Molecular Identification And Analysis Of Viruses Infecting Three Horticultural Plants

In this study,the viruses infecting three horticultural plants in Shenyang were identified by transmission electron microscopy,transcriptome sequencing,RT-PCR amplification and molecular cloning.The virus infecting Arachis hypogaea L.was identified as Liaoning isolate of bean common mosaic virus belonging to Potyvirus of Potyviridae.The virus infecting Impatiens balsamina L.was identified as a new virus in Virgaviridae.The virus infecting Rosa chinensis Jacq.was identified as the Liaoning isolate of rose yellow mosaic virus belonging to Roymovirus of Potyviridae.In this study,the viruses infecting three horticultural plants were identified accurately,which accumulated theoretical basis for the prevention and control of related horticultural plant virus diseases.(1)In September,2017,A.hypogaea L.samples with obvious mottled and faded leaves were collected in Shenyang,Liaoning Province,which were suspected to be infected by plant virus.The linear virus particles of about 15nm×800nm were observed under transmission electron microscope after negative staining.A virus belonging to Potyvirus of Potyviridae was amplified from diseased leaves by transcriptome sequencing,RT-PCR amplification and molecular cloning.The virus genome contains10,076 nucleotides,encoding a typical cleavable Polyprotein with Poly(A)tail at its 3'end.Compared with BCMV Wuhan Glycine max isolate(BCMV-[CN:WH:Glycine max],KJ807813),the homology of nucleotide and amino acid was 99.4% and 99.7%,phylogenetic analysis showed that it was clustered on the branch of BCMV A.hypogaea L.isolate.The above analysis shows that the virus is an isolate of BCMV,which is the first report of natural infection of A.hypogaea L.by BCMV in Liaoning.(2)In July,2016,I.balsamina L.with shriveled leaves were collected in Shenyang,Liaoning Province,and virus particles with a size of about 10nm×320nm were found by observing the diseased leaves of I.balsamina L.with transmission electron microscope.A Virgaviridae virus was amplified from the diseased leaves of I.balsamina L.by transcriptome sequencing,RT-PCR amplification and molecular cloning.The nearly full-length genome of the virus contains 11,363 nucleotides and four open reading frames(ORFs).According to the homology comparison between the movement protein and coat protein genes,the results showed that it had the highest homology with the Podosphaera prunicola tobamo-like virus(PPTLV),which has not been classified in Virgaviridae.The homology of nucleotide and amino acid of cp gene was 81.5% and87.2%,and that of mp gene was 77.5% and 81.5%.And in the phylogenetic tree constructed based on cp gene sequence,the virus is on the same branch as Pp TLV,and does not cluster with other virus sequences.The above analysis shows that the virus is a new member of Virgaviridae,which can not be classified into genus yet,so it is temporarily named Impatiens tobamo-like virus(ITLV).(3)In July,2019,R.chinensis Jacq.samples showing mosaic symptoms were collected in Shenyang,Liaoning Province.A virus belonging to Roymovirus of Potyviridae was amplified from the diseased leaves of R.chinensis Jacq.by transcriptome sequencing,RT-PCR amplification and molecular cloning techniques.The genome of the virus consists of 9,692 nucleotides,encoding a large polyprotein.According to the nucleotide sequence and coat protein gene homology comparison,the results showed that it had the highest homology with Rose yellow mosaic virus of American Rosa rugosa isolate(JF280796)and Japanese R.rugosa isolate(LC466941).The homology of nucleotide and amino acid of cp gene with RYMV American R.rugosa isolate(JF280796)is 84.1%,84.7% and 88.3% respectively,and the homology of nucleotide and amino acid of cp gene with RYMV Japanese R.rugosa isolate(LC466941)is 83.7%,85.9% and 89.9% respectively.The phylogenetic tree was constructed based on whole genome nucleotide sequence,and the results showed that the virus was clustered with the virus related to Roymovirus,and had the closest genetic relationship with RYMV American isolate(JF280796)and RYMV Japanese isolate(LC466941).The above analysis shows that the virus is an isolate of RYMV,which is the first report that RYMV naturally infects R.chinensis Jacq.at home and abroad.