Combined thermotherapy and cryotherapy: an effective method for the eradication of three common viruses in cultivated apples

Apple virus disease is a serious problem affecting apple quality and crop production eciency, and solutions are being actively and urgently explored. In particular, the cultivation of virus-free seedlings is being increasingly explored as an important technical method for facilitating the virus-free breeding of excellent apple cultivars. In this study, the apple cultivars ‘Ruiyang’, ‘Ruixue’, ‘Rui Xianghong’, and ‘Qinyue’ were used as source materials to compare the effectiveness of thermotherapy and combined thermo- and cryotherapy in eradicating apple chlorotic leaf spot virus (ACLSV), apple stem grooving virus (ASGV), and apple stem pitting virus (ASPV).


Results
Viral presence in the in vitro shoots of the four apple cultivars was detected using a reverse transcriptionpolymerase chain reaction. All four cultivars were found to carry ASGV and ACLSV, and 'Rui Xianghong' and 'Qinyue' additionally carried ASPV. In vitro shoots of the four cultivars were subjected to varying periods of thermotherapy (0, 2, 4, 6, and 8 weeks). Shoot tips were then removed and half were cultured directly, and the remainder were cultured after cryotherapy treatment; their survival and regeneration rates were then evaluated to determine the e ciency of virus eradication after 8 months of subculture. We showed that the eradication of viruses in each of the cultivars and their survival rate are both the highest after 4 weeks of thermotherapy. When combined with cryotherapy, application of this period of thermotherapy achieved a 100% eradication of ASPV and ACLSV, and over 50% of ASGV.

Conclusions
This study demonstrated an effective methodology for developing both new virus-free plant materials and new apple cultivars that will support sustainability of the apple production industry in China and globally.
Background Apple (Malus × domestica Borkh.) is a globally important cash crop. It is particularly valuable in China, which is both the largest producer and consumer of apples in the world. A total of 2.07 million hectares in China is dedicated to apple cultivation; the output of 39.23 million tons accounts for nearly 50% of global output [1]. In this context, apple viral diseases, which are among the main factors restricting healthy and sustainable development in apple cultivation [2], have received much attention. Viral infection of an apple tree damages normal cell proliferation, therefore seriously restricting the growth potential and other physiological mechanisms; it also reduces apple quality and yield, with consequent adverse implications for the apple industry [3][4][5]. Viruses can spread from infected plants to their offspring through asexual reproduction, or to healthy plants via insect vectors [6]. Pesticide spraying is ineffective once a plant has become infected [7,8]. The focus has therefore been on cultivating virus-free apple seedlings as an effective and core technology in the global control of apple viral diseases [9][10][11][12].
Cryotherapy, which uses liquid nitrogen as a coolant, has proved promising as a technique, playing an important role in developing virus-free plant production [27,28]. In some research, a small number of virus-free cells regenerated as healthy, detoxi ed plants after liquid nitrogen was used to kill stem tip cells [29,30]. In garlic (Allium sativum L.), cryotherapy has been shown to eradicate viral complexes successfully [31] and, in combination with STC, to eradicate ASPV and ASGV from M9 and M26 apple rootstocks [32]. The principle behind thermotherapy, which was reported in 1998 as an application suitable for virus eradication, is exposing plant tissue to a high temperature to inhibit viral spread; this minimises the extent to which meristem tissue in the stem tip carries viral particles, thus yielding virusfree seedlings [10]. However, there is growing interest in the effects of combined thermo-and cryotherapy. For example, it is more di cult to eradicate raspberry bushy dwarf virus from the in vitro shoots of raspberry when using only cryotherapy, compared with its use in combination with thermotherapy [29].
In this study, our objective was to compare and evaluate the effectiveness of thermotherapy alone and combined thermo-and cryotherapy in successfully eradicating ASPV, ASGV, and ACLSV from apple plants, using the in vitro shoots of four apple cultivars.

Virus detection in plant materials
The results for the detection of ASPV, ASGV, and ACLSV in the in vitro shoots of four apple cultivars ('Ruiyang', 'Ruixue', 'Rui Xianghong', and 'Qinyue') are shown in Fig. 1. Two latent viruses, ASGV and ACLSV, but not ASPV, were detected in 'Ruiyang' and 'Ruixue'. However, in vitro shoots of 'Rui Xianghong' and 'Qinyue' carried all three latent viruses. This therefore clari ed the viruses potentially present in each cultivar, thus making it possible to focus eradication efforts appropriately.
Survival and regrowth rates after the STC treatment are shown in Fig. 2c. In the -LN treatment, the survival rate of all four cultivars decreased with increasing thermotherapy period. When in vitro shoots were subjected to thermotherapy for up to 8 weeks, the shoot tip regrowth rate for 'Qinyue', 'Rui Xianghong', Ruixue', and 'Ruiyang' gradually decreased from 72.5%, 59.6%, 69.4%, and 75.6-19.8%, 26.3%, 21.3%, and 35.9%, respectively. In the + LN treatment, the shoot tip survival and regrowth rate decreased gradually with increasing thermotherapy period; the regrowth rate for 'Qinyue', 'Ruixue', and 'Ruiyang' decreased to zero by week 8. However, the important point is that, for any length of thermotherapy treatment, the shoot tip survival and regeneration rates are higher without than with cryotherapy treatment.

Effectiveness of different treatments in virus eradication
The results of reverse transcription-polymerase chain reaction (RT-PCR) detection of in vitro shoots subcultured 10 times are shown in Fig. 4b. Cultivars 'Qinyue' and 'Rui Xianghong' were subjected to the -LN treatment for 8 weeks, and eradication of ASPV increased from nil to 25.6% and 30%, respectively.

Discussion
In this study, we assessed two effective detoxi cation methods: thermotherapy and combined thermoand cryotherapy. These two techniques were applied to in vitro shoots of four apple cultivars subcultured at the age of 2 weeks. Our results determined that combined thermo-and hydrotherapy was more effective in virus eradication than thermotherapy alone, which is in agreement with previous studies [31,33].
The thermotherapy temperature was alternated between 38 °C in the day and 30 °C at night, rather than imposing a constant high-temperature environment. This bene ted the in vitro shoots by relieving the discomfort of the higher temperature at night while not lowering the temperature for too long [34][35][36].
The viral inhibition under thermotherapy treatment potentially reduces the transport of viral particles to the apical meristem [36,37], and expands the non-infected area at the stem tip, which is therefore virusfree [10]. The length of the thermotherapy treatment is key to the successful establishment of a detoxi cation system. When thermotherapy treatment is too short, a large number of virus particles will remain in the shoot tips, therefore preventing full detoxi cation. However, if thermotherapy is administered for too long, the heat-resistant function of the shoots will begin to decline. Tan et al. [35] found that a combined treatment of thermotherapy for 35 days (at 37 °C) and STC could produce ASGVand ACLSV-free pear plants from shoot tips of 1.0 mm in length; however, shoot tips longer than 2.0 mm could not produce virus-free plants. In our study, we were able to produce ASPV-and ACLSV-free plants using thermotherapy, a result in agreement with previous reports [35,38].
The effects of droplet-vitri cation and encapsulation-dehydration procedures on post-cryotherapy recovery patterns in apple shoot tips have been compared [39,40]. These studies have shown that shoot tip regrowth rates obtained by droplet-vitri cation is more stable than rates yielded by encapsulationdehydration throughout the year, i.e. there was no seasonal effect. In our study, we used dropletvitri cation as our cryotherapy programme.
ASGV, ACLSV, and ASPV are found in most apple cultivars in commercial orchards [41]. A previous study suggested that using cryotherapy alone could not eradicate ASGV [18]. Some studies have clearly indicated serious ASGV infection and localisation in the shoot tips and meristematic cells, which may explain why it was di cult to eradicate [18,33].
Previous reports have shown that shoot tips (about 1 mm in length) can produce virus-free plants after thermotherapy [36], but the survival rate of shoot tips of this length under cryotherapy treatment is very low [19,29,33]. In our study, we cut shoot tips to a length of 1.5 mm after thermotherapy; when then combining this treatment with cryotherapy, we showed a strong improvement in virus eradication from the cultivars 'Ruiyang' and 'Ruixue', which increased with increasing thermotherapy period, consistent with the previous studies [29,33]. We found that a combined thermo-and cryotherapy treatment of shoot tips (1 mm) in 'Ruiyang', 'Ruixue', 'Qinyue', and 'Rui Xianghong' cultivars showed an improved effectiveness in virus eradication, the rate of which increased with increasing thermotherapy period. This provides a basis for promoting healthy and sustainable development of the apple industry, so long as the development and performance of virus-free plants are strictly tested and supervised in the production process.

Conclusions
In conclusion, our study explored two effective methods of eradicating viral infections from apple plants, that is, thermotherapy and combined thermo-and cryotherapy. We demonstrated that combining the application of thermotherapy for 4 weeks with cryotherapy can effectively eradicate ASPV and ACLSV, and is effective in minimising ASGV, which is harder to eradicate completely. This study therefore provides a guaranteed technical method that supports the development of new virus-free apple plant materials and the application and promotion of new cultivars.

Plant material
We selected four apple cultivars based on their strong developmental potential as crops. removed from the trees in the eld and transported to the laboratory. There, they were soaked in a solution of 1% NaClO for 10 min, followed by hydroponic culture at 25 °C (with no further additional nutrient); water was changed daily. Shoot tip samples (length, approximately 1.5 cm) (Fig. 3a) per apple cultivar were extracted from the branches, rinsed under tap water for 30 min, treated with 75% alcohol for 30 s and then with 0.1% HgCl2 (adding 2-3 drops of surfactant [Tween 20]) for 10 min (Fig. 3b). The samples were nally rinsed in sterile water 5-7 times. Shoot tip surfaces in contact with the disinfectant solution were removed and inoculated on to the culture medium (Fig. 3c).

Virus detection
All plant materials were tested twice using RT-PCR. The in vitro shoots for each apple cultivar (Fig. 3e) were analysed to determine viral presence. The regenerated plants were then investigated to assess the virus eradication frequency. After the subculture process had been repeated 10 times (from October 2017 to June 2018), the samples were analysed again to detect the presence of viruses (Fig. 3l).

Thermotherapy and treatment
In vitro shoots of the four cultivars were subcultured for 2 weeks, and then moved into a growth chamber (RXZ-500D-LED, Ningbo, China) under light conditions of 50 µE s − 1 m − 2 (Fig. 3f). The temperature in the chamber was set to alternate between 38 °C during the day (6:00 am to 22:00 pm) and 30 °C at night (22:00 pm to 6:00 am). At 0, 2, 4, 6, and 8 weeks of thermotherapy, 100 shoot tips of each cultivar were cut under an anatomical microscope to a length of approximately 1.5 mm (four to ve leaf primordia) (Fig. 3g). The shoot tips were divided into two groups: 50 shoot tips for direct STC without cryotherapy (-LN) (Fig. 3h), and 50 for cryotherapy treatment (+ LN) (Fig. 3j). All experiments were repeated in triplicate.

Cryotherapy
Feng et al. [39] describe the procedure for droplet-vitri cation. First, shoot tips were cultured under stable conditions on a basic medium (BM) for 1 day, then transferred to the MS solution (0.8 M sucrose, 2 M glycerol), also for 1 day. The shoot tips were transferred into droplets containing 6 uL plant vitri cation solution 2 (PVS2) [47] on aluminium foil (5 × 1.5 cm) (Fig. 3i), and frozen in liquid nitrogen for 1 h. The PVS2 solution was composed of 30% glycerol (wt/vol), 15% dimethylsulfoxide (wt/vol), 15% ethylene glycol (wt/vol), and sucrose (0.4 M) in MS (pH 5.8). The shoot tips were then removed quickly from the liquid nitrogen and placed into an MS unloading solution (containing 1.2 M sucrose) for 20 min at a temperature of 20 ± 2 °C. Finally, the shoot tips were placed into a BM medium in dark culture conditions for 3 days, after which the shoot tips were transferred to light culture conditions. Within 3 days of the dark culture treatment, the shoot tips were transferred to the new medium twice, after being frozen for 12 h and 36 h, to reduce browning. After regenerating into normal shoots, they were subcultured every 4 weeks.
Virus-free plants higher than 2 cm (Fig. 3l) were selected from all four cultivars and observed to grow healthily. Prior to rooting, they were inoculated onto 1/2 MS medium with IBA concentrations of 0.9 mg L − 1 , 0.75 mg L − 1 , 0.9 mg L − 1 , and 1.1 mg L − 1 for the 'Ruiyang', 'Ruixue', 'Rui Xianghong', and 'Qinyue' cultivars, respectively. When the root length of the in vitro shoots reached approximately 2 cm, plants were transferred to the seedling re nement room. The cap of each tissue culture bottle was opened for 1-2 days, until no fungus was visible in the bottle, prior to transplanting. After the seedlings were removed from the culture medium to be transplanted, culture medium attached to the roots of the seedlings was washed with clean water (Fig. 3m) and transferred to a nutrition bowl containing peat soil, perlite, and vermiculite (volume ratio of 3:0.5:0.5). The transplanted seedlings were cultured for 7 days (Fig. 3n) in low light (< 20 µEs − 1 m − 2 ), and then gradually transitioned to normal light levels (50 µEs − 1 m − 2 ) (Fig. 3o).