Virus elimination from in vitro apple by thermotherapy combined with chemotherapy

We evaluated the effectiveness of thermotherapy at different temperatures, chemotherapy with different concentrations of ribavirin, and combinations of these two methods on virus elimination from apple. We used Malus cv. ‘Xinhongjiangjun’, an apple cultivar widely grown in China, infected with Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), and Apple stem pitting virus (ASPV). The survival and regeneration of plants were evaluated, and the efficiency of virus eradication was determined by reverse-transcription polymerase chain reaction with two primer pairs for each virus. All of the plants treated with 15 and 25 μg/ml ribavirin survived, although their proliferation was slightly inhibited. Ribavirin treatments at 15 and 25 μg/ml resulted in virus elimination rates of 74.4 and 75.0 %, respectively. Higher temperatures significantly affected the growth and proliferation of plants, and almost no axillary shoots regenerated under the highest temperature (38 ± 0.5 °C). The average virus elimination rate after 34–36 °C treatments for 20 days was no more than 45 %. The virus elimination efficiency was enhanced by combining thermotherapy and chemotherapy treatments. A treatment combining ribavirin (25 μg/ml) and thermotherapy at 36 °C (R25 + T36) resulted in high virus eradication efficiency (95.0 %). Across all of the treatments, the average survival rate of apical shoots (176/273) was 12 % lower than that of axillary shoots (266/349); the average virus elimination efficiency was 65.3 % for apical shoots and 72.7 % for axillary shoots. The results also demonstrated that it was easier to eliminate ASPV than to eliminate ACLSV and ASGV.