| TMV, CMV, TEV and PVY are great threats to tobacco production in China. To find out the distribution and molecular variation of these tobacco viruses, we conducted an investigation and analysis of tobacco viruses diseases in 12 main tobacco production areas in China. Meanwhile, the antiviral activity and mechanism of WCT-II were also studied in this study, which provides the theoretical and material basis for prevention and control technology of the environmental friendly plant virus.409(154 in 2010 and 255 in 2011) tobacco leaf samples were collected from 12 main production areas in China. In order to identify the viruses, all the infected samples were tested by using both DAS-ELISA and RT-PCR. The results confirmed that TMV, CMV, TEV and PVY distributed widely in China, and the average detection rate of these four viruses was more than 50%. For different areas, the detection rate was different. Specifically, the four viruses were most serious in Shaanxi with detection rate of 75.95%, Henan 74.78%, Hunan 74.26%, and Hubei was lowest with the average detection rate of 58.69%. For different years, the detection rate was also different. The detection rate in 2010(76.62%) was higher than that in 2011(60%). These results implied that the virus disease varied in different regions and years. As a result, It is necessary to investigate in different years and regions to accurately understand the occurrence and distribution of virus diseases.The study isolated and identified 18 TMV isolates, 21 CMV isolates, 15 TEV isolates, and 21 PVY isolates from the samples. Compared with the online data, the consistent rate of nucleotide sequences and amino acid sequences of the four kinds of virus CP were: TMV is 97% ~ 100% and 72% ~ 100%, CMV is 81% ~ 100% and 78% ~ 100%, TEV is 96% ~ 100% and 97% ~ 100%, PVY is 96% ~ 100% and 97% ~ 100%. With these isolates as the object and combined with the online database, the nucleotide sequence of the CP of these isolates was analyzed by using RDP4 software. 3 distinct recombinant events were identified, among which 1 was from CMV isolates, 1 from TEV isolates and 1 from PVY isolates. The Phylogenetic network analysis indicates that the isolates of CMV, TEV and PVY showed as non-tree like Phylogenetic networks, which means recombination plays an important role inthe genetic variation of CMV, TEV and PVY. In addition, the ratio of non-synonymous and synonymous sites(d N/d S) of these isolates was calculated by MEGA, which shows the d N values were obviously less than that of the d S values, namely dn/d S < 1, suggesting that the CP genes of TMV, CMV, TEV and PVY isolates are effected by negative selection. Negative selection is also an important factor affecting the population variability of TMV, CMV, TEV and PVY.Based on the nucleotide sequences of the CP, the genetic variation of the four viruses was analyzed by MEGA4. According to the phylogenetic tree of TMV isolates, 34 TMV isolates(16 from the published sequences online) were divided into two subgroups: Group I and Group II(bootstrap support values > 60%). 38 CMV isolates were put into Group I and 5 CMV isolates were put into Group II. 39 TEV isolates(24 from the database) were divided into four groups: Group I, Group II, Group III and Group IV(bootstrap support values > 90%). 73 PVY isolates(52 from the online database) were divided into 2 groups, Group I and Group II(bootstrap support values > 90%). The results showed that there was obvious genetic variation in TMV, CMV, TEV and PVY isolates. For the further analysis of the diversity of these isolates, the genetic distance of within-group and between-group was calculated by using MEGA4. The results showed that the genetic distance between groups was significantly greater than that of within group indicating the obvious genetic diversity of these four viruses is obvious.The effective prevention and cure of these tobacco viruses becomes a big problem. In view of this problem, the activity and mechanism of WCT-II against TMV were detected by using semi leaf spot method and quantitative PCR. After Nicotiana tabacum was treated by WCT-II, the tobacco leaves were collected, the activities of SOD, POD, PPO were measured, and the content of H2O2 and PR-1 gene expression were also calculated. The results show that the activity of SOD, POD, PPO and H2O2 are beginning to increase after WCT-II treatment for 6 h, meanwhile, the PR-1 gene is induced to express. After 12 h, the activity of PPO and SOD reached the peak. After 24 h, POD activity, the content of H2O2 and PR-1 gene expression also reached the peak, 3.16 times higher than the control group. Subsequently, they gradually decreased to control level. The experiment illustrates that under greenhouse conditions, WCT-II can effectively induce tobacco SOD, POD, and PPO activity to increase, H2O2 to accumulate and PR-1 gene to express, and finally induce tobacco to produce anti-TMV activity.TMV, CMV, TEV and PVY are widely distributed in the main tobacco production areas in China. These viral isolates have obvious genetic variation, and recombination and negative selection are the important factors that affect the genetic variation of these viral groups. Asbotanical extract, WCT-II can effectively induce the disease resistance of tobacco, which provides a theoretical and material basis for the effective prevention and control of tobacco viruses like TMV, CMV, TEV and PVY. |
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