New Disease Reports (2016) 34, 9. [http://dx.doi.org/10.5197/j.2044-0588.2016.034.009]
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Association of a 'Candidatus Phytoplasma solani'-related strain with pistachio in Iran

M. Ghayeb Zamharir

*zamharir2005@yahoo.com

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Received: 28 Jun 2016; Published: 06 Sep 2016

Pistachio (Pistachia vera) is one of the most important horticultural plants in Iran, growing in the majority of the regions throughout the country. The main cultivation centres are the provinces of Kerman, Semnan, Yazd, Qom and Qazvin. Previously, a phytoplasma of an undetermined species has been identified in pistachio showing witches’ broom symptoms in Iran (Zamharir & Mirabolfathi, 2011), whilst group 16SrI (Marzachi et al., 1999) and 16SrII (Khodaygan et al., 2014) phytoplasmas have been identified in pistachio in Italy and Iran, respectively.

In recent years, yellowing and scorch symptoms (Figs. 1-2) were observed in some pistachio-cultivation areas in Yazd. Given the increasing importance of phytoplasma-associated diseases in Iran, a survey was carried out during 2011-2015. Diseased trees developed every year during the five-year survey. Samples from twenty symptom-bearing pistachio trees and five asymptomatic trees were collected from two pistachio-growing regions in Meibod and Marvast (Yazd province). DNA was extracted from 0.5 g of leaf midribs and petioles using a CTAB-based method (Doyle & Doyle, 1987). Universal phytoplasma PCR was performed for phytoplasma detection. First round PCR used primers P1/P7 (Schneider et al., 1995), and the amplicons were re-amplified with the internal primer pair R16mF2n/R16mR1 (Gundersen & Lee, 1996), amplifying approximately 1,200 bp of 16S rRNA gene. Direct sequencing was done with primers R16mF2n/R16mR1. The sequences were assembled using MEGA6 and compared with selected nucleotide sequences in the GenBank database using BLAST. Phylogenetic analyses were done with 16S rDNA sequence from pistachio sample PM3 and from sixteen ‘Candidatus Phytoplasma’ strains. The majority of the symptom-bearing samples (90%) were positive in nested PCR, and no amplification was obtained from negative controls (without template DNA) and the asymptomatic samples. The sequence of sample PM3 was submitted to GenBank (Accession No. KX417297). The sequence showed 99% homology with many strains affiliated with ‘Candidatus Phytoplasma solani’ and with the reference strain for the species STOL11 (AF248959). The phylogenetic analysis confirmed its placement as a ‘Ca. P. solani’-related strain (Fig. 3).

On the basis of disease symptoms and molecular analyses, the pistachio scorch disease in the two regions of Yazd was associated with the presence of a phytoplasma. The epidemiology of pistachio scorch disease in Iran and the presence of possible insect vectors requires further study.

Figure1+
Figure 1: Symptoms of leaf scorch in pistachio trees from Yazd , Iran.
Figure 1: Symptoms of leaf scorch in pistachio trees from Yazd , Iran.
Figure2+
Figure 2: Symptoms of yellowing and little leaf (left) compared with a healthy branch (right) in pistachio trees from Yazd , Iran.
Figure 2: Symptoms of yellowing and little leaf (left) compared with a healthy branch (right) in pistachio trees from Yazd , Iran.
Figure3+
Figure 3: Phylogenetic tree constructed by neighbour-joining method of approximately 1,250 bp of the 16S rRNA gene sequences from 17 phytoplasmas including pistachio scorch phytoplasma (PM 3) and Acholeplasma laidlawii as an outgroup. GenBank accession numbers are in parentheses to the right of phytoplasma names, followed by phytoplasma ribosomal subgroups. Numbers on the branches are bootstrap (confidence) values of 1000 replicates. The bar indicates the number of substitutions per nucleotide position.
Figure 3: Phylogenetic tree constructed by neighbour-joining method of approximately 1,250 bp of the 16S rRNA gene sequences from 17 phytoplasmas including pistachio scorch phytoplasma (PM 3) and Acholeplasma laidlawii as an outgroup. GenBank accession numbers are in parentheses to the right of phytoplasma names, followed by phytoplasma ribosomal subgroups. Numbers on the branches are bootstrap (confidence) values of 1000 replicates. The bar indicates the number of substitutions per nucleotide position.

References

  1. Doyle JJ, Doyle JL, 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19, 11-15.
  2. Ghayeb Zamharir M, Mirabolfathi M, 2011. Association of a phytoplasma with pistachio witches’ broom disease in Iran. Journal of Phytopathology 159, 60-62. [http://dx.doi.org/10.1111/j.1439-0434.2010.01697.x]
  3. Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35, 144-151.
  4. Khodaygan P, Moslemkhani C, Shahbazi R, Moradi M, Habibi H, 2014. Molecular identification of 16S rII phytoplasma group in commercial pistachio cultivars in Iran. Archives of Phytopathology and Plant Protection 47, 1400-1407. [http://dx.doi.org/10.1080/03235408.2013.843811]
  5. Marzachi, C, Alma, A, d'Aquilio, M, Minuto, G, Boccardo, G, 1999. Detection and identification of phytoplasmas infecting cultivated and wild plants in Liguria (Italian Riviera). Journal of Plant Pathology 81, 127-136. [http://dx.doi.org/10.4454/jpp.v81i2.1057]
  6. Schneider B, Seemüller E, Smart CD, Kirkpatrick BC, 1995. Phylogenetic classification of plant pathogenetic mycoplasma-like organisms or phytoplasmas. In: Razin S, Tully, JG, eds. Molecular and Diagnostic Procedures in Mycoplasmology, Vol. I, San Diego, CA, USA: Academic Press, 369-380.

To cite this report: Ghayeb Zamharir M, 2016. Association of a 'Candidatus Phytoplasma solani'-related strain with pistachio in Iran. New Disease Reports 34, 9. [http://dx.doi.org/10.5197/j.2044-0588.2016.034.009]

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