Molecular Characterization of a Novel Enamovirus Infecting Raspberry.

Raspberry plants, valued for their fruits, are vulnerable to a range of viruses that adversely affect their yield and quality. Utilizing high-throughput sequencing (HTS), we identified a novel virus, tentatively named raspberry enamovirus 1 (RaEV1), in three distinct raspberry plants. This study provides a comprehensive characterization of RaEV1, focusing on its genomic structure, phylogeny, and possible transmission routes. Analysis of nearly complete genomes from 14 RaEV1 isolates highlighted regions of variance, particularly marked by indel events. The evidence from phylogenetic and sequence analyses supports the classification of RaEV1 as a distinct species within the Enamovirus genus. Among the 289 plant and 168 invertebrate samples analyzed, RaEV1 was detected in 10.4% and 0.4%, respectively. Most detections occurred in plants that were also infected with other common raspberry viruses. The virus was present in both commercial and wild raspberries, indicating the potential of wild plants to act as viral reservoirs. Experiments involving aphids as potential vectors demonstrated their ability to acquire RaEV1 but not to successfully transmit it to plants.

Elimination of Solanum nigrum ilarvirus 1 and Apple Hammerhead Viroid from Apple Cultivars Using Antivirals Ribavirin, Rimantadine, and Zidovudine.

Apple hammerhead viroid (AHVd) was detected in the apple cultivar 'Sampion' and in mixed infection with Solanum nigrum ilarvirus 1 (SnIV-1) in the cultivars 'Selena' and 'Jonagored Supra', using a high-throughput sequencing method. Experiments were conducted to eliminate both pathogens in apples using meristem tip cultures in combination with the antivirotics ribavirin, rimantadine, and zidovudine. Elimination of both pathogens was verified by repeated RT-PCR and qRT-PCR assays after 7-11 months. Elimination of SnIV-1 from all cultivars was successful with each of the three antivirotics at concentrations of 20, 40, and 80 mg L-1. Elimination of AHVd was also achieved, although less effectively and only with ribavirin in the concentration range of 20-160 mg L-1.

Analysis of Virus-Derived siRNAs in Strawberry Plants Co-Infected with Multiple Viruses and Their Genotypes.

Plants can be infected with multiple viruses. High-throughput sequencing tools have enabled numerous discoveries of multi-strain infections, when more than one viral strain or divergent genomic variant infects a single plant. Here, we investigated small interfering RNAs (siRNAs) in a single strawberry plant co-infected with several strains of strawberry mottle virus (SMoV), strawberry crinkle virus (SCV) and strawberry virus 1 (StrV-1). A range of plants infected with subsets of the initial viral species and strains that were obtained by aphid-mediated transmission were also evaluated. Using high-throughput sequencing, we characterized the small RNA fractions associated with different genotypes of these three viruses and determined small RNA hotspot regions in viral genomes. A comparison of virus-specific siRNA (vsiRNA) abundance with relative viral concentrations did not reveal any consistent agreement. Strawberry mottle virus strains exhibiting considerable variations in concentrations were found to be associated with comparable quantities of vsiRNAs. Additionally, by estimating the specificity of siRNAs to different viral strains, we observed that a substantial pool of vsiRNAs could target all SMoV strains, while strain-specific vsiRNAs predominantly targeted rhabdoviruses, SCV and StrV-1. This highlights the intricate nature and potential interference of the antiviral response within a single infected plant when multiple viruses are present.

First report of 'Candidatus Phytoplasma rubi' associated with Rubus stunt disease of raspberry and blackberry in the Czech Republic.

Raspberries (Rubus idaeus L.), occurring in the temperate zone of the northern hemisphere and blackberries (R. fruticosus L.), cultivated and growing all over the world, are plant species of the family Rosaceae. These species are susceptible to phytoplasma infections, which cause Rubus stunt disease. It spreads uncontrolled by vegetative propagation of plants (Linck and Reineke 2019a) and by phloem-sucking insect vectors, especially Macropsis fuscula (Hemiptera: Cicadellidae) (de Fluiter and van der Meer, 1953; Linck and Reineke 2019b). During a survey in commercial field in June 2021, over 200 raspberry bushes cv Enrosadira exhibiting typical symptoms of Rubus stunt were observed in Central Bohemia. Symptoms included dieback, leaf yellowing/reddening, stunted growth, severe phyllody and fruit malformations. Most diseased plants were growing in the edge rows of the field (about 80%). No symptomatic plants were observed in the middle of the field. Similar symptoms were observed in private gardens in South Bohemia on raspberry cv Rutrago and blackberry (unknown cultivar) in June 2018 and August 2022, respectively. DNA was extracted using the DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany) from flower stems and parts affected by phyllody of seven symptomatic plants as well as flower stems, leaf midribs, and petioles of five asymptomatic field plants. The DNA extracts were analyzed by a nested polymerase chain reaction assay using universal phytoplasma P1A/P7A primers followed by R16F2m/R1m and the group-specific R16(V)F1/R1 primers (Bertaccini et al. 2019). All samples from the symptomatic plants yielded an amplicon of expected size, while no product was amplified in asymptomatic plants. The P1A/P7A amplicons from three selected plants (two raspberries and one blackberry, each from different location) were cloned and bi-directionally Sanger sequenced (GenBank Accession Nos.OQ520100-2). The sequences spanned nearly full-length of 16S rRNA gene, 16S-23S rRNA intergenic spacer, tRNA-Ile gene, and a partial 23S rRNA gene. BLASTn search revealed the highest sequence identity (99.8-99.9%, query coverage 100%) to 'Candidatus Phytoplasma rubi' strain RS (GenBank Accession No. CP114006). To further characterize the 'Ca. P. rubi' strains, all these three samples were subjected to multigene sequence analysis. Sequences from a major portion of the tuf, rplV-rpsC, rpsH-rplR, uvrB-degV, and rplO-SecY-map genes (Acc. Nos. OQ506112-26) were obtained as described previously (Fránová et al. 2016). Comparison to GenBank sequences confirmed their highest identity (99.6-100%, query coverage 100%) with 'Ca. P. rubi' RS strain, regardless of their geographic location and host (raspberry or blackberry). Recently, Bertaccini et al. (2022) suggested the 98,65 % 'Ca. Phytoplasma' strain identity threshold within 16Sr RNA sequences. In this survey, all three strains sequenced shared ≥99.73% sequence identity of the analysed 16S rRNA gene sequences and the high identity in the other genes with the reference 'Ca. P. rubi' RS strain. To our knowledge, this is the first report of Rubus stunt disease in the Czech Republic as well as the first molecular identification and characterization of 'Ca. P. rubi' from raspberry and blackberry in our country. As Rubus stunt disease is of great economic importance (Linck and Reineke 2019a), the pathogen detection and prompt removal of the diseased shrubs are essential to mitigating the spread and impact of the disease.

The Expanding Menagerie of Prunus-Infecting Luteoviruses.

Members of the genus Luteovirus are responsible for economically destructive plant diseases worldwide. Over the past few years, three luteoviruses infecting Prunus trees have been characterized. However, the biological properties, prevalence, and genetic diversity of those viruses have not yet been studied. High-throughput sequencing of samples of various wild, cultivated, and ornamental Prunus species enabled the identification of four novel species in the genus Luteovirus for which we obtained complete or nearly complete genomes. Additionally, we identified another new putative species recovered from Sequence Read Archive data. Furthermore, we conducted a survey on peach-infecting luteoviruses in eight European countries. Analyses of 350 leaf samples collected from germplasm, production orchards, and private gardens showed that peach-associated luteovirus (PaLV), nectarine stem pitting-associated virus (NSPaV), and a novel luteovirus, peach-associated luteovirus 2 (PaLV2), are present in all countries; the most prevalent virus was NSPaV, followed by PaLV. The genetic diversity of these viruses was also analyzed. Moreover, the biological indexing on GF305 peach indicator plants demonstrated that PaLV and PaLV2, like NSPaV, are transmitted by graft at relatively low rates. No clear viral symptoms have been observed in either graft-inoculated GF305 indicators or different peach tree varieties observed in an orchard. The data generated during this study provide a broader overview of the genetic diversity, geographical distribution, and prevalence of peach-infecting luteoviruses and suggest that these viruses are likely asymptomatic in peach under most circumstances.

Mixed Infection of Blackcurrant with a Novel Cytorhabdovirus and Black Currant-Associated Nucleorhabdovirus.

A virome screen was performed on a new breeding line, KB1, of blackcurrant. Rhabdovirus-like particles were observed by electron microscopy in ultrathin sections of flower stalks, and the complete genome sequence of a novel virus, provisionally named blackcurrant rhabdovirus 2 (BCRV2), was determined and verified using high-throughput sequencing. The genomic organization of BCRV2 was characteristic of cytorhabdoviruses (family Rhabdoviridae) and included seven genes: 3 ́- N-P´-P-P3-M-G-L -5 ́. BLASTP analysis revealed that the putative L protein had the highest amino acid sequence identity (75 %) with strawberry virus 2. BCRV2 was detected in Cryptomyzusgaleopsidis, but efficient transmission by this aphid was not confirmed. Of note, we observed coinfection of the KB1 line with blackcurrant-associated rhabdovirus (BCaRV) by RT-PCR. This is likely the first evidence of the presence of a cyto- and a nucleorhabdovirus in a single host.

Detection of Apple Hammerhead Viroid, Apple Luteovirus 1 and Citrus Concave Gum-Associated Virus in Apple Propagation Materials and Orchards in the Czech Republic and Hungary.

Grafting cultivars onto rootstocks is a widely used practice by the apple industry predominantly aimed at faster fruit bearing. Using high-throughput sequencing, we revealed the presence of recently described viral agents, namely apple hammerhead viroid (AHVd), apple luteovirus 1 (ALV-1), and citrus concave gum-associated virus (CCGaV), in germplasm collections and production orchards in the Czech Republic and Hungary. The HTS results were validated with RT-(q)PCR, and Northern blotting techniques. To obtain further insight about the presence of these agents, RT-PCR based surveys were carried out and showed their widespread presence alone or in mixed infections. The pathogens were present both in production areas and in feral samples. In addition, rootstock-to-scion transmission of ALV-1 and CCGaV was confirmed using commercial rootstock materials. Phylogenetic relationships based on partial sequences of distinct variants were also investigated. Furthermore, the rosy apple aphid was found to be ALV-1-positive, suggesting that it might be a potential vector of the virus.

Transmission of Diverse Variants of Strawberry Viruses Is Governed by a Vector Species.

Advances in high-throughput sequencing methods have boosted the discovery of multistrain viral infections in diverse plant systems. This phenomenon appears to be pervasive for certain viral species. However, our knowledge of the transmission aspects leading to the establishment of such mixed infections is limited. Recently, we reported a mixed infection of a single strawberry plant with strawberry mottle virus (SMoV), strawberry crinkle virus (SCV) and strawberry virus 1 (StrV-1). While SCV and StrV-1 are represented by two and three molecular variants, respectively, SmoV has three different RNA1 and RNA2 segments. In this study, we focus on virus acquisition by individual adult aphids of the Aphis gossypii, Aphis forbesi and Chaetosiphon fragaefolii species. Single-aphid transmission trials are performed under experimental conditions. Both different viruses and individual virus strains show varying performances in single aphid acquisition. The obtained data suggests that numerous individual transmission events lead to the establishment of multistrain infections. These data will be important for the development of epidemiological models in plant virology.

Identification and Characterization of a Novel Umbra-like Virus, Strawberry Virus A, Infecting Strawberry Plants.

A novel RNA virus infecting strawberry plants was discovered using high-throughput sequencing. The analyzed plant was simultaneously infected with three different genetic variants of the virus, provisionally named strawberry virus A (StrVA). Although StrVA is phylogenetically clustered with several recently discovered, unclassified plant viruses, it has a smaller genome and several unique features in its genomic organization. A specific and sensitive qPCR system for the detection of identified StrVA genetic variants was designed. A survey conducted in the Czech Republic revealed that StrVA was present in 28.3% of strawberry samples (n = 651) from various origins (plantations, gardens, and propagation material). Sequencing of 48 randomly selected StrVA-positive strawberry samples showed that two or all three StrVA genetic variants were present in 62.5% of the samples in various proportions. StrVA was found in mixed infections with other viruses (strawberry mild yellow edge virus, strawberry crinkle virus, strawberry mottle virus, strawberry polerovirus 1, or strawberry virus 1) in 57.1% of the samples, which complicated the estimation of its biological relevance and impact on the health status of the plants.

High Incidence of Strawberry Polerovirus 1 in the Czech Republic and Its Vectors, Genetic Variability and Recombination.

In total, 332 strawberry plants from 33 different locations in the Czech Republic with or without disease symptoms were screened by RT-PCR for the presence of strawberry polerovirus 1 (SPV1) and five other viruses: strawberry mottle virus, strawberry crinkle virus, strawberry mild yellow edge virus, strawberry vein banding virus, and strawberry virus 1. SPV1 was detected in 115 tested strawberry plants (35%), including 89 mixed infections. No correlation between symptoms and the detected viruses was found. To identify potential invertebrate SPV1 vectors, strawberry-associated invertebrate species were screened by RT-PCR, and the virus was found in the aphids Aphis forbesi, A. gossypii, A. ruborum, A.sanquisorbae, Aulacorthum solani, Chaetosiphon fragaefolii, Myzus ascalonicus, and several other non-aphid invertebrate species. SPV1 was also detected in aphid honeydew. Subsequent tests of C. fragaefolii and A.gossypii virus transmission ability showed that at least 4 h of acquisition time were needed to acquire the virus. However, 1 day was sufficient for inoculation using C. fragaefolii. In conclusion, being aphid-transmitted like other tested viruses SPV1 was nevertheless the most frequently detected agent. Czech SPV1 isolates belonged to at least two phylogenetic clusters. The sequence analysis also indicated that recombination events influence evolution of SPV1 genomes.

Complete genome sequence of a mite-associated virus obtained by high-throughput sequencing analysis of an apple leaf sample.

We provide the complete sequence of a virus tentatively named "Tetranychus urticae-associated picorna-like virus 1PK13" (TuaPV1-PK13) obtained from the high-throughput sequencing of a symptomless apple leaf sample. Although the virus sequence was originally derived from apple leaves, the data suggest that the virus is associated with the two-spotted mite Tetranychus urticae.

Fragaria vesca-associated virus 1: a new virus related to negeviruses.

In this study, the complete genomic sequence of a novel virus was determined by next-generation sequencing of a sample from a symptomatic strawberry plant with severe yellow spots and mosaic on its leaves. Its genomic organization and sequence showed that this virus is related to members of the proposed insect-specific genus "Negevirus". The sample also contained sequences from the geranium aphid Acyrthosiphon malvae. Although the virus was detected repeatedly in the same plant during the three following years, no other positive samples were obtained from the surroundings or more-distant locations. Reverse transcription qPCR analysis revealed the presence of both genomic positive and complementary negative strands of the viral genome in the sample, with a 3- to 30-fold excess of the positive strand, indicating active viral replication. As the virus was not detected in any insect species collected at this location, the virus was provisionally named "Fragaria vesca-associated virus 1" (FVaV-1).

Genomic characterization of Malus domestica virus A (MdoVA), a novel velarivirus infecting apple.

Screening of apple samples using a high-throughput sequencing (HTS) approach led to the discovery of a novel virus, tentatively named "Malus domestica virus A" (MdoVA). Its genomic organisation and phylogenetic relationship showed relatedness to viruses of the genus Velarivirus in the family Closteroviridae. It is not clear whether MdoVA has any impact on its host, as the analysed apple tree contained other viruses and a viroid.

Molecular and Biological Characterization of a New Strawberry Cytorhabdovirus.

Virus diseases of strawberry present several complex problems. More than 25 viruses have been described in the genus Fragaria thus far. Here, we describe a novel rhabdovirus, tentatively named strawberry virus 1 (StrV-1), that infects F.ananassa and F.vesca plants. Genomic sequences of three distinct StrV-1 genotypes co-infecting a single F.ananassa host were obtained using combined Illumina and Ion Proton high-throughput sequencing. StrV-1 was transmitted to herbaceous plants via Aphisfabae and A.ruborum, further mechanically transmitted to Nicotianaoccidentalis 37B and sub-inoculated to N.benthamiana, N.benthamiana DCL2/4i, N.occidentalis 37B, and Physalisfloridana plants. Irregular chlorotic sectors on leaf blades and the multiplication of calyx leaves seem to be the diagnostic symptoms for StrV-1 on indexed F.vesca clones. StrV-1 was detected in asymptomatic grafted plants and in 49 out of 159 field strawberry samples via RT-PCR followed by Sanger sequencing. The bacilliform shape of the virions, which have a cytoplasm-limited distribution, their size, and phylogenetic relationships support the assignment of StrV-1 to a distinct species of the genus Cytorhabdovirus. Acyrthosiphonmalvae, A.fabae, and A.ruborum were shown to transmit StrV-1 under experimental conditions.

Red clover-associated luteovirus - a newly classifiable member of the genus Luteovirus with an enamo-like P5 protein.

This study reports the complete genomic sequence of a novel virus isolated from red clover. According to its genomic organization, its similarity to luteoviruses, and a greater than 10% difference in all genes, this virus isolate likely represents a new luteovirus species. As seen in nectarine stem pitting-associated virus (NSPaV) and NSPaV-South Korea (SK) luteoviruses, it differs from typical luteoviruses through the absence of ORF3a and ORF4 encoding movement proteins. Furthermore, its P5 protein (responsible for aphid transmission) is more similar to the P5 of enamoviruses than that of luteoviruses. The virus isolate has been named red clover-associated luteovirus (RCaV).

Molecular Characterization of Divergent Closterovirus Isolates Infecting Ribes Species.

Five isolates of a new member of the family Closteroviridae, tentatively named blackcurrant leafroll-associated virus 1 (BcLRaV-1), were identified in the currant. The 17-kb-long genome codes for 10 putative proteins. The replication-associated polyprotein has several functional domains, including papain-like proteases, methyltransferase, Zemlya, helicase, and RNA-dependent RNA polymerase. Additional open reading frames code for a small protein predicted to integrate into the host cell wall, a heat-shock protein 70 homolog, a heat-shock protein 90 homolog, two coat proteins, and three proteins of unknown functions. Phylogenetic analysis showed that BcLRaV-1 is related to members of the genus Closterovirus, whereas recombination analysis provided evidence of intraspecies recombination.

Complete genome sequences of two divergent isolates of strawberry crinkle virus coinfecting a single strawberry plant.

Strawberry crinkle disease is one of the major diseases that threatens strawberry production. Although the biological properties of the agent, strawberry crinkle virus (SCV), have been thoroughly investigated, its complete genome sequence has never been published. Existing RT-PCR-based detection relies on a partial sequence of the L protein gene, presumably the least expressed viral gene. Here, we present complete sequences of two divergent SCV isolates co-infecting a single plant, Fragaria x ananassa cv. Cacanská raná.

Identification and molecular characterization of a novel varicosa-like virus from red clover.

During aetiological study of diseased red clover (Trifolium pratense L.) using high throughput sequencing, a novel virus with a 10 kb genome divided into two segments was discovered. The virus, tentatively named red clover associated varicosavirus (RCaVV), is phylogenetically related to classifiable members of the genus Varicosavirus (family Rhabdoviridae, order Mononegavirales). Analysis of mRNA levels from the individual RCaVV genes suggested possible differences in transcription regulation between rhabdoviruses with divided and undivided genomes.

Variability Studies of Two Prunus-Infecting Fabaviruses with the Aid of High-Throughput Sequencing.

During their lifetime, perennial woody plants are expected to face multiple infection events. Furthermore, multiple genotypes of individual virus species may co-infect the same host. This may eventually lead to a situation where plants harbor complex communities of viral species/strains. Using high-throughput sequencing, we describe co-infection of sweet and sour cherry trees with diverse genomic variants of two closely related viruses, namely prunus virus F (PrVF) and cherry virus F (CVF). Both viruses are most homologous to members of the Fabavirus genus (Secoviridae family). The comparison of CVF and PrVF RNA2 genomic sequences suggests that the two viruses may significantly differ in their expression strategy. Indeed, similar to comoviruses, the smaller genomic segment of PrVF, RNA2, may be translated in two collinear proteins while CVF likely expresses only the shorter of these two proteins. Linked with the observation that identity levels between the coat proteins of these two viruses are significantly below the family species demarcation cut-off, these findings support the idea that CVF and PrVF represent two separate Fabavirus species.

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).