New Disease Reports
New Disease Reports (2017) 35, 8. [http://dx.doi.org/10.5197/j.2044-0588.2017.035.008]
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First report of a 'Candidatus Phytoplasma phoenicium'-related strain associated with Bidens alba phyllody in Iran

C. Hemmati 1, M. Nikooei 2, A. Bagheri 3 and M.M. Faghihi 3*

*mm.faghihi@yahoo.com

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Received: 30 Jan 2017; Published: 10 Feb 2017

Keywords: Hormozgan, shepherd’s needle, 16SrIX phytoplasma group

Bidens alba (family Asteraceae), commonly known as shepherd’s needle, is grown in tropical and subtropical regions of North and South America, Asia and Africa (Hall et al., 2009). Some studies indicate that it can be used as a remedy for cuts, swelling, hypertension, jaundice and diabetes (Ong et al., 2008).

In July 2016, typical symptoms of phytoplasma diseases including phyllody, virescence, witches' broom and little leaf (Figs. 1-2) were observed in Bidens alba growing as weeds in citrus orchards of Seyahoo district in Hormozgan province, Iran. Total DNA was extracted separately from five symptom-bearing and two symptomless plants (Fig. 3) using the CTAB method (Doyle & Doyle, 1990). Samples were tested for the presence of phytoplasma DNA by direct PCR using the universal primer pair P1/P7 (Deng & Hiruki, 1991; Schneider et al., 1995), followed by nested PCR using primer pair R16F2n/R16R2 (Gundersen & Lee, 1996). Amplicons of c. 1.8 (direct PCR) and 1.25 kb (nested PCR) were obtained from all symptom-bearing but not from symptomless plants. PCR products from both PCR rounds obtained from one infected plant were directly sequenced on both strands. The sequences were edited, assembled and deposited in GenBank (Accession No. KY358007). BLAST analysis of this sequence revealed that the phytoplasma associated with B. alba phyllody (BaP) shared 99% identity with phytoplasmas related to the 16SrIX phylogenetic group, 'Candidatus Phytoplasma phoenicium', such as Iranian Lactuca serricola phyllody phytoplasma (DQ889749; Iran), Eggplant big bud (JX483702; Iran) and Periwinkle virescence (HQ589191; Germany). Phylogenetic analysis using the neighbour-joining method (MEGA 6 software) based on the sequenced fragment of the BaP phytoplasma and other phytoplasmas confirmed that the BaP phytoplasma is a 'Ca. P. phoenicium'-related strain, with 99.2% sequence similarity to the reference strain for the species (AF515636) (Fig. 4).

In Iran, 16SrIX group phytoplasmas have been reported on several hosts such as aubergine, chrysanthemum, grapevine and lucerne. Results obtained from our findings suggest that B. alba may act as a natural intermediate host for the 16SrIX phytoplasma in the Hormozgan province of Iran. To our knowledge, this is the first report of a phytoplasma associated with B. alba phyllody in Iran and most probably in the world; the potential for B. alba to act as a host for 'Ca. P. phoenicium' is a concern.

Figure1+
Figure 1: Symptoms of virescence and phyllody in diseased Bidens alba.
Figure 1: Symptoms of virescence and phyllody in diseased Bidens alba.
Figure2+
Figure 2: Symptoms of witches' broom in diseased Bidens alba.
Figure 2: Symptoms of witches' broom in diseased Bidens alba.
Figure3+
Figure 3: Healthy plants of Bidens alba.
Figure 3: Healthy plants of Bidens alba.
Figure4+
Figure 4: Phylogenetic tree of partial 16S rDNA sequences from Bidens alba phyllody phytoplasma (bold text) and reference sequences. GenBank Accession Nos. in brackets and 16Sr groups annotated to the right. The tree was constructed by the neighbour-joining method with 1000 replicates using MEGA 6 software. Bar indicates number of nucleotide substitutions per site; bootstrap values shown at nodes with >50% support. Acholeplasma laidlawii was used as an outgroup to root the tree.
Figure 4: Phylogenetic tree of partial 16S rDNA sequences from Bidens alba phyllody phytoplasma (bold text) and reference sequences. GenBank Accession Nos. in brackets and 16Sr groups annotated to the right. The tree was constructed by the neighbour-joining method with 1000 replicates using MEGA 6 software. Bar indicates number of nucleotide substitutions per site; bootstrap values shown at nodes with >50% support. Acholeplasma laidlawii was used as an outgroup to root the tree.

References

  1. Deng S, Hiruki C, 1991. Amplification of 16Sr RNA 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. Doyle JJ, Doyle JL, 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13-15.
  3. Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer sets. Phytopathologia Mediterranea 35, 144-151.
  4. Hall DW, Vandiver VV, Ferrell JA, 2009. Common Beggar's-tick (Hairy Beggar's-tick), Bidens alba (L.) DC., Weeds in Florida, SP 37. Institute of Food and Agricultural Sciences, University of Florida.  http://ufdcimages.uflib.ufl.edu/IR/00/00/70/37/00001/FW00500.pdf (Accessed 30 January 2017).
  5. Ong PL, Weng BC, Lu FJ, Lin ML, Cang TT, Hung RP, Chen, CH. 2008. The anticancer effect of protein-extracted from Bidens alba in human colorectal carcinoma SW480 cells via the reactive oxidative species and glutathione depletion-dependent apoptosis. Food and Chemical Toxicity 46, 1535-1547. [http://dx.doi.org/10.1016/j.fct.2007.12.015 ]
  6. Schneider B, Seemüller E, Smart CD, Kirkpatrick BC, 1995. Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In: Razin S, Tully JG, eds. Molecular and Diagnostic Procedures in Mycoplasmology. New York, USA: Academic Press, 369-380. [http://dx.doi.org/10.1016/B978-012583805-4/50040-6]
To cite this report: Hemmati C, Nikooei M, Bagheri A, Faghihi MM, 2017. First report of a 'Candidatus Phytoplasma phoenicium'-related strain associated with Bidens alba phyllody in Iran. New Disease Reports 35, 8. [http://dx.doi.org/10.5197/j.2044-0588.2017.035.008]

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