Broad bean and sweet pepper: two new hosts associated with Candidatus Phytoplasma asteris (16SrI phytoplasma group) in Cuba

Broad bean ( Vicia faba ) and sweet pepper ( Capsicum annuum) are two important vegetable crops grown to supply both local and tourist markets in Cuba. During a survey in 2005, 12% of broad bean plants at the CAI “Osvaldo Sánchez” farm in Havana province showed symptoms of leaf yellowing, short internodes and stunting. Similar symptoms but here also with little leaf were observed on around 15% of sweet pepper plants growing in Holguín province at the CAI “Cristino Naranjo” farm. Phytoplasmas have been reported as affecting both crops in Europe (Castro & Romero, 2002 & 2004; Schneider et al , 1995) and sweet pepper from Mexico ( Genbank Accession No. DQ092321 ). Leaf samples from 56 plants with symptoms and 21 without (broad bean), and from 73 with and 32 without (sweet pepper), were collected at random from the fields. DNA was extracted using the CTAB method of Doyle & Doyle (1990) and used as template in a nested PCR assay to amplify the phytoplasma 16S rRNA gene using primers P1 /P7 and R16F2n/R16R2. Amplicons of expected size (1250 bp) were produced from all plants with symptoms but not from symptomless plants. The amplicons were compared by RFLP with restriction endonucleases Hae III, Rsa I and Alu I; each pattern of which was identical for all samples. Two amplicons of each plant species were direct sequenced using primers P1/P7 (The Sequencing Service University of Dundee, Scotland, using Applied Biosystems Big-Dye Version 3.1 chemistry on an Applied Biosystems model 3730). The sequences were subjected to BLAST analysis and showed the highest similarity (97% broadbean; 99% sweet pepper) to that of watercress yellows phytoplasma (AY665676), an isolate of Candidatus Phytoplasma asteris (16SrI Aster Yellows group). The sequences were deposited in GenBank: Accession No. DQ286953 (broadbean) and DQ286947 (sweet pepper). This is the first report of a phytoplasma infection of broadbean and sweet pepper plants in Cuba.