New Disease Reports (2015) 32, 26. [http://dx.doi.org/10.5197/j.2044-0588.2015.032.026]
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First report of 'Candidatus phytoplasma ulmi' (16SrV-A) associated with Ulmus cultivar Morfeo (elm) in the United Kingdom

J. Hodgetts*, L.J. Flint and A. Fox

*jennifer.hodgetts@fera.co.uk

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Received: 18 Sep 0000; Published: 07 Dec 2015

Young trees of Ulmus (elm) hybrid cultivar Morfeo were imported into a commercial nursery in the south of the United Kingdom from Italy. Approximately one year after importation the trees began to show symptoms typical of phytoplasma infection, primarily pronounced foliar proliferation and stunting. DNA was extracted from 0.3 g of leaves or roots from five symptom-bearing trees using a CTAB method (Doyle & Doyle, 1990) and was tested by real-time PCR using the universal phytoplasma assay (Hodgetts et al., 2009), all being found to be positive. Three samples were randomly selected for further analysis to identify the phytoplasma present. Nested PCR of the 16S rRNA gene was performed using universal phytoplasma primers P1 and P7, followed by R16F2n and R16R2 as described in Hodgetts et al. (2007), where the samples produced the specific 1,250 bp amplicon. The PCR amplicon was cloned into the pGEM®-T Easy Vector System (Promega, USA), three randomly selected clones sequenced and the sequence was submitted to NCBI (GenBank Accession No. KT381877). Phylogenetic analysis was undertaken which identified the phytoplasma as a member of the 16SrV phytoplasmas (Fig. 1), but did not provide adequate discrimination to the species level. 

To further discriminate the phytoplasma, conventional PCR to amplify the secY and elongation factor Tu (tuf) genes were performed as described in Foissac et al. (2013). The presence of a phytoplasma was confirmed by the amplification of the expected amplicon for both phytoplasma genes in all of the trees sampled. DNA sequencing was undertaken for both genes and the sequence was submitted to NCBI (KT381878 for secY and KT381876 for tuf) and phylogenetic analysis undertaken. Both genes indicated the phytoplasma to be a member of 'Candidatus Phytoplasma ulmi', group 16SrV-A (Figs. 2, 3). 

This is the first report of 'Ca. P. ulmi' in elm in the UK, a highly significant finding as it is an EPPO (European and Mediterranean Plant Protection Organization) A1 listed organism. Therefore all plants from the importation have been destroyed, and a wider survey of elm has been undertaken to identify if earlier imports have allowed the establishment of'Ca. P. ulmi' within the UK. Follow-up surveys for visual symptoms were carried out in nurseries stocking Ulmus and recently planted sites across the UK and Channel Islands. Over 11,000 trees were inspected and no symptoms typical of phytoplasma infection were observed. A survey of the UK National Elm Collection was also undertaken which included the testing of over 50 asymptomatic trees by real-time PCR; all were found to be negative for the presence of phytoplasmas. Therefore this is believed to be an isolated importation and the phytoplasma has been eradicated from the UK.

Figure1+
Figure 1: Phylogenetic tree of partial 16S rRNA gene sequences from the 'Ca P. ulmi'-related strain identified in this study shown by red diamond (Ulmus Morfeo proliferation phytoplasma, KT183877) and phytoplasma type species. (GenBank Accession Nos. are shown in brackets.) The tree was constructed by the neighbour-joining method, and bootstrap values greater than 70% (expressed as percentages of 500 replications) are shown on branches. The bar indicates the number of substitutions per nucleotide position.
Figure 1: Phylogenetic tree of partial 16S rRNA gene sequences from the 'Ca P. ulmi'-related strain identified in this study shown by red diamond (Ulmus Morfeo proliferation phytoplasma, KT183877) and phytoplasma type species. (GenBank Accession Nos. are shown in brackets.) The tree was constructed by the neighbour-joining method, and bootstrap values greater than 70% (expressed as percentages of 500 replications) are shown on branches. The bar indicates the number of substitutions per nucleotide position.
Figure2+
Figure 2: Phylogenetic tree of partial secY gene sequences from the 'Ca. P. ulmi'-related strain identified in this study (Ulmus Morfeo proliferation phytoplasma, KT381877) and other 16SrV phytoplasmas. (GenBank Accession Nos. are shown in brackets. Methodology and diamond symbol as in Fig. 1.)
Figure 2: Phylogenetic tree of partial secY gene sequences from the 'Ca. P. ulmi'-related strain identified in this study (Ulmus Morfeo proliferation phytoplasma, KT381877) and other 16SrV phytoplasmas. (GenBank Accession Nos. are shown in brackets. Methodology and diamond symbol as in Fig. 1.)
Figure3+
Figure 3: Phylogenetic tree of partial elongation factor Tu (tuf) gene sequences from the 'Ca. P. ulmi'-related strain identified in this study (Ulmus Morfeo proliferation phytoplasma, KT381877) and other 16SrV phytoplasmas. (GenBank Accession Nos. are shown in brackets. Methodology and diamond symbol as in Fig. 1.)
Figure 3: Phylogenetic tree of partial elongation factor Tu (tuf) gene sequences from the 'Ca. P. ulmi'-related strain identified in this study (Ulmus Morfeo proliferation phytoplasma, KT381877) and other 16SrV phytoplasmas. (GenBank Accession Nos. are shown in brackets. Methodology and diamond symbol as in Fig. 1.)

Acknowledgements

Phytoplasmas were held under Defra Plant Health licence nº. PHSI 23/6501 and its predecessors. This work was funded by the Department for Environment, Food and Rural Affairs (Defra) and Ulmus surveys were conducted by the Plant Health and Seeds Inspectorate (APHA).


References

  1. Doyle JJ, Doyle JL, 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13-15.
  2. Foissac X, Danet JL, Malembic-Maher S, Salar P, Šafářová D, Válová P, Navrátil M. 2013. Tuf and secY PCR amplification and genotyping of phytoplasmas. In: Dickinson M, and Hodgetts J, eds. Phytoplasma Methods and Protocols. New York, NY, USA: Humana Press, 205-215. [http://dx.doi.org/10.1007/978z1-62703-089-2]
  3. Hodgetts J, Ball T, Boonham N, Mumford R, Dickinson M, 2007. Use of terminal restriction fragment length polymorphism (TRFLP) for identification of phytoplasmas in plants. Plant Pathology 56, 357-365. [http://dx.doi.org/10.1111/j.1365-3059.2006.01561.x]
  4. Hodgetts J, Boonham N, Mumford R, Dickinson M, 2009. Panel of 23S rRNA gene-based real-time PCR assays for improved universal and group-specific detection of phytoplasmas. Applied and Environmental Microbiology 75, 2945-2950. [http://dx.doi.org/10.1128/AEM.02610-08]

To cite this report: Hodgetts J, Flint LJ, Fox A, 2015. First report of 'Candidatus phytoplasma ulmi' (16SrV-A) associated with Ulmus cultivar Morfeo (elm) in the United Kingdom. New Disease Reports 32, 26. [http://dx.doi.org/10.5197/j.2044-0588.2015.032.026]

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