Plant Pathology (2010) 59, 796 Doi: 10.1111/j.1365-3059.2010.02263.x First report of ‘Candidatus Phytoplasma asteris’ (16Sr I group) associated with yellows and little leaf diseases of Hibiscus rosa-sinensis in India Y. Chaturvedi a , M. Singh a , S. K. Snehi b , S. K. Raj b and G. P. Rao a * a Sugarcane Research Station, Kunraghat, Gorakhpur-273 008, UP; and b Plant Molecular Virology Laboratory, National Botanical Research Institute, Lucknow-226 001, UP, India Hibiscus rosa-sinensis (family Malvaceae) is an ornamental perennial shrub and native of warm temperate, subtropical and tropical regions. It is widely used for pain treatment related to menstruation, cystitis, venereal disease, feverish illnesses, bronchial catarrh and cough (Chopra et al., 1986). Yellows and little leaf symptoms were observed on H. rosa- sinensis plants growing in different gardens and nurseries of Gorakhpur, Eastern UP, India, during June 2008. Hibiscus isolate GKP-1 (from Betiahata garden, Gorakhpur) showed excessive yellowing, shortening of leaves and vein banding symptoms, while isolate GKP-2 (from Parijat nursery, Gorakhpur) exhibited little leaf, curling, puckering and stunting of the entire plant. Leaf samples from plants with and without symptoms were collected and total DNA extracted from 100 mg using a phytoplasma enrichment procedure (Ahrens & Seemu ¨ ller, 1992). Direct and nested PCR for Hibiscus phytoplasma 16S rRNA were performed using P1 / P7 (Deng & Hiruki, 1991) and R16F2n / R2 primers (Gundersen & Lee, 1996), respectively. A nested amplicon of expected size (ca. 1250 bp) was obtained for both GKP isolates, but not from symptomless plants. Ampli- cons were purified using a PCR clean-up system (Promega, USA) and sequenced (Genei Pvt. Ltd, India); data were deposited in GenBank for GKP-1 (Accession No. FJ939287) and GKP-2 (FJ939288). BLAST analysis showed a 98% 16S rRNA sequence identity between GKP-1 and GPK-2, which suggest that these are two different 16SrI-related isolates that may be associated with different symptom manifestations in the same host. GKP-1 and GKP-2 exhibited 99% 16S rRNA sequence identity with those of members of ‘Candidatus Phytoplasma asteris’ (16SrI group), which was confirmed by phylogenetic analysis (MEGA 4.0). Hibiscus sp. has been reported as a host for ‘Ca. Phytoplasma brasil- iense’ (16SrXV group) in Brazil associated with a witches’-broom disease (Montano et al., 2001), but there are no reports of Hibiscus sp. being a host for ‘Ca. Phytoplasma asteris’-related strains. This is the first report of a‘Ca. Phytoplasma asteris’ isolate associated with yellows and little leaf disease of H. rosa-sinensis. References Ahrens U, Seemu ¨ ller E, 1992. Detection of DNA of plant pathogenic mycoplasma- like organism by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82, 828–32. Chopra RN, Nayar SL, Chopra IC, 1986. Supplement to Glossary of Indian Medicinal Plants. New Delhi, India: Council of Scientific and Industrial Research. Deng S, Hiruki C, 1991. Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. Journal of Microbiological Methods 14, 53–61. Gundersen DE, Lee I-M, 1996. Ultrasensitive detection of phytoplasmas by nested- PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35, 144–51. Montano HG, Davis RE, Dally EL, Hogenhout S, Pimentel JP, Brioso PST, 2001. ‘ Candidatus Phytoplasma brasiliense’, a new phytoplasma taxon associated with Hibiscus witches’ broom disease. International Journal of Systematic and Evolutionary Bacteriology 51, 1109–18. *E-mail: gprao_gor@rediffmail.com. Accepted 16 December 2009 at http://www.bspp.org.uk/ndr where figures relating to this paper can be viewed. Plant Pathology (2010) 59, 796 Doi: 10.1111/j.1365-3059.2009.02245.x First report of a stolbur phytoplasma associated with witches’ broom of Japanese spindle (Euonymus japonicus) M. Rashidi a *, N. Habili b and A. Ghasemi a a Department of Plant Pathology, Iranian Research Institute of Plant Protection, P.O. Box 19395-1454, Tehran, Iran; and b Waite Diagnostics, School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond 5064, South Australia 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, the name ‘Japanese spindle witches’ broom’ (JSWB) was adopted. In October 2008, samples from seven affected 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 utilized. DNA from healthy trees served as a neg- ative 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 Gen- Bank (Accession No. GQ273961.) In Iran the stolbur group of phytoplas- mas has been detected in potatoes, plums, peaches and almonds (Salehi et al., 2005), and the sequence of JSWB shows 99% homology to the Ira- nian potato purple top phytoplasmas (EU661607). Since the 16S rDNA sequence of JSWB also shows 99% homology to Bois Noir (BN), a poten- tial 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. References Ahrens U, Seemuller E, 1992. Detection of DNA of plant pathogenic mycoplasma- like organism by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82, 828–32. Deng S, Hiruki C, 1991. Amplification of 16S rRNA genes from culturable and nonculturable Mollicutes. Journal of Microbiological Methods 14, 53–61. Karimi M, Contaldo B, Mahmoudi B, Duduk B, Bertaccini A, 2009. Identification of stolbur-related phytoplasmas in grapevines showing decline symptoms in Iran. 16th Meeting of the International Council for the Study of Virus and Virus-like Diseases of the Grapevine (ICVG). 31 August–4 September 2009, Dijon, France. Poster No. 60. Salehi M, Izadpanah K, Heydarnejad J, 2005. Molecular characterization and grouping of 35 phytoplasmas from central and southern provinces in Iran. Iranian Journal of Plant Pathology 41, 62–5. Smart CD, Schneider B, Blomquist CL et al., 1996. Phytoplasma-specific PCR primers based on sequence of the 16S-23S rRNA spacer region. Applied and Environmental Microbiology 62, 2988–93. *E-mail: rashidi_m642@yahoo.com. Accepted 6 October 2009 at http://www.bspp.org.uk/ndr where figures relating to this paper can be viewed. 796 ª 2010 The Authors Journal compilation ª 2010 BSPP