Pooja Thapa’s research while affiliated with Indian Agricultural Research Institute and other places

Novel viruses in plants can be detected through transcriptome data mining. In this study, a novel cytorhabdovirus, pear rhabdovirus 1 (PRV-1) was identified through reanalysis of RNA-seq data of pear (P. communis 'Bartlett'). The genomic RNA of PRV-1, with complete coding region, measured 15,628 nucleotides (nts) and encompassed six open reading frames (ORF). Homology analysis of PRV-1 genome showed sequence identity of 33.18-56.75% with the existing cytorhabdovirus sequences. Phylogenetic analysis based on genome sequences showed that PRV-1 clustered in the same clade of cytorhab-doviruses. Based on the sequence demarcation criteria, PRV-1 represents a newly discovered species within the Cytorhab-dovirus genus of the Rhabdoviridae family. Identification of a novel virus in pear will enhance our understanding on the diversity of plant cytorhabdoviruses.

Background The choice of de novo assembler for high-throughput sequencing (HTS) data remains a pivotal factor in the HTS-based discovery of viral pathogens. This study assessed de novo assemblers, namely Trinity, SPAdes, and MEGAHIT for HTS datasets generated on the Illumina platform from 23 apple samples, representing 15 exotic and indigenous apple varieties and a rootstock. The assemblers were compared based on assembly quality metrics, including the largest contig, total assembly length, genome coverage, and N50. Results MEGAHIT was most efficient assembler according to the metrics evaluated in this study. By using multiple assemblers, near-complete genome sequences of citrus concave gum-associated virus (CCGaV), apple rubbery wood virus 1 (ARWV-1), ARWV-2, apple necrotic mosaic virus (ApNMV), apple mosaic virus, apple stem pitting virus, apple stem grooving virus, apple chlorotic leaf spot virus, apple hammerhead viroid and apple scar skin viroid were reconstructed. These viruses were further confirmed through Sanger sequencing in different apple cultivars. Among them, CCGaV, ARWV-1 and ARWV-2 were recorded from apples in India for the first time. The analysis of virus richness revealed that ApNMV was dominant, followed by ARWV-1 and CCGaV. Moreover, MEGAHIT identified novel single-nucleotide variants. Conclusions Our analyses highlight the crucial role of assembly methods in reconstructing near-complete apple virus genomes from the Illumina reads. This study emphasizes the significance of employing multiple assemblers for de novo virus genome assembly in vegetatively propagated perennial fruit crops.

A novel grapevine viroid was discovered in an asymptomatic grapevine of Indian rootstocks. The whole genome sequence of the viroid (370 nt) was determined by high-throughput sequencing as well as RT-PCR followed by cloning and Sanger sequencing. The terminal conserved region (TCR), central conserved region (CCR) upper strand, and CCR lower strand are conserved regions found in the viroid that are unique to the members of the genus Apscaviroid. Based on our findings and the demarcation criteria for viroids, the novel viroid, which we have tentatively named "grapevine yellow speckle viroid 3" is a putative new member of the genus Apscaviroid.

Background Transcriptome data from a plant sample frequently include numerous reads originating from RNA virus genomes that were concurrently isolated during RNA preparation. These high-throughput sequencing reads from the virus can be assembled to form a new sequence for the plant RNA genome. Methods and results Here, we identify putative novel mitovirus, grapevine mitovirus 1 (GMV1) through high-throughput sequencing (HTS) of grapevine rootstocks (Vitis spp.), and the identified virus was confirmed using virus-specific primers in RT-PCR assay. The genomic RNA of GMV1 encodes complete open reading frame (ORF) of 2,496 nucleotides (nts) in length. RNA-dependent RNA polymerase (RdRp) encoded by the viral genome contained one RdRp conserved domain. BLASTx analysis of GMV1 genome showed sequence identity of 33.18–56.75% with the existing mitovirus sequences. Phylogenetic analysis based on genome sequences showed that GMV1 clustered in a distinct clade to other mitoviruses. Conclusion Grapevine mitovirus 1 represents a newly discovered species within the Unuamitovirus genus of the Mitoviridae family, targeting fungal mitochondria. While the majority of recognized mitoviruses typically lack a functional RdRp as per the plant mitochondrial genetic code, GMV1 encodes a complete RdRp in accordance with both fungal and plant mitochondrial genetic codes.

Grapevine stocks are generally vegetatively propagated for the commercial cultivation. The propagation is predominantly done on rootstocks with >90 % of plant stocks propagated on Dogridge rootstock alone in India. Three additional rootstocks i.e. Paulsen, SO4 and Ramsey are also used for grapevine propagation on a limited scale. However, no information is available on the health status of these rootstocks being used by nurserymen and growers. Therefore, these rootstocks were collected from nurseries of five different locations and subjected to high-throughput sequencing (HTS) to determine the spectrum of viruses and viroids. The de novo assembly of unaligned reads revealed the presence of genome sequences of nine viruses viz. grapevine rupestris stem pittingassociated virus (GRSPaV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine leafroll-associated virus 4 (GLRaV-4), grapevine virus A (GVA), grapevine virus B (GVB), grapevine virus D (GVD), grapevine virus F (GVF), grapevine virus L (GVL), grapevine fleck virus (GFkV) and five viroids viz. hopstunt viroid (HSVd), grapevine yellow speckle viroid 1 and 2 (GYSVd-1 and GYSVd-2), Australian grapevine viroid (AGVd) and grapevine latent viroid (GLVd). The identified viruses and viroids were further confirmed through RT-PCR and Sanger sequencing. Indexing for the presence of these graft-transmissible pathogens in 43 rootstocks and 23 scion samples collected through field surveys indicated that infected rootstocks are the probable source for dissemination of these viruses and viroids in grapevine scions. As GVB was the most prevalent virus in all the rootstocks and scions as observed in RT-PCR testing, a simplified crude sap-based recombinase polymerase amplification (RPA) assay was optimized for its rapid detection. The present study highlights that grapevine rootstocks are reservoir of viruses and viroids and source for their dissemination in grapevine vineyards. Findings of the study including the optimized simplified cost-effective isothermal nucleic acid-based detection technique for GVB will be highly useful for production of certified clean rootstocks of grapevine in India.

The concurrent reports on emergence of stunting disease of rice across the Northwest Indian rice growing areas attracted attention for elucidating its etiology. Surveys of different rice fields recorded an incidence of stunting disease in the range of 1-20% in the affected fields. A systematic investigation employing three independent methods was undertaken. Under electron microscope, icosahedral virions of ~65-75 nm were observed. Based on the shape and size of virion particles and symptoms of the disease, reverse transcription-PCR and quantitative-RT-PCR of stunted rice plants and prevalent white-backed planthopper (WBPH) were performed using specific primers targeting two genomic components (S9 and S10) of Southern rice black-streaked dwarf virus (SRBSDV), a double stranded RNA virus of genus Fijivirus and the results indicated its specific association with stunting disease of rice. Sequencing of the amplified S9 and S10 genomic components showed maximum identity of 97.90-100.00% and 98.04-99.48%, respectively with SRBSDV isolates from South Korea and Vietnam. To the best of our knowledge, this is the first conclusive evidence of association of SRBSDV with stunting disease of rice from India. The findings seek urgent attention and in-depth investigation on its establishment in the Indian subcontinent and necessary interventions.