Virus Identification And Analysis Of Three Garden Ornamental Woody Plants

High-throughput sequencing also known as next-generation sequencing(NGS),compared with the traditional virus identification method,has the characteristics of fast,high sensitivity,high throughput,sequence-independent and so on.It can quickly detect unknown viruses without understanding the biological characteristics,serological characteristics and genome information of viruses,which greatly promotes the development of virus diagnostics.In this study,three common ornamental woody plant virus diseases were detected and identified by high-throughput sequencing technique.The species of infected virus and the full length of genome were determined by transmission electron microscope observation,small RNA deep sequencing,transcriptional group sequencing and RT-PCR amplification.It was determined that the virus infecting Prunus davidiana(Carr.)Franch was a new virus in the genus Gratylivirus of Tymovirales order,named as Prunus yellow spot-associated virus(PYSaV);the virus infecting Syringa reticulata var.mandshurica was a new virus in the family Tombusviridae,named as Syringa tombus-like virus(STlV);the virus infecting Forsythia mandschurica Uyeki was Ligustrum virus A(LVA)of the genus Carlavirus of Betaflexiviridae family.This study laid a theoretical foundation for exploring new plant virus resources and preventing and controlling plant virus disease.In September 2017,a yellow spot leaf disease was noted on the leaves of Prunus davidiana(Carr.)Franch.plants in Liaoning,China,and spherical virions(approx.30 nm in diameter)were later observed in preparations of symptomatic leaves.Subsequent small RNA deep sequencing revealed the presence of a virus in these symptomatic leaves.The complete genome of this viral isolate consists of 6,072 nucleotides,excluding poly(A)tails.The virus showed closest genetic relationship to the Grapevine-associated tymo-like virus reported in Colmar,France(GaTLV,MH383239),which is the sole member of the newly proposed genus Gratylivirus within the order Tymovirales that is currently unassigned to a particular family.The virus clustered closely with GaTLV in a phylogenetic tree constructed based on complete genomic sequences.On the basis of the nucleotide and amino acid sequences of the replicase and coat protein genes,this virus shares the highest(although still relatively low)sequence similarities with those of GaTLV(41.6 % / 53.3 %),indicating that the virus is a distinct member of the Tymovirales,and for which the name Prunus yellow spot-associated virus(PYSaV)is proposed.To our knowledge,this is the first report of a virus naturally infecting P.davidiana.In July 2018,Syringa reticulata var.mandshurica leaves with mottled chlorosis were collected in Jilin,China.Subsequently,transcriptional group sequencing and RT-PCR amplification showed that there was a virus in these symptomatic leaves.The complete genome of this viral isolate consists of 5,492 nucleotides.The homology of the whole genome sequence with the nucleotide sequence of Hubei tombus-like virus 1(HTlV1,KX883244)was 49.4%.The virus clustered closely with HTlV1 in a phylogenetic tree constructed based on complete genomic sequences.It means that the virus is a new virus in the family Tombusviridae,which is named Syringa tombus-like virus(STlV).To our knowledge,this is the first report of the virus naturally infecting Syringa reticulata.In May,2018,a curved leaf yellow vein disease was noted on the leaves of Forsythia mandschurica Uyeki plants in Liaoning,China.In order to identify the infected virus species,transcriptional group sequencing and RT-PCR amplification and cloning were carried out.It showed that complete genome of the virus is 8,542 nt,which encodes the six typical proteins of the Carlsvirus genus and this virus shares the highest sequence similarities with Ligustrum virus A(LVA)by the BlastX.On the basis of the nucleotide and amino acid sequences of the replicase and coat protein genes,the homology is 81.7 % / 93.1 % and 86.8 % / 95.9 %.As a result,the virus is a isolate of Ligustrum virus A,named LVA-DBLQ.This is the first report of the Forsythia mandschurica infected LVA.