Identification Of Viral Pathogens From Tomato,Potato And Watermelon

Recently,Tomato spotted wilt virus(TSWV)has been reported in several areas causing serious damage.To identify the viruses in tomato samples exhibiting virus-like symptoms from Heilongjiang Province,small RNA deep sequencing and RT-PCR were adopted.Total RNA of tomato samples was extracted and the siRNA library was constructed and sequenced,data analysis results showed that the siRNA reads aligned to TSWV account for 92.64%of the total siRNA reads to the virus sequences deposited in GenBank,which indicated that the pathogen infecting the tomato samples might be TSWV.The RT-PCR further confirmed the presence of TSWV,which was named TSWV Heilongjiang isolate(TSWV-HLJ1).The phylogenetic tree based on the whole nucleotide sequence of TSWV isolate indicated that the TSWV-HLJ1 was more closely related to TSWV Yunnan isolate(TSWV-YNgp).Vector transmission experiment showed that Frankliniella occidentalis can acquire TSWV-HLJ1 and transmit it to the healthy plants successfully.And Frankliniella occidentalis are inclined to the TSWV-infected peppers.Mechanical inoculation showed that TSWV-HLJ1 can infect Nicotiana benthamiana,Capsicum annuum L.and Solanum lycopersicum L..This was the first report of TSWV infecting tomato in Heilongjiang Province.Tomato plants exhibiting severe mottle and leaf distortion symptoms were identifed in Hainan province in 2016.To identify the viruses in tomato samples exhibiting virus-like symptoms from Hainan Province,Dot-ELISA and RT-PCR were adopted.Dot-ELISA suggested that crude saps of the infected tomato samples reacted positively with the monoclonal antibody against Tobacco mosaic virus(TMV)which indicated that one or more tobamoviruses are likely associated with the disease.RT-PCR and DNA sequence analysis results further indicated that Tomato mottle mosaic virus(ToMMV)in Tobamovirus was the pathogen causing the mottle disease in tomato.We amplified and sequenced the full-length genomic sequence of the ToMMV in tomato samples which showed the highest nucleotide identity with the ToMMV YYMLJ and ToMMV TiLhaLJ isolates,the putative virus isolate was named ToMMV-HN.Mechanical inoculation showed that ToMMV Hainan can infect Nicotiana benthamiana,Capsicum annuum and Solanum lycopersicum showing serious symptoms.This was the first identifcation and characterization of ToMMV infecting tomato in Hainan of China.In 2017,in order to investigate the occurrence of potato viral disease,we collected 53 potato samples from several potato-producing areas in China,including Inner Mongolia,Jilin,Qinghai and Sinkiang.10 potato viruses were detected by RT-PCR including PVS ~a,PVS ~b,PVX,PVM,PVY,PLRV,PVA,PMTV,PBRSV and PYDV.The RT-PCR results showed that 45 samples out of 53 were infected by viruses,mainly by PVY and PLRV.39%of the samples were infected by PVY and 6%were infected by PLRV.Moreover,20 samples were coinfected by two kinds of viruses,34%samples were infected by PVY and PLRV,4%samples were infected by PVY and PVS ~a.In 2017,the watermelon samples exhibiting mosaics and systemic crinkling sysmptoms were collected in Hainan Province.We used small RNA deep sequencing and RT-PCR methods to identify the viruses in watermelon samples.The results indicated that the diseased watermelon samples including Melon yellow spot virus(MYSV)and Watermelon silver mottle virus(WSMoV).The whole nucleotide sequences of MYSV and WSMoV were amplified and constructed the phylogenetic trees,which indicated that MYSV RNA L,RNA M and RNA S showed the highest nucleotide identity with MYSV Japan melon isolate(AB061774),Thailand cucumber isolate(KX711614)and Sanya melon isolate(GQ397254);WSMoV RNA L and RNA M showed the highest nucleotide identity with Chinese watermelon isolate RNA L(JX177647)and RNA M(JX177646),while WSMoV RNA S had the highest similarity with Japan watermelon isolate WS-Y(AB042650).