Molecular polymorphism in phytoplasmas infecting peach trees in Serbia

During survey carried out in 2008 to verify phytoplasma presence in fruit trees in Serbia, samples were collected from two single peach plants (30-Niš and 103-Radmilovac) showing yellows disease and phytoplasma detection was carried out using PCR assays with primer pair P1/P7 in direct reaction, followed by nested PCR with F1/B6, R16F2n/R2, and R16(I)F1/R1. RFLP analyses carried out on R16F2n/R2 amplicons with Tru1I, TaqI, Tsp509I, AluI, and BfaI restriction enzymes showed that in peach 30, a mixed phytoplasma infection was present. Tentatively identification of 16SrII and 16SrXII phytoplasmas was confirmed by RFLP analyses carried out on amplicons obtained with primers R16(I)F1/R1 with Tru1I and Tsp509I restriction enzymes. RFLP analyses on F1/B6 and R16F2n/R2 amplicons with Tru1I showed that 16SrII group phytoplasmas were present also in peach 103. F1/B6 and R16F2n/R2 amplified products (about 1700 bp and 1200 bp respectively) of both peach samples were purified using Qiagen PCR Purification Kit (Qiagen GmbH, cloned in DH5 alfa and a number of clones was screened by PCR with M13 primers followed in nested PCR by F1/B6 and R6F2n/R2 primers (according with amplicons). RFLP analyses with Tru1I on R16F2n/R2 amplicons of seven M13 clones obtained from peach 30 show the presence of 6 profiles referable to 16SrXII and two referable to 16SrII phytoplasmas. AluI restriction enzyme did not show polymorphisms and distinguished between groups 16SrII and 16SrXII while Tsp509I showed different profiles between the two 16SrII group phytoplasmas. Similar results were obtained with Tru1I and AluI restriction enzymes on 3 clones amplified with R16(I)F1/R1. Peach 103 clones were all identical after RFLP analyses. This is the first report of 16SrII phytoplasmas in peach and the 16Sr DNA variability detected in both 16SrXII and 16SrII phytoplasmas is an indication of phytoplasma population presence in the infected plant.