New Disease Reports
New Disease Reports (2017) 35, 24. [http://dx.doi.org/10.5197/j.2044-0588.2017.035.024]
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First report of a 'Candidatus Phytoplasma aurantifolia'-related strain associated with witches'-broom disease of limequat in Iran

M.M. Faghihi 1*, A. Bagheri 1, M. Askari Seyahooei 1, A. Pezhman 2 and G. Faraji 2

*mm.faghihi@yahoo.com

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Received: 23 Jan 2017; Published: 03 May 2017

Keywords: Citrus aurantifolia, Fortunella, Hormozgan

Witches'-broom disease of lime (WBDL) caused by 'Candidatus Phytoplasma aurantifolia' was found first in Mexican lime (Citrus aurantifolia) in a small area in southern Iran in 1997 (Bové et al., 2000). Since then WBDL has spread to many areas devastating plantations in the major lime-growing provinces of Fars, Hormozgan, Kerman and Sistan-Baluchista.

The prevalence of WBDL in southern Iran has necessitated regular monitoring of this phytoplasma disease in citrus orchards. Following these monitoring programmes in 2009, we found typical symptoms of phytoplasma disease including witches' broom, little leaf and internode shortening on limequat trees (Citrus aurantifolia × Fortunella sp.) in private gardens in the region of Hasht Bandi of Minab (Hormozgan province). Four leaf samples were collected from one symptomless and three symptom-bearing trees (Fig. 1). Total DNA was extracted from the midrib tissue using a modified CTAB method. Samples were tested for phytoplasma DNA by direct-PCR using the phytoplasma universal primer pair P1/P7 (Deng & Hiruki, 1991; Schneider et al., 1995) and nested-PCR using primer pairs P1/P7 and R16F2n/R16R2 (Gundersen & Lee, 1996). Amplicons of c. 1.8 and 1.25 kb were amplified from all symptom-bearing trees with direct and nested-PCR, respectively, but not from symptomless plants or sterile distilled water included as negative controls.

Amplified PCR products (1.25 kb) from symptom-bearing limequat trees were digested with endonucleases AluI, TaqI, HpaII, HhaI, MseI and RsaI, and showed identical patterns to 'Ca. Phytoplasma aurantifolia'. The 1.25 kb amplicon was cloned using the commercial kit #K1214 (Thermo Fisher Scientific Inc., USA), sequenced, and deposited in GenBank (Accession No. KY171947). BLAST analysis revealed that the partial 16S rDNA sequence of the phytoplasma associated with witches' broom disease of limequat shared 99% identity with strains of group 16SrII (peanut witches' broom group), including Hishimonus phycitis phytoplasma (KX828609, Iran) as well as several phytoplasmas associated with witches' broom disease on Mexican lime (LN873017 and LN872963 from Oman and Iran, respectively), grapefruit and citron (JF518980 and JN967029, Iran). Phylogenetic analysis using the neighbour-joining method (MEGA 5 software) based on the 16S rRNA gene sequence of the phytoplasma isolated from limequat and other phytoplasmas demonstrated that the limequat witches'-broom phytoplasma is a member of the 16SrII phytoplasma clade (Fig. 2).

Natural occurrence of witches' broom disease related to 16SrII group of phytoplasma has been reported on Mexican lime, bakraee (a citrus hybrid), citron (C. medica) and grapefruit (C. paradisi) in Iran (Djavaheri & Rahimian, 2004; Bagheri et al., 2010). To our knowledge, this is the first report of witches' broom disease of limequat associated with 'Ca. P. aurantifolia' in Iran and probably globally. Our findings suggest that limequat may act as a natural host for the 'Ca. P. aurantifolia' and may be a potential reservoir for the pathogen in southern Iran.

Figure1+
Figure 1: Symptoms of little leaf and witches' broom on diseased limequat (shown by a red circle).
Figure 1: Symptoms of little leaf and witches' broom on diseased limequat (shown by a red circle).
Figure2+
Figure 2: Phylogenetic tree of partial 16S rRNA gene sequences from limequat witches'-broom phytoplasma (marked by a red rectangle) and reference sequences belonging to different 16S rRNA groups and 'Candidatus Phytoplasma' species. The tree was constructed by the neighbour-joining method using MEGA 5 software. Bootstrap values >50% (expressed as percentage of 1000 replications) are shown above nodes. Acholeplasma laidlawii was used as an outgroup to root the tree.
Figure 2: Phylogenetic tree of partial 16S rRNA gene sequences from limequat witches'-broom phytoplasma (marked by a red rectangle) and reference sequences belonging to different 16S rRNA groups and 'Candidatus Phytoplasma' species. The tree was constructed by the neighbour-joining method using MEGA 5 software. Bootstrap values >50% (expressed as percentage of 1000 replications) are shown above nodes. Acholeplasma laidlawii was used as an outgroup to root the tree.

References

  1. Bagheri A, Faghihi MM, Salehi M, Samavi S, Khanchezar A, 2010. First report of natural infection of grapefruit trees to lime witches'-broom phytoplasma. In: Manzari S, ed. Proceedings of the Nineteenth Iranian Plant Protection Congress. Tehran, Iran: Iranian Research Institute of Plant Protection Press, 409.
  2. Bové JM, Danet JL, Bananej K, Hassanzedeh N, Taghizadeh M, Salehi M, Garnier M, 2000. Witches' broom disease of lime (WBDL) in Iran. In: da Graça JV, Lee RF, Yokomi RK, eds. Proceedings of the Fourteenth Conference of the International Organization of Citrus Virologists. Riverside, USA: International Organization of Citrus Virologists, 207-212.
  3. 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]
  4. Djavaheri M, Rahimian H, 2004. Witches'-broom of bakraee (Citrus reticulata hybrid) in Iran. Plant Disease 88, 683. [http://dx.doi.org/10.1094/PDIS.2004.88.6.683A]
  5. Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer sets. Phytopathologia Mediterranea 35, 144-151.
  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: Faghihi MM, Bagheri A, Askari Seyahooei M, Pezhman A, Faraji G, 2017. First report of a 'Candidatus Phytoplasma aurantifolia'-related strain associated with witches'-broom disease of limequat in Iran. New Disease Reports 35, 24. [http://dx.doi.org/10.5197/j.2044-0588.2017.035.024]

©2017 The Authors