| Tomato chlorosis virus(ToCV)disease is one of the most serious diseases that have recently occurred in tomato production.There are few studies related to it,especially about the genome structure of chlorosis virus,the detection of chlorosis virus in infected plants,the prevention and treatment measures against the outbreak of chlorosis virus are still unclear.It is urgent to accurately identify and timely control ToCV diseases.In this study,six pairs of ToCV RT-PCR detection primers including selected from the literature and redesigned were compared in order to screen primer combinations with high sensitivity and stability,and the conditions of affecting RT-PCR reaction were optimized to determine the optimal reaction conditions.It is used to detect ToCV in tomato plants in the greenhouse from Xingping City in Xianyang,Jingyang County in Xianyang,Sanyuan County in Xianyang,Fuping County in Weinan,Taigong Town in Baoji,Wugong Town in Xianyang,Wuquan Town in Xia nyang,Yangling District in Xianyang,Yangling District in Xianyang,Gaoliang District in Xian,Gaoling District in Xian,Yehupo Village in Tongchuan,Shuiyu Village in Tongchuan.As well as detected the mixed infection of ToCV and Tomato yellow leaf curl virus(TYLCV).By sequencing the nucleotide and amino acid sequences of the ToCV genome CP(Coat protein)and HSP70(Heat-shock protein 70 family),compared with the ToCV nucleotide and amino acid sequences in the database of Genbank and analyzed for phylogeny.The ToCV types,occurrence and distribution of the disease and the complex infection with TYLCV in tomato planting areas of Shaanxi province were preliminarily explored.In addition,real-time fluorescence quantitative detection method is also used to detect the infection of ToCV in oder to accurately determine the virus content in different parts of susceptible plants,which laid a foundation for further research on the pathogenesis of Tomato chlorosis virus disease.Preliminarily resarched the effects of enzymies fermentted from environmental waste on the growth and disease resistance for tomato plants,trying to explore a new cultivation measures to prevent the occurrence of ToCV.The main results are as follows:(1)The best sensitivity and specificity primers were To-CP-F/R-1 and SDLC-HSP-F/R.The optimum conditions for amplification of primer To-CP-F/R-1 were as follows: annealing temperature was between 48.0 and 64.0°C,primer concentration range was 4 to 10 μM.The optimum amplification annealing temperature for primer SDLC-HSP-F/R was in the range of 40.9~64.3°C,and the optimal primer concentration was 4~10 μM.(2)The detection rate of ToCV in some tomato producing in Guanzhong areas of Shaanxi Province was 40.94%.The nucleotide sequences of the obtained isolates were aligned with the registered isolates sequences,the sequence identity of the isolates was 96% or more,except from Taiwan and Guangdong.The phylogenetic tree was constructed for the nucleotide sequence and amino acid sequence of the isolates and the registered isolates in Genbank.The analysis showed that the isolates in this study were clustered in the same group.Except for the isolates from Taiwan and Guangdong,which were close to domestic isolates.It can be inferred that domestic ToCV have a common source of communication.Also,it is closest to United States and farthest for Spain.(3)Simultaneous detection of TYLCV was performed on the sample subjected to ToCV detection,and the detection rate was 69.79%.The detection rate of the disease was found to be higher than the former.It indicated that there were two kinds of diseases on tomato complex infection in the tomato greenhouse in Guanzhong areas of Shaanxi Province.(4)This study used the real-time fluorescence quantitative PCR detection method to perform rapid identification while stabilizing the RT-PCR detection method.Supplementary explanations are given for the results of the RT-PCR method.By detecting the leaves of different parts of the plant,found that there were differences about the relative contents of virus in different parts of the same plant.(5)A certain dilution ratio of enzymies fermentted from environmental waste not only promoted the growth of tomato seedlings but also improved the activities of superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD)in tomato leaves.It indicated that the plant resistance can be enhanced.However,from the results of the investigation and test,the effect of enzymies fermentted from environmental waste on enhancing the resistance of tomato plants to ToCV was not significant. |
