Molecular Characteristics And RT-PCR Detection Of Jujube Yellow Mottle-Associated Virus Infecting Jujube Plants Grown At Aksu In Xinjiang Uygur Autonomous Region

Jujube is an economically important fruit tree at Aksu in Xinjiang Uygur Autonomous Region.In recent years,it has been found that a large number of jujube trees at this area showed virus-like symptoms,including mosaic and mottle.To identify the pathogen causing the disease,small RNA(s RNA)sequencing and RNA sequencing(RNA-seq)combined with conventional RT-PCR technology were carried out in this study.A virus(jujube yellow mottle-associated virus,JYMa V)belonging to genus Emaravirus was identified from jujube samples collected from Aksu.Furthermore,the virus was molecularly characterized and RT-PCR methods were established for the specific detection of the virus.The obtained results are helpful for understanding the molecular characteristics of JYMa V and controlling jujube virus disease.The results are as follows:1.The genome sequence of an isolate JYMa V-HZ were derived from a sample of jujube cv.huizao by using s RNA sequencing and RT-PCR technology.The genome of JYMa V-HZ contains six negative-sense single-stranded RNAs(-ss RNAs).The sizes of RNAs 1-6 are 7160 nts,2221 nts,1228 nts,1493 nts,1241 nts and 941 nts,respectively.The complementary strand of each genomic RNA contains one open reading frame(ORF).The encoded proteins of these ORFs have 91.2%-97.3% amino acid(aa)identity with the corresponding proteins of JYMa V isolate SY(JYMa V-SY)reported recently.AKS-6 obtains nearly full-length sequences of five RNAs(RNAs1-5)and six RNAs(RNAs 1-6)of JYMa V were obtained from sample AKS-15 by RNA-seq.The ORFs of the two isolates shared 90.2%-99.3% nt and 90.3%-99.4% aa identitites with the corresponding ORFs of JYMa V-SY,respectively.2.Three pairs of primers for the specific detection of JYMa V were designed basing on the the sequences of JYMa V genome RNAs 3-6.Totally,86 leaf and fruit samples of jujube,of which 61 samples were symptomatic collected from Aksu,and ten asymptomatic samples collected from Shanxi province were subjected to RT-PCR detection for JYMa V.Results showed that the positive rate of JYMa V symptomatic samples was 91.8%,confirming that JYMa V infection is associated with the mosiacand mottle disease of jujube trees.All the three pairs of primers can be used to detect JYMa V,and the primers designed on RNA5 showed a relatively high positive rate.3.Totally,17 JYMa V-positive samples were tested by RT-PCR using primer set5H/3C targeting to the conserved sequences at the ends of emaravirus genome RNAs.Cloning and sequencing of amplified products revealed 6,17,7,17,and 13 sequences of the viral RNAs2-6,respectively.Moreover,21 and 17 sequences of partial fragments of the viral RNA1 and RNA4 were obtained.These sequences were compared with the corresponding sequences of three JYMa V isolates HZ,AKS-6 and AKS-15 identified by deep sequencing and a reported SY isolate.Results showed that the virus population identified from jujube trees at Aksu were molecularly variable.The ORF sizes of RNA2 and RNA4 were different from those of isolate SY.There are insertion or deletion sites in the 5’ untranslated region of the viral RNAs 2-6,in contrast,the sequence of the 3’ untranslated region is relatively conserved.In addition,the 3’ untranslated region of the viral RNA1 contains repeated fragments in diffrent isolates.Phylogenetic analysis revealed that diffrent JYMa V isolates formed different evolutionary branches.4.The ultra-thin sections of diseased jujube leaves and petioles are observed under a transmission electron microscopy.It was found that there were many spherical virus particles with a double membrane structure in the cytoplasm of the diseased leaves.The sizes of particles are between 100-110 nm,but the virions was rarely observed in the petiole sections,indicating the uneven distribution of the virus in host plants.