Production of virus-free plants is crucial for vegetative propagation. In in vitro conditions, thermotherapy treatments are the most common method for virus elimination. However, shoot-tip necrosis and hyperhydricity are the most common physiological problems in thermotherapy-treated shoots, causing an important loss of virus-free plants. In the present study Apple chlorotic leaf spot virus (ACLSV, Trichovirus genus), Apple stem pitting virus (ASPV, Foveavirus genus) and Apple mosaic virus (ApMV, Ilarvirus genus) infected ‘Abbasi’ apple (Malus domestica Borkh.) shoots were treated through two in vitro thermotherapy strategies, constant (37°C) and alternating (32/38°C) cycles. Plant growth, shoot-tip necrosis, meristem establishment, hyperhydricity and total chlorophyll content (SPAD-502 meter readings) were recorded in virus-infected and virus-free shoots. Results showed that duration of high temperatures exposure caused shoot-tip necrosis decreasing shoot-tip survival. Meristem hyperhydricity showed two different trends related to length of treatment, increasing up to 20 d and decreasing up to 70 d applying heat treatments at constant and alternating cyclic temperatures, respectively. Treatment at constant 38°C for 5-15 d led to 100% eradication of ACLSV and ASPV, while all treated plants were still infected with ApMV. The alternating cycles of 32/38°C treatment was effective in eliminating ApMV, as well as ACLSV and ASPV. Virus-free plants had 3.9-fold growth in comparison with virus-infected plants. In addition, SPAD-502 meter readings (total chlorophyll index) showed significant differences between virus-infected and virus-free plants. Suggesting that rapid assessment of physiological, growth and sanitary state of apple shoots after thermotherapy treatments, should be performed by chlorophyll content evaluation using SPAD-502 meter. Higher SPAD-502 meter readings in apple virus-free shoots correlated with better growth and reduction of shoot-tip necrosis and hyperhydricity, confirming that photosynthetic rate is affected by virus infection via decreasing leaf chlorophyll content.

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