Cross Protection Of A Citrus Tristeza Virus Mild Strain Against A Severe Strain Detected By Real-time RT-PCR

Citrus is one of the most important fruit in the world and in Southern China. The disease caused by Citrus tristeza virus (CTV) has damaged a lot of citrus trees in the world. CTV occurs almost everywhere on citrus in China. The most effective control method is Mild Strain Cross Protection (MSCP). In this study, the dynamic population changes of a mild strain and severe strain are detected by real-time RT-PCR, which might help us understand more on MSCP.In the MSCP experiment,7 months later after the mild strain was graft-inoculated onto sweet orange seedlings that became pre-immunized plants when they were infected,25 brown citrus aphids with the severe strain were challenge-inoculating into each pre-immunized plant. The total RNA was extracted by small scale Trizol method after 15,30,45,60 and 90 days, respectively. The experimental plants (Jincheng sweet orange seedlings) in this study were monitored by real-time RT-PCR (real-time fluorescence quantitative RT-PCR). and 18S rRNA and nad5 were used as internal control to calibrate the relative expression of CTV.The main results were as follows:1) The expression peaks of p25 gene of the mild strain among different Jincheng sweet orange seedlings varied within 6 months. The contents of the mild strain in each plant had three stages: increasing stage, decreasing stage and increasing stage again.2) Each of the two-year-old Jincheng sweet orange seedlings was inoculated by 25 aphids with the severe strain CT14. The earliest detectable time of CTV in leaf and bark of each plant was not consistent, ranging from 15 to 60 dpi (days post inoculation). In most plants, virus content in bark was higher than in leaf. However, when the new shoots of the positive control were inoculated by aphids, CTV in half of the young leaves was detected earlier than the young barks, and the volume of CTV in most young leaves reached peak earlier than in young barks. But finally, the peak volume of CTV in most young barks was higher than in young leaves.3) The challenge-inoculated plants were CT14 negative until the 10th month, suggesting that, the mild strain CT11 in pre-immunized plants can cross-protect the invasion or multiplication of the severe strain CT14 under inoculation of 25 aphids/plant.4) The relative content of the mild strain CT11 in most samples (10/14, bark and leaves in the seven plants) of the challenge-inoculated plants reached the peak on 45 dpi, witch was higher than that in the mock control plants at the same time point.5) The expression of 18S rRNA and nad5 internal control was stable to some extent. The trend of CT11 content was consistent after calibrating the relative expression of CT11 by the two internal control, respectively.