Apple stem grooving virus (ASGV) and apple chlorotic leaf spot virus (ACLSV) are two major viruses of pear. In this study, in vitro thermotherapy was carried out at 37°C for 25, 30 and 35 days followed by subculturing of meristem tips of different sizes to eliminate ASGV and ACLSV from pear plants. Virus titers in heat-treated shoot tips were evaluated by ELISA testing of regenerated plants. Results showed that thermotherapy for 35 days significantly decreased the titer of ASGV and ACLSV in cultures regenerated from tips of main and axillary shoots, especially in those from explants 1 mm in length from the tip of meristems. Dot-blot hybridization of biotinylated cDNA probes derived from ACLSV and ASGV was used to detect these viruses in crude tissue extracts of in vitro-grown pear plants. Intense signals were consistently detected in untreated plant samples equivalent to less than 0.5 mg tissue. Comparison of signals from dot-blot hybridization and ELISA absorbance values (A405) confirmed that dot-blot hybridization had a higher sensitivity than PAS-ELISA. Dot-blot hybridization could detect viruses with a titer below the threshold level of ELISA. These results indicate that dot-blot hybridization is a useful tool for large-scale surveys of viruses, which facilitates the production of virus-free propagation materials in certification and sanitation programs. Results of PAS-ELISA and dot-blot hybridization showed that high virus elimination efficiency was achieved by a combination of thermotherapy for 35 days and in vitro culture of 1 mm meristem tips.
In vitro Translation of Apple Chlorotic Leaf Spot Virus RNA