Identification And Molecular Characterization Of Two Unknown Viruses Infected With Areca Palm

Areca catechu L.is the second largest cash crop in Hainan,with a planting area of 99,661 hectares.Areca catechu forests are mostly pure forest structure with single species,which is very conducive to the spread and spread of pests and diseases,leading to the occurrence of Areca catechu diseases and insect pests more and more serious in recent years.Recently,two new viral diseases have been found in the main areca plam growing area in Hainan,we named them areca plam necrotic spindle-spot disease(ANSSD)and areca plam necrotic ring spot disease(ANRSD).This study focuses on the epidemiology of ANSSD and ANRSD,the identification of viral pathogens,the genetic diversity of virus isolates and the analysis of virus-disease correlation.The main results are as follows:1.Epidemiological study of ANSSD and ANRSD in main areca plam producing areas in HainanThe incidence of ANSSD and ANRSD was investigated from July 2017 to September 2018 in the main areca plain producing areas of Hainan.The results showed that ANRSD was an epidemic disease,causing serious harm to areca plam production.The average incidence of ANRSD was 19%in the survey area,and the highest in Wanning,Ding'an and Qionghai,with 46%,45%and 36%respectively.ANSSD was only found in Baoting,with an average incidence of only I%.2.Pathogen identification of ANSSD and ANRSDThrough transmission electron microscope(TEM),deep sequencing of siRNA and biological characteristics,we found that there were viral pathogens in ANRSD and ANSSD samples,named as areca necrotic spindle spot virus(ANSSV)and areca necrotic ring spot virus(ANRSV).Curved linear virus particles were observed in the extracts of ANSSV and ANRSV leaf samples under transmission electron microscope,which were about 15*780 nm in size.ANSSV and ANRSV were successfully inoculated into Nicoliana henthamiana by juice friction method.After 7 days,the infected plants showed typical symptoms of virus diseases.A total of 44 and 24 contigs with high homology to Potyviridae virus were obtained from ANSSV and ANRSV samples by siRNA deep sequencing and NCBI Blastx.We speculate that both ANRSV and ANSSV are new members of Potyviridae family.3.Genome determination and annotation of ANSSV and ANRSVThe whole genome sequences of ANSSV and ANRSV were obtained by cloning and sequencing.By NCBI Blastx comparison,they have the highest homology with open reading frame(ORF)nucleotide sequences of members of Polyviridae family,with 44.6%(ANSSV)and 42.40%(ANRSV),respectively,which were lower than the threshold of genus classification of Potyviridae family(46%).Therefore,ANSSV and ANRSV were considered as unreported genus of Potyviridae family.Both of them contain a large open reading frame(ORF)with 5' and 3' untranslated regions on both sides.ORF were cut into 11 mature proteins,and N-terminal contains two cascaded cysteine proteases HC-Prol and HC-Pro2.At present,the Koch postulates of two diseases has not been completed.Therefore,it is uncertain whether ANSSD and ANRSD are related to these two newly discovered viruses respectively.4.Phylogenetic analysis of ANSSV and ANRSVPhylogenetic tree analysis of amino acid sequences of ANSSV,ANRSV and representative members of Potyviridae families.The results showed that ANSSV and ANRSV were classified as a separate branch and belonged to the "Arepavirus" genus of Potyviridae family.Therefore,ANSSV and ANRSV are members of the newly discovered "Arepavirus" genus in the Potyviridae family."Arepavirus" is closely related to Machtravirus,Bymovirus and Bevemovirus.5.Characterization of virus-derived small RNA(vsiRNA)of ANRSVIt was found that that no obvious hotspots of generating vsiRNAs in the whole genome of ANRSV,and the frequency of covering the just genome was significantly higher than that of covering the anti-sense genome.Regardless of the length of vsiRNA,the number of vsiRNAs derived from the righteous genome of ANRSV was larger than that derived from the anti-sense genome of ANRSV,and it was speculated that the secondary structure of single-stranded RNA genome can be used as a matrix for DCL-mediated cleavage,leading to asymmetric distribution of vsiRNA in the polarity of the strand.ANRSV is only 21 nt rich in vsiRNA,so it is presumed that DCL4 is the main dimer ribonuclease involved in the synthesis of ANRSV vsiRNA in areca plain.Moreover,the 5'-end of the vsiRNAs of ANRSV usually has a strong preference for A/U,which presumed that the vsiRNA of ANRSV is preferentially absorbed by AGO I,AG02 and AG04.6.Analysis of genetic diversity of ANRSVThe nucleotide homology and amino acid homology of CP genes of ANRSV isolates were 91.6%-100%and 95.0%-100%respectively,but the nucleotide and amino acid homology of ANSSV and ANRSV were low(80.1%-81.5%and 86.8%-88.6%).Accordingly,phylogenetic tree analysis showed that all ANRSV were significantly different from ANSSV.7.RT-PCR Detection TechnologyUsing the CP gene of ANRSV to design primers,the samples with ANRSD symptoms and asymptomatic in Areca catechu leaves were detected by RT-PCR,with the high detection rate in 95.6%.It is proved that ANRSV is closely related to ANRSD,and that the detection primers and methods designed are efficient,which can be used as a means for the detection and identification of ANRSV in the future.