Virome analysis deciphered the infection of American plum line pattern virus, little cherry virus 1 and plum bark necrosis stem pitting-associated virus in plum from India

As very limited information is available on viruses and viroids infecting plum in India, virome analysis was undertaken to determine the virus and viroid spectrum infecting plum. High throughput sequencing (HTS) analysis of five plum samples (PM1-PM5) revealed the presence of near complete genome sequences of prunus necrotic ringspot virus (PNRSV), American plum line pattern virus (APLPV), apple chlorotic leaf spot virus (ACLSV) and hop stunt viroid (HSVd) in PM1; little cherry virus 1 (LChV-1), plum bark necrosis stem pitting-associated virus (PBNSPaV), ACLSV and HSVd in PM2. No virus or viroid sequence was obtained in PM3, PM4 and PM5. Near complete genome sequences of these viruses were obtained using different assemblers. APLPV, LChV-1, PBNSPaV and HSVd are being reported here for the first time from plum in India. The presence of these viruses was also confirmed by reverse transcription polymerase chain reaction and Sanger sequencing in HTS samples as well as in additional 36 plum samples (PM6-PM41) collected from the same orchard from where PM1 and PM2 samples were obtained. The validation test in an additional 36 samples indicated predominance of APLPV and PNRSV which were detected in all the samples, while other viruses were present only in some of the samples. Moreover, fragments per kilobase of transcript per million reads mapped and virus accumulation studies showed dominance of PNRSV RNA3 in PM1 and LChV-1 in PM2. Analysis of single nucleotide variants (SNVs) showed that PNRSV RNA3 had the highest SNVs (99) and PBNSPaV had the lowest SNVs (1). The phylogenetic analysis revealed that these viruses are phylogenetically related to viruses from different countries. These findings advance our knowledge of the viral pathogens of plum, and would help in virus indexing and certification of clean plant for commercial plum cultivars.