Virus elimination from in vitro apple by thermotherapy combined with chemotherapy

Abstract

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 lg/ml ribavirin survived, although their proliferation was slightly inhibited. Ribavirin treatments at 15 and 25 lg/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 lg/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.

Exogenous application of melatonin improves eradication of apple stem grooving virus from the infected in vitro shoots by shoot tip culture

“…So far, ASGV has been successfully eradicated from infected apple and pear plants by thermotherapy‐based methods such as thermotherapy followed by shoot tip culture (Hu et al ., ; Vivek & Modgil, ; Wang et al ., ), chemotherapy followed by thermotherapy (Hu et al ., , ; Wang et al ., ), thermotherapy followed by shoot tip grafting (Hu et al ., , ; Wang et al ., ) and thermotherapy followed by shoot tip cryotherapy (Wang et al ., ; Zhao et al ., ). Contrary to these findings, Paprstein et al .…”

“…Secondly, virus eradication by exogenous application of melatonin does not require extra facilities and can be conducted in any tissue culture laboratory, whereas ASGV eradication by thermotherapy and cryotherapy requires expensive growth chambers in the former, and cryogenic procedures which may not be available in some laboratories in the latter. Thirdly, ASGV eradication frequencies obtained in the present study (95%) were comparable with those reported in previous studies (Hu et al ., , ; Vivek & Modgil, ; Zhao et al ., ). Furthermore, 100% survival and 85% shoot regrowth levels of the shoot tips were obtained in the present study, which were higher than 62.5% (Paprstein et al ., ) and 62.4% (Vivek & Modgil, ) of shoot tip survival levels in thermotherapy followed by shoot tip culture, 65% of shoot tip survival when combining thermotherapy with chemotherapy (Hu et al ., ), 63.0% of shoot tip grafting success in thermotherapy followed by shoot tip grafting (Hu et al ., ) and 33–76% of shoot regrowth levels in thermotherapy followed by cryotherapy (Zhao et al ., ).…”

Efficacy of virus elimination from apple by thermotherapy coupled with in vivo shoot‐tip grafting and in vitro meristem culture

“…Thermotherapy is an efficient technique for virus elimination from affected plants. Although prolonged treatments at high temperatures have a severe negative effect on the regeneration and survival of plants, it is necessary for apple virus elimination (Cordewener et al., ; Hu, Dong et al., ). The heat treatment of plants can reduce movement of virus particles into the apical meristem through the inhibition of viral RNA synthesis (Paprstein et al., ; Wang et al., ).…”

“…Although ELISA is routinely used to detect plant viruses (Valero, Ibañez, & Morte, ), it is not sensitive enough to detect plant viruses at very low concentrations in plant tissues. To overcome this problem, RT‐PCR has been used for virus detection (Hu, Dong et al., ; Hu, Zhang et al., ; Manganaris et al., ). To ensure accuracy, we used two primer pairs for each apple virus, and plants were judged as virus‐free when there were no target products amplified by RT‐PCRs with ten primer pairs, which increased the specificity and sensitivity of detection.…”

“…At present, use of virus‐free planting materials is pivotal for the control of apple viral diseases, which proved to promote plant growth vigour and increase fruit yield and quality (Cieslińska & Rutkowski, ; Koike, Makita, Tsukahara, & Sukahara, ), thus bringing great benefits to the apple growers. Heat treatment of plants in vivo or in vitro reduces virus titres and improves the efficiency of virus eradication (Hu, Dong et al., ; Hu, Zhang et al., ; Mink, Wample, & Howell, ). Thermotherapy followed by meristem culture (Hu, Hong, Wang, Hu, & Wang, ; Lassois, Lepoivre, Swennen, van den Houwe, & Panis, ; Manganaris, Economou, Boubourakas, & Katis, ; Zilka et al., ) and shoot‐tip grafting (Hoffman et al., ; Sharma et al., ) can markedly enhance virus eradication.…”