First Report of a 16SrVI Group Phytoplasma Associated with Witches'-Broom Disease on Withania somnifera.

Abstract

Withania somnifera (L.) Dunal is cultivated in India as an important medicinal cash crop. The whole plant is of great importance in the Indian system of medicine and pharmaceutical industries, but the roots are the main source of active alkaloids. Some of the important alkaloids are tro-pine, pseudotropine, somniferine, colin, withaferin A, withanoides, and a few flavanoides. Typical disease symptoms include phyllody, little leaf, dense clusters of highly proliferating branches with shortened internodes, and resulting witches'-broom. The disease was first observed in and around Lucknow, Uttar Pradesh Province, India during January and February 1992. On the basis of symptoms, transmission electron microscopy (TEM), and antibiotic treatment, the causal organism was identified as a phytoplasma (4). The disease is now spreading to other parts of the country (Gujrat, Haryana, Madhya Pradesh, Punjab, and Rajasthan provinces) with a high disease incidence (70%). In this report, molecular characterization and taxonomic position of the associated phytoplasma is reported. Total genomic DNA was extracted from healthy and infected plants with a modified cetyltrimethylammoniumbromide (CTAB) buffer method. The samples were assayed for the presence of phytoplasma using polymerase chain reaction (PCR) with universal phytoplasma primers P1/P6 (2) for amplification of ribosomal 16S rDNA. PCR product was diluted by 1:200 and used directly as DNA template for nested PCR with primers R16F2n and R16R2 (1). Results showed the presence of an expected 1.5-kb rDNA fragment amplified with the direct PCR and a 1.2-kb product of the nested PCR from infected W. somnifera samples. No PCR product was observed in the healthy counterparts. The PCR assay confirmed the presence of phytoplasma as causal agent. The PCR product was cloned with TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and isolated plasmids were again assessed by restriction enzyme (EcoRI) digestion before sequencing. Purified plasmids were sequenced. Partially sequenced nucleotide sequence analysis of 16SrRNA gene cloned from W. somnifera phytoplasma showed high similarity with several isolates of the 16SrVI group of phytoplasmas. The highest nucleotide matching (99 and 98%) was observed with Centaurea solstitialis virescence phytoplasma (Genbank Accession No. AY270156) and Periwinkle little leaf phytoplasma (PPL-Bd; Genbank Accession No. AF 228053) reported in Italy and Bangladesh, respectively. In restriction fragment length polymorphism (RFLP) analysis, AluI, EcoRI, HhaI, HincII, KpnI, and Sau3AI (Promega, Madison, WI; 5 U per reaction) were used for comparison of restriction pattern of present/reference phytoplasma and with that previously reported (3). The present phytoplasma produced identical restriction profile to those of periwinkle infected by PPL-Bd (periwinkle little leaf phytoplasma, Bangladesh, group 16SrVI). On the basis of PCR studies, absence of virus particles under TEM in infected samples, RFLP analysis and nucleotide sequence matching with previously characterized phytoplasma, this phyto-plasma is classified as a member of Clover proliferation group (16SrVI). To our knowledge, this is the first report of a phytoplasma belonging to 16Sr VI group from W. somnifera.