New Disease Reports
New Disease Reports (2017) 35, 4. [http://dx.doi.org/10.5197/j.2044-0588.2017.035.004]
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First report of a 'Candidatus Phytoplasma aurantifolia'-related strain associated with leaf roll symptoms on eucalyptus in Iran

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

*mhdmhrb@gmail.com

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Received: 12 Nov 2016; Published: 19 Jan 2017

Keywords: Eucalyptus camaldulensis, nested PCR

The eucalyptus (Eucalyptus camaldulensis) is a common tree in many parts of the world including Iran. During 2015-16, symptoms of leaf roll, yellowing, little leaf and short internodes (Figs. 1-2) were observed on eucalyptus trees in Ahvaz, southwestern Iran. Leaf samples from three diseased trees growing in the downtown regions and eastern and northern suburbs, and one symptomless tree from each region were tested for phytoplasmas. The leaf midribs were separated and freeze-dried, then powdered in liquid nitrogen. Total DNA was extracted using CTAB (Maixner et al., 1995) and tested for the presence of phytoplasmas using a nested PCR assay that amplifies part of the 16S rDNA using the universal primers P1/P7 (Deng & Hiruki, 1991) followed by R16F2n/R16R2 (Gundersen & Lee, 1996). The amplicons of c. 1.2 kb produced from all symptomatic samples were directly sequenced, and one (Ahvaz-3) was deposited in GenBank (Accession No. KX088462). Using a BLAST search the consensus sequence showed 99% identity to members of group 16SrII 'Candidatus Phytoplasma aurantifolia'. A maximum likelihood phylogenetic tree based on the 16S rDNA sequences, constructed with MEGA 6.0 software (Tamura et al., 2013) showed that the E. camaldulensis leaf roll phytoplasma isolate, Ahvaz-3, clustered with the 16SrII phytoplasma group (Fig. 3). A virtual RFLP with 17 typical endonucleases (Lee et al., 1998) using Restriction-Mapper V.3 software (http://www.restrictionmapper.org), and confirmed by iPhyClassifier (Zhao et al., 2009) revealed that this phytoplasma is closely related to the reference strain of the 16SrII-D subgroup (Y10097).

The association of 'Candidatus Phytoplasma aurantifolia' strains with diverse symptoms in herbaceous and woody plants has been reported from different climates of Iran in recent years. To our knowledge, this is the first report of a 16SrII phytoplasma infecting eucalyptus trees in Iran. The E. camaldulensis leaf roll phytoplasma is affecting approximately 5% of eucalyptus trees in Ahvaz and its detection will contribute to improving management of the disease.

Figure1+
Figure 1: Leaf roll and little leaf symptoms on shoots of a eucalyptus tree growing in the campus of Shahid Chamran University, Ahvaz.
Figure 1: Leaf roll and little leaf symptoms on shoots of a eucalyptus tree growing in the campus of Shahid Chamran University, Ahvaz.
Figure2+
Figure 2: Leaf roll and little leaf symptoms on shoots of eucalyptus (left and middle) compared to a symptomless shoot (furthest right).
Figure 2: Leaf roll and little leaf symptoms on shoots of eucalyptus (left and middle) compared to a symptomless shoot (furthest right).
Figure3+
Figure 3: Phylogenetic tree based on the 16S rDNA sequences of Eucalyptus camaldulensis leaf roll phytoplasma (Ahvaz-3 isolate) and reference sequences from GenBank (accession numbers in brackets). Bootstrap values >50% (percentages of 1000 replications) are shown above branches and the tree was rooted with Acholeplasma laidlawii.
Figure 3: Phylogenetic tree based on the 16S rDNA sequences of Eucalyptus camaldulensis leaf roll phytoplasma (Ahvaz-3 isolate) and reference sequences from GenBank (accession numbers in brackets). Bootstrap values >50% (percentages of 1000 replications) are shown above branches and the tree was rooted with Acholeplasma laidlawii.

References

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  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. Lee IM, Gundersen-Rindal DE, Davis RE, Bartoszyk IM, 1998. Revised classification scheme of phytoplasmas based on RFLP analysis of 16S rRNA and ribosomal protein gene sequences. International Journal of Systematic Bacteriology 48, 1153–1169. [http://dx.doi.org/10.1099/00207713-48-4-1153]
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  6. Zhao Y, Wei W, Lee IM, Shao J, Suo X, Davis RE, 2009. Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII). International Journal of Systematic and Evolutionary Microbiology 59, 2582–2593. [http://dx.doi.org/10.1099/ijs.0.010249-0]
To cite this report: Azimi M, Farokhi-Nejad R, Mehrabi-Koushki M, 2017. First report of a 'Candidatus Phytoplasma aurantifolia'-related strain associated with leaf roll symptoms on eucalyptus in Iran. New Disease Reports 35, 4. [http://dx.doi.org/10.5197/j.2044-0588.2017.035.004]

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