'Candidatus Phytoplasma pini' in pine species in Croatia

Seven conifer species: Cupressus sempervirens, Juniperus communis, J. oxycedrus, Picea abies, Pinus halepensis, P. mugo and P. nigra were tested for the presence of phytoplasma DNA. Using nested PCR (primer pairs P1/P7 followed by R16F2n/R16R2), 'Candidatus Phytoplasma pini' was detected in five out of 10 sampled P. halepensis trees and in one out of 11 sampled P. mugo trees. Symptoms like yellowing, proliferations and shedding of the needles were observed on sampled P. halepensis trees, while yellowing and dieback of shoots and branches were observed on P. mugo. Phytoplasma positive P. halepensis trees were found along the Adriatic coast, whereas the only positive P. mugo was found approximately 50 km inland. All 'Ca. P. pini' isolates obtained from conifers in Croatia had the same 16S rDNA sequence. The sequence obtained from the amplified fragments of 16S rDNA had a 100% level of identity with the 16S rDNA sequences of 'Ca. P. pini' previously found in Spain, Poland and Czech Republic.