First report of pear decline phytoplasma on pear in Serbia

In August 2004, pear (Pyrus communis L.) plants with typical symptoms of Pear Decline (PD) were observed in orchards in central Serbia. The affected plants showed premature reddening and upward rolling of leaves that often show down-shaped petioles. In some cases premature defoliation was observed. Although similar decline of pear was observed earlier, till now no identification of causal agent was performed. Nucleic acids were extracted separately from fresh leaf midribs and branch phloem scrapes of 4 symptomatic and 1 asymptomatic pear plants. A nested polymerase chain reaction assay (PCR) was used for phytoplasma detection: first PCR round with P1/P7 (3) phytoplasma universal primer pair, followed by nested PCR with f01/r01 (2) 16SrX group specific primer pairs were carried out obtaining the expected products from both phloem scrapes and midribs extracts of symptomatic plant samples. RFLP analyses with RsaI and SspI restriction enzymes, discriminating among 16SrX subgroup phytoplasmas, showed profiles corresponding to those of Apple proliferation phytoplasma group, 16SrX-C subgroup, Candidatus Phytoplasma pyri (4). A 1155 bp sequence of 16S rDNA gene for one of the PA2f/r (1) amplicons obtained in nested PCR on P1/P7 products from one of the leaf midrib samples was deposited in GenBank (AY949984); both strands of the fragment were sequenced using the Big Dye Terminator reaction kit; the sequences were analysed with Chromas 1.55 (Technelysium, Queensland, Australia) and aligned with BLAST softwares available at http://www.ncbi.nlm.nih.gov; the blast search showed 100% homology of this sequence with those of strain Y16392 confirming the identity with PD of the phytoplasma detected. To our knowledge this is the first report of pear decline phytoplasmas in Serbia