A phytoplasma is associated with witches’ broom disease of Tabebuia pentaphylla in Brazil

R. G. Mafia¹, R. W. Barreto^1*, C. A. Vanetti¹ , J. Hodgetts², M. Dickinson² and A. C. Alfenas¹

¹ Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, CEP 36571-000, Brazil
² School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK

*rbarreto@ufv.br

Accepted for publication 29/06/07

Tabebuia pentaphylla Hemsl. (syn Tabebuia pallida) is a plant native to El Salvador that produces spectacular white to pink flower panicles and has become a popular ornamental in Brazil. Severe witches’ broom symptoms, plant deformation, weakening, stunting and death was first observed in Brazil at the site of the plant’s introduction (Rio de Janeiro, Parque do Flamengo) (Figs. 1 and 2). The symptoms suggested that a fungus similar to Crinipellis perniciosa (Stahel) Singer might be involved but none could be isolated. The disease had previously been reported in Puerto Rico (Cook, 1938) and Venezuela (Ciferri, 1949) where viruses were implicated as the cause.

Figure 1: Severely diseased  Tabebuia pentaphylla in the park (Parque do Flamengo) in Rio de Janeiro. Note growth distortion, stunting and large brooms on plant on the front and on the background	Figure 2: Close-up of a broom on a diseased Tabebuia pentaphylla to which the phytoplasma was associated

Samples of healthy and diseased shoots of T. pentaphylla were collected from two sites in Rio de Janeiro. Fresh, unfixed petioles and midribs from diseased and healthy leaves were hand-sectioned (0.1-0.5 mm thick), mounted in water and examined by epifluorescence microscope equipped with an exciter filter BG 12, a dichroic mirror 510, and a barrier filter G 247 (Namba et al., 1981). TEM observations were made using the procedure of Matsuoka & Carvalho (1987).

Figure 3: Micrograph of a transverse section petiole of a diseased Tabebuia pentaphylla. Note strongly fluorescing infected sieve cells (Bar = 100 µm)	Figure 4: Micrograph of a transverse section of a petiole of a healthy Tabebuia pentaphylla. Note the absence of fluorescence (Bar = 100 µm)

Fluorescence microscopy showed a yellow-green fluorescence associated with diseased phloem cells (Fig. 3) but not healthy tissue (Fig. 4). TEM observations showed typical phytoplasmas (appearing as spherical to ovoid structures of variable sizes) consistently present in phloem sieve tubes from diseased plants (Fig. 5), but not symptomless plants. DNA was extracted from samples and used as a template for nested PCR analysis using primers P1/P7 and R16F2n/R16R2 (Arocha et al., 2005), and bands of the expected size (1.25kb) were produced in 3/5 diseased samples. These amplicons were cloned and sequenced (GenBank Accession No: EF647744), the sequences aligned and subject to BLAST analysis. The highest similarity (98% identity) was with a 16SrII group isolate from lime (Citrus aurantifolia), Candidatus Phytoplasma aurantifolia (Accession No. U15442). These observations are the first to implicate phytoplasma as the likely etiological agent of T. pentaphylla witches’ broom disease worldwide.

Figure 5: Phytoplasma colonizing phloem sieve tube elements of Tabebuia pentaphylla (Bar = 1 µm)

Acknowledgements

The authors would like to thank Charles F. Robbs for first raising the issue and suggesting the possible etiology of this disease and Julio C. M. Cascardo for raising the issue of etiology elucidation for this disease more recently.

References

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