDHN has a 22- to 24-nt insertion in the RNA1 5' untranslated region, and there was 19.1-20.1 % and 11.7 %-13.0 % sequence divergence in RNA1, and 15.5 %-20.5 % and 8.5-13.5 % in RNA2, at the nt and amino acid level, respectively. Phylogenetic analysis revealed that the origins of GFLV-SDHN are distinct from those of other GFLV isolates. One recombination event was identified in the 2AHP region of RNA2 in GFLV-SDHN.

Complete sequence and variability of a new subgroup B nepovirus infecting potato in central Peru.

The complete bipartite genome (RNA1 and RNA2) of a new nepovirus infecting potato was obtained using small RNA sequencing and assembly complemented by Sanger sequencing. Each RNA encodes a single polyprotein, flanked by 5' and 3' untranslate regions (UTR) and followed by a poly (A) tail. The putative polyproteins encoded by RNA1 and RNA2 had sets of motifs which are characteristic of viruses in the genus Nepovirus. Sequence comparisons using the Pro-Pol region and the coat protein, including phylogenetic analysis of these regions, showed closest relationships with nepoviruses. The data obtained support the taxonomical status of this new virus (putative named Potato virus B, PVB) as a member of the genus Nepovirus, subgroup B.

Comparative detection of a large population of grapevine viruses by TaqMan® RT-qPCR and ELISA.

Grapevine (Vitis spp.) can be infected by numerous viruses that are often widespread and of great economic importance. Reliable detection methods are necessary for sanitary selection which is the only way to partly control grapevine virus diseases. Biological indexing and ELISA are currently the standard methods for screening propagation material, and PCR-methods are becoming increasingly popular. Due to the diversity of virus isolates, it is essential to verify that the tests allow the detection of the largest possible virus populations. We developed three quadruplex TaqMan® RT-qPCR assays for detecting nine different viruses that cause considerable damage in many vineyards world-wide. Each assay is designed to detect three viruses and the grapevine Actin as an internal control. A large population of grapevines from diverse cultivars and geographic location was tested for the presence of nine viruses: Arabis mosaic virus (ArMV), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated viruses (GLRaV-1, -2, -3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), and Grapevine virus B (GVB). In general, identical results were obtained with multiplex TaqMan® RT-qPCR and ELISA although, in some cases, viruses could be detected by only one of the two techniques.

High-Throughput Optical Sensing Immunoassays on Smartphone.

We present an optical sensing platform on a smartphone for high-throughput screening immunoassays. For the first time, a designed microprism array is utilized to achieve a one-time screening of 64 samples. To demonstrate the capability and the reliability of this optical sensing platform on smartphone, human interleukin 6 (IL-6) protein and six types of plant viruses are immunoassayed. The ability of quantification is shown by a sigmoidal dose-response curve fitting to analyze IL-6 protein. The accuracy in measuring the concentrations of IL-6 protein achieves 99.1%. On the other hand, to validate on-field immunoassays by our device, a total of 1030 samples are assayed using three immunoassay methods to detect six types of plant viruses. The accuracy is up to 96.2-99.9%; in addition, there is a high degree of agreement with lab instruments. The total cost for this high-throughput optical screening platform is ∼$50 USD. The reading time is only 2 s for 64 samples. The size is just as big as a portable hard drive. Our optical sensing platform on the smartphone offers a route toward in situ high-throughput screening immunoassays for viruses, pathogens, biomarkers, and toxins by decentralizing laboratory tests. With this mobile point-of-care optical platform, the spread of disease can be timely stopped within a very short turnaround time.

Comparison of diagnostic techniques for the detection and differentiation of Cherry leaf roll virus strains for quarantine purposes.

Some strains of Cherry leaf roll virus (CLRV) are considered as quarantine pests in New Zealand. CLRV was detected in seven plant host species: Actinidia chinensis, Hydrangea macrophylla, Malus domestica, Plantago major, Ribes rubrum, Rubus idaeus and Rumex sp. collected from New Zealand between 2005 and 2012. Biological, serological and molecular techniques were compared for the detection and differentiation of CLRV isolates. The biological analysis revealed differences in symptomatology and disease severity among the isolates. The five isolates tested by ELISA were serologically related to each other using polyclonal antisera with only one out of four commercially-available antisera successfully detecting all of them. The phylogenetic analysis of sequences obtained from parts of the coat protein, polymerase and 3'-untranslated regions revealed that the New Zealand CLRV isolates clustered into two closely related but distinct phylogenetic groups with some isolates grouping differently depending on the gene studied. The New Zealand CLRV isolates were clearly distinct to overseas isolates found in phylogenetic groups A, D and E. The conventional RT-PCR using primers targeting the CLRV coat protein coding region is recommended for determining sequence differences between strains. These findings will be useful in making regulatory decisions with regard to the testing requirements and the CLRV strains to be regulated in New Zealand.

Rapid detection of genetically diverse tomato black ring virus isolates using reverse transcription loop-mediated isothermal amplification.

A reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) has been developed for detection of tomato black ring virus (TBRV) isolates collected from different hosts. One-step RT-LAMP was performed with a set of four primers, the design of which was based on the coat protein gene. Results of RT-LAMP were visualized by direct staining of products with fluorescent dyes, agarose gel electrophoresis, and analysis of amplification curves. The sensitivity of RT-LAMP was 100-fold greater than that of RT-PCR. The RT-LAMP assay developed here is a useful and practical method for diagnosis of TBRV.