Cherry Virus Research Articles

Diversity of the virome associated with alfalfa (Medicago sativa L.) in the U.S. Pacific Northwest

Alfalfa (Medicago sativa L.) is one of the most extensively cultivated forage legumes in the world. It is currently the third most valuable field crop in the United States with an estimated value of over $9.3 billion. Alfalfa productivity is limited by various infectious diseases that can reduce forage yield and quality and shorten stand life. The crop can frequently be infected with a diverse array of pathogens and other organisms that have distinct life cycles, biology, and mode of action. Among them are many coinfecting viruses, that greatly contribute to the heterogeneity of within-host pathogenic communities, representing a ubiquitous and abundant background for all other host–pathogen interactions. Regrettably, the impact of viral diseases, their role in alfalfa health and involvement in the severity of multi-pathogen infections are often underestimated and not well understood. As high-throughput sequencing approaches have been developed, opportunities to delve into these complex interactions can be realized. In this work, we have characterized a diversity of viral populations in several commercial alfalfa production fields located in the U.S. Pacific Northwest. At least 45 distinct viruses have been identified in all alfalfa samples. Among them some were known to infect the crop prior to this study, and others were designated as emerging, novel and viruses integrated into the alfalfa genome. Known viruses included alfalfa mosaic virus, pea streak virus and bean leafroll virus, while among emerging and novel agents were alfalfa virus S, cherry virus Trakiya, several rhabdoviruses and others. Additional biological and impact studies will be needed to determine if newly identified viruses, especially those that have not been reported from alfalfa before, should be considered pathogens of this crop.

Nearly Complete Genome Sequence of Cherry Virus A, Isolated from Prunus armeniaca in Jammu and Kashmir, India.

ABSTRACTA nearly complete genome sequence of cherry virus A (CVA), isolated from the Prunus armeniaca plant, is presented in this publication. The genome is 7,380 bases in length and is divided into two open reading frames, with a 54-nucleotide (nt) 5′ noncoding region (NCR) and a 297-nt 3′ NCR.

An Advanced One-Step RT-LAMP for Rapid Detection of Little cherry virus2 Combined with High-Throughput Sequence-Based Phylogenomics Reveal Divergent Flowering Cherry Isolates.

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 (<15 min), more analytically sensitive (100-fold), and robust broad-range diagnosis of LChV-2 isolates from sweet cherry, ornamental flowering cherry displaying heterogenous viral etiology and, for the first time, newly identified potential insect vectors. Moreover, use of Sanger and total RNA high-throughput sequencing as complementary metaviromics approaches confirmed the LChV-2 RT-LAMP detection of divergent LChV-2 isolates in new hosts and the relationship of their whole-genome was exhaustively inferred using maximum-likelihood phylogenomics. This entails unprecedented critical understanding of a novel evolutionary clade further expanding LChV-2 viral diversity. In conclusion, this highly effective diagnostic platform facilitates strategical support for early in-field testing to reliably prevent dissemination of new LChV-2 outbreaks from propagative plant stocks or newly postulated insect vectors. Validated results and major advantages are herein thoroughly discussed, in light of the knowledge required to increase the potential accuracy of future diagnostics and the essential epidemiological considerations to proactively safeguard cherries and Prunus horticultural crop systems from little cherry disease.

Cherry virus A

This datasheet on Cherry virus A covers Identity, Overview, Distribution, Dispersal, Hosts/Species Affected, Diagnosis, Biology &amp; Ecology, Impacts, Prevention/Control, Further Information.

Cherry virus A

Cherry virus A

First Report of Little Cherry Virus 1 Infecting Sweet Cherry in Ontario, Canada.

The Niagara fruit belt is one of the richest fruit-producing areas in Canada, contributing to 90% of Ontario's tender fruits such as peach, plum and sweet cherry. Little cherry virus 1 (LCV1) of the genus Velarivirus is a causal agent of little cherry disease which has devastated cherry crops in many regions (Eastwell and Bernardy 1998, Jelkmann and Eastwell, 2011). From 2013 to 2018, foliar symptoms indicative of viral infection such as leaf deformation, ringspot, mottling, vein clearing, and reddening were found on sweet cherry trees grown in the Niagara region. To determine if these trees were infected by a virus, small RNAs (sRNAs) were isolated from separately pooled asymptomatic and symptomatic leaves using the mirPremier microRNA isolation kit (Sigma Aldrich Canada, Oakville, ON). The sRNAs were used to create two libraries (four leaves per library) with the TruSeq Small RNA Sample Prep Kit (Illumina, San Diego, CA). The sRNA libraries were separately sequenced with the MiSeq Desktop Sequencer (Illumina, San Diego, CA). In total, 5,380,196 reads were obtained and Trimmomatic (Bolger et al. 2014) was used to remove adaptors. The remaining 4,733,804 clean reads were assembled into contigs using Velvet 0.7.31 (Zerbino and Birney, 2008) and Oases 0.2.09 (Schulz et al. 2012) with minimum length of 75 nt (Supplementary Table 1). A BLASTn search (Altschul et al. 1997) of the contigs identified the presence of Cherry virus A (genus: Capillovirus), two members of the Ilarvirus genus (Prunus necrotic ringspot virus and Prune dwarf virus) in both libraries. LCV1 was only found in contigs derived from the symptomatic library. Of the clean reads, 22,016 were assembled into six contigs (with lengths ranging from 86 to 116 nt, Supplementary Table 1) mapping to LCV1, covering 7.07% of the viral genome. To confirm LCV1 infection, primers were designed from the assembled contigs and used for reverse transcription polymerase chain reaction (RT-PCR). Amplicons were sequenced and the terminal sequences were determined using 5' and 3' RACE Systems (Invitrogen, Burlington, ON). Degenerate primers were designed from multiple sequence alignments of published LCV1 genomes for amplification and primer walking to obtain the sequence of LCV1 (Table S2). The complete genome sequence of LCV1 has a length of 16,934 nt and was deposited in GenBank (accession no. MN508820). A BLASTn search showed that this isolate is nearly identical (99.6% sequence identity) to an isolate from California (accession no. MN131067). To determine the incidence of infection, a field survey was performed at the same location during spring months of 2014 to 2018 using RT-PCR with primers specific to the viral coat protein gene (Supplementary Tale 2). Among 46 cherry trees sampled, two (4.3%) trees were infected with LCV1 and showed negative results with CVA, PNRSV and PDV. Both trees displayed mild suturing of primary and secondary veins (Supplementary Figure 1). LCV1 has been identified in Western stone fruit producing regions (British Columbia in Canada, and Washington, California, and Oregon in the United States of America). To the best of our knowledge, this is the first report of LCV1 in any eastern region of Canada. The low incidence of LCV1 suggests that this virus is not widespread in this region. Routine monitoring and detection of LCV1 is required to prevent this devastating cherry disease from spreading in this region.

First report of Cherry virus A infecting sweet cherry in Ontario, Canada

First report of <i>Cherry virus A</i> infecting sweet cherry in Ontario, Canada

Molecular characterization of cherry virus a and prunus necrotic ringspot virus and their variability based on nucleotide polymorphism

A survey was conducted to know the incidence of viruses in apricot growing in the Srinagar and Shopian regions of Jammu and Kashmir during May 2015. A total of 52 leaf samples were obtained and virus infection was confirmed using nucleic acid spot hybridization (NASH) and reverse transcription-polymerase chain reaction (RT-PCR). The infectivity of cherry virus A (CVA) and prunus necrotic ringspot virus (PNRSV) was found to be 44% and 98% by NASH and 36% and 77% by RT-PCR, respectively. Based on the sequence variability of RdRp, CVA isolates found globally have been grouped into six phylogroups (Group 1–6), whereas PNRSV isolates from India have been divided into three phylogroups (Group 1, Group 2A and Group 2B) based on coat protein gene. Our findings prove that PNRSV and CVA are the two significant viruses of apricot, with PNRSV being the leading cause of apricot necrosis in old orchards.

Titer and distribution of little cherry virus 2 in Prunus avium.

Little cherry virus 2 (LChV-2) is a causal agent of little cherry disease, which produces small, misshapen fruit with poor color and taste. As LChV-2 symptoms are only present near harvest, molecular detection is essential for effective control. Therefore, we determined the titer and distribution of this virus in infected trees over time. While initial infections were found to be basipetal, in field trees, early-stage infection was characterized by uneven distribution and low titer, concentrated in woody stems. In contrast, established infections were systemic, and detection was consistent across tissues. These data provide improved sampling recommendations for the detection of LChV-2.

First report of Cherry virus F infecting Japanese plum in Korea.

Cherry virus F (CVF) is a tentative member of the genus Fabavirus in the family Secoviridae, consisting of two RNA segments (Koloniuk et al. 2018). To date, CVF has been documented in only sweet cherry (Prunus avium) in the Czech Republic (Koloniuk et al. 2018), Canada, and Greece. In May 2014, we collected leaf samples from four symptomatic (leaf spots and dapple fruits) and two asymptomatic Japanese plum cultivars (Sun and Gadam) grown in an orchard in Hoengseong, South Korea, to identify viruses and viroids infecting plum trees. Total RNA from individual plum trees was extracted using two commercial kits: Fruit-mate for RNA Purification Kit (Takara, Shiga, Japan) and RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). We generated six mRNA libraries from the six different plum cultivars for RNA-sequencing using the TruSeq RNA Library Preparation Kit v2 (Illumina, CA, U.S.A.) as described previously (Jo et al. 2017). The mRNA libraries were paired-end (2 X 100 bp) sequenced with a HiSeq 2000 system (Macrogen, Seoul, Korea). The raw sequence reads were de novo assembled by Trinity program v. 2.8.6, with default parameters (Haas et al. 2013). The assembled contigs were subjected to BLASTX search against the non-redundant protein database in NCBI. Of the two asymptomatic cultivars, the transcriptome of asymptomatic plum cv. Gadam contained five contigs specific to CVF. Two and three contigs were specific to CVF RNA1 (2,571 reads, coverage 42.15%) and RNA2 (2,025 reads, coverage 53.04%), respectively. The size of these five contigs ranged from 241 to 5,986 bp. Contigs of 5,986 and 3,867 bp in length, referred to as CVF isolate Gadam RNA1 (GenBank MN896996) and RNA2 (GenBank MN896995), respectively, were subjected to BLASTP search against NCBI's non-redundant protein database. The results showed that the polyprotein sequences of RNA1 and RNA2 shared 95.3% and 93.11% amino acid identities with isolates SwC-H_1a from the Czech Republic (GenBank acc. no. AWB36326) and Stac-3B_c8 from Canada (AZZ10055), respectively. To confirm the infection of CVF in cv. Gadam, RT-PCR was conducted using CVF RNA1-specific primers designed based on the CVF reference genome sequences (MH998210 and MH998216), including 5'-CCACCAAATAGGCAAGAGGTCAC-3' (position 3190-3212) and 5'-CACAATCACCATCAATGGTCTCTGC-3' (position 3742-3766), and CVF RNA2-specific primers, including 5'-CTGCTTTATGATGCTAGACATCAAGATG-3' (position 1015-1042) and 5'-ACAATAGGCATGCTCATCTCAACCTC-3' (position 1594-1619). We amplified 577-bp RNA1-specific and 605-bp RNA2-specific amplicons that were cloned and then performed Sanger sequencing. Sequencing of the cloned amplicons for isolate Gadam RNA1 (GenBank MN896993) and RNA2 (GenBank MN896994) revealed values of 99.48% and 99.17% nucleotide identity to that of RNA1 and RNA2 determined by high-throughput sequencing, respectively. Additionally, we tested five plants for each of the six plum cultivars grown in the same orchard. The detection of CVF was carried out through PCR using the primers and protocol described above. Of the 30 trees, CVF was detected in three trees of cv. Gadam by both primer pairs. To our knowledge, this is the first report of CVF infecting Japanese plum and the first report of the virus in Korea. However, its prevalence in other Prunus species, including apricot, European plum, and peach, should be further elucidated.

First Report of Apricot vein clearing-associated virus (AVCaV) infecting Prunus domestica revealed by High-Throughput Sequencing in Morocco.

Plum (Prunus domestica L., Rosaceae) trees, like many stone fruit trees, are known to be infected by numerous plant viruses, predominantly as consequence of their clonal mode of propagation and perennial cultivation (Jelkmann and Eastwell, 2011). Apricot vein clearing-associated virus (AVCaV) is a member of the genus Prunevirus in the family Betaflexiviridae. AVCaV was first reported in Italy infecting apricot (P. armeniaca L.) associated with foliar vein clearing symptoms (Elbeaino et al. 2014). It has also been detected in various Prunus species, like plum, Japanese plum (P. salicina L.), sour cherry (P. cerasus L.), and Japanese apricot (P. mume L.), apricot and peach (P. persica L.) sourced from Asian and European countries (Marais et al. 2015), as well as in the ornamental Myrobolan plum (P. cerasifera L.) in Australia (Kinoti et al. 2017). In 2018, during the vegetative season, a survey was carried out in two different apricot and plum orchards in the southern region of Agdez (Agadir, Morocco) where stone fruit trees are grown. Five branches with leaves were sampled from three apricot and three plum trees of unknown cultivars, all asymptomatic. Total RNA was extracted from 100 mg plant tissue (leaves and cambial scrapping) using RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) and separate samples (one per species) were used for library preparation (NEBNext Ultra RNA library kit; New England BioLabs, MA, USA), and sequencing (Illumina NextSeq v2, totRNA sequencing) at Admera Health (New Jersey, USA). All generated reads (6,756,881) from the plum sample were quality filtered and submitted to the VirusDetect pipeline (Zheng et al., 2017). The plum cDNA library, a total of 20 viral contigs (68-1928 bp) mapped to several AVCaV accessions in GenBank. A reference mapping (CLC Genomics Workbench 12, Qiagen, Denmark) was conducted against all four available AVCaV full genomes (KM507062-63, KY132099 and HG008921), revealing 100% coverage of the full sequence (8358 nt) with 97-98 % nucleotide (nt) identities (BLASTn). Analysis of the derived sequences allowed to identify the location of the four predicted ORFs i.e. (ORF1: 6066 nt/2,021 aa), (ORF2: 1383 nt/460 aa), (ORF3: 666 nt/221 aa) and (ORF4: 420 nt/139 aa), previously described for the AVCaV genome (Elbeaino et al. 2014). The amino acid sequences of the encoded proteins of AVCaV isolate from Morocco also shared 97-98% identities with the corresponding sequences of complete genome AVCaV isolates in GenBank. To confirm the detection of AVCaV in the three plum samples, specific RT-PCR primers (VC37657s: 5'-CCATAGCCACCCTTTTTCAA-3' / VC28239a: 5'-GTCGTCAAGGGTCCAGTGAT-3') (Elbeaino et al. 2014) were used and the expected 330 bp fragment from the replicase gene was amplified in all three samples and subsequently sequenced (MT980794-96). Sanger sequences were 100% identical to corresponding HTS derived sequence. This is the first report of AVCaV infecting plum in Africa. The incidence of AVCaV in Moroccan Prunus species is unknown. Plum trees from the surveyed orchards were also confirmed to be co-infected with little cherry virus 1 (LChV-1) using HTS. Further investigation is required to determine the impact of AVCaV on these asymptomatic plum trees and other stone fruits species.

First report of peach leaf pitting-associated virus (PLPaV), plum bark necrosis stem pitting-associated virus (PBNSPaV), and mume virus A (MuVA) from Mei (Prunus mume) in China.

Mei (Prunus mume Sieb. et Zucc.), widely distributed in East Asian countries for both fruiting- and flowering-purposes, is susceptible to viral infections (Marais et al. 2018). Infection by plum bark necrosis stem pitting-associated virus (PBNSPaV) or little cherry virus 2 (LChV-2) possibly caused overall yield loss in mei in Japan due to incomplete flower development, low fruit bearing rate, and interveinal chlorosis (Numaguchi et al. 2019). Virus-like disease showing mosaic, interveinal chlorosis, vein clearing, or necrotic spot on leaf was observed in mei trees in Beijing, Wuhan, Wuxi, and Nanjing in spring and early summer from 2017 to 2018. Symptomatic leaves collected from the four regions were pooled as two samples for RNA-sequencing (RNA-seq) analysis. After ribosomal RNA (rRNA)-depletion, total RNA extracted by TRNzol reagent (TIANGEN, China) was subjected to library construction using NEBNext Ultra RNA Library Prep Kit (NEB, MA, USA) and sequenced on an Illumina Hiseq 4000 (Novogene, China). Sequencing data was filtered, screened, and assembled as described previously (Zhou et al. 2020) to generate contigs, following by BLAST-x/n search in viral genomes in GenBank. We identified >300 contigs (208-10756 nt) homologous to Asian prunus virus 1 and Asian prunus virus 2 (APV1 and 2), mume virus A (MuVA), PBNSPaV, and peach leaf pitting-associated virus (PLPaV), with 71-100% of nucleotide sequence identity values. APV1 and 2 have been reported in mei in China (Wang et al. 2018), here, we focused on the other three viruses. Contigs homologous to these three viruses were further assembled into three scaffolds of 14,224 nt, 1107 nt, and 753 nt for PBNSPaV, MuVA, and PLPaV, respectively. The scaffold of PBNSPaV (MW217574) nearly covered the whole genome of the isolate VIC3 from Australia (LC523039.1) (Kinoti et al. 2020) with 92.30% of sequence identity; the scalffold of MuVA (MW217572) covered 14.50% of the genome of the isolate pm14 from Japan (NC 040568.1) (Marais et al.2018) with 98.47% sequence identity; the scaffold of PLPaV (MW217573) covered 15.26% of the genome of the isolate XJ-6 from peach (KY867750.1) (He et al. 2017) with 85.23% sequence identity. Presence of the three viruses were verified by RT-PCR detection using designed specific primers for PBNSPaV (Forward: 5'-CAACAAAACTCCCACAGCGG-3 [positions 4014-4033, NC_009992.1] / Reverse: 5'-GCCAAAAGAAGTGCTGGTGG-3' [positions 4659-4640, NC_009992.1]), MuVA (Forward: 5'-AAGAGAATTACTTCAATGCCCTC-3' [positions 171-194, NC_040568.1] / Reverse: 5'-GATATCCAAGATACGATTAGCCAG-3' [positions 533-510, NC_040568.1]), and PLPaV (Forward: 5'-GCTATATCTCAACAACTGCAAGAA-3 [positions 5798-5821, KY867750.1] / Reverse: 5'- GAGTGATACATAGTCCACAGAGAT-3'[ positions 6045-6022, KY867750.1]). The amplified 626, 350 and 251 bp fragments of PBNSPaV, MuVA and PLPaV had 91.47%, 98.07% and 81.89% sequence identity to their respective reference sequences. This is the first report of PBNSPaV and MuVA infecting mei in China, and more importantly, the first report of a new host for PLPaV. In addition, 30 collected leaf samples from Nanjing and Wuhan were analyzed by RT-PCR and 15, 6, and 5 samples tested positive to PLPaV, PBNSPaV, and MuVA, respectively. Although it is difficult to link a particular virus with the observed symptoms due to mixed infections, the symptoms were significantly associated with viral infection because almost all symptomatic leaf samples were virus(es)-positive. Further studies would be required to determine the distribution and impact of these viruses on mei trees and other stone fruits species and to understand the possibility that mei trees may play a role in PLPaV epidemiology.

Establishment of in vitro transcription system based on RNA-dependent RNA polymerase (RdRp) of Little cherry virus 1.

Establishment of in vitro transcription system based on RNA-dependent RNA polymerase (RdRp) of Little cherry virus 1.

Zdravstvena selekcija višnje Maraske (Prunus cerasus var. Marasca) u najvećoj Hrvatskoj plantaži “Vlačine”

Sour cherry cv. Marasca is considered a native Croatian variety with the best fruit quality in the Northern Dalmatia region. The current production is not sufficient to cover the demand, therefore new orchards should be established with pathogen-tested planting material. To select virus- and bacteria-free mother trees, a survey was conducted in the largest Croatian Marasca plantation "Vlačine". ELISA on 205 trees (51 elite, 103 average, 51 below average) confirmed the presence of prunus necrotic ringspot virus (PNRSV) on 101 trees (49.3%); petunia asteroid mosaic virus (PeAMV) and prune dwarf virus (PDV) on 10 trees each (4.9%); raspberry ringspot virus (RpRSV) on 7 trees (3.4%); cherry leafroll virus (CLRV), arabis mosaic virus (ArMV) and plum pox virus (PPV) on 3 trees each (1.5%); and apple mosaic virus (ApMV) on 1 tree (0.5%). For PNRSV, significant ELISA detection sensitivity was found, with twigs in dormant period as more reliable virus source compared to leaves during the growing season. In addition, six trees were positive on little cherry virus 2 (LChV-2) by RT-PCR. Along with very frequent latent infections, following symptoms were observed during pre-harvest period: yellow spots on leaves (ApMV); yellowing, stunted growth, leafless twigs with leaves only at the tip (PDV), bark splitting (PeAMV), reduced leaf size (RpRSV), chlorotic/necrotic ring spots (PNRSV), and uneven fruit ripening (LChV-2). Eleven trees were free of all viruses and bacteria defined by EPPO cherry certification scheme, including PPV. The selected virus- and bacteria-free elite trees represent a valuable genetic source for propagation and further clonal selection.

First Report of Mume Virus A Infection of Prunus persica in China

Mume virus A, belonging to Capillovirus in the family Betaflexiviridae, was discovered recently from Japanese apricot (Prunus mume) and exhibited diffuse chlorotic spots on leaves (Marais et al. 2018). To date, Japanese apricot is the only known host of Mume virus A (MuVA) (Maliogka et al. 2018). Peach (Prunus persica) is a delicious and healthy summer fruit grown in most temperate regions of the world. More than 20 different viruses infecting peach trees have been reported (Jo et al. 2018). In April 2019, the leaves from four peach trees showing severe yellow mosaic and leaf curling were collected from peach orchards in Xinjiang, China. The pooled total RNA was extracted from these four trees leaves with TRIzol reagent (Life Technologies Corporation, Carlsbad, CA) and subjected to high-throughput sequencing (HTS) using a PE150 sequence strategy on an Illumina HiSeq2000 (Novogene Corporation, Tianjin, China). Approximately 39.3 million of 150-bp paired-end reads were obtained and used for de novo transcriptome assembly by SPAdes Genome Assembler (Bankevich et al. 2012). A total of 89,163 contigs with N50 of 866 bp were obtained and then were annotated by BLASTX analysis against the NCBI nr virus database (Altschul et al. 1997). Six known viruses (apple chlorotic leaf spot virus, Asian prunus virus 2, cherry green ring mottle virus, nectarine stem pitting-associated virus, peach associated luteovirus, and plum bark necrosis stem pitting-associated virus) and one known viroid (hop stunt viroid) were identified. Moreover, a 7,623-bp contig with a coverage of 84.4-fold was identified as capillovirus-related sequence. To classify the capillovirus accurately, pairwise genome alignments analysis was performed between all members of the Capillovirus genus using PASC online tool (Bao et al. 2014). The closest match was the genome of MuVA (NC_040568.1), sharing 78.36% nucleotide identity, followed by those of some isolates of cherry virus A, sharing less than 58.5% nucleotide identity. This result suggests that the capillovirus from peach belongs to the same species with the MuVA from apricot but should be a different isolate. We designated it as isolate pp, and its HTS-derived viral genome sequence was deposited in GenBank (accession no. MN412555). To confirm the HTS experiment results, from the same peach orchards, 23 peach leaf samples from different trees were collected, and total RNA was extracted with TRIzol reagent with standard procedures following the instructions. The RNA was subsequently used to test for pp isolate by reverse transcription polymerase chain reaction (PCR) using specific primers DT-MVAF5716 (5′-CTTCACATCGGAGCTGTCCT-3′) and DT-MVAR6277 (5′-TGCTGATTGCTTCAGACCCT-3′), targeting a portion of the open reading frame 2 (putative movement protein). Eleven samples produced the expected 560-bp PCR fragment. Three PCR products randomly selected were cloned into pMD19-T Vector (TaKaRa) for Sanger sequencing. All sequences of amplicons shared nearly 100% sequence identity with the corresponding region of the HTS-derived viral genome sequence. However, at least the other two viruses and viroid had been detected in all 11 MuVA-positive samples, so the symptoms of the initial sample cannot be explained by MuVA infection. To our knowledge, this is the first report of MuVA infecting peach trees. This report suggests that MuVA may have a broad host range in the genus Prunus.

Development of a novel polyprobe for simultaneous detection of six viruses infecting stone and pome fruits

A biotin-labeled, non-isotopic, novel polyprobe was developed for the simultaneous detection of six viruses viz. apple chlorotic leaf spot virus (ACLSV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV), cherry virus A (CVA), prunus necrotic ringspot virus (PNRSV) and plum pox virus (PPV) infecting stone and pome fruit trees through dot-blot hybridization assay. The sensitivity of the polyprobe was checked by serial dilutions of total RNA extracted from the tissues of infected trees. ACLSV was detected up to a dilution of 5-5, whereas ApMV, ASGV, CVA, PPV and PNRSV up to 5-4. The developed assay was validated following testing a total of 45 symptomatic leaf samples collected from different geographical regions of Jammu and Kashmir (India), and the presence of the viruses was further confirmed by RT-PCR and sequencing. The polyprobe, designed for performing molecular hybridization assay can be developed quickly and avoid the tedious transformation and cloning procedures. Apart from simultaneously detecting viruses in stone and pome fruit trees, it holds great potential for virus indexing programmes to ascertain the supply of virus-free plant materials to the growers.

First Report of Cherry Virus Turkey in Sweet Cherry in Greece

The number of viruses identified in sweet cherry has been constantly increasing over the last few years, following the broad application of high-throughput sequencing (HTS). Some of these were reported to cause leaf symptoms and yield reduction. In 2009 to 2013, surveys were performed on sweet cherry orchards located in northern Greece for the presence of Betaflexiviridae viruses using generic and specific molecular assays (Foissac et al. 2005). Sanger sequencing of a generic reverse transcription polymerase chain reaction (RT-PCR) product originated from a sample collected from a symptomless sweet cherry (cv. Ferrovia) revealed a virus sequence sharing 75% nucleotide (nt) similarity with a highly conserved portion of the betaflexivirus polymerase gene. Total RNA, extracted with the TRIzol reagent (Invitrogen, Carlsbad, CA) from leaves of this sample, was subjected to HTS analysis on an Illumina Hi-seq 4000 platform (Novogene, Tiangen, China). The run, after quality control, yielded 26,002,319 of 150-nt-long paired-end reads. De novo assembly of these reads using Geneious Prime 2019.2.3 and Velvet and subsequent BLAST analysis (BLASTn/x) of 82 contigs revealed, apart from several known sweet cherry viruses (cherry virus A, Prunus virus F, little cherry virus 1), the presence of a newly described Robigovirus species. More specifically, the BLAST results revealed that eight de novo contigs ranging from 277 to 2,287 nt long were similar to the sequence of the recently reported cherry virus Turkey (CVTR) (Caglayan et al. 2019). The nearly complete genome sequence consisting of 8,362 nt (99.5%) of the Greek CVTR isolate (CVTR-C18GR, accession no. MT043307) was reconstructed using iterative mapping of the reads to the de novo contigs, with 4,690 reads mapped in total and Sanger sequencing of RT-PCR amplicons to fill the sequence gaps. Similarity analysis of the CVTR-C18GR sequence with the full virus sequences from GenBank CVTR-BUR12 (MK600387) and CVTRAZQ (MH177869) showed 90.47 and 90.60% nt identities, respectively. Phylogenetic analyses confirmed the clustering of C18GR with other CVTR isolates. To investigate for the virus presence in Greece, primers F2Rob (5′-GTCAGAGAGAGGTATCTATGTC-3′) and RT1Rob (5′-CAGGCTGTTCATAACCT-3′), which amplify 577 nt of CVTR replicase, were designed. Sixty-six samples from sweet cherry trees showing no virus-like symptoms were collected from various orchards and screened for the presence of CVTR. Eight of them were found to be infected with the new virus. Sanger sequencing of sample C11GR amplicon confirmed the presence of CVTR in Greek fields (96% nt similarity with C18GR, accession no. MT043308). Sequence similarity between C11GR and C18GR isolates was also high (97% nt) in partial TGB2, TGB3, and CP genomic regions amplified with primer pair 7006 F (5′-GAAAAGTGATTATTCAGCRCCAGT-3′) and 7708 R (5′-CTTTCACCCACTCATCACCTATCTCC-3′) (accession no. MT461407). This is the first report of this virus in Greece, thus extending the information on its geographical distribution. Robigoviruses cause variable symptoms and diseases in sweet and sour cherries, and for that reason they are included in the European Commission Directive concerning official inspections (2019/2072/EU). Additional screenings are necessary to evaluate the presence and impact of CVTR in sweet cherry orchards in Greece.

Molecular characterisation of little cherry virus 1 infecting apricots in the Czech Republic

Little cherry virus 1 is one of the pathogens associated with little cherry disease of cherries. The host range of the virus is widening and recently it has been detected in apricot trees in the Czech Republic, Hungary, and Morocco. To obtain a view about the importance of apricots in the epidemiology of the virus, a survey was conducted in Czech apricot-growing areas. Little cherry virus 1 was detected in seven out of 13 locations, and the infection was noted predominantly in old orchards, but its frequency was low. During the survey, an infected almond tree developing severe leaf mosaic was also detected. Using high-throughput sequencing and Sanger sequencing, five near-complete genome sequences and another 20 partial ORF8 sequences were obtained. Phylogenetic analysis showed a close relationship of most of the apricot isolates to the phylogenetic group G3 with UW2, UW1, and ITMAR isolates. Three apricot isolates, including the almost complete Apr 184R isolate and almond isolate Alm138, showed homology within the phylogenetic group G5. The analysis clearly demonstrates the presence of isolates of this type in Europe. The 4-year-long survey of selected pomological characteristics showed that infection with the virus is generally latent, but the infection could significantly affect the ripening of the fruit of some apricot genotypes. A significant decrease in the fruit yield was noticed for ‘Magyar Kajszi’. The potential routes of the transmission of little cherry virus 1 to apricots are discussed in the context of propagation practice.

Complete genome sequence of cherry virus T, a novel cherry-infecting tepovirus.

Double-stranded RNA and total RNA purified from sour cherry leaves (Prunus cerasus, cv. Amarelka Chvalkovicka) was analyzed by high-throughput sequencing. BLAST annotation identified contigs with homology to several already known cherry-infecting viruses (prune dwarf virus, prunus necrotic ringspot virus, prunus virus F, little cherry virus 1) as well as contigs with sequences more distantly related to those of members of the family Betaflexiviridae and in particular to prunus virus T of the genus Tepovirus. The full genome sequence of a putative virus (6,847 nucleotides [nt]; GenBank no. MT090966) was assembled and completed at the genome ends. The genome has a typical tepovirus organization, containing three overlapping open reading frames (ORFs), encoding a replication-associated protein, a movement protein and a capsid protein, respectively. Both its genome organization and its phylogenetic relationships show that the virus belongs to the genus Tepovirus, but considering the species demarcation criteria for the family Betaflexiviridae, it appears to represent a novel virus species, and we propose the name "cherry virus T" (ChVT) for this virus.

Little cherry virus 1. [Distribution map

Abstract A new distribution map is provided for Little cherry virus 1 (Closteroviridae: Velarivirus). Hosts: Prunus spp. Information on the geographical distribution in Europe (Belgium, Czech Republic, France, Germany, Greece, Hungary, Italy, Mainland Italy, Poland, Romania, Slovakia, Spain, Switzerland, UK, England and Wales), Asia (China, Hebei, Liaoning, Shandong, India, Japan, Honshu, Korea Republic, Turkey), Africa (Morocco), North America (Canada, British Columbia, USA, California, Oregon, Washington), South America (Chile) and Oceania (Australia, Queensland, Tasmania, New Zealand) is also given.