First report of a new Carlavirus provisionally named rose virus B and apple mosaic virus in mixed infections of Rosa sp. in Mexico.

Rose (Rosa sp.) is an important ornamental plant in the cut flower industry around the world. This species is prone to hosting several viruses since it is propagated vegetatively, mainly by grafting (Mollov et al., 2013). In 2021, rose plants of unidentified variety with mosaic, vein yellowing, chlorotic line patterns, and interveinal chlorosis were observed in a rose plantation established in open field in Temixco, Morelos (Supplementary Figure 1). To determine the cause of symptoms was due to viral infection, nucleic acids were extracted from leaves by in-house CTAB procedure and DNase treated. A pooled RNA sample extracted from 4 symptomatic plants was sent to BGI Genomics (China) for high-throughput sequencing (HTS). A stranded mRNA library was prepared and sequenced on the DNBSEQ platform (BGI). A total number of 13,646,715 paired 150-bp clean reads were generated. The reads were assembled de novo into 79,309 contigs ranging from 78 to 15,817 nucleotides (nt) using SPAdes (Prjibelskiet et al., 2020). The contigs were subjected to BLASTx and BLASTn for annotation. A contig with a length of 8,842 nt (208x average coverage per nt) showed 90.6% identity to rose virus B (RVB) (MT473961), and was deposited in GenBank under accession number ON165234. Additionally, three contigs (ON165235-ON165237) corresponding to RNA1 (3,443 nt; 154x coverage), RNA2 (2,938 nt; 231x coverage), and RNA3 (1,897 nt; 232x coverage) of apple mosaic virus (ApMV) were identified. These contigs showed up to 98.4%, 89.7%, and 98.6% identity, respectively, to each corresponding RNA sequences of ApMV. No other viral sequence was identified from the constructed contigs. Subsequently, the presence of RVB was confirmed by RT-PCR performed with an aliquot of the pooled RNAspan style="font-family:'Times New Roman'; font-size:11pt"> with specific primers targeting the replicase and CP (Diaz-Lara et al., 2021). For ApMV, a new set of primers were designed: ApMV_RNA1F (5'-AAATCTCCCGAAAGGGCCTG-3')/ApMV_RNA1R (5'-TCACTCGTCGCATGGATGGATAGC-3'), ApMV_RNA2F (5'-TTGGTACGAGTCGTGGTTGGTTGG-3')/ApMV_RNA2R (5'-GGAAAACTGACCGCAAACCC-3'), and ApMV_RNA3F (5'-GGAGGTTAGAGGCCCGAATG-3')/ApMV_RNA3R (5'-CGCACAGGTGGTAACTCACT-3') which amplify segments of 444 bp, 546 bp, and 434 bp, respectively. The amplicons obtained for both viruses were subjected to Sanger sequencing, confirming the identity of RVB and ApMV. The sequences from the RVB replicase (ON165241) and CP (ON165240) showed 93.9% and 97.0% nt identity with an RVB isolate reported in the USA (MT473961). On the other hand, sequences from RNA1 (ON165238), RNA2, (OP413436), and RNA3 (ON165239) of ApMV had 99.2%, 89.2%, and 99% nt identity, respectively. Finally, the four symptomatic plants were individually tested by RT-PCR to identify RVB and ApMV. Interestingly, both viruses were detected in all the plants analyzed. ApMV (genus Ilarvirus) is associated with mosaic and mottling symptoms in rose (Thomas, 1984). It has been accepted that ApMV is present in rose plants in Mexico (Cardenas-Alonso, 1994), with no evidence to confirm it. RVB was identified in rose in USA, and this virus was classified as a new species of the genus Carlavirus (Diaz-Lara et al., 2021). In addition to RVB, rose virus A and rose virus C have also been reported in rose; however, the symptomatology linked to these viruses is unknown (Xing et al. 2021; Diaz-Lara et al., 2020). Recently, RVB and ApMV were reported in rose plants in Taiwan (Chen et al., 2022). To our knowledge, this is the first report of RVB and ApMV in a mixed infection in rose in Mexico.

Integration of Rubus yellow net virus in the raspberry genome: A story centuries in the making.

Rubus yellow net virus (RYNV) belongs to genus Badnavirus. Badnaviruses are found in plants as endogenous, inactive or activatable sequences, and/or in episomal (infectious and active) forms. To assess the state of RYNV in Rubus germplasm, we sequenced the genomes of various cultivars and mined eight raspberry whole genome datasets. Bioinformatics analysis revealed the presence of a diverse array of endogenous RYNV (endoRYNV) sequences that differ significantly in their structure; some lineages have nearly complete, yet non-functional genomes whereas others have rudimentary, short sequence fragments. We developed assays to genotype the main lineages as well as the only known episomal lineage present in the United States. This study discloses the widespread presence of endoRYNVs in commercial raspberries, likely because breeding efforts have focused on a limited pool of germplasm that harbored endoRYNVs.

New Host Plant Species of Grapevine Virus A Identified with Vector-Mediated Infections.

Grapevine virus A (GVA) is an economically important virus and a member of the genus Vitivirus (family Betaflexiviridae) that causes a range of symptoms with qualitative and quantitative effects on grape production. Wild and domesticated species of Vitis, including hybrids used as rootstocks, are considered important natural hosts of GVA. Mechanical transmission to some herbaceous plant species, graft transmission, and vector transmission from grape to grape by various mealybugs and soft scale insects have been reported. Under laboratory and greenhouse conditions, this study demonstrates the transmission of GVA from grapes to alternative hosts by the vine mealybug (Planococcus ficus). Results of ELISA, end-point one-step RT-PCR, and real-time RT-PCR, and in some cases electron microscopy and genome sequencing, confirmed successful transmission to three new plant species commonly found in Croatian vineyards: velvetleaf (Abutilon theophrasti), redroot pigweed (Amaranthus retroflexus), and field poppy (Papaver rhoeas), along with Chenopodium murale and the previously known host Nicotiana benthamiana, with variable infection rates. Depending on the host species, symptoms in the form of leaf reddening, yellow spots, reduced growth of lateral shoots, systemic vein clearing, foliar deformation and rugosity, and dwarfism were observed in GVA-infected plants, whereas no symptoms were observed in infected plants of A. theophrasti. Reverse transmission from these new hosts to grapevines by Pl. ficus was not successful. These results confirm four new GVA host species and open new research venues.

High-Throughput Sequencing of Grapevine in Mexico Reveals a High Incidence of Viruses including a New Member of the Genus Enamovirus.

This is the first viral metagenomic analysis of grapevine conducted in Mexico. During the summer of 2021, 48 plants displaying virus-like symptoms were sampled in Queretaro, an important grapevine-producing area of Mexico, and analyzed for the presence of viruses via high-throughput sequencing (HTS). The results of HTS were verified by real-time RT-PCR following a standardized testing scheme (Protocol 2010). Fourteen different viruses were identified, including grapevine asteroid mosaic-associated virus (GAMaV), grapevine Cabernet Sauvignon reovirus (GCSV), grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine Pinot gris virus (GPGV), grapevine red globe virus (GRGV), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), grapevine virus B (GVB), and grapevine leafroll-associated viruses 1, 2, 3, 4 (GLRaV1, 2, 3, 4). Additionally, divergent variants of GLRaV4 and GFkV, and a novel Enamovirus-like virus were discovered. This is the first report of GAMaV, GCSV, GLRaV4, GPGV, GRGV, GRVFV, and GSyV-1 infecting grapevines in Mexico; the impact of these pathogens on production is unknown.

Senna multiglandulosa, a new natural host for Tobacco etch virus in Mexico.

Plants of Senna multiglandulosa (family Fabaceae), an ornamental shrub, growing adjacent to tomato and chrysanthemum greenhouses located in San Diego, Texcoco, Estado de Mexico, had leaves with putative virus symptoms, consisting of annular or irregular chlorotic spots of different sizes (Supplementary Fig. 1a). To investigate the presence of a virus, high-throughput sequencing (HTS) was performed. Total RNA was extracted from symptomatic leaves of S. multiglandulosa plants using the SV Total RNA Isolation System Kit (Promega, USA). A portion of the RNA was sent to BGI Genomics (China) for cDNA library construction and sequencing on the DNBSEQ platform (BGI Genomics). HTS yielded 14,673,469 clean paired reads (150x2), which were assembled de novo into 91,879 contigs using SPAdes v3.15 software (Prjibelski et al. 2020). The contigs ranged from 78 to 14,534 nucleotides (nts), which were subjected to BLASTx and BLASTn analyses. A single viral contig of 9,501 nts was detected (average coverage: 56,716x per nt) representing the nearly complete genome of tobacco etch virus (TEV). The highest identity was 79.26% at the nt level (92% query coverage) with TEV isolate TEV7DA (GenBank: DQ986288; length: 9,539 nts) from the USA, and 86.67% at the amino acid (aa) level considering the polyprotein, which are higher than the species demarcation threshold (<76% nt and <82% aa) for the genus Potyvirus (Inoue-Nagata et al. 2022). Additionally, the sequence obtained from S. multiglandulosa revealed 79.21-79.37% nt identities with different TEV isolates from Solanaceae plants (Capsicum annuum, MW748496; Solanum lycopersicum, OM471966.1; Nicotiana tabacum, OL311684.1). The new TEV genome was deposited in GenBank under accession number ON110203. The results obtained by HTS were confirmed by RT-PCR with the original isolated RNA using a pair of specific primers designed from the TEV sequence (TEV-NIb-F, 5'- GCGCTTAAATGCAGACTCGG-3' and TEV-NIb-R, 5'-GTGAAAGTTCAGCAGCAAGCGCA-3') that amplify a 550-bp fragment of the RNA-dependent RNA polymerase. The obtained amplicon was sequenced by the Sanger method, and was 100% identical to the sequence generated by HTS. Subsequently, N. tabacum and N. glutinosa plants were mechanically inoculated using TEV-positive S. multiglandulosa leaves as the inoculum source. Twenty days after inoculation, light chlorotic spots and necrotic lesions were observed on N. tabacum and mosaic on N. glutinosa (Supplementary Fig. 1b-c). RT-PCR analysis confirmed the presence of TEV infection in these indicator plants. To determine the incidence of S. multiglandulosa plants showing TEV-infection symptoms, a survey (n=16) was carried out on two farms in Texcoco; the survey showed a 100% incidence of symptoms. Five survey samples were randomly selected, and the presence of TEV was confirmed by RT-PCR. The discovery of Tobacco etch virus (family Potyviridae: genus Potyvirus) in tobacco was reported in Kentucky, USA in 1928 (Valleau and Johnson, 1928), one of the most common and damaging viruses for the chili crop in Mexico (Delgado, 1974). TEV causes heavy yield loss in several Solanaceae plants and infects more than 120 species in 19 families of dicotyledons (Holmes, 1946). S. obtusifolia (originally Cassia obtusifolia) was the first legume reported as a natural host of TEV in Florida, USA (Anderson, 1954). To our knowledge, this is the first report of the natural infection of S. multiglandulosa by TEV in Mexico and the first TEV genome isolated and sequenced from a legume. S. multiglandulosa is widely distributed in 16 states in Mexico, both cultivated and naturalized, however, it is not considered native to the country (Rzedowski and Calderón, 1997). The occurrence of TEV in S. multiglandulosa represents an alternative reservoir of the virus, with an important role in the epidemiology of the disease.

A Novel, Divergent Member of the Rhabdoviridae Family Infects Strawberry.

Strawberry (Fragaria × ananassa) is the most important berry crop worldwide and viruses pose a constant threat to the industry. In this communication, we describe a novel virus in the family Rhabdoviridae referred to as strawberry virus 3 (StrV-3). The virus does not show significant homology when compared with recognized rhabdoviruses and, therefore, the establishment of a new genus should be considered. A triplex reverse-transcription PCR test was developed and successfully employed in a survey of the National Clonal Germplasm Repository Fragaria collection. A CRISPR-Cas-based protocol was also developed and shown to detect the virus in as little as 1 fg of total RNA, a protocol to be used in the detection of the virus in candidate G1 plants. The strawberry aphid (Chaetosiphon fragaefolii) was evaluated-alas, unsuccessfully-as a potential vector of the virus. This work broadens our understanding of the family Rhabdoviridae and assists in the quest of releasing plant material free of viruses.

Incidence and Genetic Diversity of Grapevine Virus G in Croatian Vineyards.

Grapevine virus G (GVG) is a recently discovered vitivirus infecting grapevines. Historically, viruses in the genus Vitivirus have been associated with the grapevine rugose wood disease. Based on new and previously reported GVG isolates, primers and probes were developed for real-time RT-PCR. The developed assay successfully detected the virus in infected plants during dormancy and the growing season. A field study of 4327 grapevines from Croatian continental and coastal wine-growing regions confirmed the presence of GVG in 456 (~10.5%) grapevines from three collection plantations and 77 commercial vineyards, with infection rates ranging from 2% to 100%. Interestingly, the virus was confirmed only in vines considered to be Croatian autochthonous cultivars, but not in introduced cultivars. A 564-nucleotide long portion of the coat protein gene from previously known and newly characterized GVG isolates had nucleotide and amino acid identities ranging from 89% to 100% and from 96.8% to 100%, respectively. Phylogenetic analysis revealed five distinct groups, with isolates originating from the same site being close to each other, indicating possible local infection. The information presented in this manuscript sets the stage for future studies to better understand the ecology and epidemiology of GVG and the possible need for inclusion in certification schemes.

Grapevine Badnavirus 1: Detection, Genetic Diversity, and Distribution in Croatia.

Grapevine badnavirus 1 (GBV-1) was recently discovered in grapevine using high throughput sequencing. In order to carry out large-scale testing that will allow for better insights into virus distribution, conventional and real-time PCR assays were developed using sequences both from previously known, and four newly characterized isolates. Throughout the growing season and dormancy, GBV-1 can be detected by real-time PCR using available tissue, with the possibility of false-negative results early in vegetation growth. GBV-1 real-time PCR analysis of 4302 grapevine samples from the Croatian continental and coastal wine-growing regions revealed 576 (~13.4%) positive vines. In the continental wine-growing region, virus incidence was confirmed in only two collection plantations, whereas in the coastal region, infection was confirmed in 30 commercial vineyards and one collection plantation. Infection rates ranged from 1.9 to 96% at the different sites, with predominantly autochthonous grapevine cultivars infected. Conventional PCR products obtained from 50 newly discovered GBV-1 isolates, containing the 375 nucleotides long portion of the reverse transcriptase gene, showed nucleotide and amino acid identities ranging from 94.1 to 100% and from 92.8 to 100%, respectively. The reconstructed phylogenetic tree positioned the GBV-1 isolates taken from the same vineyard close to each other indicating a possible local infection event, although the tree nodes were generally not well supported.

Virus and Virus-like Pathogens in the Grapevine Virus Collection of Croatian Autochthonous Grapevine Cultivars.

Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of "flavescence dorée" phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of "flavescence dorée", makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years.

Viruses of Economic Impact on Tomato Crops in Mexico: From Diagnosis to Management-A Review.

Tomato is the most economically important vegetable crop worldwide and the second most important for Mexico. However, viral diseases are among the main limiting factors that affect the productivity of this crop, causing total losses in some cases. This review provides key information and findings on the symptoms, distribution, transmission, detection, and management of diseases caused by viruses of major importance in tomato crops in Mexico. Currently, about 25 viruses belonging to nine different families have been reported infecting tomato in Mexico, but not all of them cause economically significant diseases. Viruses of economic importance include tomato brown rugose fruit virus (ToBRFV), tomato spotted wilt virus (TSWV), tomato yellow leaf curl virus (TYLCV), pepino mosaic virus (PepMV), and tomato marchitez virus (ToMarV). The topics discussed here will provide updated information about the status of these plant viruses in Mexico as well as diverse management strategies that can be implemented according to the specific circumstances of each viral pathosystem. Additionally, a list of tomato-affecting viruses not present in Mexico that are continuous threats to the crop health is included.

Olea europaea geminivirus is present in a germplasm repository and in California and Texas olive (Olea europaea L.) groves.

Olea europaea geminivirus (OEGV) from olive accessions in Italy was characterized recently. OEGV was also detected during routine high-throughput sequencing screening of olive (cv. Leccino) material, and its complete bipartite genome segments were sequenced and shown to be 100% identical to those of the isolate from Italy. Using two pairs of newly designed primers targeting the AV1 and BV1 genes, OEGV was detected in randomly sampled olive trees from the U.S. Department of Agriculture National Clonal Germplasm Repository (USDA-NCGR) (21.4% or 6/28), commercial and residential settings in California (47.6% or 10/21), and an orchard in Texas (60% or 30/50). The cuttings for the USDA-NCGR-positive trees originated from the former Serbia and Montenegro, Spain, Italy, and Greece. Comparative analysis of the directly sequenced gene fragments from randomly selected samples showed that OEGV isolates from the different sources were 100% identical to each other. The results indicate that OEGV spread was likely facilitated by inadvertent movement of contaminated olive germplasm.

Sequencing a Strawberry Germplasm Collection Reveals New Viral Genetic Diversity and the Basis for New RT-qPCR Assays.

Viruses are considered of major importance in strawberry (Fragaria × ananassa Duchesne) production given their negative impact on plant vigor and growth. Strawberry accessions from the National Clonal Germplasm Repository were screened for viruses using high throughput sequencing (HTS). Analyses of sequence information from 45 plants identified multiple variants of 14 known viruses, comprising strawberry mottle virus (SMoV), beet pseudo yellows virus (BPYV), strawberry pallidosis-associated virus (SPaV), tomato ringspot virus (ToRSV), strawberry mild yellow edge virus (SMYEV), strawberry vein banding virus (SVBV), strawberry crinkle virus (SCV), strawberry polerovirus 1 (SPV-1), apple mosaic virus (ApMV), strawberry chlorotic fleck virus (SCFaV), strawberry crinivirus 4 (SCrV-4), strawberry crinivirus 3 (SCrV-3), Fragaria chiloensis latent virus (FClLV) and Fragaria chiloensis cryptic virus (FCCV). Genetic diversity of sequenced virus isolates was investigated via sequence homology analysis, and partial-genome sequences were deposited into GenBank. To confirm the HTS results and expand the detection of strawberry viruses, new reverse transcription quantitative PCR (RT-qPCR) assays were designed for the above-listed viruses. Further in silico and in vitro validation of the new diagnostic assays indicated high efficiency and reliability. Thus, the occurrence of different viruses, including divergent variants, among the strawberries was verified. This is the first viral metagenomic survey in strawberry, additionally, this study describes the design and validation of multiple RT-qPCR assays for strawberry viruses, which represent important detection tools for clean plant programs.

Identification and characterization of a novel virus associated with an eriophyid mite in extracts of fruit trees leaves.

Eriophyid mites are commonly found on the leaf surface of different plant species. In the present study, a novel virus associated with an eriophyid mite species was detected using high-throughput sequencing (HTS) of total RNA from fruit tree leaves, primarily growing under greenhouse conditions. The complete genome sequence was characterized using rapid amplification of cDNA ends followed by Sanger sequencing, revealing a genome of 8885 nucleotides in length. The single positive-stranded RNA genome was predicted to encode typical conserved domains of members of the genus Iflavirus in the family Iflaviridae. Phylogenetic analysis showed this virus to be closely related to the unclassified iflavirus tomato matilda associated virus (TMaV), with a maximum amino acid sequence identity of 59% in the RNA-dependent RNA polymerase domain. This low identity value justifies the recognition of the novel virus as a potential novel iflavirus. In addition to a lack of graft-transmissibility evidence, RT-PCR and HTS detection of this virus in the putative host plants were not consistent through different years and growing seasons, raising the possibility that rather than a plant virus, this was a virus infecting an organism associated with fruit tree leaves. Identification of Tetra pinnatifidae HTS-derived contigs in all fruit tree samples carrying the novel virus suggested this mite as the most likely host of the new virus (p-value < 1e-11), which is tentatively named "eriophyid mite-associated virus" (EMaV). This study highlights the importance of a careful biological study before assigning a new virus to a particular plant host when using metagenomics data.

Identification of grapevine Pinot gris virus in free-living Vitis spp. located in riparian areas adjacent to commercial vineyards.

Grapevine Pinot gris virus (GPGV) is a recently identified pathogen of grapevines in California. To advance our knowledge about the epidemiology of GPGV, we investigated if free-living Vitis spp. can represent a source of virus infection. In 2019 a field survey of GPGV infection was conducted in Napa County. During the inspection 60 free-living vines in riparian habitats near commercial vineyards with GPGV infection were sampled. Samples were tested by real-time reverse transcription PCR (RT-PCR), identifying 23 free-living Vitis spp. positive for GPGV. Later, GPGV infection was confirmed in these plants via end-point RT-PCR and Sanger sequencing. Based on sequence analysis, detected GPGV isolates are more related to the asymptomatic variant of the virus. Vitis species ancestry was determined by DNA fingerprinting. GPGV-infected material included V. californica, V. californica × V. vinifera hybrids and hybrid rootstock cultivars. Here, GPGV is reported for the first time in free-living Vitis spp. The results of this study will support the development of management strategies for GPGV in California and beyond.

Detection and characterization of a second carlavirus in Rosa sp.

A new virus resembling members in the genus Carlavirus was identified in an Out of Yesteryear rose (Rosa sp.) by high-throughput sequencing. The virus was discovered during the screening of a rose virus collection belonging to Foundation Plant Services (UC-Davis). The full genome of the virus is 8825 nt long, excluding a poly(A) tail, and includes six predicted genes coding for replicase, triple gene block, coat protein (CP), and nucleic acid binding protein. The closest relative of the putative virus is rose virus A (RVA; genus Carlavirus), with 75% and 78% aa sequence identity in the replicase and CP, respectively. The relationship with RVA and other carlaviruses was supported by phylogenetic analyses using replicase and CP sequences. Based on genome organization, sequence identity, and phylogenetic analysis, the virus found in the Out of Yesteryear plant represents a new member of the genus Carlavirus and is provisionally named "rose virus B" (RVB). Further testing by reverse transcription PCR confirmed the presence of RVB in the original source and seven additional rose selections from the same collection.

Development of a universal RT-PCR assay for grapevine vitiviruses.

The genus Vitivirus in the family Betaflexiviridae includes eleven viruses known to infect grapevine: grapevine vitiviruses A, B, D, E, F, G, H, I, J, L and M (GVA-GVM). Three of these viruses, GVA, GVB and GVD, have been associated with the etiology of rugose wood disease in grapevine and cause agronomically significant losses. The other vitiviruses were more recently discovered and their effects on grapevine are undetermined. To certify grape material for propagation as virus tested, an updated reverse transcription PCR (RT-PCR) assay to detect all known vitiviruses is desirable. To accomplish this, multiple grapevine vitivirus sequences were aligned at the amino acid level to search for conserved motifs. Two highly conserved motifs were found at an ideal distance for RT-PCR detection in the RNA-dependent RNA polymerase region of the replicase protein. The amino acid motifs were back translated to create degenerate primers and used to successfully amplify all eleven grapevine vitiviruses. The RT-PCR primers were used to test a panel of vitivirus-infected vines for inclusivity as well as vines infected with closely related viruses in the Betaflexiviridae family (i.e. grapevine pinot gris virus and grapevine rupestris stem pitting-associated virus) for exclusivity. Broader use of these primers to detect vitiviruses in other plant hosts was investigated. In summary, an end-point RT-PCR assay that detects all the known grapevine vitiviruses and potentially other members of the genus Vitivirus has been developed. The universal assay represents an alternative to individual assays to reduce the work associated with the diagnosis of vitiviruses, including for regulatory purposes.

Evidence of Rubus Yellow Net Virus Integration into the Red Raspberry Genome.

Rubus yellow net virus (RYNV) infects Rubus spp., causing a severe decline when present in mixed infections with other viruses. RYNV belongs to the family Caulimoviridae, also known as plant pararetroviruses, which can exist as episomal or integrated elements (endogenous). Most of integrated pararetroviruses are noninfectious; however, a few cases have been reported where they excised from the plant genome and formed infectious particles. Graft transmission onto indicator plants R. occidentalis "Munger" has been the standard test method for RYNV detection in certification programs. Previously, it was noticed that some RYNV PCR-positive plants did not induce symptoms on "Munger", suggesting an integration event. In this study, bio-indexing and different molecular techniques were employed to differentiate between integrated and episomal RYNV sequences. Reverse transcription-PCR using RYNV-specific oligonucleotides after DNase treatment generated positive results for the virus in graft transmissible isolates (episomal) only. To confirm these results, rolling circle amplification on DNA preparations from the same samples resulted in amplicons identified as RYNV only from plants with graft transmissible RYNV. High-throughput sequencing was used to identify the RYNV-like sequences present in the host DNA. These results indicate the integration of RYNV into the red raspberry genome and highlight the necessity to recognize this phenomenon (integration) in future Rubus quarantine and certification programs.

Development of RT-PCR degenerate primers to overcome the high genetic diversity of grapevine virus T.

Grapevine virus T (GVT) is a new member of the genus Foveavirus and has been reported to infect grapevines in several European countries. In 2018, GVT was detected for the first time in California in a domestic selection of wine grape, cv. Lambrusca di Alessandria, via high-throughput sequencing (HTS). To further investigate the presence of GVT in other grapevine plants, a two-step reverse transcription (RT)-PCR assay involving degenerate primers was developed. In order to cover the high genetic diversity of GVT, the sequences of available isolates were aligned to identify a conserved region in the coat protein gene that was a suitable target for the assay. The results of the RT-PCR assay showed that GVT was present in three additional grapevine selections among 416 plants integrating the Foundation Plant Services introduction pipeline; all were later confirmed by HTS. A complete and three near-complete genomes of the four GVT isolates were characterized and found to be divergent, sharing an overall 81 % pairwise identity in their nucleotide sequences. This suggested that the new RT-PCR assay was effective in detecting a broad range of GVT variants. The RT-PCR detection method developed in this study would be useful for routine virus testing.

Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting Prunus spp.

Viruses can cause economic losses in fruit trees, including Prunus spp., by reducing yield and marketable fruit. Given the genetic diversity of viruses, reliable diagnostic methods relying on PCR are critical in determining viral infection in fruit trees. This study evaluated the broad-range detection capacity of currently available real-time RT-PCR assays for Prunus-infecting viruses and developed new assays when current tests were inadequate or absent. Available assays for 15 different viruses were exhaustively evaluated in silico to determine their capacity to detect virus isolates deposited in GenBank. During this evaluation, several isolates deposited since the assay was designed exhibited nucleotide mismatches in relation to the existing assay's primer sequences. In cases where updating an existing assay was impractical, we performed a redesign with the dual goals of assay compactness and comprehensive inclusion of genetic diversity. The efficiency of each developed assay was determined by a standard curve. To validate the assay designs, we tested them against a comprehensive set of 87 positive and negative Prunus samples independently analyzed by high throughput sequencing. As a result, all the real-time RT-PCR assays described herein successfully detected the different viruses and their corresponding isolates. To further validate the new and updated assays a Prunus germplasm collection was surveyed. The sensitive and reliable detection methods described here will be used for the large-scale pathogen testing required to maintain the highest quality nursery stock.

Complete genome sequence of rose virus A, the first carlavirus identified in rose.

A novel virus was discovered in a Rosa wichuraiana Crep. by high-throughput sequencing and tentatively named "rose virus A" (RVA). Based on sequence identity and phylogenetic analysis, RVA represents a new member of the genus Carlavirus (family Betaflexiviridae). The genome of RVA is 8,849 nucleotides long excluding the poly(A) tail and contains six open reading frames (ORFs). The predicted ORFs code for a replicase, triple gene block (TGB), coat protein, and nucleic acid binding protein, as in a typical carlavirus. RVA is the first carlavirus identified in rose and has the highest nucleotide sequence similarity to poplar mosaic virus. Reverse transcription-PCR-based assays were developed to confirm the presence of RVA in the original source and to screen additional rose plants.