Identification And Genetic Diversity Analysis Of Potyviruses

In this paper,viruses belong to the Potyvirus(Potyviridae)were identified from four plants by transmission electron microscope(TEM)and RT-PCR.Viruses infecting pumpkin,Ailanthus,Solanum nigrum and tomato,are Zucchini yellow mosaic virus(ZYMV),Watermelon mosaic virus(WMV),Chilli veinal mottle virus(ChiVMV)and Potato vitus Y(PVY),respectively.The research enriched resources of the plant virus in Liaoning Province and can provide the theoretical foundation for the control of the viruses.(1)Zucchini yellow mosaic virus,ZYMVSamples from the pumpkin(Cucurbita moschata)showing yellow mosaic symptoms were collected from Shenyang and Gaizhou,Liaoning Province,in August 2016.Virions measuring about 750×13 nm with a typical morphology of Potyvirus were observed by transmission electron microscope(TEM).The universal degenerate primers PVY-Legpoty-F/R was used for the detection of Potyvirus from three symptomatic leaf samples from Shenyang(LNsyA,LNsyB and LNsyC)and Gaizhou(LNgzA,LNgzB and LNgzC)by RT-PCR.With the reported specific primers ZYMV-F/ZYMV-R(701bp),the expected fragments about 1.2kb which contain complete coat protein gene(CP)was obtained.Based on nucleotide sequence of CP,the homology analysis results showed that they had the highest homology with the Shanxi isolate ZYMV-[CN:SX:Chrysanthemum:14](KP742962),99.1%-99.4%.The phylogenetic tree results showed that they were also clustered with ZYMV-[CN:SX:Chrysanthemum:14] in a branch.According to the classification criteria of Potyvirus,they are considered as the Liaoning isolate of ZYMV.(2)Watermelon mosaic virus,WMVSamples from the Ailanthus altissima showing yellow mosaic symptoms were collected from Gaizhou,Liaoning Province,China,in 2015.Virions measuring about 800×18 nm with a typical morphology of Potyvirus were observed by transmission electron microscope(TEM).The universal degenerate primers PVY-Legpoty-F/R was used for the detection of Potyvirus from three symptomatic leaf samples by RT-PCR.The expected fragments about 750 bp in size were amplified.By blast,it shared the highest nucleotide sequence identity with the published Watermelon mosaic virus(WMV).WMV-5NF/WMV-5NR,WMV-MNF/WMV-MNR and WMV-3NF/ WMV-3NR primers were then used to amplify the whole viral genome.It was determined to be 10070 nucleotide(MF418043).Based on the cp,it showed most closed to another WMV isolate from Ailanthus altissima.(3)Chilli veinal mottle virus,ChiVMVSamples from Solanum nigrum L.showing yellow mottled symptoms were collected from Shenyang,Liaoning Province,in 2016.The 737 bp positive fragment was amplified by PCR using PVY-Legpoty-F/M4.It shared the highest with the Chilli veinal mottle virus(ChiVMV).Specific primers ChiVMV-F/ChiVMV-R was designed and used to amplified the 1103 fragment,which contained the complete cp gene.The cp was 863 bp in size and encoded 287 amino acids.Based on the cp gene,ChiVMV-LNsy-Longkui isolate shared the highest homology with the ChiVMV-[CN:SC:CD:Lycopersicon esculentum:13](KF738253),for 98.6%.The virus was first isolated from Solanum nigrum L..(4)Potato virus Y,PVYIn August 2015,tomato samples showing yellow leaves were collected from Gaizhou,Liaoning Province.The cDNA was amplified by RT-PCR using the universal primer PVY-Legpoty-F/R of Potyvirus.The positive fragment was 675 bp.The sequence was most closed to Potato mosaic virus Y(PVY).The homology relationship confirmed that the virus on tomato was PVY.