New Disease Reports
New Disease Reports (2015) 32, 17. [http://dx.doi.org/10.5197/j.2044-0588.2015.032.017]
Get pdf (594 KB)
Next > < Prev

First report of Tragopogon dubius witches' broom disease associated with a subgroup 16SrI-B phytoplasma in Iran

M. Salehi 1* and E. Salehi 2

*salehi_abarkoohi@yahoo.com

Show affiliations

Received: 12 May 2015; Published: 07 Nov 2015

Keywords: virescence, phyllody, PCR

Tragopogon dubius (yellow salsify, Asteraceae) is a perennial herb in Iran. In a survey conducted for phytoplasma diseases in 2014, T. dubius witches' broom disease was observed in vineyards and green areas of Bajgah and Zarghan localities (Fars province, Iran). The characteristic symptoms of T. dubius witches' broom were flower virescence and phyllody, crown proliferation and witches' broom (Fig. 1). Since the symptoms were suggestive of phytoplasma infection, plants were assayed for presence of phytoplasma by PCR amplification. Total DNA was extracted from 0.3 g of midrib tissue of four witches' broom affected and two symptomless T. dubius plants (two plants per location), using the procedure of Zhang et al. (1998). Total DNA samples were tested by both direct and nested PCR assays using phytoplasma universal 16S rDNA primer pairs P1/P7 (Deng & Hiruki, 1991; Schneider et al., 1995) and R16F2n/R16R2 (Gundersen & Lee, 1996) that amplify fragments of 1800 bp and 1250 bp, respectively. Expected fragments were amplified following direct and nested PCR from all symptom-bearing but not from symptomless plants. 

P1/P7-primed PCR products amplified from four witches' broom-affected T. dubius plants were separately cloned and sequenced. The full-length 16S rDNA sequences displayed 100% sequence identity with each other. A consensus sequence corresponding to the Bajgah T. dubius witches' broom was deposited in GenBank (Accession No. KR262955). A Blast search showed that this sequence had 100% identity with Oenothera phytoplasma ('Candidatus Phytoplasma asteris') (M30790), representative of subgroup 16SrI-B. Virtual RFLP analysis using iPhyClassifier online tool (http://www.ba.ars. usda.gov/data/mppl/iphyclassifier), carried out with 17 restriction enzymes, indicated the presence of a 16SrI-B phytoplasma in witches' broom-affected T. dubius plants (Fig. 2). Phylogenetic analysis of full length 16S rDNA sequences using MEGA version 5 (Tamura et al., 2011) grouped T. dubius witches' broom phytoplasma with onion yellows phytoplasma (GenBank Accession No. D12569), a member of 16SrI-B subgroup (Fig. 3). To our knowledge this is the first report of natural occurrence and partial molecular characterisation of a phytoplasma associated with T. dubius witches' broom disease. A 16SrI-B subgroup phytoplasma has previously been reported as causal agent of rapeseed phyllody disease in Zarghan (Salehi et al., 2011). The significance of diseased T. dubius as natural reservoir of rapeseed phyllody phytoplasma remains to be determined. Aster yellows phytoplasmas were reported in grapevine in several countries. However, possible transmission of T. dubius witches' broom phytoplasma to grapevine is yet to be tested.

Figure1+
Figure 1: (a) Symptoms of crown proliferation and witches' broom in Tragopogon dubius plant in Zarghan (Fars province, Iran); (b) healthy T. dubius plant.
Figure 1: (a) Symptoms of crown proliferation and witches' broom in Tragopogon dubius plant in Zarghan (Fars province, Iran); (b) healthy T. dubius plant.
Figure2+
Figure 2: Computer-simulated virtual RFLP patterns derived from in silico digestions of phytoplasma 16S rRNA gene (1.2 kb fragment) of Tragopogon dubius witches' broom phytoplasma with 17 enzymes (AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI). MW: virtual ØX174 HaeIII digest size standard.
Figure 2: Computer-simulated virtual RFLP patterns derived from in silico digestions of phytoplasma 16S rRNA gene (1.2 kb fragment) of Tragopogon dubius witches' broom phytoplasma with 17 enzymes (AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI). MW: virtual ØX174 HaeIII digest size standard.
Figure3+
Figure 3: Phylogenetic tree based on 16S rDNA gene sequences from Tragopogon dubius witches' broom phytoplasma identified in this study and reference phytoplasmas from GenBank. The tree was constructed by the neighbour-joining method. Acholeplasma laidlawii was used as an outgroup. GenBank accession numbers are shown in brackets.
Figure 3: Phylogenetic tree based on 16S rDNA gene sequences from Tragopogon dubius witches' broom phytoplasma identified in this study and reference phytoplasmas from GenBank. The tree was constructed by the neighbour-joining method. Acholeplasma laidlawii was used as an outgroup. GenBank accession numbers are shown in brackets.

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 pairs. Phytopathologia Mediterranea 35, 144-151.
  3. Salehi M, Izadpanah K, Siampour M, 2011. Occurrence, molecular characterization and vector transmission of a phytoplasma associated with rapeseed phyllody in Iran. Journal of Phytopathology 159, 100-105. [http://dx.doi.org/10.1111/j.1439-0434.2010.01731.x]
  4. 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, Vol.1. Academic Press, San Diego, CA, USA, 369-380. [http://dx.doi.org/10.1016/B978-012583805-4/50040-6]
  5. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S, 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology Evolution 28, 2731-2739. [http://dx.doi.org/10.1093/molbev/msr121]
  6. Zhang YP, Uyemoto JK, Kirkpatrick BC, 1998. A small-scale procedure for extracting nucleic acids from woody plants infected with various phytoplasmas for PCR assay. Journal of Virological Methods 71, 45-50. [http://dx.doi.org/10.1016/S0166-0934(97)00190-0]
To cite this report: Salehi M, Salehi E, 2015. First report of Tragopogon dubius witches' broom disease associated with a subgroup 16SrI-B phytoplasma in Iran. New Disease Reports 32, 17. [http://dx.doi.org/10.5197/j.2044-0588.2015.032.017]

©2015 The Authors