First Report of 'Candidatus Phytoplasma asteris'-Related Strain Associated with Peach Rosette in Canada.

Prunus persica (L.) Bastch (family Rosaceae) is currently represented by 83 accessions at the Canadian Clonal Genebank. Approximately 3,200 ha are devoted to peach cultivation in Canada where Ontario Province accounts for 82% of the national production. The clonal peach accessions, also located in Ontario, are monitored routinely for symptoms of phytoplasma infection, including rosette-like symptoms (3) that are characterized by new shoots with very short internodes, loss of older shoot leaves leaving only bunches of young leaves on the tips of naked shoots, and flowers that rarely set fruit. From June to August 2009, peach accessions PRU0382 and PRU0445 showed typical peach rosette symptoms, while another 14 accessions exhibited either short internodes or no symptoms. Leaf midrib samples were collected from 16 peach accessions, including 17 symptomatic (from which 8 corresponded to accession PRU0382, 6 for PRU0445, 1 for PRU0335, 1 for PRU0179, and 1 for PRU0451) and 16 asymptomatic (from which 5 corresponded to a representative of each accession PRU0382, PRU0445, PRU0335, PRU0179, and PRU0451 and 11 to other peach accessions). Total DNA was extracted (DNeasy Plant Extraction Mini Kit, QIAGEN, Valencia, CA) from 100 mg of each sample and used as a template in a nested PCR with phytoplasma universal primers R16mF2/R1 (1) and fU5/rU3 (2). Nested PCR products of the expected size (~880 bp) were obtained from all symptomatic samples (14 of 14) of accessions PRU0382 (peach-almond cv. Kando from the Czech Republic) and PRU0445 (peach cv. HW271 from Canada) only. All other plants with or without symptoms yielded no PCR products. Amplicons were purified (Wizard PCR Clean-up, Promega, Madison, WI), cloned in pGEM-T Easy Vector (Promega), and sequenced (Robarts Institute, London, Canada). The resulting 16S rDNA sequences were identical; one of each was archived in GenBank as Accession No. GU223904. BLAST analysis determined that the P. persica phytoplasma sequence shared 99% identity with 16S rDNA sequences of 'Candidatus Phytoplasma asteris'-related strains. This relationship was also supported by restriction fragment length polymorphism analysis (RFLP) of rDNA amplicons using AluI, RsaI, and MseI endonucleases that yielded fragment profiles indicative of phytoplasmas belonging to group 16SrI (Aster Yellows), subgroup B (16SrI-B). Among phytoplasma diseases, those attributed to group 16SrI strains are most numerous and affect the widest plant host range. They include peach rosette in the United States and Europe (3) as well as diseases of various horticultural crops in Canada, including grapevine (4). To our knowledge, this is the first report of a subgroup 16SrI-B phytoplasma affecting peach in Canada. Early detection of phytoplasmas by PCR in accessions with both European and Canadian origins underscores the importance of prompt identification of infected plants for subsequent thermotherapy treatment to maintain the health of the collection and prevent further disease spread. References: (1) D. E Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:1441, 1996. (2) K. H. Lorenz et al. Phytopathology 85:771, 1995. (3) C. Marcone et al. Acta Hortic. 386:471, 1995. (4) C. Y. Olivier et al. Plant Dis. 93:669, 2009.