Cestrum species are being used as hedge and ornamental plants in Turkey. In this study, a
Cestrum elegans
plant showing chlorotic spots, rings and reddening was subjected to high throughput ...sequencing of small RNAs, in order to clarify its etiology. Results suggested the presence of tomato chlorosis virus (ToCV), which was further confirmed by RT-PCR using two specific primers amplifying RNA-dependent RNA polymerase (RdRp) and coat protein (CP) of the virus. When 25 Cestrum samples were tested using CP primers, one symptomatic
C. elegans
and four symptomless
C. nocturnum
were infected by ToCV. The obtained sequences shared 83–99.9% nucleotide identity with ToCV isolates available in the GenBank. Phylogenetic relationship among Turkish tomato isolates of ToCV and those available in the GenBank showed that two
Cestrum
spp. isolates of ToCV were closely related to Turkish tomato isolates, while the other three clustered with isolates from different countries.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
•A novel Foveavirus was identified in raspberry by high throughput sequencing.•Complete genome of the virus obtained by HTS and confirmed by overlapping RT-PCR.•The genome ends were determined by ...Rapid Amplification of Complementary DNA Ends.•RuV1 shares the highest identity with Peach chlorotic mottle virus.•The virus was detected from both raspberry and blackberry plants.
A novel plant virus was identified by high-throughput sequencing analysis from a raspberry plant showing slight mottling symptom. The complete genome sequence of this virus is 8645 nucleotides long, including the 5′ and 3′ UTRs. Its genome contains five ORFs and is very close to members of the genus Foveavirus (Quinvirinae, Betaflexiviridae) in terms of genome organization, TGB presence and the sizes of the RdRp and CP proteins. The novel virus shares 33.5–51.3 % and 23.3–41.3 % nucleotide identity to other genera of the Betaflexifiviridae family based on polymerase (RdRp) and CP genes, respectively. Compared to other foveavirus species, the RdRp protein showed the highest sequence identity (45.3 %) to the RdRp of peach chlorotic mottle virus (PCMV) while the maximal sequence identity for the CP protein was 33.9 % with grapevine rupestris stem pitting-associated virus (GRSPaV). The low nucleotide and amino acid sequence identity with known foveaviruses indicated that it was a novel virus, for which the provisional name “rubus virus 1 (RuV1)” is proposed. The phylogenetic analysis supports the assignment of this virus as a new species of the genus Foveavirus. A survey of 537 Rubus spp. samples grown in six provinces of Turkey, including some symptomatic samples, showed a RuV1 prevalence of 2.2 %, confirming its presence in both raspberry and blackberry plants in a single province, although no obvious association between virus infection and specific symptoms was found.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Passive “vector” plants can harbor insect viruses without supporting their replication and may have epidemiological consequences. We report complete sequence of Aphid lethal paralysis virus (ALPV, ...Cripavirus, Dicistroviridae) from small RNA sequencing of a new passive “vector” plant, Cestrum elegans in Turkey using de novo assembly and annotating of contigs. Its genome is 9837 nt in length which encodes for 2 ORFs (including an intergenic untranslated region (IGR) of 196 nt) flanked by a 525 nt 5′ UTR and a 596 nt 3′ UTR. Multiple alignment and phylogenetic analysis showed that the isolate shares highest identity with ALPV-An (JX480861) isolated from oleander aphid, Aphis nerii and was placed into the same clade with other isolates of passive “vector” plants comprising cucumber and maize isolates.
•Aphid lethal paralysis virus (ALPV) was detected in Cestrum elegans by means of high throughput sequencing of small RNAs.•Complete genome of the virus obtained from reference guided assembly via VirusDetect.•ALPV-CE shares highest identity with ALPV-An (JX480861) isolated from oleander aphid, Aphis nerii.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Grapevine virus A (GVA), the type species of the
genus, is one of the causal agents of the Kober stem grooving disease of the rugose wood complex and one of the most frequently detected viruses in ...grapevine. There is little information on GVA gene(s) marker useful for phylogenetic analysis. To this aim, a total of 403 leaf samples were collected from vineyards of East and West Azarbaijan provinces in the Northwestern provinces of Iran during 2014-2016 and tested by DAS-ELISA and RT-PCR using ORF5-specific primers. GVA was detected in 56 symptomatic samples, corresponding to 14% of infection, while it was not detected in asymptomatic samples. The ORF5 (p10) protein sequence of eight Iranian isolates was compared to other vitiviruses, showing that the most conserved region resides in the N-terminus, carrying an arginine-rich motif followed by a zinc-finger motif. Next, to define a robust phylogenetic marker representative of the whole genome sequence suitable for phylogenetic and evolutionary studies, phylogenetic trees based on the full genome sequences of all the available GVA isolates and on individual genomic regions were constructed and compared. ORF1, which encodes the RNA-dependent RNA polymerase, was found to be the best phylogenetic marker for GVA classification and evolution studies. These results can be used for further research on phylogenetic analyses, evolution history, epidemiology, and etiology of rugose wood complex, and to identify control measures against GVA and other vitiviruses.
We investigated the presence of Hop stunt viroid (HSVd) in apple samples, showing dappling fruit symptoms, in the Maragheh area (Northwest Iran) by means of RT-PCR. The viroid was detected only in ...leaves collected from symptomatic trees and a 298bp amplicon (IR-Gala) was directly sequenced in both directions. Multiple sequence alignment and Blast analyzes revealed that IR-Gala isolate shares the highest identity with grapevine isolates from Brazil and China. Amongst Iranian isolates of HSVd available in the GenBank, this isolate had the highest identity with grapevine isolate of HSVd from Maragheh region. In Phylogenetic analysis by MrBayes, IR-Gala was clustered with grapevine isolates from Brazil, China and Iran and may suggest that HSVd-apple isolate could be originated from grapevine.
Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the ...viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed.
Pomegranate is an important crop in the Mediterranean Basin that can be affected by a range of pathogens. With the aim to better understand the impact of viral diseases on pomegranate, two leaf ...samples from Turkey showing virus-like symptoms such as chlorotic spots and oak-leaf patterns were subjected to high throughput sequencing (HTS). Data analysis indicated the presence of passiflora edulis symptomless virus (PeSV: genus Roymovirus, Potyviridae family) in these two pomegranate samples, consistent with the observation by electron microscopy of flexuous filamentous viral particles 760 to 780 nm long. Further analysis of HTS reads revealed the presence of five PeSV variants in one of the samples and another single variant in the other. PeSV occurrence was also identified from publicly available SRA pomegranate RNA-Seq transcriptomic data from India and China. The genome of these PeSV-pomegranate variants share 78.0–86.8% nucleotide identity with that of the reference isolate from passionfruit (MH379332). The presence of PeSV in pomegranate was confirmed by specific RT-PCR assays targeting either the coat protein (CP) or Nla-Pro genes in 37 cultivated and one ornamental pomegranate out of 133 samples collected from the Eastern Mediterranean region of Turkey. To our knowledge, this is the first application of HTS to assess virus occurrence in pomegranate and the first recognition of pomegranate as a new host for PeSV.
High throughput sequencing of total RNA isolated from symptomatic leaves of a sweet cherry tree (
cv. 0900 Ziraat) from Turkey identified a new member of the genus
designated cherry virus Turkey ...(CVTR). The presence of the virus was confirmed by electron microscopy and overlapping RT-PCR for sequencing its whole-genome. The virus has a ssRNA genome of 8464 nucleotides which encodes five open reading frames (ORFs) and comprises two non-coding regions, 5' UTR and 3' UTR of 97 and 296 nt, respectively. Compared to the five most closely related robigoviruses,
,
,
,
and
share amino acid identities ranging from 43-53%, 44-60%, 39-43%, 38-44% and 45-50%, respectively. Unlike the four cherry robigoviruses, CVTR lacks ORFs 2a and 5a. Its genome organization is therefore more similar to African oil palm ringspot virus (AOPRV). Using specific primers, the presence of CVTR was confirmed in 15 sweet cherries and two sour cherries out of 156 tested samples collected from three regions in Turkey. Among them, five samples were showing slight chlorotic symptoms on the leaves. It seems that CVTR infects cherry trees with or without eliciting obvious symptoms, but these data should be confirmed by bioassays in woody and possible herbaceous hosts in future studies.
► Unknown viral symptoms were observed in cv. Pinot gris. ► Small RNAs from grapevine tissue were sequenced by Illumina technology. ► Viruses and viroids infecting Pinot gris plants and properties of ...their viral small RNAs were described. ► De novo assembling of small RNAs allowed assembling the genome of a new virus. ► This new virus is taxonomically related to trichoviruses. ► Limited field survey for the presence of this virus did not clearly associate the virus to the observed symptoms.
Field symptoms of chlorotic mottling and leaf deformations were observed on the cv Pinot gris (PG) in the Trentino region (Italy). Extensive assays excluded the presence of widely distributed nepo-, ampelo- and vitiviruses. An analysis of small RNA populations from two PG grapevines showing or not symptoms was carried out by Illumina high throughput sequencing. The study disclosed the virus and viroids contents of the two vines that was composed by Grapevine rupestris stem pitting-associated virus (GRSPaV), two viroids Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1), the marafiviruses Grapevine rupestris vein feathering virus (GRVFV) and Grapevine Syrah virus 1 (GSyV-1), and a hitherto unrecorded virus. This virus had a genome organization identical to that of Grapevine berry inner necrosis virus (GINV), a trichovirus reported only from Japan, with which it grouped in phylogenetic trees constructed with sequences of the RdRp domain and the coat protein gene. However, molecular differences with GINV are wide enough to warrant classification of the virus in question as a new species, for which the provisional name of Grapevine Pinot gris virus (GPGV) is proposed. A limited field survey for the presence of GPGV in diseased and symptomless plants from three different cultivars did not allow to clearly associating the virus to the observed symptoms.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Abstract
Raspberry bushy dwarf virus (RBDV), recently renamed to
Idaeovirus rubi
, is one of the most common viruses infecting
Rubus
species worldwide but there is still a limited number of genome ...sequences available in the GenBank database and the majority of the sequences include partial sequences of RNA‐1 and RNA‐2. The distribution and incidence of RBDV in main raspberry and blackberry growing provinces in Turkey were monitored during 2015–2019 and 537
Rubus
spp. samples were tested by both DAS‐ELISA and RT‐PCR. Among the tested samples, 36 samples tested positive for RBDV by DAS‐ELISA and 67 samples by RT‐PCR. There was relatively low nucleotide diversity among the Turkish isolates. Turkish isolates shared 93%–97.7%, 84.3%–98.9%, and 85%–99.2% nucleotide sequence identities with available sequences in the GenBank, in partial RNA‐1, movement protein (MP) and coat protein (CP) genes, respectively. In the phylogenetic tree constructed for RNA‐1, MP, and CP sequences, all Turkish raspberry isolates were clustered in a distinct clade. However, the blackberry isolates showed considerable variation in nucleotide sequences and were placed in three distinct groups. The divergent blackberry isolates showed high variability in MP (84.5%–89.3%) and CP (85.5%–89.7%) regions and were placed in a distinct group. The rest of blackberry isolates clustered together with sweet cherry RBDV isolates adjacent to the grapevine clade or together with raspberry isolates. The comparative analysis conducted on three RNA segments of RBDV highlighted the high sequence diversity of Turkish RBDV isolates. This study also emphasizes the importance of regular monitoring of RBDV infections in Turkey, with special regard to those
Rubus
spp. and grapevine accessions employed in conservation and selection programmes. In particular, the presence of new RBDV genetic variants and infection of
Rubus
species must be taken into account to choose a correct detection protocol and management strategy.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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