Viromic Approaches Of Pumpkin Major Producing Regions In China Through Transcriptomic And Metagenomics

China is the leading country for pumpkin production in the world.As with other cucurbits,diseases caused by viruses are among the serious threats to pumpkin production,but our knowledge of the virus species infecting pumpkin plants is fragmentary.To determine the geographical distribution characteristics,relative abundance and evolutionary relationship of pumpkin infecting viruses,159 samples that exhibited typical viral symptoms collected across China were subjected to metatranscriptome sequencing(RNA-seq)and viromic analysis in this study.19 highly confident viral contigs were retained from 2048 viral contigs in the seven sequencing libraries from different sampling regions,which could be identified as 11 known and three new viruses,including DNA and RNA viruses,as well as single-segment and multi-segmented viruses.RT-PCR using newly designed primers for five known confirmed the results of the meta-transcriptome analysis.Interestingly,three new viruses identified in this study should be single-stranded positive-sense RNA viruses whose hosts are prokaryotes actually.The known viruses identified in different sampling regions exhibit significant variations in term of virus species and relative abundance.However,our data demonstrate there is a consistently high proportion of Potyviruses across all samples.Mixed infections are common in tested samples;meta-transcriptome data analysis reveals that,apart from the samples collected in Chengdu,only PRSV was detected.However,at least three plant viruses were identified in all other samples,with the highest number of 7 plant viruses found in the Kaifeng sample.The results obtained in this study provide valuable information for understanding the virus species and their diversity in cultivated pumpkin plants across major pumpkin-growing regions of China.Next,the complete genomic sequence of a novel phage with a positive-sense single-stranded RNA(+ss RNA)genome was obtained in pumpkin leaves by RT-PCR and rapid amplification of c DNA ends(RACE)techniques.The complete genome of this novel phage is 3,552 nucleotides(nt)in length and encodes three proteins,including a maturation protein of 394 amino acids,a coat protein of 144 amino acids,a RNA replicase protein of 524 amino acids.The complete nucleotide sequence of the genome was found to be 43.30%-45.50% identical to other Fiersviridae phages,while the maturation protein(Mat P),capsid protein(CP),and RNA replicase(RP)amino acid sequences were 17.60%-28.20%,21.10%-39.30%,and 28.50%-34.20% identical,respectively,to the corresponding sequences of other Emesvirus members.Phylogenetic analysis based on the amino acid sequences of the polyproteins of the new Chengdu Emesvirus(NG)phage and other Fiersviridae phages confirmed that this phage is a member of the genus Emesvirus,family Fiersviridae.Subsequent experiments revealed that the phage is derived from an endophytic Enterobacteria within the pumpkin plant.