Detection and identification of phytoplasmas affecting blackcurrant and redcurrant

During October-November/2011, blackcurrant (Ribes nigrum L.) and redcurrant (Ribes rubrum L.) shrubs exhibiting peach symptoms of leaf redness and downward curling in Harrow, Ontario, Canada, were tested for phytoplasma. Total DNA was extracted (Fast DNA Spin kit, MP Biomedicals, USA) and used in a nested PCR assay with universal primers that target the phytoplasma 16S rRNA gene, P1/P7 (Deng and Hiruki, 1991; Smart et al, 1996) followed by either R16F2n/R2 (Gundersen and Lee, 1996) or fU5/rU3 (Lorenz et al, 1995) for the nested reaction. R16F2n/R2 PCR amplicons were obtained for all the symptomatic plants collected, purified (Cycle Pure kit, Omega, USA) and sequenced bi-directionally (University of Health Network, Toronto, Canada). Sequences were compared to those of reference phytoplasmas in GeneBank. BLAST analysis showed a 100% of 16S rDNA sequence identity between the two phytoplasmas detected in both blackcurrant and redcurrant plants, and a 99% of sequence identity to those of members of group 16SrX, subgroup 16SrX-A. Virtual and actual RFLP of the R16F2n/R2 and fU5/rU3 amplicons with AluI, RsaI and MseI restriction endonucleases yielded RFLP patterns similar to those of phytoplasma members of the group 16SrX ‘Apple Proliferation’, subgroup 16SrX-A. Phylogenetic analysis (MEGA version 4.1, USA) based on the 16S rDNA sequences confirmed RFLP and BLAST results. Phytoplasmas of group 16SrX have been previously reported in Harrow, Ontario, affecting Prunus and Pyrus species. However, results represent the first record of a 16SrX-A infecting blackcurrant and redcurrant in Canada, and provide a valuable tool for further epidemiological studies of a 16SrX phytoplasma in these two plant species.