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Little cherry virus 2 (LChV-2, genus Ampelovirus) is considered to be the main causal agent of the economically damaging little cherry disease, which can only be controlled by removal of infected trees. The widespread viral disease of sweet cherry (Prunus avium L.) is affecting the survival of long-standing orchards in North America and Europe, hence the dire need for an early and accurate diagnosis to establish a sound disease control strategy. The endemic presence of LChV-2 is mainly confirmed using laborious time-consuming reverse-transcription (RT-PCR). A rapid reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting a conserved region of the coat protein was developed and compared with conventional RT-PCR for the specific detection of LChV-2. This affordable assay, combined with a simple RNA extraction, deploys desirable characteristics such as higher ability for faster (

The roles of proteins encoded by members of the genus Ampelovirus, family Closteroviridae are largely inferred by sequence homology or analogy to similarly located ORFs in related viruses. This study employed yeast two-hybrid and bimolecular fluorescence complementation assays to investigate interactions between proteins of grapevine leafroll-associated virus 3 (GLRaV-3). The p5 movement protein, HSP70 homolog, coat protein, and p20B of GLRaV-3 were all found to self-interact, however, the mechanism by which p5 interacts remains unknown due to the absence of a cysteine residue crucial for the dimerisation of the closterovirus homolog of this protein. Although HSP70h forms part of the virion head of closteroviruses, in GLRaV-3, it interacts with the coat protein that makes up the body of the virion. Silencing suppressor p20B has been shown to interact with HSP70h, as well as the major coat protein and the minor coat protein. The results of this study suggest that the virion assembly of a member of the genus Ampelovirus occurs in a similar but not identical manner to those of other genera in the family Closteroviridae. Identification of interactions of p20B with virus structural proteins provides an avenue for future research to explore the mechanisms behind the suppression of host silencing and suggests possible involvement in other aspects of the viral replication cycle.

To identify the viruses in tree peony plants associated with the symptoms of yellowing, leaf rolling, stunted growth, and decline, high-throughput sequencing of small RNA and mRNA was conducted from a single symptomatic plant. Bioinformatic analyses and reconstruction of viral genomes indicated mixed viral infections involving cycas necrotic stunt virus (CNSV), apple stem grooving virus (ASGV), lychnis mottle virus (LycMoV), grapevine line pattern virus (GLPV), and three new viruses designated as peony yellowing-associated citrivirus (PYaCV, Citrivirus in Betaflexiviridae), peony betaflexivirus 1 (PeV1, unclassified in Betaflexiviridae), and peony leafroll-associated virus (PLRaV, Ampelovirus in Closteroviridae). PYaCV was 8,666 nucleaotides (nt) in length, comprising three open reading frames (ORFs) and shared 63.8-75.9% nucleotide sequence identity with citrus leaf blotch virus (CLBV) isolates. However, the ORF encoding the replication-associated protein (REP) shared 57% and 52% sequence identities at the nt and amino acid (aa) level, respectively, with other reported CLBV isolates, which were below the criterion for species classification within the family Betaflexiviridae. Recombination analysis identified putative recombination sites in PYaCV, which originated from CLBV. PeV1, only identified from the transcriptome data, was 8,124 nt in length with five ORFs encoding the REP (ORF1), triple gene block (TGB, ORF2-4) and coat protein (CP, ORF5) proteins. Phylogenetic analysis and sequence comparison showed that PeV1 clustered with an unassigned member, the garlic yellow mosaic-associated virus (GYMaV) within the Betaflexiviridae family, into a separate clade. Partial genome sequence analysis of PLRaV (12,545 nt) showed it contained seven ORFs encoding the partial polyprotein 1a, the RNA-dependent RNA polymerase (RdRp), two small hydrophobic proteins p11 and p6, HSP70h, p55, and a CP duplicate, which shared low aa sequence identity with Closteroviridae family members. Phylogenetic analysis based on the aa sequences of RdRp or HSP70h indicated that PLRaV clustered with grapevine leafroll-associated virus 1 (GLRaV-1) and GLRaV-13 in the Ampelovirus genus. Field investigation confirmed the wide distribution of these viruses, causing mixed infections of peony plants in Beijing.

International audience; The epidemiology of yam viruses remains largely unexplored. We present a large-scale epidemiological study of yam viruses in Guadeloupe based on the analysis of 1124 leaf samples collected from yams and weeds. We addressed the prevalence of cucumber mosaic virus (CMV), Cordyline virus 1 (CoV1), Dioscorea mosaic associated virus (DMaV), yam asymptomatic virus 1 (YaV1), yam mosaic virus (YMV), yam mild mosaic virus (YMMV), badnaviruses, macluraviruses and potexviruses, and the key epidemiological drivers of these viruses. We provide evidence that several weeds are reservoirs of YMMV and that YMMV isolates infecting weeds cluster together with those infecting yams, pointing to the role of weeds in the epidemiology of YMMV. We report the occurrence of yam chlorotic necrosis virus (YCNV) in Guadeloupe, the introduction of YMMV isolates through the importation of yam tubers, and the absence of vertical transmission of YaV1. We identified specific effects on some cropping practices, such as weed management and the use of chemical pesticides, on the occurrence of a few viruses, but no crop-related factor had a strong or general effect on the overall epidemiology of the targeted viruses. Overall, our work provides insights into the epidemiology of yam viruses that will help design more efficient control strategies.

The genome sequence of a closterovirus (genus Closterovirus, family Closteroviridae), tentatively named Thesium chinense closterovirus 1 (TcCV1), was identified by performing high-throughput RNA-sequencing of the haustoria and root tissues of Thesium chinense, a parasitic plant. The TcCV1 genome was predicted to encode nine proteins, eight of which have orthologs in previously identified closteroviruses. The TcCV1 RNA-dependent RNA polymerase (RdRp) and heat shock protein 70 homolog (Hsp70h) showed 27.8-68.2% and 23.8-55.1% amino acid identity, respectively, to orthologous proteins of known closteroviruses. The putative +1 ribosomal frameshifting site required for producing RdRp was identified as GUUUAGC with UAG stop codon and the skipped nucleotide U. Phylogenetic trees based on RdRp and Hsp70h show that TcCV1 is a novel member of the genus Closterovirus, forming a subclade with a group of known closteroviruses, including mint virus 1 and carnation necrotic fleck virus. The genome sequence of TcCV1 may be useful for studying the genome evolution of closteroviruses. Keywords: Thesium chinense closterovirus 1; Closterovirus; Closteroviridae; Thesium chinense.

Citrus aurantifolia (acid lime) trees exhibiting yellowing, stem pitting and decline symptoms in Oman were found to be infected by Citrus tristeza virus (CTV) (genus; Closterovirus, family; Closteroviridae). The sequence of the 3′ approximately 9.8 Kb genome, encompassing the partial RNA dependent RNA polymerase to P23 genes including the 3′ UTR region was determined. Pairwise nucleotide comparisons revealed that the Oman isolate had the highest nucleotide identity with those from Mexico and Egyptian isolates. Phylogenetic analysis showed that the Oman isolate clustered with the Egyptian isolate and was identified as T36 genotype. Recombination analysis showed the presence of three potential recombination events and the findings of this study are discussed. Further, extensive studies on CTV species mainly in Oman and as a whole in the Arabian Peninsula is needed, especially after investigating the recombinant 3′ CTV genome isolate.

Grapevine leafroll disease (GLD) constrains wine production worldwide. In New Zealand, the main causal agent of GLD is grapevine leafroll-associated virus 3 (GLRaV-3). To control GLD, an integrated management program is used and includes removing (roguing) GLRaV-3-infected vines from the vineyard. The classical foliar symptoms from virus-infected red-berry cultivars are leaves with dark red intervein, green veins, and downward rolling of margins. Growers use these phenotypic cues to undertake visual symptom identification (VSI) for GLD. However, the influence of the known large genetic variation among GLRaV-3 isolates on the foliar symptoms from different grapevine cultivars remains undescribed, especially in cool-climate growing environments, such as New Zealand. Over three vintages (2015, 2016, and 2017), VSI for GLD was undertaken at three field sites in New Zealand (Auckland, Hawke’s Bay, and Marlborough), each including four cultivars (Merlot, Pinot noir, Sauvignon blanc, and Pinot gris) infected with three GLRaV-3 genotypes (Groups I, VI, and X) or GLRaV-3-uninfected control plants. Throughout this study, no visual symptoms were observed on white-berry cultivars infected with GLRaV-3. For red-berry cultivars, the greatest variability in observed foliar symptoms among regional study sites, cultivars, and GLRaV-3 genotypes was observed early in the growing season. In particular, Group X had significantly delayed symptom expression across all three sites compared with Groups I and VI. As the newly infected, young vines matured in years 2 and 3, the GLRaV-3 genotype, cultivar, region, and environmental conditions had minimal influence on the accuracy of VSI, with consistently high (>95%) within-vintage identification by the end of each vintage. The results from this study strongly support the use of VSI for the GLD management of red-berry cultivar grapevines, Merlot and Pinot noir, as a reliable and cost-effective tool against GLD.

Grapevine-infecting ampelo- and vitiviruses are transmitted by several scale insect species, including the Bohemian mealybug

The whiteflies of the Bemisia tabaci (Genn.) complex (Hemiptera: Aleyrodidae) are serious agricultural pests that cause severe losses to vegetable, ornamental and fiber crops, including potato plants, mainly as a vector of economically important viruses. Among the most important viruses affecting potato is tomato chlorosis virus (ToCV) (Closteroviridae: Crinivirus), which is semi-persistently transmitted by whiteflies of the genera Bemisia and Trialeurodes. Chemical control is the main method used to manage B. tabaci and ToCV; however, this whitefly is resistant to most commercially available insecticides, and some products may not effectively prevent the vector stylet activities associated with virus transmission. The effective management of B. tabaci is crucial to reduce the spread of vector-borne diseases, and to minimize economic losses. We evaluated the effects of the foliar spraying with the systemic insecticides acetamiprid, flupyradifurone and cyantraniliprole on the probing behavior of non-viruliferous and ToCV-viruliferous B. tabaci MEAM1 and ToCV transmission in potato plants. To evaluate ToCV transmission in greenhouse conditions, viruliferous whiteflies were released on potato plants at different time points (3, 24 and 72 h and 7 days) after insecticide spraying. The EPG assay showed that at 3 h after insecticide application, the probing behavior differed, depending mainly on the state of the insects (viruliferous or not), whereas 72 h after application, the probing behavior differed only on plants treated with acetamiprid and flupyradifurone, for both viruliferous and non-viruliferous whiteflies. ToCV transmission was reduced mainly in plants treated with flupyradifurone and acetamiprid, likely as a result of phloem activity disruption.

Background: the most widely distributed and virulent Grapevine leafroll-associated viruses (GLRaV) that affect grapevine are GLRaV-1 and GLRaV-3, transmitted semi-persistently by different mealybugs and soft scales, mainly causing downward rolling of the leaf margins and interveinal reddening. Methods: the main objectives of this study were to investigate the genetic structure and molecular diversity of GLRaV-1 and GLRaV-3 in 617 samples from 11 autochthonous Sicilian grapevine cultivars, ascertaining their presence and spread. The detection was implemented by serological and molecular analyses and subsequently phylogenetic analyses on selected Sicilian isolates were conducted. Results: in total, 33 and 138 samples resulted positive to GLRaV-1 and GLRaV-3, with an incidence of 5.34% and 22.36%, respectively; 9 out of the 11 cultivars resulted positive, while the presence of both viruses was not found in ‘Grillo’ and ‘Moscato’ cultivars. Conclusions: phylogenetic analyses of the coat protein (CP) gene of 12 GLRaV-1 selected sequences showed a close relationship with European isolates; the discrete nucleotide differentiation and positive selection could demonstrate a current increase in population fitness. The phylogenetic analyses of the CP gene of 31 GLRaV-3 Sicilian CP sequences demonstrates a close relationship between Sicilian and different countries isolates; a certain stability of GLRaV-3 in the different cultivars analyzed is suggested by the discrete differentiation nucleotide and negative selection of the Sicilian isolates.

ObjectiveGrapevine (Vitisspp.) viral infections, including those byGrapevine leafroll-associated virus 3(GLRaV-3) andGrapevine red blotch virus(GRBV), greatly reduce fruit yields and quality. Evidence exists that host chemistry shifts result in reductions in fruit quality. However, changes over the season in foliar chemistry has not been well examined. Therefore, phenolic and amino acid levels were examined in leaves collected in grapevines with different rootstocks that were healthy or were infected with GLRaV-3 or GRBV. This was part of an effort to assess changes that different pathogens cause in grapevine tissues.ResultsMonth and year appeared to account for the greatest variability in grapevine foliar phenolic or amino acid levels, followed by differences in rootstock, and then differences in infection status. GLRaV-3 infection significantly lowered levels of total and individual hydroxycinnamic acid derivatives, and GRBV lowered total phenolic levels, total and individual hydroxycinnamic acids. Amino acid levels were increased over controls in vines infected by GLRaV-3, but not with GRBV. Overall, changes within grapevine leaves due to viral infection were likely too small to overcome variability due to sampling time or rootstock cultivar, and therefore such factors should be considered in determining infection effects on plant foliar chemistry.

Yellow leaf disease (YLD) is an economically important disease affecting betel palm in several countries, the cause of which remains unclear despite associations with putative agents, including phytoplasmas. In this study, we screened the potential casual agents associated with YLD in Hainan, China using next-generation sequencing and revealed the association of areca palm velarivirus 1 (APV1) with the YLD-affected palm. The complete genome of the APV1-WNY isolate was determined to be 17,546 nucleotides in length, approximately 1.5 kb longer than the previously reported APV1_HN genome. Transmission electron microscopy showed that APV1 particles are flexuous and filamentous, a typical morphology of species in the Closteroviridae family. Comparison of symptomatic and symptomless tree populations showed a strong association between APV1 and YLD. APV1 was detected in Pseudococcus sp. mealybugs sampled from YLD-affected trees in many locations, suggesting that mealybugs are a potential transmission vector for APV1. Although further studies are needed to confirm a causal relationship, these results provide timely information for the prevention and management of YLD associated with APV1.

Grapevine leafroll disease (GLD) is a virus disease present in all grapevine-growing regions of the world. Mealybugs and scale insects have been reported as vectors of some grapevine leafroll-associated viruses belongs to the ampeloviruses (family Closteroviridae ) in particular with grapevine leafroll-associated virus-1 (GLRaV-1) and GLRaV-3. Both grape ( Pseudococcus maritimus ) and Gill’s mealybugs are commonly present in Virginia vineyards, but we have limited information on acquisition and transmission of GLRaV-3 by Gill's mealybug ( Ferrisia Gilli ). We conducted acquisition and transmission assays in the greenhouse to examine the threshold for shorter acquisition time of GLRaV-3 with F. gilli . Approximately 67% and 58% rates of GLRaV-3 acquisition by F. gilli following 24 and 48 hours, respectively, of feeding period were documented. F. gilli first instars fed on a GLRaV-3-positive grapevine for 24 and 48 hours successfully transmitted GLRaV-3 to healthy grapevines after 24 hours of feeding/transmission time. The quick acquisition demonstrated in this study could be one of the factors that promoted the rapid expansion of GLRaV-3-infected vines in vineyards documented in previous studies.

The Pineapple mealybug wilt-associated virus (PMWaV) provokes a disease that causes considerable losses to pineapple growers due to the reduced leaf tissue turgescence, resulting in leaf necrosis and death in severe cases. In this work, we describe the comparison of shoot tip culture and cryotherapy methods to eradicate the pineapple wilt disease-associated ampeloviruses. Plants from the accessions Ananas comosus var. comosus (AGB-009), var. bracteatus (AGB-119), and var. parguazensis (AGB-376) were indexed by RT-PCR, confirming mixed infections of PMWaV-1, PMWaV-2, and PMWaV-3. The accessions were cultured in vitro and their shoot tips treated by cryotherapy following a droplet vitrification protocol. The regeneration rate from shoot tip culture was 93% for AGB-376 and 100% for the other two accessions. After freezing, AGB-376 had 100% regeneration subsequent to exposure to PVS2 for 45 min, followed by 95% for AGB-009, while for AGB-119 the optimal exposure time was 60 min, with plant regeneration from nearly 80% of the shoot tips. For the accessions AGB-009 and AGB-376, all the recovered plants were virus free by the two methods, while 50% of the plants from accession AGB-119 remained infected. These results indicate that shoot tip culture alone or in association with cryotherapy is a promising routine method for virus removal from pineapple plant tissues and is useful to ensure that backup reserves of pineapple germplasm, conserved by in vitro bank and cryobank, are formed with virus-free plants.

Plants usually suffer from parasitic and non parasitic diseases in the world. One of the major groups that cause infections in plants belong to viruses, which is obligate parasite and replicate only in living cells. They are generally insect or seed transmitted and changes associated with global warming may affect both their incidence and severity. New virus diseases that are threatening the world’s agricultural production are called “emerging diseases” which are commonly detected on different crop plants. Recent emerging viruses have RNA or DNA genome and belong to Potyviridae, Bunyaviridae, Geminiviridae, Closteroviridae, Betaflexiviridae and Secoviridae families. Plant virus diseases, like diseases caused by other pathogens, appear to be proliferating at ever increasing rates. Scientific and popular media abound with terms such as new, emerging, re-emerging and threatening plant diseases. In the present paper, the emerging viral diseases reported in cotton were discussed in detail.

Eighty-six viruses have been isolated to date from grapevines worldwide. Some of these viruses are associated with economically damaging diseases such as leaf mottling and deformation, vein clearing, leafroll, degeneration and red blotch. They belong to the families Betaflexiviridae, Caulimoviridae, Closteroviridae, Secoviridae and Geminiviridae, and are transmitted by diverse vectors such as mealybugs (Hemiptera: Pseudococcidae), soft scale insects (Hemiptera: Coccidae), an aphid (Hemiptera: Aphididae), dagger nematodes (Nematoda: Longidoridae), a treehopper (Hemiptera: Membracidae) and eriophyid mites (Acari: Eriophyidae). Management of these viruses primarily relies on preventive measures to limit their presence in the propagation and planting material. In the vineyard, specific disease scenario-based strategies such as rogueing in combination with agrochemical applications to limit vector populations, if appropriate, and the removal of entire parcels and their replacement with clean planting material, including vector tolerant rootstocks, if opportune, are implemented to reduce their incidence, prevent their spread and mitigate their impact. These solutions are simple but their implementation is often suboptimal and their adoption is largely low. Some of the uncertainties that hinder their endorsement are captured here, and options to refine them and to enhance their adoption are discussed.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses of grapevine but, despite this, there remain several gaps in our understanding of its biology. Because of its narrow host range - limited to Vitis species - and because the virus is restricted to the phloem, most GLRaV-3 research has concentrated on epidemiology and the development of detection assays. The recent discovery that GLRaV-3 can infect Nicotiana benthamiana, a plant model organism, makes new opportunities available for research in this field. We used RNA-seq to compare both V. vinifera and P1/HC-Pro N. benthamiana host responses to GLRaV-3 infection. Our analysis revealed that the majority of DEGs observed between the two hosts were unique although responses between the two hosts also showed several shared gene expression results. When comparing gene expression patterns that were shared between the two hosts, we observed the downregulation of genes associated with stress chaperones, and the induction of gene families involved in primary plant physiological processes. This is the first analysis of gene expression profiles beyond Vitis to mealybug-transmitted GLRaV-3 and demonstrates that N. benthamiana could serve as a useful tool for future studies of GLRaV-3-host interactions.

To investigate the occurrence of little cherry virus 1 (LChV-1), little cherry virus 2 (LChV-2), cherry green ring mottle virus (CGRMV), cherry necrotic rusty mottle virus (CNRMV), and cherry virus A (CVA) in stone fruit trees in Poland, leaf samples were collected from sweet and sour cherry, peach, and apricot trees. Two sets of primers were used to increase the effectiveness of virus detection. The RT-PCR results indicated that the most frequently detected virus in all of the tested samples was CVA (60%), followed by CGRMV (13%), CNRMV (12%), LChV-1 (11%), and LChV-2 (4%). CVA and CNRMV were not detected in peaches. Mixed infections of these viruses were frequently detected. Keywords: little- cherry virus 1; little cherry virus 2; cherry green ring mottle virus; cherry necrotic rusty mottle virus; cherry virus A; RT-PCR.

The

Cordyline virus 1 (CoV1) is a velarivirus that has so far only been reported in ornamental Ti plants (Cordyline fruticosa). Using high-throughput sequencing, we identified CoV1 infection in yam accessions from Vanuatu. Using a specific RT-PCR assay, we found that CoV1 is also present and highly prevalent in Dioscorea alata, D. cayenensis, and D. trifida in Guadeloupe. Phylogenetic analysis showed that CoV1 isolates infecting yam in Guadeloupe display a low level of molecular diversity. These data provide insights into the transmission of CoV1 in yam in Guadeloupe.

In July 2019, 11 watermelon (Citrullus lanatus) plants (six ‘Augusta negra’ and five ‘Kasmira’) and seven zucchini (Cucurbita pepo) ‘Marcado’ plants showing yellowing and vein clearing on the leaves were collected in Arico on Tenerife Island of the Canary Islands. Analysis with enzyme-linked immunosorbent assay (ELISA) with polyclonal antibodies using kits from LOEWE Biochemica GmbH (Sauerlach, Germany) detected cucurbit chlorotic yellows virus (CCYV) in four watermelon (two ‘Augusta negra’ and two ‘Kasmira’) plants and one zucchini plant.

Se ha descrito por primera vez la secuencia completa de 16700 nt del virus asociado al amarilleamiento de la hoja de olivo (OLYaV), un closterovirus que no estaba asignado a género y de cuyo genoma solo se conocían 4605 nt. El descubrimiento de la organización del genoma y las relaciones filogenéticas con todos los miembros de la familia Closteroviridae en las proteínas compartidas (ORF1a, ORF1b, HSP70h, HSP90h y CP) ha permitido la propuesta y creación del nuevo género Olivavirus compuesto por este virus y otras dos especies virales no asignadas a género, que comparten su proximidad filogenética en todas las proteínas, la codificación de una proteína de la familia de las taumatinas y la ausencia de la proteína menor de la cápsida (CPm).

The 3’-most genes in RNA-2 of the Crinivirus genus members (family Closteroviridae) code for non-structural p26 proteins that share amino acid sequence similarity [Stewart LR, Hwang MS, Falk BW (2009) Virus Res 145:293-299]. In this study, sensitive bioinformatic tools have been used to identify the homologous p26 proteins encoded by the 3’ genes in monopartite genomes of the members of Velarivirus, another Closteroviridae genus, and mint vein banding-associated virus, an unassigned member of the family. The p26 proteins showed similarity in their predicted secondary structures, but an amino acid sequence alignment showed no strictly conserved positions, thus indicating a high plasticity of these non-structural proteins. The implications of the sequence analysis for possible functions of the crinivirus and velarivirus p26 proteins are discussed. Electronic supplementary material The online version of this article (10.1007/s00705-019-04491-8) contains supplementary material, which is available to authorized users.

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.

The complete genome sequence of peony leafroll-associated virus (PLRaV) was determined by deep sequencing of ribosomal-RNA-depleted total RNA extracted from a peony plant exhibiting leafroll symptoms. Further PCR and RACE analysis showed that the PLRaV genome consists of 15,406 nucleotides and contains 10 putative open reading frames, with an organization typical of members of the genus Ampelovirus, family Closteroviridae. Amino acid sequence comparisons showed that the viral heat shock protein 70 homolog (HSP70h) shared the highest sequence identity (41.7%) with the corresponding region of grapevine leafroll-associated virus 1, and the coat protein (CP) and RNA-dependent RNA polymerase (RdRp) shared the highest sequence identity (32.1% and 52.3%, respectively) with grapevine leafroll-associated virus 13. Phylogenetic analysis of the HSP70h, CP, and RdRp aa sequences showed that PLRaV clustered with members of subgroup I of the genus Ampelovirus.

The use of high throughput sequencing (HTS) for the analysis of Spanish olive trees showing leaf yellowing discoloration, defoliation, and/or decline has provided new insights into the olive viruses present in Spain and has opened discussions about the pros and cons of these technologies for diagnostic purposes. In this study, we report for the first time in Spanish orchards the presence of olive leaf yellowing-associated virus (OLYaV), for which the second full coding sequence has been determined. This virus has also been detected in a putative vector, the psyllid Euphyllura olivina. In addition, the presence in Spain of Olea europaea geminivirus (OEGV), recently reported in Italy, has been confirmed, and the full-length sequence of two isolates was obtained by HTS and Sanger sequencing. These results, as well as the detection of other viral sequences related to olive latent virus 3 (OLV-3) and olive viral satellite RNA, raises questions on the biological significance of the findings, about the requirement of standardization on the interpretation of HTS results, and the necessity of additional tests to confirm the relevance of the HTS detection of viral sequences.

Background Areca palm (Areca catechu L.) is an important commercial crop in southeast Asia, but its cultivation is threatened by yellowing leaf disease (YLD). Areca palm velarivirus 1 (APV1) was recently associated with YLD, but little is known regarding its population and genetic diversity. To assess the diversity of YLD, the APV1 genome was sequenced in YLD samples collected from different sites in Hainan. Results Twenty new and complete APV1 genomes were identified. The APV1 isolates had highly conserved sequences in seven open reading frames (ORFs; > 95% nucleotide [nt] identity) at the 3′ terminal, but there was diversity (81–87% nt identity) in three ORFs at the 5′ terminal. Phylogenetic analysis divided the APV1 isolates into three phylogroups, with 16 isolates (> 70%) in phylogroup A. Mixed infections with different genotypes in the same tree were identified; this was closely correlated with higher levels of genetic recombination. Conclusions Phylogroup A is the most prevalent APV1 genotype in areca palm plantations in Hainan, China. Mixed infection with different genotypes can lead to genomic recombination of APV1. Our data provide a foundation for accurate diagnostics, characterization of etiology, and elucidation of the evolutionary relationships of APV1 populations.

The cottony grape scale Pulvinaria vitis is a scale insect colonizing grapevine; however, its capacity as a vector of grapevine viruses is poorly known in comparison to other scale species that are vectors of viral species in the genera Ampelovirus and Vitivirus. The ability of P. vitis to transmit the ampeloviruses Grapevine leafroll-associated viruses [GLRaV]−1, −3, and −4, and the vitivirus Grapevine virus A (GVA), to healthy vine cuttings was assessed. The scale insects used originated from commercial vine plots located in Alsace, Eastern France. When nymphs sampled from leafroll-infected vineyard plants were transferred onto healthy cuttings, only one event of transmission was obtained. However, when laboratory-reared, non-viruliferous nymphs were allowed to acquire viruses under controlled conditions, both first and second instar nymphs derived from two vineyards were able to transmit GLRaV−1 and GVA. This is the first report of GLRaV−1 and GVA transmission from grapevine to grapevine by this species.

Little cherry virus 2 (LChV-2, genus

Yellow leaf disease (YLD) is the most destructive disease of betel palm (

Sisal is a common name for different plant varieties in the genus Agave (especially Agave sisalana) used for high-quality natural leaf fiber extraction. Despite the economic value of these plants, we still lack information about the diversity of viruses (virome) in non-tequilana species from the genus Agave. In this work, by associating RNA and DNA deep sequencing we were able to identify 25 putative viral species infecting A. sisalana, A. fourcroydes, and Agave hybrid 11648, including one strain of Cowpea Mild Mottle Virus (CPMMV) and 24 elements likely representing new viruses. Phylogenetic analysis indicated they belong to at least six viral families: Alphaflexiviridae, Betaflexiviridae, Botourmiaviridae, Closteroviridae, Partitiviridae, Virgaviridae, and three distinct unclassified groups. We observed higher viral taxa richness in roots when compared to leaves and stems. Furthermore, leaves and stems are very similar diversity-wise, with a lower number of taxa and dominance of a single viral species. Finally, approximately 50% of the identified viruses were found in all Agave organs investigated, which suggests that they likely produce a systemic infection. This is the first metatranscriptomics study focused on viral identification in species from the genus Agave. Despite having analyzed symptomless individuals, we identified several viruses supposedly infecting Agave species, including organ-specific and systemic species. Surprisingly, some of these putative viruses are probably infecting microorganisms composing the plant microbiota. Altogether, our results reinforce the importance of unbiased strategies for the identification and monitoring of viruses in plant species, including those with asymptomatic phenotypes.

Grapevine leafroll‐associated virus (GLRaV) infections are accompanied by symptoms influenced by host genotype, rootstock, environment, and which individual or combination of GLRaVs is present. Using a dedicated experimental vineyard, we studied the responses to GLRaVs in ripening berries from Cabernet Franc grapevines grafted to different rootstocks and with zero, one, or pairs of leafroll infection(s). RNA sequencing data were mapped to a high‐quality Cabernet Franc genome reference assembled to carry out this study and integrated with hormone and metabolite abundance data. This study characterized conserved and condition‐dependent responses to GLRaV infection(s). Common responses to GLRaVs were reproduced in two consecutive years and occurred in plants grafted to different rootstocks in more than one infection condition. Though different infections were inconsistently distinguishable from one another, the effects of infections in plants grafted to different rootstocks were distinct at each developmental stage. Conserved responses included the modulation of genes related to pathogen detection, abscisic acid (ABA) signalling, phenylpropanoid biosynthesis, and cytoskeleton remodelling. ABA, ABA glucose ester, ABA and hormone signalling‐related gene expression, and the expression of genes in several transcription factor families differentiated the effects of GLRaVs in berries from Cabernet Franc grapevines grafted to different rootstocks. These results support that ABA participates in the shared responses to GLRaV infection and differentiates the responses observed in grapevines grafted to different rootstocks., GLRaVs elicit changes in gene expression, ripening‐related metabolites, and hormone metabolism in berries; one set of responses is observed across infections and host genotypes while other responses differ based on rootstock. ​

Grapevine leafroll disease (GLD) is one of the most economically important viral diseases of grapevines. GLD is caused by a complex of several ssRNA (+) viruses referred to as Grapevine leafroll-associated viruses (GLRaVs). To date, five different GLRaV species have been identified. One of those species, GLRaV-7, was first reported from a symptomless white-fruited wine grape cultivar from Albania. Since its discovery, GLRaV-7 has been reported from 14 countries. Although serological assays have been developed to detect GLRaV-7, commercially available antibodies produce high background signals making them unsuitable for regulatory testing. Furthermore, while molecular detection assays have been shown to be more sensitive when compared to the serological assays, published molecular assays, except the one Reverse Transcription-quantitaive Polymerase Chain Reaction (RT-qPCR) assay based on heat shock protein 70 homologue (HSP70h) gene, have been reported to be inadequate in detecting all reported isolates of GLRaV-7. Availability of multiple assays provides flexibility to diagnostic laboratories in cases where the chosen assay fails to detect a strain or an isolate of a pathogen due to variation in its targeted region or where additional confirmation of the results is required. In this study, we developed a sensitive and specific RT-qPCR assay, based on a region of p61 gene of GLRaV-7, which detected all available isolates.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source–sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development.

Tomato chlorosis virus (ToCV) is an emergent plant pathogen that causes a yellow leaf disorder in tomato and other solanaceous crops. ToCV is a positive-sense, single stranded (ss)RNA bipartite virus with long and flexuous virions belonging to the genus Crininivirus (family Closteroviridae). ToCV is phloem-limited, transmissible by whiteflies, and causes symptoms of interveinal chlorosis, bronzing, and necrosis in the lower leaves of tomato accompanied by a decline in vigor and reduction in fruit yield. The availability of infectious virus clones is a valuable tool for reverse genetic studies that has been long been hampered in the case of closterovirids due to their genome size and complexity. Here, attempts were made to improve the infectivity of the available agroinfectious cDNA ToCV clones (isolate AT80/99-IC from Spain) by adding the hepatitis delta virus (HDV) ribozyme fused to the 3′ end of both genome components, RNA1 and RNA2. The inclusion of the ribozyme generated a viral progeny with RNA1 3′ ends more similar to that present in the clone used for agroinoculation. Nevertheless, the obtained clones were not able to infect tomato plants by direct agroinoculation, like the original clones. However, the infectivity of the clones carrying the HDV ribozyme in Nicotiana benthamiana plants increased, on average, by two-fold compared with the previously available clones.

Little cherry disease (LCD) threatens the long-term economic sustainability of the Pacific Northwest sweet cherry (Prunus avium) industry. Results from a series of partial budget analyses indicate that additional investments in monitoring, testing, spraying to control for insect vectors, and removing infected trees are lower than the reduced profit losses compared with the do-nothing scenario. Also, management can prevent or lessen the negative impacts of higher little cherry virus (Velarivirus little cherry virus 1, Ampelovirus little cherry virus 2) spread rates. Our findings illustrate the importance of prevention, correct identification, and controlling for insect vectors in preventing the dissemination of LCD, for which the only known treatment is tree removal.

The decline of cultivated strawberry (Fragaria × ananassa Duchesne ex Rozier; Rosaceae) observed in the province of Quebec, Canada, between 2012 and 2014 was mostly caused by persistent viruses: strawberry mild yellow edge virus (SMYEV) (Potexvirus; Alphaflexiviridae) and strawberry crinkle virus (SCV) (Cytorhabdovirus; Rhabdoviridae); and semi-persistent viruses: strawberry mottle virus (SmoV) (Secoviridae), strawberry vein banding virus (SVBV) (Caulimovirus; Caulimoviridae), and strawberry pallidosis virus (SPaV) (Crinivirus: Closteroviridae) transmitted by insect vectors. The objective of this study was to determine the sources of viral contamination in commercial strawberry fields in Quebec. Specifically, we wished to 1) determine the prevalence of persistent viruses in winged strawberry aphid Chaetosiphon fragaefolii (Cockerell) (Hemiptera: Aphididae) specimens captured; 2) determine the prevalence of all viruses in wild strawberry Fragaria virginiana Miller plants near commercial plantings; and 3) evaluate the viral contamination of strawberry transplants obtained from nurseries and tested before and after planting in commercial strawberry fields. Results indicated high percentage (38%) of the aphids (n = 205) and high percentage (67%) of F. virginiana patches (n = 12) were infected by strawberry viruses. Ultimately, our results showed a low percentage (5%) of the plants from various nurseries (n = 56) were infected before planting, whereas a third (29%) of the healthy exposed plants in the fields (n = 96) became rapidly infected by insect vectors within a year of having been planted. This study provides significant insights on the relative importance of the various sources of contamination in Quebec strawberry fields: C. fragaefolii versus F. virginiana versus nurseries versus post-nursery infections through exposure to virus-carrying insects.

Tomato chlorosis virus (ToCV) causes an important disease that primarily affects tomato, although it has been found infecting other economically important vegetable crops and a wide range of wild plants. First described in Florida (USA) and associated with a 'yellow leaf disorder' in the mid-1990s, ToCV has been found in 35 countries and territories to date, constituting a paradigmatic example of an emergent plant pathogen. ToCV is transmitted semipersistently by whiteflies (Hemiptera: Aleyrodidae) belonging to the genera Bemisia and Trialeurodes. Whitefly transmission is highly efficient and cases of 100% infection are frequently observed in the field. To date, no resistant or tolerant tomato plants are commercially available and the control of the disease relies primarily on the control of the insect vector. Taxonomy Tomato chlorosis virus is one of the 14 accepted species in the genus Crinivirus, one of the four genera in the family Closteroviridae of plant viruses. Virion and genome properties The genome of ToCV is composed of two molecules of single-stranded positive-sense RNA, named RNA1 and RNA2, separately encapsidated in long, flexuous, rod-like virions. As has been shown for other closterovirids, ToCV virions are believed to have a bipolar structure. RNA1 contains four open reading frames (ORFs) encoding proteins associated with virus replication and suppression of gene silencing, whereas RNA2 contains nine ORFs encoding proteins putatively involved in encapsidation, cell-to-cell movement, gene silencing suppression and whitefly transmission. Host range In addition to tomato, ToCV has been found to infect 84 dicot plant species belonging to 25 botanical families, including economically important crops. Transmission Like all species within the genus Crinivirus, ToCV is semipersistently transmitted by whiteflies, being one of only two criniviruses transmitted by members of the genera Bemisia and Trialeurodes. Disease symptoms Tomato 'yellow leaf disorder' syndrome includes interveinal yellowing and thickening of leaves. Symptoms first develop on lower leaves and then advance towards the upper part of the plant. Bronzing and necrosis of the older leaves are accompanied by a decline in vigour and reduction in fruit yield. In other hosts the most common symptoms include interveinal chlorosis and mild yellowing on older leaves. Control Control of the disease caused by ToCV is based on the use of healthy seedlings for transplanting, limiting accessibility of alternate host plants that can serve as virus reservoirs and the spraying of insecticides for vector control. Although several wild tomato species have been shown to contain genotypes resistant to ToCV, there are no commercially available resistant or tolerant tomato varieties to date.

Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically significant virus of grapevines, with secondary spread mediated by several species of mealybug and soft scale insects. To better understand virus-vector interactions, sensitive virus detection in these insects is a key tool. In this research, two new hydrolysis-probe-based real-time assays for GLRaV-3 detection were developed and compared to three existing assays. Of the five assays compared, the one-step RT-qPCR probe-based assay was the most sensitive and reliable, with as few as 10 virus RNA copies detected. This is the first description of a real-time molecular assay for virus detection in mealybugs with such sensitivity.

Tristeza, caused by citrus tristeza virus (CTV; Closterovirus, Closteroviridae), is of significant economic importance. Tristeza epidemics have caused severe declines in productivity, and even death, of millions of citrus trees on sour orange rootstock in many regions all over the world. In the field, CTV is most efficiently vectored by the brown citrus aphid (Toxoptera citricida (Kirkaldy)) in a semipersistent manner. The transmission efficiency of the vector is influenced by its acquisition access period (AAP) for CTV. A real-time RT-PCR assay using SYBR Green fluorescent dye was used to estimate the CTV titers in groups of 15 aphids under AAPs after 0.5 to 48 h for three CTV isolates (CT11A, CT16-2, and CTLJ). Similar trends for CTV titer in viruliferous aphids were displayed for the three isolates. The maximum CTV titer was at AAP 6 h for isolates CT11A and CT16-2, and at 4 h for isolate CTLJ. During the AAPs from 0.5 to 6 h, the mean CTV titer of CT16-2 increased from 7.8 × 104 to 1.71 × 107 copies per 15 aphids, and was correlated with an increase in transmission rate from 20 to 90.9%. This suggests that the transmission efficiency is positively correlated with viral titer in the insect from 0.5 h until 6 h AAPs. While a downward trend in CTV titer was observed after a 6-h AAP, the transmission rate remained higher than 90% up to 48 h. These results indicate that factors other than the virus titer in the vector contribute to successful transmission under long acquisition conditions. This is the first detailed quantitative analysis of CTV in its main vector species following different AAPs and its association with transmission efficiency, and should enhance our understanding of T. citricida-CTV interactions.

Mealybug wilt of pineapple (MWP) disease has been associated with the infection by a complex of ampeloviruses (Pineapple mealybug wilt-associated virus 1 (PMWaV-1), PMWaV-2, PMWaV-3, PMWaV-4 and PMWaV-5)) (Closteroviridae) and badnaviruses (Pineapple bacilliform CO virus (PBCOV) and Pineapple bacilliform ER virus (PBERV) (Caulimoviridae)). In Cuba, PMWaV-1, PMWaV-2, PMWaV-3 and PBCOV are widespread in commercial pineapple fields, but the association of these viruses with the aetiology of the disease remains unknown. During 2009 to 2012, a survey to detect PMWaV-1, PMWaV-2 and PMWaV-3 by RT-PCR, and PBCOV by non-radioactive Dot Blot hybridization was carried out on samples collected from asymptomatic or MWP symptomatic pineapple plants from 24 commercial fields in 10 provinces and Isla de la Juventud. PMWaV-1, PMWaV-2, PMWaV-3, and PBCOV occurred in simple or mixed infections, whereas PMWaV-2 was the viral species most frequently detected in symptomatic plants. The positive or negative diagnostic results of the RT-PCR to detect PMWaV-2 was more reliably associated with the MWP symptomatic or asymptomatic status of the sampled plants, respectively, than those from PMWaV-1, PMWaV-3 or PBCOV detection techniques. This work discloses the correlation between the infection by PMWaV-2 and the presence of MWP symptoms in Cuba, supporting previous studies indicating the major role of this virus triggering MWP disease.

Little cherry virus 1 (LChV-1) belongs to the genus Velarivirus, family Closteroviridae, is an economically important pathogen affecting mainly cherry around the world emphasizing the impetus for its efficient and accurate on-site detection. This study describes the development of a reliable diagnostic protocol of LChV-1 based on a one-step reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The protocol detects LChV-1 isolates in less than 10 min by fluorescence monitoring using a mobile detection device and is most optimal when performed at 67 °C. Sharp melting curves and unique melting temperatures (Tm) were obtained for the positive samples. Both the RT-LAMP and classical RT-PCR methods are capable of specifically detecting LChV-1 in infected leaf tissues. In addition, the RT-LAMP has remarkable advantages in comparison to RT-PCR. It is at least hundred fold more sensitive, significantly faster (allowing on-field leaf-to-result diagnostic) and efficient at minimal cost. In conclusion, this innovative RT-LAMP approach can contribute to the implementation of sustainable integrated management strategies for detection of LChV-1 in commercial orchards or for horticultural research stations. It is also suitable for decision support in phytosanitary epidemiological programs.

Grapevine leafroll disease (GLD) is one of the most economically damaging virus diseases in grapevine, with grapevine leafroll-associated virus 1 (GLRaV-1) and grapevine leafroll-associated virus 3 (GLRaV-3) as the main contributors. This study complements a previously published transcriptomic analysis and compared the impact of two different forms of GLD to a symptomless control treatment: a mildly symptomatic form infected with GLRaV-1 and a severe form with exceptionally early leafroll symptoms (up to six weeks before veraison) infected with GLRaV-1 and GLRaV-3. Vine physiology and fruit composition in 17-year-old Pinot noir vines were measured and a gradient of vigor, yield, and berry quality (sugar content and berry weight) was observed between treatments. Virome composition, confirmed by individual RT-PCR, was compared with biological indexing. Three divergent viromes were recovered, containing between four to seven viruses and two viroids. They included the first detection of grapevine asteroid mosaic-associated virus in Switzerland. This virus did not cause obvious symptoms on the indicators used in biological indexing. Moreover, the presence of grapevine virus B (GVB) did not cause the expected corky bark symptoms on the indicators, thus underlining the important limitations of the biological indexing. Transmission of GLRaV-3 alone or in combination with GVB by Planococcus comstocki mealybug did not reproduce the strong symptoms observed on the donor plant infected with a severe form of GLD. This result raises questions about the contribution of each virus to the symptomatology of the plant.

Japanese apricot (Prunus mume Sieb. et Zucc.) is one of the most popular fruit tree species in Japan and is most densely cultivated in Wakayama Prefecture. Since the 1980s, graft-transmissible symptoms, such as leaf-edge necrosis and incomplete flower development, have been observed in Nanko, the major Japanese apricot cultivar in Wakayama. Two ampeloviruses, plum bark necrosis stem pitting-associated virus (PBNSPaV) and little cherry virus 2 (LChV-2) were detected in Nanko trees showing typical symptoms. However, there is currently insufficient epidemiological data to infer that these viruses cause the symptoms. In this study, we conducted a large-scale survey of symptoms and RT-PCR detection of the two viruses in midwestern districts, the major cultivation areas in Wakayama. Among 208 surveyed Nanko trees, two viral infections were significantly associated with incomplete flower development, low fruit bearing rate, and interveinal chlorosis. LChV-2 infection also affected Nanko fruit and stone sizes. Additionally, we showed that the two viruses were already widely distributed throughout the main cultivation area. These results imply that the two ampeloviruses cause overall yield loss in Japanese apricot in Wakayama Prefecture.

Vineyard surveys were conducted for three consecutive seasons in eastern Washington State, the major grapevine-growing region in the state, to document the occurrence of Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine red blotch virus (GRBV). The majority of samples were collected from red-berried wine grape (Vitis vinifera) cultivars exhibiting symptoms of or suspected for grapevine leafroll (GLD) and red blotch (GRBD) diseases. A limited number of samples from white-berried cultivars were collected randomly due to the lack of visual symptoms. Samples were collected from a total of 2,063 grapevines from 18 red-berried cultivars and seven white-berried cultivars planted in eight American Viticultural Areas and tested for GLRaV-3 and GRBV using RT-PCR and PCR, respectively. The results showed 67.77% and 6.01% of total samples positive for GLRaV-3 and GRBV, respectively, and 9.06% of samples positive for both viruses. About 17% of samples tested negative for the two viruses, but some of these samples were positive for GLRaV-2 and GLRaV-4. Overall results indicated that GLRaV-3 was more common than GRBV, independent of cultivars and the geographic origin of samples. Due to variability in symptoms in red-berried cultivars, virus-specific diagnostic assays were deemed necessary for reliable identification of GLRaV-3 and GRBV and to differentiate GLD and GRBD symptoms from those induced by biotic and abiotic stresses in vineyards. A multiplex PCR protocol was developed for simultaneous detection of GLRaV-3 and GRBV in grapevine samples. A global phylogenetic analysis of GRBV genome sequences revealed segregation of virus isolates from Washington State vineyards into two distinct clades, with the majority of isolates belonging to clade II.

Grapevine leafroll is one of the most widespread and highly destructive grapevine diseases that is responsible for great economic losses to the grape and wine industries throughout the world. Six distinct viruses have been implicated in this disease complex. They belong to three genera, all in the family Closteroviridae. For the sake of convenience, these viruses are named as grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4, -7, and -13). However, their etiological role in the disease has yet to be established. Furthermore, how infections with each GLRaV induce the characteristic disease symptoms remains unresolved. Here, we first provide a brief overview on each of these GLRaVs with a focus on genome structure, expression strategies and gene functions, where available. We then provide a review on the effects of GLRaV infection on the physiology, fruit quality, fruit chemical composition, and gene expression of grapevine based on the limited information so far reported in the literature. We outline key methodologies that have been used to study how GLRaV infections alter gene expression in the grapevine host at the transcriptomic level. Finally, we present a working model as an initial attempt to explain how infections with GLRaVs lead to the characteristic symptoms of grapevine leafroll disease: leaf discoloration and downward rolling. It is our hope that this review will serve as a starting point for grapevine virology and the related research community to tackle this vastly important and yet virtually uncharted territory in virus-host interactions involving woody and perennial fruit crops.

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

Nineteen samples from members of the plant genera Agapanthus, Clivia, Hippeastrum, and Scadoxus were collected from gardens in the Gauteng and Western Cape provinces of South Africa. The plants displayed highly variable symptoms of viral disease, including chlorosis, necrosis, streaking, and ringspot. RNAtag-seq was used to characterize the associated viral populations. Plants of the genus Agapanthus were found to be associated with three novel viruses from the families Caulimoviridae, Closteroviridae, and Betaflexiviridae; plants of the genus Clivia were associated with novel members of the families Potyviridae and Betaflexiviridae; and plants of the genus Scadoxus were associated with a novel member of the family Tospoviridae. Nerine latent virus was associated with plants of the genera Agapanthus, Clivia, and Hippeastrum, while hippeastrum mosaic virus was associated exclusively with a Hippeastrum cultivar.

Our knowledge of citrus viruses is largely skewed toward virus pathology in cultivated orchards. Little is known about the virus diversity in wild citrus species. Here, we used a metatranscriptomics approach to characterize the virus diversity in a wild citrus habitat within the proposed center of the origin of citrus plants. We discovered a total of 44 virus isolates that could be classified into species Citrus tristeza virus and putative species citrus associated ampelovirus 1, citrus associated ampelovirus 2, and citrus virus B within the family Closteroviridae, providing important information to explore the factors facilitating outbreaks of citrus viruses and the evolutionary history of the family Closteroviridae. We found that frequent horizontal gene transfer, gene duplication, and alteration of expression strategy have shaped the genome complexity and diversification of the family Closteroviridae. Recombination frequently occurred among distinct Closteroviridae members, thereby facilitating the evolution of Closteroviridae. Given the potential emergence of similar wild-citrus-originated novel viruses as pathogens, the need for surveillance of their pathogenic and epidemiological characteristics is of utmost priority for global citrus production., Author summary Closterovirids are principal plant pathogens for citrus trees and other plants, as they sometimes cause new or re-emerging diseases. However, the closterovirid diversity in natural plant hosts, especially citrus plants, is unclear. Here, we describe three novel species and Citrus tristeza virus within the family Closteroviridae that were sampled from wild citrus trees growing in their natural habitat in southwestern China. The presence of three different taxon classes of the family Closteroviridae indicates the geographical uniqueness of the sampling region for citrus closterovirid evolution. Our analysis shows that frequent horizontal gene transfer, gene duplication, alteration of expression strategy, and recombination have been important evolutionary processes in the diversification of the family Closteroviridae. Our study also shows the significance of natural reserves as potential sources of disease agents endangering cultivated crop plants.