First report of ‘Candidatus Phytoplasma asteris’ (16Sr I group) associated with yellows and little leaf diseases of Hibiscus rosa‐sinensis in India

Abstract

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 & Seemüller, 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. Amplicons 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 brasiliense’ (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.