New plant hosts for group 16SrII, ‘Candidatus Phytoplasma aurantifolia’, in India

Abstract

Lettuce (Lactuca sativa), carrot (Daucus carota) and French bean (Phaseolus vulgaris) are important staple food crops in India. Symptoms of leaf yellowing, chlorosis, and little leaf were recently observed in these species at the Vegetable Research Center, GBPUA&T, Uttarakhand, Pantnagar, India. Plants of Amaranthus sp. growing in adjacent hedges also exhibited leaf yellowing symptoms. Three leaf samples of each plant species with symptoms were collected, including healthy looking plants. Total DNA was extracted and indexed in a nested PCR assay with universal primers R16mF2/R1 and fU5/rU3 that target the phytoplasma 16S rRNA. PCR amplicons (880 bp) were produced in all samples with, but not without symptoms. PCR products were subjected to RFLP analyses with RsaI and HpaII restriction enzymes, and further purified, cloned (pGEM-T Easy Vector, Promega), and sequenced in both directions using M13 forward and reverse sequencing primers (http://www.dnaseq.co.uk). The 16S rRNA sequences were compared with those of reference strains from GenBank using blast, and aligned with clustal W (Thompson et al., 1994) to produce a phylogenetic tree using the neighbour-joining method and 1000 replicates with mega 3·1 (Kumar et al., 2004). RFLP profiles were all similar to those of group 16SrII, ‘CandidatusPhytoplasma aurantifolia’. Sequences of phytoplasmas (GenBank) identified in lettuce (EU362630), carrot (EU362628), French bean (EU362629) and Amaranthus sp. (EU362627) shared 99% identity to each other, and to a member of group 16SrII, ‘CandidatusPhytoplasma aurantifolia’ (EU099570). Phylogeny supported RFLP results and sequence comparisons, and indicates that the Pantnagar phytoplasmas cluster in the 16SrII group branch more closely related to the cactus witches’ broom phytoplasma. ‘CandidatusPhytoplasma aurantifolia’ has been previously reported in acid lime in India (Ghosh et al., 1999) and chickpea (Ghanekar et al., 1988), but this is the first record of a 16SrII phytoplasma isolate in lettuce, carrot, French bean, and Amaranthussp. in this country. Work in the UK was done under Defra licence No. PHF 174D/5185(08/2005). Sequencing was done by the Sequencing Service (School of Life Sciences, University of Dundee, Scotland, http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3·1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer.