Phytoplasma caused diseases pose a potentially serious threat to the production of fruit trees worldwide. In recent years stone fruit trees with characteristic symptoms of phytoplasma associated disease were noticed in Isfahan and Chaharmahal-o-Bakhtiari provinces. Symptoms included witches'broom, chlorosis, leaf stunting, rosette, leaf rolling and dieback of branches in almond and yellowing, rosette and rolling leaves of plum peach and cherry trees. During 2003-2005 symptomatic and apparently healthy leaves of affected trees were sampled from different sites in Isfahan , Chaharmahal-o-Bakhtiari, East Azerbaijan (Shabestar) and Tehran (Sahriar-karadj). The midribs and peduncle of the species were retained for DNA extraction. First-round PCR analysis was performed with all samples with primer sets P1/P7, P1/Tint and PA2F/R. Three additional primer pairs 16F2/R, fU5/rU3 and NPA2F/R which prime the amplification of definite regions within 16Sr-23S rRNA were utilized for Nested PCR reaction. PCR products were detected and their sizes estimated by electrophoresis on 1. 2% agarose gel in TBE buffer . Except few DNA samples, no DNA amplification was observed when the primer pairs P1/P7, P1/Tint and PA2F/R were used in first-round of PCR. However employing Nested PCR, using R16F2/R2, primed a DNA fragment of 1239bp from products of first-round PCR by P1/P7 and P1/Tint. Also primer pair NPA2F/R primed amplification of a DNA band of approximately 485bp from products of first-round PCR with PA2F/R. Taken together the results of detection of phytoplasma from symptomatic stone fruit trees without amplification of DNA fragment in healthy plants confirmed the capability of Nested PCR technique to identify phytoplasma associated trees in the region. It is worthwhile to mention that Nested PCR with combination of PA2F/R in first-round and NPA2F/R in the second run of PCR was evaluated as best method for the detection of phytoplasma agents in diseased trees. Restriction enzymes of a part of 16S rRNA gene sequence of the isolates amplified, using R16F2/R2 and then NPA2F/R primer sets in Nested PCR, indicated considerable differences in RFLP patterns of isolates obtaine from peach, cherry and plum trees. Using restriction enzyme Taq I for PCR-RFLP analyses of phytoplasmas, detected from almond trees showing witches broom symptom exhibited the same pattern which was different from those associated with branching and die bach syndromes . Although, Hpa II digestion showed noticeable divergance within each group. Based on restriction analyses, five subgroup were distinguished within phytoplasmas isolated from various stone fruits. Few strains from each subgroup were
