First report of a phytoplasma associated with Periploca aphylla witches' broom in Iran

Periploca aphylla (Asclepiadaceae), is a large erect branched shrub, grown on different altitudes, in desert or in dry, insolated grounds, on rocky and sandy slopes throughout Southwest Asia. P. aphylla is commonly used for feeding camels and goats. Its latex is applied as a poultice in tumours and swellings, while the decoction of the bark is used as a purgative or against fever (Kazimierz, 1966). In September 2008, symptoms of witches' broom, proliferation and shoot dieback were observed on P. aphylla in Hormozgan province, Iran. The affected plants developed dense clusters of highly proliferating branches with shortened internodes (Fig. 1). Total DNA was extracted separately from 0.5g of shoots of ten symptom-bearing and five symptomless plants using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle & Doyle, 1990). The samples were analysed for phytoplasma DNA by both direct and nested PCR assays using phytoplasma universal 16S rDNA gene primer pairs P1/P7 and R16F2n/R16R2 that amplify fragments of 1800 bp and 1250 bp, respectively. A 1250 bp PCR product was amplified with nested PCR from all symptom-bearing, but not from symptomless, plants. Two amplicons were cloned using cloning commercial kit #K1214 (Fermentas, Lithuania), and sequenced. The partial 16S rDNA sequence (GenBank Accession No. GQ249159) of the phytoplasma associated with P. aphylla witches' broom was most closely related (98% sequence identity) to salt cedar witches' broom phytoplasma, ‘Candidatus Phytoplasma tamaricis’ (FJ432664) (Zhao et al., 2009). Moreover, P. aphylla witches' broom phytoplasma showed a 96% sequence identity with strains ofgroup 16SrX, apple proliferation, including a ‘Ca. Phytoplasma pyri’-related strain from subgroup 16SrX-C, previously associated with pear decline in Iran (Salehi et al., 2008), which has been reported in almost all pear growing areas of Fars, Isfahan, Tehran and Khorasan provinces. It is the first report of a phytoplasma associated with P. aphylla witches' broom, this being a real concern regarding its potential as a host for a 16SrX phytoplasma isolate.