Detection and characterization by PCR/RFLP analyses of phytoplasmas in plum orchards in i̇zmi̇r provi̇nce and determination of possible vectors

In this study, the presence of phytoplasmas in plum trees in Izmir province was determined, the possible phytoplasma vector insects were collected from phyoplasma infected orchards and morphologically identified and the presence of phytoplasma in the insect was investigated by molecular methods. The most common symptoms observed in plum trees were chlorosis and inward curling in the leaves, small leaves, reddening and shoot proliferation. As a result of field surveys, a total of 110 plum samples from 9 different plum varieties and 30 insects identified as Psammotettix provincialis belonging to the Cicadellidae family were collected from these orchards. Phytoplasma was detected in 29 of the plum samples tested by Nested-PCR analyses and 8 PCR products were cut with RsaI and SspI restriction endonuclease enzymes for RFLP analysis and 4 were found to be infected with Apple proliferation phytoplasma (Candidatus Phytoplasma mali) and 4 other plum samples were infected with Pear decline phytoplasma (Candidatus Phytoplasma pyri). 16 of the 30 P. provincialis individuals tested by Nested-PCR analyses were found to be positive for phytoplasma. In the phylogenetic analyzes performed, it was determined that these individuals were infected with Candidatus Phytoplasma asteris (16SrI). 22 out of 29 phytoplasma positive isolates were sent for sequence analysis and the obtained sequences were subjected to virtual RFLP analysis with 17 restriction enzymes [AluI, BamHI, BfaI, BstUI (ThaI), DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI (MboI), MseI, RsaI, SspI and TaqI] were cut and the obtained band profiles were compared with reference isolates. As a result of virtual RFLP analysis, 11, 4, 3, 2 and 2 isolates gave a similar enzyme cut profile with Ca. P. mali, Ca. P. prunorum, Ca. P. pyri, Ca. P. asteris and Ca. P. trifolii, respectively.