DISEASE NOTE Detection and characterization of a phytoplasma associated with witches’-broom disease of Salvadora persica in India Sachin Kumar • Vibhuti Singh • Suman Lakhanpaul Received: 13 January 2011 / Accepted: 14 March 2012 / Published online: 27 April 2012 Ó The Phytopathological Society of Japan and Springer 2012 Abstract Salvadora persica trees with symptoms of witches’-broom, little leaf and severe leaf curling were observed in the Biodiversity Park, North Delhi (India) during February 2010. Using a nested-PCR assay targeting two phytoplasma specific loci (16S ribosomal RNA and the ribosomal protein gene) and sequence analysis of the amplified products, we identified the associated phyto- pathogen as a phytoplasma of group 16SrI. RFLP-mediated characterization revealed that the phytoplasma belongs to subgroup 16SrI-B. Phylogenetic reconstruction also grouped the phytoplasma within group 16SrI. The study reports a new host of phytoplasma in India and worldwide. Keywords Witches’-broom Á ‘Candidatus Phytoplasma asteris’ Á 16S rDNA Á Ribosomal protein Introduction Commonly known as the toothbrush tree, miswak, Salv- adora persica (family Salvadoraceae) has the habit of an evergreen shrub or small tree. The plant is a native of subtropical to tropical Africa with a wide distribution across arid and semi-arid areas of the globe. Therapeutic extracts from its roots, twigs and stems are important ingredients in dental care products and have been recom- mended for oral hygiene by the World Health Organization (1978). Miswak trees suffering from severe witches’-broom disease were observed in North Delhi (India) region during February 2010 with a disease incidence of 60 %. Infected plants suffered severe losses in flower production, accom- panied by common features like little leaf, where the size of the leaves was highly reduced (Fig. 1). Almost every leaf had leaf roll, where the apical end of leaf lamina turned downward. Such reduction in leaf size and rolling of leaves tended to increase as the disease progressed. Extreme shortening of internodes led to shorter branches and clus- tering of leaves. This phenotype along with excessive proliferation of axillary buds resulted in a witches’-broom appearance of the branches throughout the tree. Such symptoms are usually associated with phloem-inhabiting, wall-less bacteria, phytoplasmas that are transmitted via sap-sucking insect vectors (viz. leafhoppers and psyllids). To investigate this possibility, we collected young stem tissues from four affected and two symptomless miswak trees. Genomic DNA, isolated from stem tissues of miswak and from the positive control, periwinkle infected with onion yellows (OY) phytoplasma according to Saghai- Maroof et al. (1984), was PCR amplified using phytopl- asma-specific universal 16S rDNA primers P1/P7 (Deng and Hiruki 1991), followed by nested-PCR with primer pair R16F2/R2n (Lee et al. 1993). The PCR products from the first round were diluted 1:30 and used as the template (1 lL/reaction) in a second round. The rp gene region (consisting of rps19, rpl22, rps3) was amplified with the first round primers rpF1/R1 (Lim and Sears 1992) along with a nested primer pair specific for group 16SrI, rp(I)F1A/rp(I)R1A (Lee et al. 2004). For both genes, amplicons were obtained only with the symptomatic and positive control samples and were of the expected size (*1.25 and *1.2 kb, respectively). PCR products were gel extracted and purified by QIAquick gel extraction kit (Qiagen, Chatsworth, USA). Sequences of 16S rDNA fragments were ligated in pGEMT-Easy vector (Promega, S. Kumar (&) Á V. Singh Á S. Lakhanpaul Plant Molecular Genetics Laboratory, Department of Botany, University of Delhi, Delhi 110007, India e-mail: sach_inom@yahoo.co.in 123 J Gen Plant Pathol (2012) 78:294–297 DOI 10.1007/s10327-012-0381-y