Identification Of Virus Species In Alfalfa And Surrounding Weeds In Partial Regions Of Tibet By Deep Sequencing

In recent years,Tibet has actively promoted the construction of high-quality artificial grassland and promoted the development of animal husbandry.Alfalfa(Medicago sativa)has been introduced and planted as a high-quality forage.With the expansion of alfalfa planting area and the accumulation of planting years,virus diseases have gradually occurred and become more and more serious,which has affected the development of animal husbandry in Tibet.In order to identify the virus types of alfalfa virus diseases in Tibet,this paper carried out a virus disease investigation on alfalfa and its surrounding weeds in Zhanang County,Dazi County and Linzhi City,Tibet Autonomous Region,collected 211 alfalfa samples and 139 weed samples with suspected virus disease symptoms,and identified their virus pathogens by deep sequencing combined with RT-PCR technology,which preliminarily identified the main virus diseases of alfalfa in Tibet,the harm and distribution of viruses in the above-mentioned investigation areas,and provided data for the prevention and control of alfalfa virus diseases in Tibet.The specific results are as follows:Viral diseases have occurred in alfalfa planting areas in Zhanang County,Dazi County and Linzhi City,Tibet Autonomous Region,and there are many kinds of viruses.A total of 11 viruses were detected in 211 alfalfa samples with suspected symptoms of viral diseases,including medicago sativa amalgavirus 1(MsAV1),alfalfa mosaic virus(AMV),lucerne transient streak virus(LTSV),cucumber mosaic virus(CMV),cnidium vein yellowing virus(CnVYV),medicago sativa alphapartitivirus 1(MsAPV1),bean yellow mosaic virus(BYMV),bidens mottle virus(BiMoV),tobacco mosaic virus mosaic virus(TMV),brassica yellows virus(BrYV)and medicago sativa secovirus 1(MsSV1).Among them,medicago sativa secovirus 1 is a new member of Comovirus of Secoviridae,which was first discovered and temporarily named in this paper.Among the 11 viruses,the highest detection rate is BYMV,with a detection rate of 30.3%,followed by BrYV,with a detection rate of 18.5%.The most widely distributed viruses are MsAV1,AMV,LTSV and BrYV,which occur in three areas.In addition,this is the first report of MsAV1,AMV,LTSV,CnVYV,MsAPV1,BYMV,BiMoV and BrYV in Tibet,and the first report of TMV on Tibetan alfalfa,and the first report of CMV,BiMoV and BrYV on China alfalfa.The detection of 139 samples of weeds with suspected symptoms of virus disease showed that there were four kinds of virus infections,namely,AMV and MsAV1 were detected in Trifolium repens,MsAPV1 was detected in Vicia sativa and Taraxacum Mongolicum,TMV was detected in Pisum sativum.The detection rates of the four viruses were all low,which are 4.3%,2.9%,5% and 1.4% respectively.This is the first report of AMV on Trifolium repens in Tibet,the first report of MsAV1 on Trifolium repens,the first report of MsAPV1 on Vicia sativa and Taraxacum mongolicum,and the first report of TMV on Pisum sativum in Tibet.The detection of AMV,TMV,MsAV1 and MsAPV1 on weeds around alfalfa indicates that the four viruses may spread in the field through weeds.Members of the genus Comovirus contain two RNA sequences.The whole genome sequence of MsSV1 was analyzed,and it was found that there was no poly(A)tail,and the full length of RNA1 was 5784 nucleotides(nt)and RNA2 was 3343 nt,which contained an Open Reading Frame(ORF)and encoded 1820 amino acids(aa)and 1009 aa respectively.The results showed that the amino acid sequence of CP(large coat protein and small coat protein)of MsSV1 was 20.04%-31.81% homologous to other members of Comovirus.Pro-Pol(Pro: proteinase;The amino acid sequence of Pol:RNA-dependent RNA polymerase has 21.75%-33.78% homology with other members of Comovirus at Pro-Pol amino acid level,so it should be a new member of Comovirus.The phylogenetic tree was constructed by using MsSV1,some viruses in9 virus genera of the associated Secoviridae,and some unknown viruses based on polyprotein,which showed that MsSV1 was a member of Comovirus genus together.The healthy seedlings of Pisum sativum、Glycine max、Vigna unguiculata、Vicia faba、Luffa cylindrica and Solanum lycopersicum were inoculated with MsSV1 by friction inoculation and injection inoculation.It was found that all the above plants could not be infected by MsSV1,and their host range needed further study.