First Report of Pear Decline Phytoplasmas on Pear in Serbia.

ABSTRACT

During August of 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 showed down-turned petioles. In some cases, premature defoliation was observed. Although a similar decline of pear was observed earlier, until now, the causal agent had not been identified. DNA was extracted with a chloroform/phenol procedure from fresh leaf midribs and branch phloem scrapes of four symptomatic and one asymptomatic pear plants separately. A nested polymerase chain reaction assay (PCR) was used for phytoplasma detection (first PCR round with P1/P7 (4) phytoplasma universal primer pair, followed by nested PCR with group 16SrX specific primers f01/r01) (3). With these primers, the expected products from phloem scrapes and midrib extracts of symptomatic plant samples were obtained. Restriction fragment length polymorphism (RFLP) analyses of the f01/r01 amplicon, with RsaI and SspI restriction enzymes, discriminating among 16SrX subgroup phytoplasmas, showed profiles corresponding to those of the apple proliferation phytoplasma group, 16SrX-C subgroup, "Candidatus Phytoplasma pyri" (2). A 1,155-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 (Accession No. AY949984); both strands of the fragment were sequenced with the Big Dye Terminator reaction kit (Applied Biosystems, Foster City, CA). The sequences were analyzed with the Chromas 1.55 DNA sequencing software (Technelysium, Queensland, Australia) and aligned with BLAST software ( http//www.ncbi.nlm.nih.gov ). The blast search showed 100% homology of this sequence with that of PD 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. References (1) M. Heinrich et al. Plant Mol. Biol. Rep. 19169, 2001. (2) IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group. Int. J. Syst. Evol. Microbiol. 541243, 2004. (3) K.-H. Lorenz et al. Phytopathology 85771, 1995. (4) Schneider et al. Pages 369-380 in Molecular and Diagnostic Procedures in Mycoplasmology. Vol I. S. Razin and J. G. Tully, eds. The American Phytopathological Society, 1995.