Identification And Molecular Characterization Of Lily And Rose Viruses

Lily(Lilium spp.)and China rose(Rosa chinensis Jacq.)are important ornamental and garden flowers which are mainly propagated by asexual means.Virus disease is an important factor affecting their quality and yield.Cultivating and planting virus-free seedlings is an effective way to prevent the virus disease of horticultural plants.Clarifying the virus species infesting lily and China rose and establishing and perfecting the accurate and rapid detection system of virus are the premise of cultivating virus-free seedlings.In this study,high-throughput sequencing(HTS)and reverse transcription-polymerase chain reaction(RT-PCR)were conducted to identify and molecularly characterize the viruses in lily and China rose samples,the main results are as following:1.Virus-infected lily samples collected from Yunnan and Zhejiang Provinces were analyzed by using high-throughput sequencing(HTS)technique.Nine known viruses including Lily mottle virus(LMo V),Dahlia mosaic virus(DMV),Cucumber mosaic virus(CMV),Figwort mosaic virus(FMV),Plantago asiatica mosaic virus(Pl AMV),Rose yellow vein virus(RYVV),Dahlia common mosaic virus(DCMV),Cassava vein mosaic virus(Cs VMV)and Lily amalgavirus 1(LAV1),and one new viruse were identified through sequence alignment and assembly.Then 48 lily samples collected from Yunnan,Zhejiang,Jiangxi,Guangdong,Hunan Province and Shanghai City were detected by RT-PCR.The results showed that DMV,RYVV,Pl AMV and LMo V were predominant and the positive detection rates of them were above 95%.The positive rate of FMV was higher than 85%,while that of CMV was the lowest,which was only positive in the samples from Kunming,Yunnan Province.DCMV and Cs VMV were not detected in these samples.The mixed infection rate of lily virus was 100%,and 3-8 viruses were positive in a single sample.The mixed infection of lily virus in Kunming,Yunnan Province was the most complicated,with 8types of virus complex infection.The complete sequence of lily amalgavirus 2(LAV2),a new virus of lily,was obtained with a complete length of 3432 nucleotide(nt).Sequence analysis showed that the full-length nucleotide sequence similarity between LAV2 and other members of the genus Amalgavirus ranged from 46.04% to51.59%,phylogenetic analysis based on Rd Rp encoded by LAV2 showed that LAV2 belongs to a new virus of the genus Amalgavirus.2.Virus-infected China rose samples collected from Nanyang city,Henan Province,Anyi County,Jiangxi Province and Guangzhou City,Guangdong Province were analyzed by using HTS.Nine known viruses including RYVV,Apple mosaic virus(ApMV),Blueberry fruit drop associated virus(BFDa V),Prunus necrotic ringspot virus(PNRSV),Rose partitivirus(Ro PV),Apple stem grooving virus(ASGV),Rose spring dwarf-associated virus(RSDa V),Rose leaf rosette-associated virus(RLRa V)and Blackberry chlorotic ringspot virus(BCRV),and two new viruses were identified through sequence alignment and assembly.The two new viruses were tentatively named Rose latent virus(Ro LV)and Rose latent virus 1(Ro LV1).The complete sequence of Ro LV genome was 7528 nt,encoding two ORFs.Sequence alignment analysis showed that the complete genome nucleotide sequence,ORF1 and ORF2 amino acid sequences of Ro LV had the highest similarity with the corresponding sequences of Pe LV,which were 50.72%,42.43% and 44.82%,respectively.Phylogenetic analysis showed that Ro LV was most closely related to Pe LV(NC＿023983)and belonged to the unclassified ds RNA virus.The complete sequence of Ro LV1 was 8832 nt,encoding two ORFs,ORF1 encoded a putative285.6 k Da replicase,with the highest amino acid sequence similarity of 25.03% to the replicase encoded by rubber tree latent virus 1(RTLV1)(QXO33012),and ORF2 encoded a putative protein of 27.7 k Da.Ro LV1 complete sequence showed the highest nucleotide similarity of 31.88% with opuntia virus 2(Genbank no.NC＿040685.2),and the phylogenetic analysis showed that Ro LV1 belonged to Virgaviridae.3.The genetic variation of CP and MP gene sequences of ApMV,PNRSV and ASGV isolates of China rose was studied.The results showed that the CP gene and MP gene sequences of three viruses isolated from China rose had high similarity and little genetic variation.Phylogenetic analysis showed a correlation between the phylogeny of ApMV and the hosts,with most viruses isolated from the same hosts clustered in the same subgroup;The PNRSV rose isolates from Nanyang city of Henan Province showed molecular variation.In the phylogenetic tree of CP and MP nucleotide sequences,the rose isolates were distributed in two subgroups.The ASGV rose isolates had a closely genetic relationships,while the ASGV derived from other countries and hosts had a long genetic distance,indicating that ASGV clustering was correlated with hosts and regions.4.Complete genome sequence of ApMV was obtained from China rose for the first time,the infectious clone of ApMV was successfully constructed with infesting activity against Nicotiana benthamiana and cucumber(’Suyo’).ApMV was positive detected by RT-PCR in N.benthamiana and cucumber systemic leaves via agroinfiltration at 28 dpi(days post-inoculation),and in N.benthamiana systemic leaves via sap inoculation at 7 dpi,but no obvious symptoms were observed.