Molecular Characterization And RT-PCR Detection Of Three Viruses Infecting Jujube Trees Grown At Aksu In Xinjiang Uygur Autonomous Region

Chinese jujube(Ziziphus jujuba Mill.)is a fruit crop widely grown at Aksu in Xinjiang Uygur Autonomous Region of China.Viral disease-like symptoms are common on jujube trees in this region,but the etiologies are not yet clear.Previously,our laboratory conducted field investigation and performed high-throughput sequencing(HTS)for RNAs of three diseased-jujube samples collected from the area and identified a jujube yellow mottle associated virus(JYMa V)belonging to the genus Emaravirus from samples AKS6 and AKS15.Meanwhile,contig sequences matched other three viruses were identified.In this study,these sequences were analyzed and the genome of three viruses were constructed.The infection status of the three viruses in the collected samples were determined by RTPCR assays.The main results were as follows:(1)By analyzing the HTS data of samples AKS15 and AKS6,two contig sequences with lengths of 14,142 nt and 14,229 nt were identified from the AKS15 library,which matched the genomic sequence of persimmon ampelovirus(PAmp V)in the family Closteroviridae.In the AKS6 library,four contigs of 6,062 nt,3,786 nt,3,276 nt and 1,405 nt,which matched the genomic RNA1～4 chain sequence of a blunervirus in the family Kitaviridae,were identified.In AKS6 and AKS15 libraries,two contigs of 5,786 nt and3,361 nt,and three contigs of 5,878 nt,5,918 nt and 3,068 nt matching the genomic sequence of the Chinese jujube virus F(CJVF)BJ isolate registered in NCBI were identified,respectively.The full-length genome sequences of the three viruses had been determined by RT-PCR.(2)Two molecular variants(AKS15-17 and AKS1520)of the PAmp V jujube isolate(PAmp V-Ju)had a genome size of 14,209 nt,shared nucleotide(nt)sequence identity of83.7% with each other and of 79.1% with PAmp V persimmon isolates.In the phylogenetic tree based on the amino acid(aa)sequences of Rd Rp,CP and HSP70 h in the family Closteroviridae,AKS15-17 and AKS15-20 were clustered in the same evolutionary branch with the two persimmon isolates of PAmp V.The infection of jujube by a closterovirus was reported for the first time.(3)The full genomic sequences of the four RNA chains of blunervirus were 6,105 nt,3,808 nt,3,528 nt and 1,398 nt in length,respectively.RNA1 and RNA2 each contained an ORF encoding replication-related proteins(Rep1 and Rep2).RNA3 contained five ORFs(ORF3a-e),of which ORF3 c detected a SP24 structural protein domain.RNA4 encoded a hypothetical movement protein.The genome organization of this virus was the same as that blunervirus.The highest nt sequence identity of the virus RNA1～4 to the corresponding gene chain of virus in the genus Blunervirus was 51.9%,54.2%,49.1% and 56.6%,respectively,which was closely related to the phylogeny of apple blunervirus 1(Ap BV1)and blueberry necrotic ring blotch virus(BNRBV).These results indicated that the virus was a new member in the genus Blunervirus,named jujube blunervirus 1(Ju BV1).Ju BV1RNA3 encoding five proteins were revealed subcellular localization characteristics.(4)The RNA1 and RNA2 of CJVF isolate AKS6 were 5,917 nt and 3,327 nt in length respectively;two sequences of 5,937 nt and 5,942 nt in length were obtained from isolate AKS15 RNA1,with nt sequence identity of 84.3% and RNA2 of 3,345 nt in length.The RNA1 of AKS6 and AKS15 shared 79.7% nt and 90.5% aa sequence identities with each other,and 78.1%-79.3% nt and 88.9%-90.1% aa corresponding sequence identities with isolate BJ,respectively.Their RNA2 showed 79.6% nt and 94.9% aa sequence identities with each other,and 79.7%-80% nt and 92.8%-94.3% aa corresponding sequence identities with isolate BJ,respectively.The RNA1 and RNA2 of CJVF each encoded a polyprotein protein.The study showed that MP,LCP and SCP encoded of CJVF RNA2 were located in the nucleus,plasma membrane,endoplasmic reticulum and plasmodesmata.(5)Based on the genomic sequences of PAmp V-Ju,Ju BV1 and CJVF,5,3 and 2 pairs of primers were designed for RT-PCR detection of these three viruses in 62 jujube leaf samples collected grown at Aksu.According to the detection results of each primer,the detection rates of PAmp V-Ju,Ju BV1 and CJVF were 37.1%,51.6% and 53.2%,respectively.The sequencing and phylogenetic analysis of the amplified fragments showed that the molecular variation of Ju BV1 was low,whereas PAmp V-Ju and CJVF had high molecular variation.The nt and aa sequence identity the Pol gene clones of PAmp V-Ju were 79.6%-99.7% and 84.4%-99.0%,respectively.The sequence identity the CP gene clones of the CJVF were 79.7%-100% and 95.0%-100% at nt and aa level,respectively.In conclusion,this study identified these three viruses based on high-throughput sequencing of leaf samples from diseased jujube plants.RT-PCR amplification provided the complete genome sequence of these three viruses,identification of their molecular characteristics,and analysis of the infection status of these three viruses in 62 collected jujube leaf samples.The results provide reference information for the establishment of detection systems,prevention and control of these three viruse.