Genome analysis of viruses of Phenuiviridae, Betaflexiviridae and Bromoviridae, and apple scar skin viroid in pear by high-throughput sequencing revealing host expansion of a rubodvirus and an ilarvirus

Pear (Pyrus spp.) is an important perennial fruit crop of India. During May 2022, pear leaf samples were collected from the orchard of ICAR-Indian Agricultural Research Institute, Regional Station, Dhanda, Shimla, Himachal Pradesh, India. RNA sequencing of two pooled samples, Pe1 and Pe2, was done using NovaSeq 6000. Host unaligned reads were de novo assembled into contigs and annotated through BLAST using the virus database. Near complete genome sequences of apple rubbery wood virus 1 (ARWV-1), apple necrotic mosaic virus (ApNMV), citrus virus A (CiVA), apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), and a genetic variant of ASGV named as ASGV-Shimla, and a partial sequence of apple scar skin viroid (ASSVd) were assembled. ARWV-1 and ApNMV belong to the genus Rubodvirus and Ilarvirus having tripartite negative sense and positive sense single stranded RNA genomes, respectively, and their presence in pear trees was observed for the first time indicating the host expansion. Presence of CiVA, ASGV and ACLSV in pear in India was also recorded for the first time in India. The fragments per kilobase of transcript per million reads mapped estimation showed the dominance of ASPV and CiVA in Pe1 and Pe2, respectively. Single nucleotide variants (SNVs) analysis revealed that ASPV had the highest number of SNVs (477), while ACLSV had the lowest number of SNVs (1). The presence of ARWV-1, ApNMV, CiVA, ASPV, ASGV, ACLSV, ASGV-Shimla, and ASSVd was also confirmed in pear samples through Sanger sequencing. The present study widens our understanding of the pear virome and host range expansion of viruses in the genus Rubodvirus and Ilarvirus. This information will be useful in biological study of these viruses and virus indexing programs in pear.