Molecular Identification Of The Pathogen Of Tomato Virus Disease From Province Or Autonmous Region In Northern China And Complete Sequences Analysis Of Tomato Mosaic Virus

Tomato is widely grown across the world,and it is popular as edible fruits and vegetables.But in recent years,with the market demand for tomato increases and the tomato acreage expanding rapidly,tomato virus diseases have become a major limiting factor in the tomato production.In order to investigate the pathogenic species of virus disease,mixed infection and primary and secondaryrelations between virus diseases in the main producing area of tomato in Shandong,Inner Mongolia Hohhot and Shanxi jinzhong,425 tomato samples collected from 9 growing regions were tested using 15 specific primer pairs by polymerase chain reaction.In addition,23 isolates of Tomato mosaic virus from Shandong,Shanxi,Inner Mongolia were cloned and sequenced,compared with the related ToMV sequences in the Genbank database,and phylogenetic analysis of movement protein gene,coat protein gene and complete nucleotide sequences were carried out.The main conclusions are as follows:(1)Using the 15 specific primer pairs to detect the 425 susceptible tomato samples.The results showed that 7 viral pathogens were detected.Respectively as: ToMV(Tomato mosaic virus),ToCV(Tomato chlorosis virus),TMV(Tobacco mosaic virus),CMV(Cucumber mosaic virus),PVY(Potato virus Y),TYLCVN β(Tomato yellow leaf curl vietnam betasatellite),TYLCV(Tomato yellow leaf curl virus).No sample positive to TICV(Tomato infectious chlorosis virus),TSWV(Tomato spot wilt virus),PMMoV(Pepper mild mottle virus),ToMMV(Tomato mottle mosaic virus),TYLCVN α,PVX(Potato virus X),ToTV(Tomato torrado virus),PaLCuCNV(Papaya leaf curl China virus).Among the viral pathogens,ToMV,TYLCV and ToCV have the higher infection rate.The total virus infection rate was 84%.The detection rate of TYLCV,ToMV,ToCV,CMV,TMV,PVY,TYLCVN β were 62.59%,61.41%,51.76%,39.76%,35.76%,4.00%,and 1.41% respectively.The results showed that the main viruses in most tomato growing areas in the three provinces were TYLCV,ToMV,ToCV,followed by CMV,TMV,and other viruses were less harmful.(2)The full genome sequence of ToMV 23 isolates ware acquired by overlap PCR.Nucleotide sequence and amino acid consensus analysis were carried out of the complete sequence.The results showed that 23 isolates consisted of 6383 nucleotides sequences contained four open reading frames,with the typical characteristic of ToMV genome.23 nucleotide sequences of the ToMV genome were compared with the nucleotide sequences of 15 ToMV genome sequences from GenBank,and their consistency was higher than 91%,butthe consistency with other members of the genus of Tobamovirus is less than 85%,thisindicates that ToMV sequence is conservative.The phylogenetic tree was constructed using MEGA 5 with the neighbour-joining method.The results showed that the 23 complete genome sequences gathertogether in the subgroup I of the Tobamovirus genus,all ToMV isolates form a large clade,clustered into a cluster with three major clades,Clade I,Clade II,and Clade III.The viruses isolates of Clade I come from China,Kazakhstan,Japan,Australia,Oman,Japan,Germany and other countries;the viruses isolates of Clade II and Clade III are from countries such as Egypt,the United States,and other countries,the relationship with subgroup II was far away,the relationship with the subgroup III was the farthest,the results show that ToMV isolates have obvious regional specificity.