New Disease Reports
New Disease Reports (2008) 18, 36.
Next > < Prev

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

M. Kamińska* and H. Berniak

*maria.kaminska@insad.pl

Show affiliations

Accepted: 09 Dec 2008

Symptoms of growth abnormalities including shoot proliferation, leaf rosetting and malformation of two-to-three years old ash trees (Fraxinus excelsior) used as rootstock, as well as on plants of cv. Aurea (Figs. 1-3), 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 symptomatic and two symptomless trees, and healthy Catharanthus roseus plants, using a DNeasy Plant Mini Kit (Qiagen, USA). 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 were 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, USA).

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 one symptomatic and the symptomless ash trees or healthy C. roseus. RFLP profiles indicated that ash trees were infected with a 16SrI phytoplasma ('Ca. Phytoplasma asteris') (Fig. 4). 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 (Fig.5).

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 and Śliwa, 2006), associated with symptoms of stunting, shoot proliferation and dieback. To our knowledge, this is the first record on the occurrence of ‘Ca. Phytoplasma asteris’ in ash trees.

Figure1+
Figure 1: Shoot proliferation and leaf rosetting of two years old ash tree
Figure 1: Shoot proliferation and leaf rosetting of two years old ash tree
Figure2+
Figure 2: Apical leaf malformation and size reduction of ash tree
Figure 2: Apical leaf malformation and size reduction of ash tree
Figure3+
Figure 3: Leaf malformation and necrosis of ash tree ‘Aurea’
Figure 3: Leaf malformation and necrosis of ash tree ‘Aurea’
Figure4+
Figure 4: HhaI, MseI and RsaI RFLP patterns obtained for phytoplasma detected in ash plant (J) and phytoplasma reference strains (‘Ca. Phytoplasma asteris’ AY1, ‘Ca. Phytoplasma mali’ AP15, ‘Ca. Phytoplasma ulmi’ ULW). M – molecular marker Ф174 DNA/HinfI (Promega Symbios).
Figure 4: HhaI, MseI and RsaI RFLP patterns obtained for phytoplasma detected in ash plant (J) and phytoplasma reference strains (‘Ca. Phytoplasma asteris’ AY1, ‘Ca. Phytoplasma mali’ AP15, ‘Ca. Phytoplasma ulmi’ ULW). M – molecular marker Ф174 DNA/HinfI (Promega Symbios).
Figure5+
Figure 5: Phylogenetic tree based on 16S rRNA gene sequences of of phytoplasma isolates from ash plants (acc. no. EU211911 and EU219612) and reference phytoplasma sequences from GenBank: (acc no. M86340 and M30790), Ca. Phytoplasma ulmi (AY197655), Ca. Phytoplasma mali (AJ542541), Ca. Phytoplasma fraxini (AF092209), Western X-disease phytoplasma (L04682).
Figure 5: Phylogenetic tree based on 16S rRNA gene sequences of of phytoplasma isolates from ash plants (acc. no. EU211911 and EU219612) and reference phytoplasma sequences from GenBank: (acc no. M86340 and M30790), Ca. Phytoplasma ulmi (AY197655), Ca. Phytoplasma mali (AJ542541), Ca. Phytoplasma fraxini (AF092209), Western X-disease phytoplasma (L04682).

References

  1. Griffiths HM, Sinclair WA, Smart CD, Davis RE, 1999. The phytoplasma associated with ash yellows and lilac witches’ broom: ‘Candidatus Phytoplasma fraxini’. International Journal of Systematic Bacteriology 49, 1605-1614.
  2. Kamińska M, Śliwa H, Rudzińska-Langwald A, 2001. The association of phytoplasma with stunting, leaf necrosis and witches’ broom symptoms in magnolia plants. Journal of Phytopathology 149, 719-724.
  3. Kamińska M, Śliwa H, Malinowski T, Skrzypczak Cz, 2003. The association of aster yellows phytoplasma with rose dieback disease in Poland. Journal of Phytopathology 151, 469-476.
  4. Kamińska M, Śliwa H, 2006. First report of a decline of ashleaf maple (Acer negundo) in Poland, associated with ‘Candidatus Phytoplasma asteris’. Plant Pathology 55, 293.
This report was formally published in Plant Pathology

©2008 The Authors