Application of Nanopore-Based Sequencing to Identify Virus Infections in Woody Plants.

Plant viruses threaten the yield and quality of crops. Efficient and affordable pathogen diagnosis is crucial to regulate the trade of plant materials and for disease management and control. Sequencing technology based on Illumina platform is a powerful tool for the identification of plant viruses, but it requires long and expensive protocols, cumbersome equipment, and significant cost per library. Nanopore sequencing technology, developed by Oxford Nanopore Technologies (ONT), is a recent sequencing system very easy to use, suitable for onsite-field detection, and associated with low costs. Coupled with its portability, nanopore technology has great application prospects in the field of quick detection of plant viruses. In this protocol, we expose in detail the application of cDNA-PCR nanopore-based sequencing for the detection of plant viruses.

First Identification of Fig Virus A and Fig Virus B in Ficus carica in Italy.

Extracts of double-stranded RNA from three fig cultivars from an Apulian (Southern Italy) germplasm collection were used for high-throughput sequencing and revealed the presence of two distinct, recently described closteroviruses. Sequences obtained from these Apulian isolates belong to fig virus A and fig virus B and cover 38 and 25% of their RNA genome, respectively. Primer sets designed on selected contigs confirmed the presence of each virus in infected plants. A close phylogenetic relationship, investigated in a fragment of HSP70h protein, occurs among these isolates and the reference genomes. A nucleotide divergence (ranging from 10 to 30% along the different genes) was observed among our isolates and the reference genomes. This is the first finding of these virus species in autochthonous fig accessions in Europe.

Nanopore Technology Applied to Targeted Detection of Tomato Brown Rugose Fruit Virus Allows Sequencing of Related Viruses and the Diagnosis of Mixed Infections.

Tomato (Solanum lycopersicum) plants from a commercial glasshouse were identified with symptoms compatible with a tomato brown rugose fruit virus (ToBRFV) infection. Reverse transcription-PCR and quantitative PCR confirmed the presence of ToBRFV. Subsequently, the same RNA sample and a second from tomato plants infected with a similar tobamovirus, tomato mottle mosaic virus (ToMMV), were extracted and processed for high-throughput sequencing with the Oxford Nanopore Technology (ONT). For the targeted detection of ToBRFV, the two libraries were synthesized by using six ToBRFV sequence-specific primers in the reverse transcription step. This innovative target enrichment technology enabled deep coverage sequencing of ToBRFV, with 30% of the total reads mapping to the target virus genome and 57% mapping to the host genome. The same set of primers applied to the ToMMV library generated 5% of the total reads mapping to the latter virus, indicating that sequencing of similar, non-target viral sequences was also allowed. Further, the complete genome of pepino mosaic virus (PepMV) was also sequenced from the ToBRFV library, thus suggesting that, even using multiple sequence-specific primers, a low rate of off-target sequencing can usefully provide additional information on unexpected viral species coinfecting the same samples in an individual assay. These results demonstrate that targeted nanopore sequencing can specifically identify viral agents and has sufficient sensitivity towards non-target organisms to provide evidence of mixed virus infections.

A New Jasmine Virus C Isolate Identified by Nanopore Sequencing Is Associated to Yellow Mosaic Symptoms of Jasminum officinale in Italy.

Some plants of Jasminum officinale were selected in a nursery for investigation of sanitary status of candidate mother plants before vegetative propagation. The presence of yellow spots and leaf discoloration symptoms pushed for a generic diagnosis through deep sequencing to discover systemic pathogens. Either dsRNA or total RNA were extracted and used in nanopore and Illumina platform for cDNA-PCR, direct RNA and total RNA rRNA-depleted sequencing. A few single reads obtained by nanopore technology or assembled contigs gave unequivocal annotation for the only presence of a jasmine virus C (JaVC, a putative member of genus Carlavirus) isolate. The full-length genome of this isolate was reconstructed, spanning 8490 nucleotides (nt). This isolate shared 90.9% similarity with coat protein sequences and 84% with the entire ORF1 polyprotein, with the other two available JaVC full genomes, isolated from infections in J. sambac in Taiwan and China. The overall nucleotide identity shared by the newly discovered Italian isolate with the Chinese JaVC full genomes was 76.14% (Taiwan) and 75.60% (Fujian). The application of quick nanopore sequencing for virus discovery was assessed. The identification of the virus in a new ornamental host species, largely used in gardening, creates a concern for the potential virus spread and need of testing for production of clean vegetative material.

High throughput sequencing from Angolan citrus accessions discloses the presence of emerging CTV strains.

BACKGROUND: Citrus industry is worldwide dramatically affected by outbreaks of Citrus tristeza virus (CTV). Controls should be applied to nurseries, which could act as diversity hotspots for CTV. Early detection and characterization of dangerous or emerging strains of this virus greatly help to prevent outbreaks of disease. This is particularly relevant in those growing regions where no dedicated certification programs are currently in use. METHODS: Double-stranded RNA extracted from Citrus spp. samples, collected in two locations in Angola, were pooled and submitted to a random-primed RNA-seq. This technique was performed to acquire a higher amount of data in the survey, before the amplification and sequencing of genes from single plants. To confirm the CTV infection in individual plants, as suggested by RNA-seq information from the pooled samples, the analysis was integrated with multiple molecular marker amplification (MMM) for the main known CTV strains (T30, T36, VT and T3). RESULTS: From the analysis of HTS data, several assembled contigs were identified as CTV and classified according to their similarity to the established strains. By the MMM amplification, only five individual accessions out of the eleven pooled samples, resulted to be infected by CTV. Amplified coat protein genes from the five positive sources were cloned and sequenced and submitted to phylogenetic analysis, while a near-complete CTV genome was also reconstructed by the fusion of three overlapping contigs. CONCLUSION: Phylogenetic analysis of the ORF1b and CP genes, retrieved by de novo assembly and RT-PCR, respectively, revealed the presence of a wide array of CTV strains in the surveyed citrus-growing spots in Angola. Importantly, molecular variants among those identified from HTS showed high similarity with known severe strains as well as to recently described and emerging strains in other citrus-growing regions, such as S1 (California) or New Clade (Uruguay).

Sixty Years from the First Disease Description, a Novel Badnavirus Associated with Chestnut Mosaic Disease.

Although chestnut mosaic disease (ChMD) was described several decades ago, its etiology is still not clear. Using classical approaches and high-throughput sequencing (HTS) techniques, we identified a novel Badnavirus that is a strong etiological candidate for ChMD. Two disease sources from Italy and France were submitted to HTS-based viral indexing. Total RNAs were extracted, ribodepleted, and sequenced on an Illumina NextSeq500 (2 × 150 nt or 2 × 75 nt). In each source, we identified a single contig of ≈7.2 kb that corresponds to a complete circular viral genome and shares homologies with various badnaviruses. The genomes of the two isolates have an average nucleotide identity of 90.5%, with a typical badnaviral genome organization comprising three open reading frames. Phylogenetic analyses and sequence comparisons showed that this virus is a novel species; we propose the name Chestnut mosaic virus (ChMV). Using a newly developed molecular detection test, we systematically detected the virus in symptomatic graft-inoculated indicator plants (chestnut and American oak) as well in chestnut trees presenting typical ChMD symptoms in the field (100 and 87% in France and Italy surveys, respectively). Datamining of publicly available chestnut sequence read archive transcriptomic data allowed the reconstruction of two additional complete ChMV genomes from two Castanea mollissima sources from the United States as well as ChMV detection in C. dentata from the United States. Preliminary epidemiological studies performed in France and central eastern Italy showed that ChMV has a high incidence in some commercial orchards and low within-orchard genetic diversity.

Pest categorisation of non-EU viruses of Rubus L.

The Panel on Plant Health of EFSA conducted a pest categorisation of 17 viruses of Rubus L. that were previously classified as either non-EU or of undetermined standing in a previous opinion. These infectious agents belong to different genera and are heterogeneous in their biology. Blackberry virus X, blackberry virus Z and wineberry latent virus were not categorised because of lack of information while grapevine red blotch virus was excluded because it does not infect Rubus. All 17 viruses are efficiently transmitted by vegetative propagation, with plants for planting representing the major pathway for entry and spread. For some viruses, additional pathway(s) are Rubus seeds, pollen and/or vector(s). Most of the viruses categorised here infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Cherry rasp leaf virus, raspberry latent virus, raspberry leaf curl virus, strawberry necrotic shock virus, tobacco ringspot virus and tomato ringspot virus meet all the criteria to qualify as potential Union quarantine pests (QPs). With the exception of impact in the EU territory, on which the Panel was unable to conclude, blackberry chlorotic ringspot virus, blackberry leaf mottle-associated virus, blackberry vein banding-associated virus, blackberry virus E, blackberry virus F, blackberry virus S, blackberry virus Y and blackberry yellow vein-associated virus satisfy all the other criteria to be considered as potential QPs. Black raspberry cryptic virus, blackberry calico virus and Rubus canadensis virus 1 do not meet the criterion of having a potential negative impact in the EU. For several viruses, the categorisation is associated with high uncertainties, mainly because of the absence of data on biology, distribution and impact. Since the opinion addresses non-EU viruses, they do not meet the criteria to qualify as potential Union regulated non-quarantine pests.

ICTV Virus Taxonomy Profile: Closteroviridae.

Viruses in the family Closteroviridae have a mono-, bi- or tripartite positive-sense RNA genome of 13-19 kb, and non-enveloped, filamentous particles 650-2200 nm long and 12 nm in diameter. They infect plants, mainly dicots, many of which are fruit crops. This is a summary of the ICTV Report on the family Closteroviridae, which is available at ictv.global/report/closteroviridae.

Surface Dielectric Barrier Discharge plasma: a suitable measure against fungal plant pathogens.

Fungal diseases seriously affect agricultural production and the food industry. Crop protection is usually achieved by synthetic fungicides, therefore more sustainable and innovative technologies are increasingly required. The atmospheric pressure low-temperature plasma is a novel suitable measure. We report on the effect of plasma treatment on phytopathogenic fungi causing quantitative and qualitative losses of products both in the field and postharvest. We focus our attention on the in vitro direct inhibitory effect of non-contact Surface Dielectric Barrier Discharge on conidia germination of Botrytis cinerea, Monilinia fructicola, Aspergillus carbonarius and Alternaria alternata. A few minutes of treatment was required to completely inactivate the fungi on an artificial medium. Morphological analysis of spores by Scanning Electron Microscopy suggests that the main mechanism is plasma etching due to Reactive Oxygen Species or UV radiation. Spectroscopic analysis of plasma generated in humid air gives the hint that the rotational temperature of gas should not play a relevant role being very close to room temperature. In vivo experiments on artificially inoculated cherry fruits demonstrated that inactivation of fungal spores by the direct inhibitory effect of plasma extend their shelf life. Pre-treatment of fruits before inoculation improve the resistance to infections maybe by activating defense responses in plant tissues.

Resistance to Sharka in Apricot: Comparison of Phase-Reconstructed Resistant and Susceptible Haplotypes of 'Lito' Chromosome 1 and Analysis of Candidate Genes.

Sharka, a common disease among most stone fruit crops, is caused by the Plum Pox Virus (PPV). Resistant genotypes have been found in apricot (Prunus armeniaca L.), one of which-the cultivar 'Lito' heterozygous for the resistance-has been used to map a major quantitative trait locus (QTL) on linkage group 1, following a pseudo-test-cross mating design with 231 individuals. In addition, 19 SNP markers were selected from among the hundreds previously developed, which allowed the region to be limited to 236 kb on chromosome 1. A 'Lito' bacterial artificial chromosome (BAC) library was produced, screened with markers of the region, and positive BAC clones were sequenced. Resistant (R) and susceptible (S) haplotypes were assembled independently. To refine the assembly, the whole genome of 'Lito' was sequenced to high coverage (98×) using PacBio technology, enabling the development of a detailed assembly of the region that was able to predict and annotate the genes in the QTL region. The selected cultivar 'Lito' allowed not only to discriminate structural variants between the two haplotypic regions but also to distinguish specific allele expression, contributing towards mining the PPVres locus. In light of these findings, genes previously indicated (i.e., MATHd genes) to have a possible role in PPV resistance were further analyzed, and new candidates were discussed. Although the results are not conclusive, the accurate and independent assembly of R and S haplotypes of 'Lito' is a valuable resource to predict and test alternative transcription and regulation mechanisms underpinning PPV resistance.

Molecular variability of apple hammerhead viroid from Italian apple varieties supports the relevance in vivo of its branched conformation stabilized by a kissing loop interaction.

In the absence of protein-coding ability, viroid RNAs rely on direct interactions with host factors for their infectivity. RNA structural elements are likely involved in these interactions. Therefore, preservation of a structural element, despite the sequence variability existing between the variants of a viroid population, is considered a solid evidence of its relevant role in vivo. In this study, apple hammerhead viroid (AHVd) was first identified in the two apple cultivars 'Mela Rosa Guadagno' (MRG) and 'Agostinella' (AG), which are cultivated since long in Southern Italy, thus providing the first solid evidence of its presence in this country. Then, the natural variability of AHVd viroid populations infecting MRG and AG was studied. The sequence variants from the two Italian isolates shared only 82.1-87.7% sequence identity with those reported previously from other geographic areas, thus providing the possibility of exploring the impact of this sequence divergence on the proposed secondary structure. Interestingly, all the AHVd sequence variants considered in this study preserved a branched secondary structure stabilized by a kissing-loop interaction, resembling the conformation proposed previously for variants from other isolates. Indeed, most mutations did not modify the proposed conformation because they were co-variations, conversions of canonical into wobble base-pairs, or vice versa, as well as changes mapping at loops. Importantly, a cruciform structural element formed by four hairpins, one of which is implicated in the proposed kissing-loop interaction, was also preserved because several nucleotide changes actually resulted into two, three and up to five consecutive co-variations associated with other changes that did not affect the secondary structure. These data provide very strong evidence for the relevance in vivo of this cruciform structure which, together with kissing-loop interaction, likely contribute to further stabilizing the branched AHVd secondary structure.

A new emaravirus discovered in Pistacia from Turkey.

High throughput sequencing was performed on total pooled RNA from six Turkish trees of Pistacia showing different viral symptoms. The analysis produced some contigs showing similarity with RNAs of emaraviruses. Seven distinct negative-sense, single-stranded RNAs were identified as belonging to a new putative virus infecting pistachio. The amino acid sequence identity compared to homologs in the genus Emaravirus ranged from 71% for the replicase gene on RNA1, to 36% for the putative RNA7 gene product. All the RNA molecules were verified in a pistachio plant by RT-PCR and conventional sequencing. Although the analysed plants showed a range of symptoms, it was not possible to univocally associate the virus with a peculiar one. The possible virus transmission by mite vector needs to be demonstrated by a survey, to observe spread and potential effect on yield in the growing areas of the crop.

Pest categorisation of non-EU viruses of Ribes L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses of Ribes L. determined as being either non-EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. Alaska vitivirus 1 and Ribes virus F were excluded from categorisation because these are very poorly characterised viruses. The pest categorisation was completed for seven viruses with clear identity and for which detection methods are available. All these viruses are efficiently transmitted by vegetative propagation techniques, with plants for planting representing the major pathway for long-distance dispersal and thus considered as the major pathway for entry. Depending on the virus, additional pathway(s) can also be Ribes seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but tomato ringspot virus (ToRSV) has a wide host range, thus extending the possible entry pathways. ToRSV meets all the criteria evaluated by EFSA to qualify as potential Union quarantine pest (QP). With the exception of impact in the EU territory, on which the Panel was unable to conclude, Actinidia virus X, blackcurrant leaf chlorosis-associated virus, blackcurrant leafroll-associated virus, black currant-associated rhabdovirus, blackcurrant waikavirus A and Ribes americanum virus A satisfy all the other criteria to be considered as potential Union QPs. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically the non-EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non-quarantine pests.

List of non-EU viruses and viroids of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L.

The Panel on Plant Health performed a listing of non-EU viruses and viroids (reported hereinafter as viruses) of Cydonia Mill., Fragaria L., Malus Mill., Prunus L., Pyrus L., Ribes L., Rubus L. and Vitis L. A systematic literature review identified 197 viruses infecting one or more of the host genera under consideration. Viruses were allocated into three categories (i) 86 non-EU viruses, known to occur only outside the EU or having only limited presence in the EU (i.e. reported in only one or few Member States (MSs), known to have restricted distribution, outbreaks), (ii) 97 viruses excluded at this stage from further categorisation efforts because they have significant presence in the EU (i.e. only reported so far from the EU or known to occur or be widespread in some MSs or frequently reported in the EU), (iii) 14 viruses with undetermined standing for which available information did not readily allow to allocate to one or the other of the two above groups. Comments provided by MSs during consultation phases were integrated in the opinion. The main knowledge gaps and uncertainties of this listing concern (i) the geographic distribution and prevalence of the viruses analysed, in particular when they were recently described; (ii) the taxonomy and biological status of a number of poorly characterised viruses; (iii) the host status of particular plant genera in relation to some viruses. The viruses considered as non-EU and those with undetermined standing will be categorised in the next steps to answer a specific mandate from the Commission to develop pest categorisations for non-EU viruses. This list does not imply a prejudice on future needs for a pest categorisation for other viruses which are excluded from the current categorisation efforts.

Pest categorisation of non-EU viruses and viroids of Cydonia Mill., Malus Mill. and Pyrus L.

Following a request from the EU Commission, the Panel on Plant Health performed a pest categorisation of 17 viruses and viroids, herein called viruses, of Cydonia Mill., Malus Mill. and Pyrus L. determined as being either non-EU or of undetermined standing in a previous EFSA opinion. These viruses belong to different genera and are heterogeneous in their biology. They can be detected by available methods and are efficiently transmitted by vegetative propagation techniques, with plants for planting representing a major long-distance spread mechanism and, potentially, a major entry pathway. Depending on the viruses, additional pathway(s) can also be seed, pollen and/or vector transmission. Most of the viruses categorised here are known to infect only one of few related plant genera, but some of them have a wider host range, thus extending the possible entry pathways. Three viruses (apple necrotic mosaic virus, cherry rasp leaf virus, temperate fruit decay-associated virus) and one viroid (apple fruit crinkle viroid) satisfy all the criteria to be considered as Union quarantine pests. Five viruses (apple green crinkle-associated virus, blackberry chlorotic ringspot virus, eggplant mottled crinkle virus, tobacco ringspot virus and tomato ringspot virus) and one viroid (apple scar skin viroid), satisfy the criteria to be considered as Union quarantine pests with the possible exception of being absent from the EU territory or having a restricted presence and being under official control. The remaining six viruses (apple geminivirus, apple latent spherical virus, apple-associated luteovirus, Pyrus pyrifolia cryptic virus, Pyrus pyrifolia partitivirus 2 and Tulare apple mosaic virus) and one viroid (apple hammerhead viroid) were not found to satisfy one or more of these criteria. The Panel highlights that for several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly linked to the absence of data on biology and distribution. Since this opinion addresses specifically the non-EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as a potential Union regulated non-quarantine pests.

Pest categorisation of non-EU viruses and viroids of Vitis L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Vitis L. determined as being either non-EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of grapevine virus 101-14.N.23.9.1/South Africa/2009 for which very limited information exists, the pest categorisation was completed for 30 viruses or viroids having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative propagation techniques, with plants for planting representing the major pathway for long-distance dispersal and thus considered as the major pathway for potential entry. Depending on the virus, additional pathway(s) can also be seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Grapevine yellow speckle viroid 2, blueberry leaf mottle virus, grapevine Ajinashika virus, grapevine Anatolian ringspot virus, grapevine berry inner necrosis virus, grapevine deformation virus, grapevine fabavirus, grapevine red blotch virus, grapevine stunt virus, grapevine Tunisian ringspot virus, grapevine vein-clearing virus, temperate fruit decay-associated virus, peach rosette mosaic virus, tobacco ringspot virus, tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). With the exception of impact for the EU territory, on which the Panel was unable to conclude, blackberry virus S, grapevine geminivirus A, grapevine leafroll-associated virus 7, grapevine leafroll-associated virus 13, grapevine satellite virus, grapevine virus E, grapevine virus I, grapevine virus J, grapevine virus S, summer grape enamovirus, summer grape latent virus satisfy all the other criteria to be considered as potential Union QPs. Australian grapevine viroid, grapevine cryptic virus 1, grapevine endophyte endornavirus and wild vitis virus 1 do not meet all the criteria evaluated by EFSA to be regarded as potential Union QPs because they are not known to cause an impact on Vitis. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically non-EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as a potential Union regulated non-quarantine pests.

Pest categorisation of non-EU viruses and viroids of Prunus L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Prunus L. determined as being either non-EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of Ilarvirus S1 and Ilarvirus S2, for which very limited information exists, the pest categorisation was completed for 26 viruses and 1 viroid having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative plant propagation techniques, with plants for planting representing the major pathway for long-distance dispersal and thus considered as the major pathway for entry. Depending on the virus, additional pathway(s) can also be Prunus seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Apple scar skin viroid, American plum line pattern virus, cherry mottle leaf virus, cherry rasp leaf virus, cherry rosette virus, cherry rusty mottle-associated virus, cherry twisted leaf-associated virus, peach enation virus, peach mosaic virus, peach rosette mosaic virus, tobacco ringspot virus and tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). With the exception of impact in the EU territory, on which the Panel was unable to conclude, apricot vein clearing virus, Asian prunus virus 1, Asian prunus virus 2, Asian prunus virus 3, Caucasus prunus virus, cherry virus B, Mume virus A, nectarine stem pitting-associated virus, nectarine virus M, peach chlorotic mottle virus, peach leaf pitting-associated virus, peach virus D, prunus virus F and prunus virus T satisfy all the other criteria to be considered as potential Union QPs. Prunus geminivirus A does not meet the criterion of having negative impact in the EU. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically the non-EU viruses, in general these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non-quarantine pests.

Pest categorisation of non-EU viruses of Fragaria L.

Following a request from the EU Commission, the Panel on Plant Health addressed the pest categorisation of the viruses and viroids of Fragaria L. determined as being either non-EU or of undetermined standing in a previous EFSA opinion. These infectious agents belong to different genera and are heterogeneous in their biology. With the exclusion of strawberry latent virus and strawberry latent C virus for which very limited information exists, the pest categorisation was completed for 12 viruses having acknowledged identities and available detection methods. All these viruses are efficiently transmitted by vegetative propagation techniques, with plants for planting representing the major pathway for long-distance dispersal and thus considered as the major pathway for entry. Depending on the virus, additional pathway(s) can also be Fragaria seeds, pollen and/or vector(s). Most of the viruses categorised here are known to infect only one or few plant genera, but some of them have a wide host range, thus extending the possible entry pathways. Strawberry chlorotic fleck-associated virus, strawberry leaf curl virus, strawberry necrotic shock virus, strawberry pallidosis-associated virus, strawberry vein banding virus (SVBV) and tomato ringspot virus meet all the criteria evaluated by EFSA to qualify as potential Union quarantine pests (QPs). For SVBV, the Panel considered that following its entry and establishment into the EU territory, an impact of uncertain magnitude is expected mainly because a synergistic effect may occur in strawberry in case of mixed infections with viruses already present in the EU . Strawberry crinivirus 3, strawberry crinivirus 4 and strawberry polerovirus 1 meet all criteria for being considered as potential Union QPs, except for the impact in the EU territory, on which the Panel was unable to conclude. Fragaria chiloensis cryptic virus, Fragaria chiloensis latent virus and strawberry pseudo mild yellow edge virus do not meet the criterion of having potential negative impact in the EU. For several viruses, especially those recently discovered, the categorisation is associated with high uncertainties mainly because of the absence of data on their biology, distribution and impact. Since this opinion addresses specifically the non-EU viruses, in general, these viruses do not meet the criteria assessed by EFSA to qualify as potential Union regulated non-quarantine pests.

Recent Advances on Detection and Characterization of Fruit Tree Viruses Using High-Throughput Sequencing Technologies.

Perennial crops, such as fruit trees, are infected by many viruses, which are transmitted through vegetative propagation and grafting of infected plant material. Some of these pathogens cause severe crop losses and often reduce the productive life of the orchards. Detection and characterization of these agents in fruit trees is challenging, however, during the last years, the wide application of high-throughput sequencing (HTS) technologies has significantly facilitated this task. In this review, we present recent advances in the discovery, detection, and characterization of fruit tree viruses and virus-like agents accomplished by HTS approaches. A high number of new viruses have been described in the last 5 years, some of them exhibiting novel genomic features that have led to the proposal of the creation of new genera, and the revision of the current virus taxonomy status. Interestingly, several of the newly identified viruses belong to virus genera previously unknown to infect fruit tree species (e.g., Fabavirus, Luteovirus) a fact that challenges our perspective of plant viruses in general. Finally, applied methodologies, including the use of different molecules as templates, as well as advantages and disadvantages and future directions of HTS in fruit tree virology are discussed.