Molecular Identification, Complete Sequences Analysis And Detcetion Method Development Of Tomato Chlorosis Virus

Tomato is one of the Main vegetables in daily life, but the tomato virus disease often results in its low production and poor quality, which seriously restricts the development of tomato planting industry. Tomato Chlorosis Virus(To CV) is a newly discovered virus in China in recent years, and it is threatening the production of tomato in North China.The To CV was used as the research object in this study, based on molecular biology technology. The complete genome sequence of Shouguang isolate in Shandong was determined, and PCR molecular biological technology and RT-LAMP detection technology were established. The detection and molecular identification for To CV incidence of the main tomato producing areas of Shandong province were carried out by the optimized detection technology. The purpose of this study is to establish a rapid detection technology for To CV, in order to clarify the occurrence situations of To CV and mixed infection situation in Shandong vegetable producing areas by investigating fields. In addition, through the detection and phylogenetic analysis of the complete genome sequence of Shouguang isolate in Shandong, and combine the molecular identification results of different isolates from different sources, and carried out the phylogenetic analysi of To CV isolate in Shandong.1. 401 tomato samples were collected from tomato producing areas of Shandong province(Weifang, Tai’an, Linyi, Rizhao, and Liaocheng). After the detection, 355 positive samples were screened out. The obtained isolate sequences(Gen Bank accession number KC812620/KC812625, KC812623/KC812627, KC812622/KC812626, KC812619/KC812624, and KC812621) are used for consistency analysis and phylogenetic analysis. The results showed that the isolates of the study were gathered into a cluster.2. The Detection for TYLCV on the above positive samples screened out 56 positive samples. Through the phylogenetic analysis, the TYLCV isolates(Gen Bank accession number KJ546418) were belonged to a group of Israel strain(TYLCV-IL), this result showed that the mix infection of Tocv and TYLCV was truly exist in Shandong’s tomato producing area. The nucleotide sequences of TYLCV isolates(Gen Bank accession number KJ546418) were used for phylogenetic analysis. The results showed that the TYLCV isolates were belonged to a group of Israel strain(TYLCV-IL). The mixed infection of To CV and TYLCV in Shandong province was confirmed in this study.3. Established the reverse transcription loop mediated isothermal amplification detection technology on To CV, and optimized the reaction parameters(Mg2+, d NTP, reaction temperature) of this technology. The final optimal detection system was determined: 10 × Thermopol buffer 2.5 μL, 10 m M primer(F3 and B3) 0.5 μL, 10 m M primer(FIP and BIP) 2.0 μL, 10 m M d NTP 2.0 μL, 25 m M Mg Cl2 4.0 μL, 200 U/μL M-MLV 0.5 μL, 800 U/m L Bst DNA polymerase 1.0 μL and RNA 1.0 μL, the reaction completed within 40 min under isothermal conditions at 60°C without a thermal cycler.4. The full genomes of To CV were acquired by fusion PCR, and correlation analysis of the whole genome sequence was carried out. The obtained nucleotide sequence of To CV(Gen Bank accession number KC709509 and KC709510) in this study was consistent above 90% with the To CV reference sequence in Gen Bank. The whole genome sequences of To CV isolates were systematically analyzed, and the results showed that all the To CV isolates were gathered into a cluster.