Morphological and phylogenetic analyses of Uromyces Gagea on Gajea lutea in Iran

Typical rust symptoms were observed on leaves of Gajea lutea plants collected from wheat fields in Maragheh, Northwest of Iran, in May 2017. Black, circular to elongated or diamond-shaped telial pustules 1-3 mm long were found on both sides of the leaves. Under light microscope, stalked, one- celled teliospores 22.5-37.5×17.5-22.5 μm were observed. The ITS region of DNA was amplified from teliospores and sequenced. Based on classical and molecular analyses, we report Uromyces Gagea from Iran

Phylogenetic Marker Selection and Protein Sequence Analysis of the ORF5 Gene Product of Grapevine Virus A

Grapevine virus A (GVA), the type species of the Vitivirus 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

Identification and molecular characterization of a novel foveavirus from Rubus spp. in Turkey

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

A new grapevine virus discovered by deep sequencing of virus-and viroid-derived small RNAs in Cv Pinot gris

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 viroidsHop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1), the marafivirusesGrapevine rupestrisvein 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 proteinproteingene. 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

Identification and Characterization of a Novel <i>Robigovirus</i> Species from Sweet Cherry in Turkey

High throughput sequencing of total RNA isolated from symptomatic leaves of a sweet cherry tree (Prunus avium cv. 0900 Ziraat) from Turkey identified a new member of the genus Robigovirus 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&#8242; UTR and 3&#8242; UTR of 97 and 296 nt, respectively. Compared to the five most closely related robigoviruses, RdRp, TGB1, TGB2, TGB3 and CP share amino acid identities ranging from 43&#8722;53%, 44&#8722;60%, 39&#8722;43%, 38&#8722;44% and 45&#8722;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

Identification of Pomegranate as a New Host of Passiflora Edulis Symptomless Virus (PeSV) and Analysis of PeSV Diversity

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

Identification of Pomegranate as a New Host of Passiflora Edulis Symptomless Virus (PeSV) and Analysis of PeSV Diversity

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

Detection of single nucleotide polymorphisms in virus genomes assembled from high-throughput sequencing data: large-scale performance testing of sequence analysis strategies

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