Detection And Identification Of Plant Viruses In Environmental Water

The pathogenicity of the virus in environmental water(rivers,lakes,irrigation water,sea water,tap water,etc.),is a serious threat to human health and agricultural safety.Plant viruses have been shown to exist in environmental water,these viruses may enter the disease cycle,which can lead to the spread of plant virus diseases.In this study,plant viruses in environmental water were screened,and a technical system for detection and identification of plant viruses in environmental water was established by optimizing concentration and separation method,and the infectivity of plant virus isolates in water environment to host plants was verified by inoculation.The main results are as follows:1.After concentration and purification of the 65 water samples collected from Shandong,Hebei,Inner Mongolia and Heilongjiang area,4 pairs of generic primers and 15 pairs of specific primers were used to detect viruses.Seven viruses of Cucumber green mottle mosaic virus(CGMMV),Tobacco mosaic virus(TMV),Tomato mosaic virus(ToMV),Cucumber mosaic virus(CMV),Pepper mild mottle virus(PMMoV),Tomato chlorosis virus(ToCV)and Tomato yellow leaf curl virus(TYLCV)were detected in samples from Tai'an,Ji'nan,Yantai,Binzhou,Zibo,Weihai,Liaocheng,Jining,Dezhou and Weifang,10 regions which belong to Shandong;One virus,CGMMV were detected in the samples of the the Hohhot city,Inner Mongolia Autonomous Region and Jixi City,Heilongjiang Province;One virus,PMMoV was detected in Hebei Langfang city,Hebei Province.The detection rate of CGMMV was the highest,reaching 38.0%.2.In the 65 water samples,4 kinds of viruses contained CGMMV,TMV,ToMV and TYLCV can be detected in 6 samples at the same time;TMV and CGMMV can be detected in the 5 samples at the same time;ToCV and CGMMV can be detected in the 3 samples at the same time.In Shandong province,samples of Ji'nan are relatively rich in virus detection,totally 5 viruses of CGMMV,ToCV,TMV,ToMV and TYLCV were detected;Next is those samples of Shandong Shouguang,totally 4 viruses including CGMMV,TMV,CMV and PMMoV were detected.Among the detected plant viruses,most are RNA viruses,TYLCV was the only plant DNA viruse to be detected.3.TMV,ToMV and CGMMV,as members of the(Tobamovirus),can spread through the juice friction.Water samples containing TMV,ToMV,or CGMMV were concentrated and purified respectively to reach a certain concentration and quantity,and the virus particles then inoculated the tested host plants.Plants inoculatd with TMV,ToMV and CGMMV shown obvious symptoms,virus exists in inoculated plants was verified by RT-PCR,the results showed that TMV,ToMV and CGMMV from water can infect host successfully.4.Sequences of isolates(TMV-X1-3,ToMV-X1-3,CGMMV-BS-14)from sewage were used to sequence consistency comparison,and phylogenetic analysis;The results showed that the TMV-X1-3 isolate had the closest phylogenetic relationships with the Bei Cang Zhu(KU198186)isolate,up to 99.4%.ToMV-X1-3 isolate was the most consistent with the Xinjiang isolate XJT-1(FN985165)with the closest genetic relationship,up to 100%;The CGMMV-BS-14 isolate had the highest concordance rate and the closest genetic relationship with the Taiwan isolate BG-SB(FJ654657),up to 100%.