Identification of phytoplasmas on pomaceous fruit tree species in Hungary

In Hungary local occurrence of Apple proliferation disease(AP) on apple was described for the first time in the early sixties, since that time the disease has been found in different part of the country based on the results of biological indexing. The first suspicious sign for the presence of Pear decline disease (PD) in Hungarian pear orchard was indicated in the early 70s, but at the time there were no adequate laboratory techniques for identification. The aim of the present study was the molecular identification of phytoplasmas in symptom-showing apple trees, as well as confirmation of the occurrence of PD phytoplasma in symptomatic pear trees, testing them in parallel also on woody indicators. Symptom showing trees, indicating possible phytoplasma infection, were sampled in different growing regions in 2002 for indexing. AP was detected on 'Golden Delicious' using root and double grafting. Typical symptoms of PD did not appear on root or double grafted Pyrus communis 'Comice'. Nucleic acids were prepared from all samples by chloroform/phenol extraction from fresh, frozen or dry phloem tissue of leaves and young branches. Nested PCR procedures were employed with different sets of primers to amplify phytoplasma 16SrDNA, or 16SrDNA plus spacer region. The identity of PCR products was confirmed by RFLP as 16SrX-A (AP) and 16SrX-C (PD). It was concluded that molecular methods were useful for identification of phytoplasmas in several of the symptomatic orchard trees of apple and pear respectively. Both greenhouse indexing and nested PCR/RFLP were suitable for detection and identification of AP from bearing trees and the phytoplasma was identified by molecular methods also in the inoculated AP indicators. PD could not be detected in greenhouse indexing.