© 2006 Director NBRI 822 Journal compilation © 2006 BSPP Blackwell Publishing Ltd Basil little leaf: a new disease associated with a phytoplasma of the 16SrI (Aster Yellows) group in Cuba Y. Arocha a *, B. Piñol a , B. Picornell b , R. Almeida c , P. Jones d and E. Boa e a National Centre for Animal and Plant Health (CENSA), Apdo 10. San José de Las Lajas, Havana; b CAI ‘Osvaldo Sánchez’, Güines, Havana; c National Institute of Sugarcane Research (INICA), Havana, Cuba; d Global Plant Clinic, Plant-Pathogen Interactions Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ; and e Global Plant Clinic, CABI-Bioscience, Egham, TW20 9EY, UK In Cuba, the development of urban and peri-urban agriculture, using a combined organic and hydroponic (‘organoponic’) system of intensive vegetable production, currently plays a very important role for the sustainability of food security. Basil (Ocimum basilicum) has become a very popular barrier crop for organoponic gardens, where it is commonly used to repel undesirable pests. During a survey in 2005 of weeds from the ‘Alamar’ organoponic in Havana province, typical phytoplasma-symptoms of little leaf and witches’ broom ( Jones, 2002) were observed in all basil plants used as barrier crops. Leaf samples from 27 plants with symptoms and 16 apparently healthy (symptomless) plants were collected and the DNA extracted. This was used as the template in a nested PCR with universal phytoplasma rRNA primer pairs P1/P7 and R16F2n/R16R2. All 27 extracts from plants with symptoms produced a 1250 bp product, which gave typical phytoplasma profiles when digested with the restriction endonuclease HaeIII. No amplicons were produced when DNA of symp- tomless plants was used as the template. RFLP using RsaI and AluI gave identical profiles for all 27 samples. The P1-P7 PCR products were directly sequenced and compared by BLAST analysis with those of other phytoplasmas in GenBank. The highest similarity (99%) was to that of a 16SrI group phytoplasma (Aster Yellows; ‘Candidatus Phytoplasma asteris’) affecting watercress (GenBank accession no. AY665676). The 16S rRNA sequence from basil was deposited in GenBank (Accession no. DQ286577). This report from Cuba is the first record of a new disease for which the name basil little leaf is proposed. Acknowledgements Work in the UK was done under Defra plant health licence No PHL 174D/5186(08/2005). Sequencing was performed by The Sequencing Service, School of Life Sciences, University of Dundee, Scotland (www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3·1 chemistry on an Applied Biosystems model 3730 capillary sequencer. Reference Jones P, 2002. Phytoplasma plant pathogens In: Waller M, Lenné JM, Waller SJ, eds. Plant Pathologists Pocketbook. Wallingford, UK: CAB International, 126 –39. *E-mail: yaimaarocha@yahoo.es. Accepted 23 May 2006 at www.bspp.org.uk/ndr where figures relating to this paper can be viewed. Plant Pathology (2006) 55, 822 Doi: 10.1111/j.1365-3059.2006.01481.x First report of molecular detection of an Aster yellows phytoplasma (‘Candidatus Phytoplasma asteris’) isolate infecting chilli (Capsicum annuum) in India M. S. Khan and S. K. Raj* Molecular Virology, National Botanical Research Institute, Lucknow-2260 01, India Chilli (Capsicum annuum; family Solanaceae) is an important spice crop, being cultivated over large areas in Asia, Africa, South and Central America, parts of USA and Southern Europe. India is the largest chilli producer in the world, producing 1·2 million tonnes of dry chilli, from an area of about 880 000 hectares. During the winter of 2004, a severe phytoplasma-like disease of chilli was noticed in the Bahraich district of Uttar Pradesh with a low incidence (c. 5% of plots showed some infection). The symptom of the disease consisted of shortening of leaves, petioles & internodes and crowding of leaves and stunting of whole plant. For molecular detection of the causal pathogen, the total DNA of plant samples with or without symptoms was isolated using the protocol of Ahrens & Seemüller (1992). Direct PCR was carried out using the universal 16S rDNA-specific primers P1/P6 (Deng & Hiruki, 1991), which resulted in the production of a ∼1·5 kb product from diseased samples but not symptomless ones. Nested-PCR was further performed with primers R16F2n/R16R2 (Gundersen & Lee, 1996) which gave an amplicon of the expected size ∼1·2 kb DNA. This product was cloned, sequenced and the data deposited in GenBank (Accession DQ343288). A blast search revealed the highest level of sequence identities (98%) with 16SrI Aster yellows group members, such as Barley deformation phytoplasma (AY734453), Aster yellows phytoplasma (AY665676), Valeriana yellows phytoplasma (AY102274), Onion yellows phytoplasma (AP006628) and Silene virescence phytoplasma (AY744070). Previously, a phytoplasma associated with chilli little leaf disease has been detected in India by graft transmission and electron microscopy (Singh & Singh 2000), but the causal pathogen was not characterised at the molecular level. To our knowledge, this is the first report of molecular detection of an isolate of Aster yellows phytoplasma (‘Candidatus Phytoplasma asteris’) infecting chilli in India. References Ahrens U, Seemüller 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 D, 1991. Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. Journal of Microbiological Methods 14, 53–61. Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathologia Mediterranea 35, 144–51. Singh D, Singh SJ, 2000. Chilli little leaf – a new phytoplasma disease in India. Indian Phytopathology 53, 309–10 *E-mail: skraj2@rediffmail.com. Accepted 20 March 2006 at www.bspp.org.uk/ndr where figures relating to this paper can be viewed. Plant Pathology (2006) 55, 822 Doi: 10.1111/j.1365-3059.2006.01482.x © 2006 The Authors Journal compilation © 2006 BSPP