First report of 'Candidatus Phytoplasma fraxini' (group 16SrVII phytoplasma) associated with a peach disease in Canada

Peach (Prunus persica), family Rosaceae, is native to China . In Canada , the province of Ontario produces 82% of the nation's peach crop. The Canadian Clonal Genebank, also located in Ontario, currently holds 83 accessions of peach. The collection is routinely monitored for symptoms of phytoplasma including decline (Fig. 1), leaf reddening (Fig.2), yellowing, shortening of internodes, witches' broom and reduced fruit size.

Leaf samples from trees exhibiting symptoms and symptomless trees representing sixteen accessions were collected from June to August 2009.Total DNA was extracted (DNeasy plant extraction kit, QIAGEN). Phytoplasma universal primers specific for 16S rDNA (R16mF2/R1 and R16F2n/R2; Gundersen & Lee, 1996) were used in a nested PCR assay. Nested PCR products of expected size (~1250 bp) were obtained for accessions PRU0430 (cv. HW274, from Canada), PRU0380 (cv. GF 305 from France), PRU0334 (cv. RedSkin from USA), PRU0155 (cv. Harblaze from Canada) and PRU0375 (cv. Babygold #5 from USA). Symptomless plants yielded no PCR products. Amplicons were purified (Wizard PCR Clean-up, Promega), cloned (pGEM-T Easy Vector, Promega), and sequenced (Robarts Institute, London, Canada ). The obtained sequences shared 100% identity with each other and a representative corresponding to PRU0430 was deposited in GenBank (Accession No. GU223903).

BLAST analysis showed that the Prunus phytoplasma shared 99% 16S rDNA sequence identity with those of phytoplasmas in the group 16SrVII (ash yellows) for which 'Candidatus Phytoplasma fraxini' is the reference strain. Restriction fragment length polymorphism analysis (RFLP) of amplicons using AluI, RsaI and MseI yielded profiles similar to those of 16SrVII-A phytoplasma subgroup. Phytoplasmas of group 16SrVII have been reported in different hosts in Colombia , Argentina , Brazil and Chile (Fiore et al., 2007). Particularly in North America , including Canada , 16SrVII phytoplasmas have been associated with ash yellows and lilac witches' broom diseases (Sinclair et al., 1996; Griffiths et al., 1999). To our knowledge, this is the first report of a 16SrVII-related phytoplasma strain identified in peach after an incidental report in peach in Southern Italy (Paltrineri et al., 2003). The 16SrVII phytoplasma has causeddevastating effects in ornamentals. Therefore its presence in peach is of great phytosanitary significance due to its commercial interest, and represents a potential threat for disease spread to other fruit crops, particularly Prunus sp.