Identification of an X-disease (16SrIII) group phytoplasma (‘Candidatus Phytoplasma pruni’) infecting delphiniums in the UK

V.A Harju*, A.L Skelton, W.A. Monger, B. Jarvis and R.A. Mumford

Central Science Laboratory, Sand Hutton, York, YO41 1LZ, UK

*v.harju@csl.gov.uk

Accepted for publication 19/10/07

In 2005, a delphinium (Delphinium sp.) plant was sent to the Central Science Laboratory for diagnostic testing.  The plant was part of a small trial (25 plants in total), being conducted in eastern England.  All the plants in the trial were showing typical phytoplasma disease symptoms: severe phyllody, virescence and proliferation (Fig. 1).  A putative phytoplasma was detected by real-time (TaqMan) PCR (unpublished) and confirmed using conventional PCR, using the universal 16S ribosomal RNA gene primer pair MLO RU3 and FU5 (Lorenz et al., 1995).  The 834 bp PCR product obtained was cloned into pGEM^®-T Easy Vector (Promega, UK) and sequenced.  Analysis of the sequence obtained (GenBankAcc. No. DQ350774) identified the pathogen as a possible member of the X-disease (16SrIII) group (‘Candidatus Phytoplasma pruni’).

Figure 1: X-disease phytoplasma infected delphinium showing typical symptoms
of phyllody, virescence and proliferation.

This was confirmed by sequencing the full 16S rRNA and 16S-23S intergenic region (1701 bp), amplified using primers P1/P7 (Smart et al., 1996), from a second delphinium from the same trial.  This sequence (Acc. No. EF514210) was identical to the partial sequence obtained from the first isolate and also aligned closest with members of the X-disease group.  The closest match (>99%) was found with a Chinaberry yellows phytoplasma isolate (Acc. No. AF495657).

Because aster yellows phytoplasmas (group 16SrI; ‘Ca. Phytoplasma asteris’) had been identified previously in delphiniums (Lee et al., 2004), restriction fragment length polymorphism analysis was performed in an attempt to rule out a mixed infection.  PCR products generated using the universal primer pair R16mF2/R1, followed by the nested pair R16F2n/R2 (Gundersen & Lee, 1996) were cut with the restriction enzyme RsaI, and the products were separated on a 2100 bioanalyzer (Agilent Technologies).  The observed banding pattern was typical for a member of the 16SrIII group and not the 16SrI group (results not shown).

To our knowledge, this is the first time a member of the X-disease group has been detected in this host and is highly significant given that X-disease is an EU-listed quarantine pathogen.  As a result, all the trial plants were subsequently destroyed and the disease eradicated.

References

Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35, 144-151.

Lee IM, Gundersen-Rindal DE, Davis RE, Bottner KD, Marcone C, Seemüller E, 2004. ‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxon associated with aster yellows and related diseases. International Journal of Systematic and Evolutionary Microbiology 54, 1037-1048.

Lorenz K-H, Schneider B, Ahrens U, Seemüller E, 1995. Detection of the Apple Proliferation and Pear Decline Phytoplasma by PCR amplification of Ribosomal and Non-Ribosomal DNA. Phytopathology 85, 771-776.

Smart CD, Schneider B, Blomquist CL, Guerra LJ, Harrison NA, Ahrens U, Lorenz K-H, Seemüller E, Kirkpatrick BC, 1996. Phytoplasma-specific PCR Primers based on sequences of the 16S-23S rRNA spacer region. Applied and Environmental Microbiology 62, 2988- 2993.