First report of 'Candidatus Phytoplasma mali' in oriental lilies and its association with leaf scorch in Poland

M. Kamińska*and H. Śliwa

Department of Plant Protection, Research Institute of Pomology and Floriculture, 96-100 Skierniewice, Pomologiczna 18, Poland

*maria.kaminska@insad.pl

Accepted for publication 21/06/07

Leaf scorch or leaf burn, usually considered to be a physiological problem, is the most important disorder of lily plants occurring during production under cover. Symptoms of leaf scorch observed on oriental lily hybrids cv. Siberia in several greenhouses in Poland included leaf malformation and necrosis and flower bud abscission. Similar symptoms in lily cv. Woodriff's Memory were associated with the presence of 'Candidatus Phytoplasma asteris' (Kamińska & Dziekanowska, 2002). This phytoplasma has been reported in several lily cultivars in Poland (Kamińska & Korbin, 2000; Bertaccini et al., 2002).

Figure 1:  Lily plant cv. 'Siberia' with leaf malformation and necrosis and flower bud abscission.

Nucleic acids were extracted from bulb scales using a DNeasy Plant Mini Kit (Qiagen). In nested PCR the phytoplasma universal primer pair P1/P7 was used, followed by fA/rA, R16F2n/R16R2 or the group-16SrI (R16(I)F1/R16(I)R1) or group-16SrX (fAT/rAS) specific primer pairs. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R16R2 PCR product was done using the endonucleases MseI, HhaI, RsaI or SspI. The PCR-amplified 16S rRNA gene fragment of lily ‘Siberia’ phytoplasma was purified and sequenced.

Figure 2:  Agarose gel electrophoresis of nested PCR products obtained for samples isolated from lily ‘Siberia’ (L), AY1 – aster yellows reference strain, AP – apple proliferation reference strain, using primers: R16F2n/R16R2, fA/rA, fAT/rAS in nested PCR; L- molecular marker: 1 kb DNA Ladder (Sigma-Aldrich).

Figure 3:  Polyacrylamide gel electrophoresis of PCR products digested with enzymes MseI, HhaI, RsaI or SspI. Sib – sample from lily Siberia’, Simp – sample from lily ‘Simplon’; AY1 – aster yellows reference strain, AP – apple proliferation reference strain, M – Φ174DNA/HinfI marker (Promega).

Phytoplasma rDNA fragments were amplified by nested PCR fA/rA, R16F2n/R16R2 and fAT/rAS but not with R16(I)F1/R16(I)R1 primers from three lilies out of more than 75 (five cultivars) tested, all with disease symptoms. No products were amplified from three symptomless lilies or Catharanthus roseus plants. RFLP profiles indicated that lilies ‘Siberia’ were infected with a phytoplasma belonging to 16SrX-A subgroup ('Ca. Phytoplasma mali'). Analysis of the sequence determined in this study (GenBank acc. no. EF370450) confirmed the results of PCR-RFLP. The isolate showed the highest similarity to the members of 'Ca. Phytoplasma mali'. A BLAST analysis of the sequence from lily 'Siberia' revealed that it was most closely related to the phytoplasmas deposited in GenBank under accession numbers AY598319 and AJ542541.

Figure 4:  Grouping of phytoplasma isolates based on similarity data obtained from comparison of 16S rRNA gene fragment sequence of phytoplasma isolated from oriental lily hybrid ‘Siberia’ and selected sequences from GenBank. Sequences were compared using ClustalW program, and they were grouped in a consensus tree by the module MegAlign (Lasergene, DNASTAR,USA). EF370450 is the sequence determined in this work.

16SrX-A subgroup phytoplasmas have been reported frequently from woody plants. In Poland, they have been found in ornamental crops including rose (Kamińska & Śliwa, 2004). To our knowledge, this is the first record on the occurrence of ‘Ca. Phytoplasma mali’ in lily plants.

 Acknowledgement

This research was supported by the State Committee of Scientific Research (KBN), Grant No 2 PO6R 029 29.

References

Bertaccini A, Kamińska M, Botti S, Martini M, 2002. Molecular evidence for mixed phytoplasma infection in lily plants. Proc. 10^th IS on Virus Diseases in Ornamentals, ed. J. Hammond, Acta Horticulturae ISHS, 568, 35-41.

Kamińska M, Korbin M, 2000. Phytoplasma infection in Lilium sp. plants. Phytopathologia Polonica 20, 45-57

Kamińska M, Dziekanowska D. 2002. Molecular evidence for the presence of aster yellows-related phytoplasma in lilies with leaf scorch and flower virescence. Journal of Phytopathology 150, 90-93.

Kamińska M, Śliwa H, 2004. First report of phytoplasma belonging to apple proliferation group in roses in Poland. Plant Disease 88, 1283.