First Report of a Natural Infection of Stevia rebaudiana by a Group 16SrXXIV Phytoplasma in India

Abstract

Stevia rebaudiana Bertoni (Asteraceae) is one of the most important commercial crops in the world (4). It is known to produce glycosides that are as much as 300 times sweeter than sucrose and do not affect blood sugar levels. Unlike artificial sweeteners like saccharin, they are noncarcinogenic and safe for diabetics. An unknown disease emerged during the summers of 2007 to 2009 in a field of S. rebaudiana at CIMAP Lucknow, India, where more than 20% of the plants exhibited symptoms typical of phytoplasma infection including leaf yellowing, reduced size of leaves, shoot proliferation, flower bud deficiency, as well as bushy and stunted growth. Some of these plants were potted and kept in a glasshouse for investigation. Affected plants in the field expressed a quick decline consisting of growth cessation, bronzing of mature leaves, wilting, and death, resulting in a significant reduction in biomass and quality. Typical phytoplasma-like (pleomorphic) bodies ranging from 450 to 900 nm were observed in the phloem cells of infected plants by transmission electron microscopy (1). These bodies were always found in diseased plants, but not in asymptomatic ones. No other microorganisms were noted. Total DNA was extracted from symptomatic as well as asymptomatic plants by a CTAB method. PCR was carried out with the universal phytoplasma primers P1/P6 (P1, 5'-AAGAGTTTGATCCTGGCTCAGGATT-3'; P6, 5'-CGGTAGGGATACCTTGTTACGACTTA-3') (2) followed by nested primers R16F2n/R16R2 (R16F2n, 5'-GAAACGACTGCTAAGACTGG-3'; R16R2, 5'-TGACGGGCGGTGTGTACAAACCCCG-3') targeting the 16S rRNA gene sequence (3). The P1/P6 and R16F2n/R16R2 primers produced the expected 1.5- and 1.2-kb amplicons, respectively, from the symptomatic plants and not from the asymptomatic ones. Seventeen symptomatic and eight asymptomatic samples were analyzed through PCR. Nested PCR products were ligated into the plasmid vector using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). Transformation and selection of recombinant clones was carried out according to the manufacturer's recommended protocol. The sequence obtained from the final PCR product was deposited in the GenBank database (No. JF970603). It was analyzed through the iPhyClassifier ( http://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi ) online tool and found to share 98.2% similarity with that of the 'Sorghum bunchy shoot phytoplasma' reference strain (GenBank No. AF509322) that belongs to 16SrXXIV-A subgroup. The virtual restriction fragment length polymorphism pattern of the S. rebaudiana phytoplasma 16S rRNA gene sequence showed maximum similarity to the reference pattern of AF509322 (similarity coefficient of 0.85). Although a number of phytoplasmas have been detected on a wide range of plants in India, little is known about the leafhopper that presumably transmits them to S. rebaudiana and other medicinal crops. Infections by diverse phytoplasma strains/species underscore the need for phytoplasma-free planting stock and intensification of research efforts to reduce ecological and economic impacts of these phytoplasmas. To our knowledge, this is the first report of a natural infection of S. rebaudiana by a group of 16SrXXIV-A phytoplasma.