Detection And Identification Of Radish Virus Disease In Shenyang And Gaizhou

Transmission electron microscopy(TEM)and RT-PCR amplification techniques were used to detect and identify the viruses in the diseased Raphanus sativus L.samples in Shenyang and Gaizhou region of Liaoning province and the agent was determined to be Turnip Mosaic virus(TuMV)of Potyvirus,Potyviridae.In this paper,three full genetic sequences of TuMV and sixteen coat protein(cp)sequences of liaoning isolates were determined,which enriched the radish virus resources Liaoning province and laid a theoretical foundation for the prevention and treatment of radish virus disease.(1)In May 2017,six radish samples presenting bubble-like chlorosis symptom were collected in Shenyang city,Liaoning province.Linear virus particles(700 × 13nm)were observed by Transmission Electron Microscope(TEM),and they were initially identified as the Potyviuses.Universal primers PVY-legpoty-F/R were used to amplify the possible viral sequences and 1200 bp fragments were obtained.Blast comparison was conducted after cloning and sequencing and the results showed that they had the highest homology with Turnip Mosaic virus(TuMV).The reported specific primer TuMV-CP-F/R was used and fragments about 1000 bp including the complete coat protein(cp)were amplified from 6 diseased samples.Homology comparison showed that the nucleotide and amino acid sequence homology between the six isolates were higher than 90%,indicating that they were different isolates from the same virus.Isolate Ln-sy was selected to amplify the full using six pairs of primers,TuMV-1F/1R,TuMV-2F/2R,TuMV-3F/3R,TuMV-4F /4R,TuMV-5F/5R and TuMV-6F/6R based on the National Center for Biotechnology Information(NCBI)of the United States.The viral genome was determined to be 9855 nt.Based on the homology analysis of cp gene,Ln-sy shared the highest homology with TuMV-[China:Raphanus sativus](KR153038)and TuMV-[China:Raphanus sativus](KR153040),for 99.8%.Based on the cp gene,phylogenetic tree was constructied and results showed that TuMV Ln-sy clustered with TuMV-[China:Raphanus sativus](KR153038)and TuMV-[China:Raphanus sativus](KR153040)in the same branch.According to the classification standard of Potyvirus,Ln-sy is a isolated of TuMV isolated from Raphanus sativus L.in Shenyang,Liaoning province.(2)In May 2017,ten radish samples with dwarfing or mottled leaves symptoms were collected in Gaizhou city of Liaoning province.Lnear virus particles about 800 × 13 nm were observed in both symptomatic leaves using Transmission Electron Microscope(TEM),which preliminarily determined that the disease samples were infected by Potyviuses.The TuMV-CP-F/R primer was used to amplified possiblre cp geneand about 1000 bp fragments were all isolated from ten diseased samples.The full genome of TuMV-GZ1 and TuMV-GZ5 were found to be 9867 nt and 9856 nt,respectively.Based on the cp gene,it was found that both TuMV-GZ1 and TuMV-GZ5 were most closed to TuMV-[China:Raphanus sativus](KR153038)and tumv-[China:Raphanus sativus](KR153040),for 99.4%.Phylogenetic tree based on cp gene,two isolates clusted together with the TuMV-[China:Raphanus sativus](KR153038)and TuMV-[China:Raphanus sativus](KR153040)in the same branch.According to the classification standard of Potyvirus,TuMV-GZ1 and TuMV-GZ5 were also isolates of TuMV isolated from Gaizhou,Liaoning province.This is the first report of TuMV infected Raphanus sativus L.in Liaoning province.