New Disease Reports
New Disease Reports (2016) 34, 4. [http://dx.doi.org/10.5197/j.2044-0588.2016.034.004]
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First report of a 'Candidatus Phytoplasma aurantifolia'-related phytoplasma strain associated with yellowing symptoms on pineapple palm in Iran

M. Azimi, R. Farokhi-Nejad and M. Mehrabi-Koushki*

*mhdmhrb@gmail.com

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Received: 04 Jun 2016; Published: 29 Jul 2016

Keywords: nested PCR, pineapple palm

Pineapple palm, Phoenix canariensis, is one of the most widely grown and appreciated ornamental palms, including in the south of Iran. During the last three years, symptoms resembling nutrient deficiency were observed on pineapple palms in urban landscapes in Ahvaz in southwestern Iran. The symptoms included leaf yellowing in the lower fronds which began from the leaf tips and extended towards the blade and the rachis (Figs. 1, 2, 3). During January 2016, P. canariensis palms were surveyed for phytoplasma. Leaf samples from three diseased and one symptomless palm were collected. The midribs were freeze-dried and then powdered in liquid nitrogen. Total DNA was extracted using a CTAB-based method (Maixner et al., 1995). Total DNA was analysed by nested PCR assay using generic primers P1/P7-R16F2n/R16R2 (Deng & Hiruki, 1991; Gundersen & Lee, 1996). Amplicons of the expected size (c. 1.2 kb) were obtained from all diseased palms, and were purified (GF-1 AmbiClean Kit, Malaysia) and directly sequenced. The partial consensus 16S rDNA sequence was deposited in GenBank (Accession No. KX088466) and compared to those of reference phytoplasmas by BLAST analysis. The consensus 16S rDNA sequence showed 99% sequence identity to members of group 16SrII ‘Candidatus Phytoplasma aurantifolia’. A phylogenetic tree (Fig. 4) based on the 16S rDNA sequences of P. canariensis phytoplasma and those of reference phytoplasmas was constructed using the maximum likelihood algorithm under the TN93+G+I model of the MEGA 6 software (Tamura et al. 2013). The P. canariensis phytoplasma (Ahvaz-5 isolate) clustered within the 16SrII phytoplasma group. Restriction mapping of the partial 16SrRNA sequence (1161bp) with typical endonucleases AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI in a virtual digest using Restriction-Mapper V.3 online software (http://www.restrictionmapper.org) showed that the virtual RFLP pattern of the P. canariensis phytoplasma was identical (similarity coefficient 1.00) to the reference pattern of subgroup 16SrII-D (Y10097). Both, the virtual RFLP and phylogenetic analyses confirmed that the P. canariensis phytoplasma is a member of the 16Sr II group, subgroup D.

Phoenix canariensis is affected by the Texas phoenix palm decline phytoplasma, a member of the 16SrIV group, in Florida and Texas, USA. To our knowledge, this is the first report of a phytoplasma disease on palm in Iran. The P. canariensis phytoplasma (Ahvaz-5 isolate) is affecting approximately 20% of the pineapple palms in Ahvaz. The identification of this phytoplasma will contribute to improving the future management of the disease.

Figure1+
Figure 1: Leaf yellowing symptoms in pineapple palm in the campus of Shahid Chamran University Ahvaz: yellowing of the foliage begins with the lowest leaves and progresses upward towards the crown.
Figure 1: Leaf yellowing symptoms in pineapple palm in the campus of Shahid Chamran University Ahvaz: yellowing of the foliage begins with the lowest leaves and progresses upward towards the crown.
Figure2+
Figure 2: Leaf yellowing symptoms in pineapple palm in the campus of Shahid Chamran University, Ahvaz: leaf yellowing symptoms are localised in the rachis.
Figure 2: Leaf yellowing symptoms in pineapple palm in the campus of Shahid Chamran University, Ahvaz: leaf yellowing symptoms are localised in the rachis.
Figure3+
Figure 3: Leaf yellowing symptoms in pineapple palm in the campus of Shahid Chamran University, Ahvaz: yellow leaves eventually turn brown starting from the tip.
Figure 3: Leaf yellowing symptoms in pineapple palm in the campus of Shahid Chamran University, Ahvaz: yellow leaves eventually turn brown starting from the tip.
Figure4+
Figure 4: Phylogenetic tree based on the 16S rDNA sequences of the Phoenix canariensis phytoplasma (Ahvaz-5 isolate) and reference sequences. Accession numbers are shown in brackets. Bootstrap values greater than 50% (expressed as percentages of 500 replications) are shown above branches. The tree was rooted with Acholeplasma laidlawii ATTC 23206 (U14905).
Figure 4: Phylogenetic tree based on the 16S rDNA sequences of the Phoenix canariensis phytoplasma (Ahvaz-5 isolate) and reference sequences. Accession numbers are shown in brackets. Bootstrap values greater than 50% (expressed as percentages of 500 replications) are shown above branches. The tree was rooted with Acholeplasma laidlawii ATTC 23206 (U14905).

References

  1. Deng S, Hiruki C, 1991. Amplification of 16S rRNA genes from culturable and non-culturable mollicutes. Journal of Microbiological Methods 14, 53–61. [http://dx.doi.org/10.1016/0167-7012(91)90007-D]
  2. Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer sets. Phytopathologia Mediterranea 35, 144-151.
  3. Maixner M, Ahrens U, Seemuller E, 1995. Detection of the German grapevine yellows (Vergilbungskrankheit) MLO in grapevine, alternative hosts and a vector by a specific PCR procedure. European Journal of Plant Pathology 101, 241-250. [http://dx.doi.org/10.1007/BF01874780]
  4. Tamura K, Stecher G, Peterson D, Filipski A,  Kumar S, 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30, 2725-2729. [http://dx.doi.org/10.1093/molbev/mst197]
To cite this report: Azimi M, Farokhi-Nejad R, Mehrabi-Koushki M, 2016. First report of a 'Candidatus Phytoplasma aurantifolia'-related phytoplasma strain associated with yellowing symptoms on pineapple palm in Iran. New Disease Reports 34, 4. [http://dx.doi.org/10.5197/j.2044-0588.2016.034.004]

©2016 The Authors