Abnormal shoot branching was observed in Pinus sylvestris trees in Poland. These abnormalities resulted in the formation of dense, ball-like structures with dwarfed needles. The presence of phytoplasma in the needles of branched and surrounding symptomless shoots was demonstrated using nested-polymerase chain reaction (PCR) with universal primer pairs that amplified phytoplasma 16S rDNA, as well as using restriction fragment length polymorphic analysis of PCR products. Comparison of nucleotide sequences of DNA samples from three P. sylvestris trees with balllike structures revealed that their fragments of 16S rDNA were identical. The nucleotide sequence showed more than 99% similarity with the corresponding fragments of sequence of ÔCandidatus phytoplasma piniÕ.

Polymerase chain reaction (PCR) assay, restriction fragment length polymorphism analysis, sequencing of PCR‐amplified cDNA, graft transmission and mycological tests were used to investigate the possible association of phytoplasma infection with rose dieback symptoms and to identify any associated pathogen. Rose plants with shoot dieback symptoms, symptomless rose plants, and experimentally infected periwinkle seedlings were shown to be infected with phytoplasma, which was classified to the aster yellows (AY) group, subgroup 16SrI‐B, based on the analysis of the nucleotide sequence of a 1101‐bp fragment of 16S rRNA gene (GenBank accession no. AF450325). No evidence was obtained for the infection of rose plants with Verticillium  albo‐atrum. The rose plants cultivated under plastic cover without heating or under outdoor conditions, in contrast to greenhouse‐grown roses, developed downy mildew symptoms caused by Peronospora  sparsa.

In 1999±2000 a severe disease was observed on plants of four Magnolia spp. cultivated in a commercial nursery in Poland. Aected plants showed a progressive loss of vigour, were stunted, and had severely malformed leaves, leaf necrosis and witches' broom. Phytoplasma was detected in magnolias with severe symptoms and in dodder-inoculated Catharanthus roseus seedlings by nested polymerase chain reaction (PCR) assay with primer pair R16F1/R0 followed by universal (rA/fA) and group speci®c (R16(I)F1/R1) primer pairs which ampli®ed a fragment of phytoplasma 16S rDNA. The PCR products (560 bp or 1.1 kb) of all samples used for restriction fragment length polymorphism analysis after digestion with endonuclease enzymes AluI and MseI produced the same pro®le which corresponded to that of an aster yellows phytoplasma reference strain. Phytoplasma DNA was detected throughout the growing season in roots, stems and young but not mature leaves. Electron microscope examination of the ultra-thin sections of the leaf and stem of diseased magnolias showed collapsed and degenerated sieve tube elements with wall thickening. The reduced lumen of these sieve elements contained numerous vesicles and membranebound structures, but no typical phytoplasma cells. This is the ®rst report of aster yellows phytoplasma in magnolia identi®ed by molecular assays. U. S.

In summer 2004, five Dahlia cultorum plants of unknown cultivar, grown in garden plots in Skierniewice, Poland, exhibited bushy growth accompanied by shoot proliferation, narrowed leaf and flower bud deficiency. Research in Italy (Marzachi et al ., 1999) had indicated that stunted growth and shoot proliferation symptoms in some dahlia plants was associated with aster yellows phytoplasma infection. To detect the possible presence of phytoplasmas in dahlias in Poland, three plants showing symptoms and two symptomless plants were assayed for the presence of phytoplasma 16S rDNA by PCR. Nucleic acids were extracted from leaves and roots using DNeasy Plant Mini Kit (Qiagen). A nested PCR was done using the universal phytoplasma primer pair P1/P7, followed by primers fA/rA or R16F2n/R16R2. To detect potential mixed infection in dahlias, the phytoplasma 16SrI or 16SrX group-specific primer pairs (R16(I)F1/R16(I)R1, fAT/rAS, fAT/rPRUS or fPD/rAT) were used for nested PCR. Phytoplasma identification was accompanied by restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2-primed PCR product digested with Alu I, Mse I, Hha I, Hpa II, Ssp I or Rsa I endonucleases.Products were generated by nested PCRs with universal and groupspecific 16(I)F1/R16(I)R1 and fAT/rAS primer pairs exclusively from two diseased D. cultorum plants. No bands were amplified from one dahlia with disease symptoms or from healthy dahlia and Catharanthus roseus plants.

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