ELIMINATION AND DETECTION OF PATHOGENS FROM TISSUE CULTURES OF PRUNUS SP.

Virus infection seriously limits stone fruit production in Europe and in the Mediterranean region. The most important viral pathogen is Plum pox virus (PPV), however, other viruses, e.g. Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) also represent a major threat. Experiments on in vitro thermotherapy and subsequent meristem culture led to virus-free plant material, which could be multiplied rapidly by micropropagation techniques. In vitro cultures of peach, plum and apricot cultivars were established. Cultures initiated from different buds of the same cultivar were treated as different clones. The cultures were inoculated onto modified MS medium containing BAP, IBA and GA₃ and kept at 22°C under a 16/8-hr photoperiod. Successfully established cultures of each cultivar and clone were tested for virus infection, first by DAS-ELISA and then by IC-RT-PCR technique. The results showed major differences not only between cultivars, but also between different clones of the same cultivars. Some of the clones proved to be virus-free, while other clones (originating from the same tree) were infected by PPV or by PNRSV, and in some cases by both viruses. The peach clone (B5) of ‘Biscoe’ was found to be infected by European stone fruit yellows (ESFY) phytoplasma. Major differences were observed between the viability of cultivars and clones, which were not only due to genetic differences, but also to the different phytosanitary status of the plants. Cultures of clones infected by only one virus grew much more vigorously than cultures of clones, which were infected by two or three pathogens. During thermotherapy, cultures were kept in an environmental growth chamber at 38°C/36°C under a 16/8-hr photoperiod for 15-20 days. Astonishingly, the in vitro cultures of peach are more sensitive to high temperatures compared to apricot or plum, which leads to a loss of cultures within 2-3 weeks. After thermotherapy, meristem tips of surviving shoots were excised under a stereomicroscope and placed onto modified MS medium supplemented with BAP. One half of the meristem cultures were kept in the dark, and the other half of them were kept under a 16/8-hr photoperiod. After 3 weeks, all cultures were transferred to modified MS medium containing BAP, IBA and GA3 and placed under a 16/8-hr photoperiod. Shoots regenerated from the excised meristems of thermotherapy-treated shoots of the following peach cultivars: ‘Frederica’ (clones 2, 7, 8) and ‘Biscoe’ (clone 3). Most shoots were regenerated from meristems kept under a 16/8 hr photoperiod from the beginning. Meristem-derived cultures grew vigorously, and the number of shoots has been multiplied several times for phytosanitary and clonal control. The phytosanitary status of the regenerated shoots was controlled under in vitro conditions by IC-RT-PCR technique and in vivo by biotests. All examinations ensured the successful thermotherapy, since all the plantlets tested negative. The established and infected plum and apricot cultivars are currently being treated by thermotherapy.