Improved detection methods for fruit tree phytoplasmas

Phytoplasmas infecting fruit trees are considered quarantine organisms in E urope and North America. Detection often is hampered by their extremely irr egular distribution in host plants. A sensitive, specific and quick diagnos tic test would be highly desirable for routine detection, mainly to avoid u sing infected planting material. PCR methods require tedious preparation of DNA: also, the available primers are highly specific and exhibit some homo logy to chloroplast and plastid DNA. To address these problems, we compared several DNA preparation protocols for purity of DNA, cost and time require d. We also developed new primers using rDNA sequence information from an Au strian isolate of European Stone Fruit Yellows (ESFY). These primers operat e at high annealing temperatures and, thus, increase the specificity and de crease the risk of false positives. The primers could reliably detect the E uropean phytoplasmas (AP, ESFY and PD) within a collection of isolates main tained in micropropagated periwinkle. Thus, they are suitable as general pr imers for phytoplasma detection. The primers also can be used for strain id entification by direct PCR followed by RFLP analysis as demonstrated with m icropropagated fruit tree material. Finally, an IC-PCR method that uses the primers for AP detection was found very sensitive and suitable for large-s cale testing of apple material in vivo and in vitro.