Identification Of Novel Viruses Infecting Watermelon Through High-throughput Sequencing

Watermelon(Citrullus lanatus)represents one of the most important cucurbit crops for human consumption throughout the world.Like other crops grown in the open fields,it is frequently exposed to various pathogens including fungi,bacteria and virus.Viral diseases have led to serious yield and quality losses of watermelon in the world.However,there were few studies on new virus identification in watermelon in the past times.Recently,the developments of high-throughput sequencing and bioinformatics accelerate the entire process of novel virus discovery.In this study,the watermelon samples collected from Kaifeng,Henan province in 2015 and 2016 were used to identify the causal agents by high-throughput sequencing,combined with RT-PCR and sequences analyses.A total of eight viruses were identified: five known viruses,i.e.,Watermelon mosaic virus,Zucchini yellow mosaic virus,Melon aphid-borne yellows virus,Cucumber green mottle mosaic virus and one dsRNA virus named Citrullus lanatus cryptic virus(CiLCV)reported for the first time in China;three novel RNA viruses,tentatively named Watermelon crinkle leaf virus 1(WCLV-1),Watermelon crinkle leaf virus 2(WCLV-2)and Watermelon virus A(WVA),respectively.The molecular and biological properties of 3 novel viruses and CiLCV were studied in this work.The multipartite genomes of WCLV-1 and WCLV-2 contain three RNA molecules of approximately 6.8,1.4 and 1.3 kb,each containing a single open reading frame(ORF)in the negative polarity.The genomes and the deduced proteins of RNA1 and RNA3 show features resembling those of members in the genera Phlebovirus and Tenuivirus.However,the predicted proteins encoded by RNA2 are slightly related to the movement protein(MP)in the genera Ophiovirus and Emaravirus.Furthermore,these two viruses define a novel clade,which is phylogenetically related to the viruses in above four genera.In addition,observation of infected crude sap by transmission electron microscopy reveals the presence of tenuivirus and ophiovirus-like flexuous virions.Moreover,WCLV-1 can be mechanically transmissible to the natural host watermelon plants.Based on these combined results,we propose a novel genus named Wacrlevirus in the order Bunyavirales.The complete nucleotide sequence of WVA is 8,372 nucleotide(nt),containing a large open reading frame(ORF1)and three smaller ORFs(ORF2-4).The largest ORF1 is 5,481 nt long and encodes a putative replication-associated polyprotein(RP)of 1,826 amino acids(aa).ORF2 and ORF4 encode movement protein(MP)and coat protein(CP),respectively.The analysis of phylogeny indicates that WVA forms a well-supported and independent phylogenetic cluster,distant from the reported members in the family Betaflexiviridae.Furthermore,different from the genomic organizations of known viruses in the family Betaflexiviridae,one putative protein with estimated molecular mass of 25 kDa between MP and CP was predicted and no similarity to any recognized proteins was characterized.Therefore,we suggest WVA may represent a novel genus in the family Betaflexiviridae.The complete genome of the dsRNA virus is comprised of dsRNA-1(1,603 nt)and dsRNA-2(1,466 nt),both of which have single open reading frames and potentially encode a 54.2 kDa RNA-dependent RNA polymerase(RdRp)and a 45.9 kDa coat protein(CP),respectively.The RdRp and CP share the highest amino acid identities 85.3% and 75.4% with a previously reported Israeli strain Citrullus lanatus cryptic virus(CiLCV),respectively.Genome comparisons indicate that this virus is the same species with CiLCV,whereas the reported sequences of the Israeli strain of CiLCV are partial,and our newly identified sequences can represent the complete genome of CiLCV.Futhermore,phylogenetic tree analyses based on the RdRp sequences suggest that CiLCV is one member in the genus Deltapartitivirus,family Partitiviridae.In addition,field investigation and seed-borne bioassays show that CiLCV commonly occurs in many varieties and is transmitted through seeds at a very high rate.In this research,the investigation and characterization of watermelon virus diseases were carried out,and the types of viruses were identified,which provided important guidance for developing comprehensive control strategies.