Molecular Identification Of Tomato Chlorosis Virus And Development And Application Of Real-time PCR Assay

Tomato chlorosis virus(ToCV) is an devastating virus for growing tomato production. Study on the rapid and efficient detection methods of virus infection is helpful to timely detection and prevention of this virus, and as a result, can reduce the economic losses caused by ToCV. In this study, RT-PCR technology was applied to detect and identify ToCV in tomato plants and the surrounding potential host plants in tomato growing regions in Tianjin and Hebei. Study of mixed infection of ToCV and Tomato yellow leaf curl virus(TYLCV) in these regions were also carried out. A SYBR Green I real-time PCR for quantitative detection of ToCV was developed and applied for detecting the virus titer in different tomato varieties, different parts and different stages of tomato plant.The main results are as follows.(1) 319 tomato samples and 55 weed samples were collected from tomato growing areas in Tianjin municipality and Hebei province, respectively. 16.0% of tomato samples were positive, but none of the weeds samples. The obtained isolate sequences(GenBank accession number: KP219722/KP226698, KP217199/KP217201, KP217196/KP217197, KP217195/KP217202, KP217200/KP217198) were clustered together with those of isolates from China, Japan and the United States.(2) All the ToCV positive samples were tested for TYLCV occurrence, 27 samples were positive for mixed infection(53.0%).(3) Primers specific to heat shock protein HSP70 of ToCV for the SYBR Green I real-time RT-PCR assay were designed based on reference sequences in GenBank and recombinant plasmid was obtained after verification. Optimization of annealing temperature and primer concentration was performed using the plasmid as template(annealing temperature is 63°C, primer is 0.2?mol/L). The linear equation for the real-time RT-PCR was Ct=-3.227 Log(copy number)+49.508. What's more, melt curve appeared only one characteristic peak and it has no cross reaction with Cucurbit chlorotic yellows virus(CCYV), Cucumber mosaic virus(CMV), Tomato masaic virus(ToMV)and Tomato spotted wilt virus(TsWV). The specificity of melt curve is good. And this method is 100 times more sensitive than conventional RT-PCR. The results of the intra-assay and inter-assay variability were less than 5.0%, which suggest that this method is of good reproducibility and stability as well.Detection of virus titer in different parts, different cultivars of the diseased tomato plants and in different period after inoculation was carried out, the results showed that the virus titer in stem phloem is highest, next in old leaves, then in root, and the virus titer in upper leaves is the lowest; The T03 tomato variety has the worst virus resistance. The virus tier increased suddenly 28 days after inoculation.