First report of a stolbur phytoplasma associated with witches' broom of Japanese spindle (Euonymus japonicus)

Japanese spindle trees (Euonymus japonicus) are evergreen shrubs grown for hedges in parks. Recently, diseased trees in Tehran ( Iran ) were observed with symptoms of little leaves and a bushy appearance suspected to be caused by a phytoplasma. Since the trees showed witches’ broom in one or more of their branches (Fig. 1), we named it “Japanese spindle witches’ broom” (JSWB). In October 2008, samples from seven infected trees were collected in Tehran and the total DNA was extracted by the CTAB procedure (Ahrens & Seemuller, 1992).Amplification of the 16S rDNA was done by nested PCR using generic phytoplasma primers specific to the 16S rRNA gene. The first primer pair was P1/P7 while the nested primers were R16F2 and R16R2 (Deng & Hiruki, 1991; Smart et al., 1996). For positive controls, DNA from alfalfa witches’ broom and safflower phyllody were utilised. DNA from healthy trees served as a negative control.

All seven samples gave the expected phytoplasma specific PCR band of 1250 bp, corresponding to those of the positive controls, while healthy trees gave no bands. Amplified DNA products of nested PCR from two samples were isolated and directly sequenced with the forward and reverse primers R16F2 and R16R2. The BLAST analysis of the sequence obtained confirmed that the spindle witches’ broom phytoplasma belongs to the 16SrXII-A (stolbur) group. The sequence was deposited at GenBank (Accession No. GQ273961.) In Iran the stolbur group of phytoplasmas has been detected in potatoes, plums, peaches and almonds (Salehi et al., 2005), and the sequence of JSWB shows 99% homology to the Iranian potato purple top phytoplasmas (EU661607).Since the 16S rDNA sequence of JSWB also shows 99% homology to Bois Noir (BN), a potential grapevine yellows pathogen, grapevines in Iran may be the next target (Karimi et al., 2009). This is the first report of a phytoplasma infecting Japanese spindle trees.