Improved molecular methods for detection of European stone fruit yellows (ESFY) phytoplasmas from in vitro shoots of fruit trees

Prunus species are prone to infections by viruses and phytoplasmas, against which no effective cure exists for already infected plants in the field. Phytoplasmas infecting fruit trees can cause severe symptoms and are considered as quarantine organisms in Europe and North America. However, detection often is hampered by their irregular distribution in host plants. In the frame of phytosanitary measures a sensitive, specific and quick detection system would be highly desirable for routine detection, mainly to avoid the use of infected planting material. The development of improved strategies for the production of elite plants of phytoplasma-free stone fruit cultivars to be used for the production of certified elite propagation material was achieved. In vitro thermotherapy and meristem culture to eliminate pathogens from stone fruit plants were applied and protocols were validated for survival rates of shoots and plants and on their effectiveness for pathogen elimination. To gain time and to confirm the elimination of these pathogens from planting material by the applied in vitro treatments, improved detection methods were tested on micropropagated material soon after the treatment. The use of diverse general primers in bi-nested PCR allows to detect phytoplasmas belonging to different groups. Broad spectrum PCR are advisable for fruit tree material in micropropagation when the sanitary status of the mother plants is not known, while specific PCR primers could be employed to detect the presence of a known phytoplasma.