Identification Of Viral Causal Agent For Two Citrus Disease Through High-throughput Sequecing

Citrus is one of the most widely grown fruit crops in the world,of which China is a major citrus producer with the largest area and production.Citrus is mainly cultivated by asexual propagation and easy to infect and accumulate one or more viruses during long-term grafting and reproduction.The viruses spread through seedlings and scions for long distances.Both agricultural operations in the field and vector insects can disseminate these viruses,which results in low yields and short life span of fruit trees,resulting in major economic losses.Therefore,detection and identification of citrus virus are the basis for prevention and control of viral spread.The distribution of citrus viruses in plants is uneven,low in accumulation,seasonal variation,and latent infection.These factors limit the identification of viruses by traditional virus detection methods.Deep sequencing(such as transcriptome sequencing)has increased the identification efficiency of citrus viruses and been used in recent years to identify both unknown and known viruses of citrus.In this study,two different citrus viral diseases were identified by high-throughput sequencing.The results are described as below.Transcriptome sequencing analyses for the ‘Huangjinmiyou’ pomelo samples from Fengdu in Chongqing that exhibited mosaic symptoms,only identified a virus with high homology to the Citrus tatter leaf virus(CTLV)or Apple stem grooving virus(ASGV),indicating that ‘Huangjinmiyou’ pomelo might be infected with ASGV.To further verify the results,the complete genome sequences of three ASGV isolates(ASGV-HJY-1,ASGV-HJY-2,and ASGV-HJY-3)were obtained using segmented RT-PCR and RACE-PCR.Sequence analysis showed that the genome lengths of three ASGV isolates were all 6,496 nt,and their genome structures were the same as that of discribed CTLV and ASGV.The whole-genome sequence alignments showed that ASGV-HJY-1 and ASGV Brazilian apple isolates(KX66848)had the highest similarity(84.0%)while ASGV-HJY-2 shared a highest similarity with ASGV Chinese apple isolates(KF434636)and ASGV Japanese apple isolates(D14495)(92.4%),and ASGV-HJY-3 and CTLV isolates of Chinese orange(JQ765412)were most similar(97.5%).Based on the genome evolutionary analyses,ASGV-HJY-3 and CTLV citrus isolates were clustered in the same branch,ASGV-HJY-1 was grouped together with three ASGV isolates from apple and pear,ASGV-HJY-2 clustered with four ASGV apple isolates into the same branch.Based on the phylogenetic analyses of coat protein(CP)nucleotide sequences of related viral isolates,three virus groups clustered in the tree were reaveled that ASGV-HJY-1 and ASGV-HJY-2 were in Group 1 while ASGV-HJY-3 in Group 2.Compared viral isolates from different geography or host found sequence diversity in the CP regions was not significant.These differentiation relationships are instructive for studying the origin and evolution of CTLV and ASGV.On base of biological analyses,leaves of the virus-infected ‘Huangjinmiyou’ pomelo at room temperature(20-24 ℃)were mosaic and shrunken,whereas at high temperatures of 30-35 ℃ these symptoms were spotted disappeared.RT-PCR and RT-qPCR assays were used.The results showed that ASGV was not detected in ‘Huangjinmiyou’ pomelo leaves without symptoms,but was detected in leaves with obvious symptoms.High-temperature treatment might inactivate or deactivate the ASGV virus in ‘Huangjinmiyou’ pomelo and changed the symptoms.So at the biological level,it was a preliminary assessment that ‘Huangjinmiyou’ pomelo mosaic disease was caused by ASGV.Interestingly,the virus-infected ‘Guanximiyou’ pomelo had no obvious symptoms similar to healthy plants.However,‘Huangjinmiyou’ pomelo was a kind of ‘Guanximiyou’ pomelo from bud mutation breeding,and it proposed that some mechanisms present in ‘Guanximiyou’ pomelo was resistant to ASGV and ‘Huangjinmiyou’ pomelo as a variety was susceptible to ASGV.Through field investigation,AGSV was detected in ‘Huangjinmiyou’ pomelo with mosaic symptoms in Chongqing,Sichuan,Fujian,and Jiangxi,but the positive rates were different.Using transcriptome sequencing analysis,a positive single-stranded RNA virus(temporarily named Haruka-associated Citrivirus,HaCV)was identified in the Haruka sample expressing leaf mottle.The full genome sequence of HaCV was obtained by transcriptomics and RT-PCR techniques.The full-length genome of HaCV was 8697 nt with three open reading frames that was 62.9%(maximally)identical to Citrus leaf blotch virus(CLBV).Compared with the known CLBV isolates,HaCV had a shorter genome,mainly due to the deletion of 11 amino acids from ORF1;difference in the starting sequence of 5’UTR was CGAAAA of HaCV while GAAAA of other CLBV isolates.HaCV coded ORF2 without frameshift different from previously published CLBV.Because HaCV was similar to Citrivirus in genome structure,and clustered with CLBV isolates in the same group based on the phylogenetic trees of genome,replicase polyprotein(RP)and CP,suggesting that HaCV is a novel member of the genus Citrivirus.According to classification criteria of viruses in the family Betaflexiviridae defined by the International Committee on Taxonomy: When the amino acid sequence similarity of the RP or CP gene is less than 80% or the nucleotide sequence similarity is less than 72%,it is considered as a new species.Based on CP sequence analysis,the nucleotide and amino acid sequence identitiy bettween HaCV and CLBV isolates were 76.4%-85.6% and 87.6%-95.3% respectively,in this regard HaCV was like a divergent strain of CLBV.However,HaCV shared the low homology with CLBV citrus isolates from different countries based on the full genome,and in paticular the nucleotide sequences of the 5’UTR and 3’UTR were significantly different.In addition,the RP of HaCV and the published CLBV isolates shared the identities of nucleotide and amino acid sequences ranged from 55.0%-55.7% and 51.3%-51.9% respectively were significantly lower than the viral species classification criteria.Based on these results,we considered HaCV be a new species of the genus Citrivirus.