Acquisition of Virus Eliminated Apple Plants by Thermotherapy and the Factors Influenced the Eliminating Efficiency

Abstract

The aim of this study was to establish reliable and efficient detection and elimination methods for five apple viruses and viroids, including Apple chlorotic leaf spot virus (ACLSV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Prunus necrotic ringspot virus (PNRSV) and Apple scar skin viroid (ASSVd) from infected apple plants using thermotherapy. Based on multiple alignments of available virus or viroid characteristic genes, compatible degenerate primer pairs were designed and an efficient and sensitive detection method was developed using quantitative real-time PCR (qPCR) and was further verified by short interfering RNA (siRNA) deep sequencing. Coupled with root protecting and shoot growth promoting measures, six cultivars of potted apple plants were subjected to thermotherapy for 30 days, followed by virus detection of new emerged shoots and shoot tip grafting. The results showed that the virus elimination efficiency was over 30% for the five viruses tested for ‘Kudowu’, ‘Fuji 2001’, and ‘Devil Gala’, but only 10% for ‘Red Jonathan’ and ‘Shizuka’, while no virus-free plants were detected for ‘Shinano Gold’. Correlation analysis of the factors influencing the virus elimination showed that the elimination efficiency and the virus free plant number were significantly related to the shoot growth rate, implying a vital role of the shoot growth rate in virus elimination using thermotherapy. This study provides a new qPCR assay for five apple viruses and stresses the importance of cultivar characteristic in virus thermotherapy elimination.