Distribution of phytoplasma diseases in the Lombardy poplar tree population of Zagreb urban area

Phytoplasmas are uncultivable prokaryotic wall-less pathogens belonging to the class Mollicutes that inhabit plant phloem and insects. Their identification and classification is difficult and mainly based on the polymorphism of their 16S rRNAgene sequences. Aster yellows (AY; 'Candidatus Phytoplasma asteris') phytoplasmas (ribosomal subgroup 16SrI-P) had been previously identified in several Populus nigra L. 'Italica' (Lombardy poplar) trees from the urban area of Zagreb. The aim of this research was to examine phytoplasmosis distribution in the poplar tree population of the wider Zagreb urban area. Total nucleic acids were extracted from leaf samples of 30 symptomatic and 4 asymptomatic trees. Phytoplasma 16S rDNAwas amplified in direct and nested PCRs by using universal and group-specific primers. The pathogens were classified on the basis of 16S rDNA amplicon RFLP analyses. Phytoplasmas belonging to the 16SrI ribosomal group (AY) were detected in 12 out of 34 trees examined. In addition, a phytoplasma putative gene for aa kinase was analyzed for positive samples. RFLP profiles from 10 samples were referable to the phytoplasma 16SrI-P ribosomal subgroup previously found only in poplars from Zagreb area. In two samples, unique restriction patterns were found, showing the presence of molecular variability within this conserved gene region. In the northern and north-western part of the area the infection was equally distributed, while in the southern part of the city phytoplasmas were not detected in sampled trees. Further research, including a search for potential insect vectors, is needed in order to indentify the ecological and epidemiological implications of these diseases and their impact on the sanitary status of ornamental trees in the city of Zagreb.