‘Candidatus Phytoplasma asteris’ in Fraxinus excelsior and its association with ash yellows newly reported in Poland

Abstract

Symptoms of growth abnormalities including shoot proliferation, leaf rosetting and malformation of two-to-three year-old ash trees (Fraxinus excelsior) used as rootstock, as well as on plants of cv. Aurea, were observed in a commercial nursery in Poland. Symptoms were similar to ash yellows disease associated with phytoplasmas of Group 16SrVII, ‘Candidatus Phytoplasma fraxini’ in the USA (Griffiths et al., 1999). Nucleic acids were extracted from shoot phloem of three trees with and two without symptoms, and healthy Catharanthus roseus plants, using a DNeasy Plant Mini Kit (Qiagen). In nested PCR the phytoplasma universal primers P1/P7 were used, followed by fA/rA, R16F2n/R16R2 or the group-16SrI (R16(I)F1/R16(I)R1) specific primers. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 PCR products was done using the endonucleases HhaI, MseI or RsaI. PCR-amplified phytoplasma 16S rRNA fragments from two F. excelsior (J1, J2) were purified, sequenced, and sequences analyzed using BLAST (http://www.ncbi.nlm.nih.gov) and Lasergene v. 7·1 (DNASTAR). Phytoplasma rDNA was amplified by nested PCR with fA/rA, R16F2n/R16R2 and R16(I)F1/R16(I)R1 primers from two out of three tested ash trees with disease symptoms, but not from the other tree with symptoms, nor the symptomless ash trees or healthy C. roseus. RFLP profiles indicated that ash trees were infected with a 16SrI phytoplasma (‘Ca. Phytoplasma asteris’). Sequence analysis for J2 (GenBank Accession No. EU219611) and J1 (EU219612) confirmed PCR-RFLP results, and showed a 99·7% of identity to each other, and more than 98% with those of phytoplasma members of 16SrI-B sub-group. Phylogeny analyses of F. excelsior phytoplasma sequences revealed their closest relation to 16SrI group, particularly with that of Accession No. M86340. 16SrI group phytoplasmas seem to be most widespread in Europe and America and they have been reported frequently from woody plants. In Poland they have been found in ornamental crops including magnolias, roses (Kamińska et al., 2001, 2003) and ashleaf maple trees (Kamińska & Śliwa, 2006), associated with symptoms of stunting, shoot proliferation and dieback. This is the first record on the occurrence of ‘Ca. Phytoplasma asteris’ in ash trees.