Viromes of Plants Determined by High-Throughput Sequencing of Virus-Derived siRNAs.

Plants growing in open airfields can be infected by several viruses even as a multiple infection. Virus infection in crops can lead to a serious damage to the harvest. In addition, virus presence in grapevine, fruit trees, and tuberous vegetables, propagated vegetatively affects the phytosanitary status of the propagation material (both the rootstock and the variety) having profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Small interfering (si) RNAs produced by the RNA silencing-based host immune system during viral infection can be sequenced by high-throughput techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample and therefore opening new avenues in virus diagnostics. This method is based on Illumina sequencing and bioinformatics analysis of virus-derived siRNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, to ensure success for every user.

Clematis vitalba Is a Natural Host of the Novel Ilarvirus, Prunus Virus I.

Clematis vitalba L. is a climbing shrub and a pioneer plant in abandoned orchards or vineyards that are widespread in temperate climate zones. In past years, several viruses infecting the Clematis species have been identified, including different ilarviruses. Prunus virus I (PrVI) is a recently described ilarvirus, which has been shown to infect sweet cherries and peaches in Greece. Moreover, its presence has been detected in ornamental Clematis in Russia. In the present work, we analyzed the virome of wildly growing C. vitalba plants from Hungary, Slovakia and Croatia showing different kinds of symptoms using high-throughput sequencing (HTS) of small RNAs or ribodepleted RNAs. Applying HTS enabled us to identify the presence of PrVI in C. vitalba, and the bioinformatic analyses were further validated with RT-PCR using PrVI-specific primers and Sanger dideoxy sequencing. Nearly full genome sequences of all three viral RNAs of one Hungarian, two Slovak and one Croatian isolate were determined. Their phylogenetic analysis showed high similarity to each other and to other PrVI isolates described from Central Europe. As the sampled plants were co-infected with other viruses, it is not possible to determine a direct correlation between the infection with PrVI and the observed symptoms. Analyses of different Prunus species in stock collection showed infection of several peach and sweet cherry varieties in Hungary. Our results expand the knowledge on the natural host range of PrVI and highlight the necessity to evaluate alternative plant hosts (even non-Prunus) of PrVI and the role of the virus in the etiology of the potential diseases.

Grapevine Pinot Gris Virus Is Present in Different Non-Vitis Hosts.

Grapevine Pinot gris virus (GPGV) was described in Italy using a metagenomic approach: next-generation sequencing of the virus-derived small RNAs. Since that time, it has been reported all over the world. The presence of GPGV is associated with grapevine disease, but most of the time, the disease is asymptomatic. Although the host range of this virus has not been investigated, it has been found in the non-Vitis hosts, Silene latifolia and Chenopodium album. We investigated the presence of GPGV in grapevine and other plant species growing as weeds in the vineyard. Using RT-PCR, we identified GPGV in seven non-Vitis hosts: Ailanthus, Asclepias, Crataegus, Fraxinus, Rosa, Rubus, and Sambucus. In the case of Rosa and Rubus, this finding was supported by Northern blot detection of the virus. GPGV strains in non-Vitis hosts belong to the asymptomatic clade, and are clustered according to their original geographic locations. The presence of GPGV in species other than grapevine shows that besides well-known vector and propagating material-based infections, other possible entry sites for the virus can exist, which have to be taken into consideration when developing reliable regulation strategies.

Variable Populations of Grapevine Virus T Are Present in Vineyards of Hungary.

Grapevine virus T (GVT) is a recently described foveavirus, which was identified from a transcriptome of a Teroldego grapevine cultivar in 2017. Recently, we surveyed vineyards and rootstock plantations in Hungary using small RNA (sRNA) high-throughput sequencing (HTS), at a time when GVT had not yet been described. A re-analysis of our sRNA HTS datasets and a survey of grapevines by RT-PCR revealed the presence of GVT in most of the vineyards tested, while at rootstock fields its presence was very rare. The presence and high variability of the virus in the country was confirmed by sequence analysis of strains originating from different vineyards. In this study, we demonstrate the presence of GVT in Hungary and show its high diversity, suggesting that GVT presence may not seriously affect grapevine health and that it could have been present in European vineyards for a long time as a latent infection.

HTS-Based Monitoring of the Efficiency of Somatic Embryogenesis and Meristem Cultures Used for Virus Elimination in Grapevine.

Meristem culture and somatic embryogenesis are effective tools for virus elimination of vegetatively propagated crops including grapevine (Vitis vinifera L.). While both have been shown to be useful to eliminate the main grapevine viruses, their efficiency differs depending on the virus and grapevine variety. In our work, we investigated the efficiency of these two virus elimination methods using small RNA high-throughput sequencing (HTS) and RT-PCR as virus diagnostics. Field grown mother plants of four clones representing three cultivars, infected with different viruses and viroids, were selected for elimination via somatic embryogenesis (SE) and meristem culture (ME). Our results show for the first time that using SE, elimination in mother plants was effective for all viruses, i.e., grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), Grapevine virus T (GVT) and grapevine Pinot gris virus (GPGV). This study also confirms previous studies showing that SE is a possible strategy for the elimination of GFkV, GRSPaV, HSVd, and GYSVd-1. Our results demonstrate that the efficacy of virus elimination via SE is relatively high while the purging of viroids is lower. Our work provides evidence that the efficiency of SE is comparable to that of the technically difficult ME technique, and that SE will offer a more effective strategy for the production of virus-free grapevine in the future.

Millet Could Be both a Weed and Serve as a Virus Reservoir in Crop Fields.

Millet is a dangerous weed in crop fields. A lack of seed dormancy helps it to spread easily and be present in maize, wheat, and other crop fields. Our previous report revealed the possibility that millet can also play a role as a virus reservoir. In that study, we focused on visual symptoms and detected the presence of several viruses in millet using serological methods, which can only detect the presence of the investigated pathogen. In this current work, we used small RNA high-throughput sequencing as an unbiased virus diagnostic method to uncover presenting viruses in randomly sampled millet grown as a volunteer weed in two maize fields, showing stunting, chlorosis, and striped leaves. Our results confirmed the widespread presence of wheat streak mosaic virus at both locations. Moreover, barley yellow striate mosaic virus and barley virus G, neither of which had been previously described in Hungary, were also identified. As these viruses can cause severe diseases in wheat and other cereals, their presence in a weed implies a potential infection risk. Our study indicates that the presence of millet in fields requires special control to prevent the emergence of new viral diseases in crop fields.

Local Aphid Species Infestation on Invasive Weeds Affects Virus Infection of Nearest Crops Under Different Management Systems - A Preliminary Study.

In the present study, we conducted field surveys to detect the population density of the most important invasive weed species and their associated virus vectoring aphids in crops grown under high input field (HIF) vs. low-input field (LIF) conditions, with and without fertilizers and pesticides. The most frequent invasive weed species were annual fleabane, Erigeron annua (L.), Canadian horseweed, Erigeron canadensis (L.) and Canadian goldenrod, Solidago canadensis (L.). These species were predominantly hosts of the aphids Brachycaudus helichrysi and Aulacorthum solani under both management systems. The 13% higher coverage of E. annua under LIF conditions resulted in a 30% higher B. helichrysi abundance and ∼85% higher A. solani abundance compared with HIF conditions. To reveal the incidence of virus infection in crop plants and invasive weeds, high-throughput sequencing of small RNAs was performed. Bioinformatics analysis combined with independent validation methods revealed the presence of six viruses, but with strikingly different patterns under LIF and HIF conditions. Their presence without symptoms in invasive weeds and crop plants supports the necessity of employing new approaches to those currently employed in invasive weed management. These findings also suggest that invasive weeds could serve as hosts for local aphid species and reservoirs for plant pathogenic viruses, both under low and high input management systems. In this light, as here demonstrated, viruses transmitted by local aphid species were found to differ between the management systems; hence, the importance of B. helichrysi and A. solani as virus vectors in particular clearly needs to be re-evaluated. Altogether, we accept that the present study is a pilot one and individual virus vectoring of aphids still needs to be directly tested. Even so, it represents one of the first contributions to this particular area, and thereby paves the way for further similar applied research in the future.

Complete Sequence, Genome Organization and Molecular Detection of Grapevine Line Pattern Virus, a New Putative Anulavirus Infecting Grapevine.

: Grapevine line pattern virus (GLPV) was first described 30 years ago in Hungary. The lack of its genomic sequences and of an available antiserum made its detection impossible in other parts of the world. Three different high-throughput sequencing (HTS) protocols applied on a GLPV-infected vine allowed the construction of the full genome sequence of this virus. It includes three RNA segments, encoding four proteins: methyltransferase-helicase (1a), RNA-dependent RNA polymerase (2a), movement protein (3a) and coat protein (3b). The obtained sequences were used to design specific primers for its detection by RT-PCR and Northern blot hybridization, respectively. These diagnostic methods were used to test the presence of GLPV in graft-inoculated plants and in 220 grapevine accessions of different Mediterranean origins. The three RNAs-encoding proteins of GLPV shared a very high amino acid identity with those of hop yellow virus, a tentative member of the Anulavirus genus, leaving no doubt that both are two isolates of the same viral species. A circular RNA originating from the RNA2 was found, for which an alternative silencing suppressor role is hypothesized. Further investigation is needed to determine this possibility and also the host range and pathological significance of the virus.

Use of siRNAs for Diagnosis of Viruses Associated to Woody Plants in Nurseries and Stock Collections.

Woody perennial plants like grapevine and fruit trees can be infected by several viruses even as multiple infections. Since they are propagated vegetatively, the phytosanitary status of the propagation material (both the rootstock and the variety) can have a profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Viral derived small RNAs produced by the host immune system during viral infection can be sequenced by next-generation techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample. This method is based on Illumina sequencing of short RNAs and bioinformatics analysis of virus-derived small RNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, in order to ensure success for every user.