Virome analyses by next-generation sequencing (NGS) in chilli (Capsicum anuum L.) presented with diverse symptoms phenotype revealed the association of seven plant viruses

Abstract

Chilli is an important vegetable and spice crop, is known to be infected by several viruses. Techniques used in diagnosis of plant viral diseases before Next-generation sequencing (NGS) are having limitation of identifying the only known viruses. In the present study, virome analyses in infected chilli leaf samples was carried out using NGS to know the diversity of both known and unknown viruses associated with diseased symptoms. For virome profiling, samples from 19 fields were collected from chilli plants showing leaf curling, vein banding, mosaic, mottling, shoestring/rat tail/filiform/leathery and dull coloured leaves. Viral disease incidence in the surveyed fields varied from 26.66% to 47.50%. Total RNA was extracted from the 19 chilli leaf samples collected from fields and were pooled at equimolar concentration for virome profiling. From the rRNA-depleted pooled total RNA, mRNA and sRNA libraries were prepared and sequenced using Illumina NOVASEQ 6000 platform. Raw sequence data obtained was de novo assembled using three approaches; mRNAome with Trinity, sRNAome with Velvet and whole transcriptome (WT) with SPAdes assembly. Chilli virome, pairwise sequence identity and phylogenetic analyses revealed the presence of seven different viruses; chilli leaf curl virus (ChiLCV) along with its associated alpha and betasatellites, cucumber mosaic virus (CMV), groundnut bud necrosis orthotospovirus (GBNV), pepper cryptic virus-2 (PCV-2), pepper vein yellows virus (PeVYV), bell pepper alphaendornavirus (BPEV) and tobacco vein clearing virus (TVCV). From the virus associated contigs, complete/near-complete genomes for ChiLCV, CMV, PCV-2, PeVYV and BPEV and, partial genomes for GBNV and TVCV were reconstructed from the RNAome. Recombination breakpoint analyses revealed presence of recombination breakpoints in ChiLCV (coat protein and AC4 regions), CMV RNA2 (2a protein region) and P0, P3 and P5 protein regions of PeVYV. viruses identified in the current study are known to be originated from intra and interspecific recombination. Further, all the viruses detected in the pooled RNA sample were validated by PCR and loop mediated isothermal amplification (LAMP) using specific primers designed. Among the seven viruses identified in the study in chilli, PeVYV and BPEV are the first reports from India.