Detection And Identification Of Virus Infecting Tomato Planted In Shandong Province And Surrounding Areas Based On Small RNA Deep Sequencing

Tomato is a kind of vegetable crops cultivated in large area in China.Its yield and quality are often affected by a variety of pests and diseases.Among them,the types and quantities of viral diseases in tomato are increasing,the outbreak and epidemic of viral diseases will lead to the reduction of tomato yield,affecting farmers’ income.It is a great challenge for the development of tomato industry in China.In order to further clarify the virus species and the distribution of virus infecting tomato,this study used small RNA deep sequencing and PCR technology to detect and identify 487 tomato samples collected from Shandong Province and surrounding areas.And the virus with the highest detection rate was ToMV,three isolates of the whole genome sequence of ToMV were cloned,sequenced and analyzed.The genome sequences of ToMV virus were compared with the representative isolates of the virus in GenBank,the similarity,systematic evolution and recombination of the genome sequence of the three isolates were analyzed.Identification of Beet western yellows virus(BWYV),which was the first report of infecting tomato in China.The main results are as follows:1.Small RNA deep sequencing technology was used to analyze the small RNA sequences of mixed tomato samples from main tomato production areas of Shandong Province.The results showed that the small RNA sequences in the samples were comparable with those reported in Nucleotide Sequence Database(NT)of GenBank,and the similarity between the small RNA sequences and the nucleotides of 40 plant viruses was high.The 40 viruses belong to 8 families and 8 genera,respectively.They are Begomovirus of Geminiviridae,Cavemovirus of Caulimoviridae,Potyvirus of Potyviridae,Cucumovirus of Bromoviridae,Polerovirus of Luteoviridae,Amalgavirirus of Amalgaviridae,Crinivirus of Closteroviridae,Tobamovirus of Virgaviridae.2.487 tomato samples were detected for 40 viruses.Eight viruses were detected,including ToMV,TYLCV,PeVYV,BWYV,ToCV,STV,PVY,and CMV.To our knowledge,this is the first report of BWYV infecting tomato in China.32 viruses such as Ageratum yellow vein virus(AYVV),Chilli leaf curl virus(ChiLCuV)and Crassocephalum yellow vein virus(CYVV)were not detected.Among all the samples collected,the detection rate of the virus was 83.16%.The results showed that viral disease infection severity was in the order of ToMV(49.69%)> TYLCV(49.08%)> ToCV(29.98%)> CMV(24.02%)> PeVYV(18.89%)> STV(7.60%)> PVY(6.57%)> BWYV(0.82%).3.Among 487 samples detected,405 were positive.Among them,83 samples were infected by a single virus,while the others were infected by two or more viruses.There are 12 types of virus co-infection.The compound infection rate of the virus was 79.51%.Among them,the highest proportion was 46.67% with two viruses,22.47% with three viruses,8.64% with four viruses and 1.73% with five viruses.4.The virus with the highest detection rate was ToMV,three samples from Jiangsu Province,Shanghai City,Hebei Province were used to amplify the whole genome sequence of ToMV.Three ToMV genome sequences were obtained,the length of which were 6383 bp.5.Similarity analysis and phylogenetic tree analysis of the whole gene nucleotide sequence of ToMV showed that ToMV-HBQX isolate and Japan tobacco isolate AB355139 were clustered on one branch,and had the closest relationship.ToMV-SHPD and ToMV-JSGY isolates were clustered on one branch with China tomato isolate MF002491 and had the closest relationship.Overall,the three isolates clustered with the Asian subgroup on a large branch.Seven packages of RDP4 software were used to analyze the recombination of ToMV.The results showed that there was no gene recombination between ToMV-SHPD and ToMV-JSGY and ToMV-HBQX and other selected isolates.