Identification And Molecular Characterization Of Viral Pathogens Of Grapevine Mosaic Disease

Grapes belong to Vitaceae,Vitis L.They are woody vines.In recent years,with the continuous expansion of the grape planting industry,the irregular propagation and introduction have led to the widespread spread of viral diseases of grapevine to various places,causing large area spread of viral of grapevine.However,the traditional method of virus detection is not perfect,and the detection process is long and inefficient.Therefore,exploring fast and effective detection methods is of great significance for the identification of viral diseases of grapevine.In this study,grapevine mosaic disease was used as the research object,the species and molecular characteristics of the viral pathogens were identified by small RNA(sRNA)and RNAseq deep sequencing.Furthermore,the molecular characteristics of GFkV were analyzed.Eventually,a RT-PCR method for the detection and differentiation of viral pathogens was established,which would be served as an important analysis method for the breeding and healthy cultivation of grapevine virus-free seedlings.The main results are as follows:(1)The grape mosaic symptoms were investigated from May to October 2017 in Qing Xiling.The investigation found that there are a total of 10 grapevines in the vineyard,including 8 varieties,namely Kyoho,Fujiminori,Miyoshi 40,Giant Rose,Xikang,Margherita,Red and Black,and the grape mosaic virus in all varieties,and the grape mosaic virus in all varieties.Infection,and the highest incidence of 31.11%.It was shown that grape mosaic virus was caused by multiple infections of multiple viruses.Therefore,infection of multiple varieties of grapes can be caused and it is difficult to control.(2)In the vineyards of Qing Xiling,Baoding,the grape leaves with chlorotic mottle,floral mottle and veins were collected.Through the Illumina sequencing platform for sRNA sequencing,the sequencing results showed that the grape mosaic virus caused by the mixed infection of six viruses.Three viruses i.e.,Grapevine fleck virus(GFkV),Grapevine rupestris stem pitting-associated virus(GRSPaV),Grapevine leafroll-associated virus(GLRaV-2).Three viroids,i.e.,Grapevine yellow speckle viroid 1(GYSVd-1),Grapevine yellow speckle viroid 2(GYSVd-2),and Hopstunt viroid(HSVd).The study of grape mosaic disease caused by the combined infection of the 6 viruses has not been reported.(3)Obtain 4,735,047 sRNAs by using Illumina HiSeqTM2000/MiSeq sequencing.The length of sRNAs data is mostly concentrated between 20-24 nt,with20 nt and 21 nt dominant.After assembly by Velvet,942 contigs were obtained.The average length of contigs was 62 bp,of which 53 contigs were longer than 100 bp.(4)Near-genome full-length sequences were obtained by RNAseq sequencing assembly,with low homology to the reported GFkV isolate MT48(accession number:AJ309022)from Italy.In this study,multiple alignments of REP and CP amino acid sequences showed that there were 17 amino acid variation sites,8 simple information loci and 9 single heterotopic sites between the REP amino acid sequence and other isolates,and there were obvious molecular differences.There were 71 amino acid variation sites,38 simple information loci,33 single variant sites between the CP amino acid sequence and other isolates,and the variation was obvious.Through the phylogenetic analysis of the REP gene sequences of the GFkV isolates from the Baoding and 12 GFkV isolates obtained from the NCBI database.Phylogenetic tree analysis showed that the relationship of GFkV isolates of Baoding were closely related to the GFkV Macedonian isolate(accession number: KF594431.1).Through the phylogenetic analysis of the CP gene sequences of the GFkV isolates from the Baoding and 20 GFkV isolates obtained from the NCBI database.Phylogenetic tree analysis showed that the GFkV isolate(accession number: JN133953.1)and the Brazilian GFkV isolate(accession number: KX828706.1)of Slovakia are highly related.(5)Collect leaf samples of grape mosaic disease and extract total RNA from them.Specific primers were designed using the contigs sequence obtained from RNAseq sequencing as a template and subjected to RT-PCR.Through the RT-PCR detection of the collected samples,it was proved that the grape leaf samples showing the symptom of mosaic were with six viruses or viroids,i.e.,Grapevine fleck virus,Grapevine rupestris stem pitting-associated virus,Grapevine leafroll-associated virus,Grapevine yellow speckle viroid 1,Grapevine yellow speckle viroid 2,and Hop Stunt viroid,were consistent with the types of viruses detected by sRNA and RNAseq deep sequencing.In this study,a RT-PCR method suitable for the detection of pathogens of this type of virus was established,which provides an important analysis method for virus-free seedling propagation and healthy cultivation of grapes.