Identification and Molecular Characterization of a 16SrV-F Phytoplasma Associated with Little Leaf and Witches’ Broom Disease in Jackal Jujube (Ziziphus oenoplia L.) in Kerala, India

Ziziphus oenoplia (Jackal jujube), a perennial thorny shrub in the Rhamnaceae family, is widely distributed in India, particularly in Kerala, Karnataka, Andra Pradesh, Telangana, and Maharashtra, specifically in the Western Ghats, renowned for its medicinal properties, attributed to alkaloids known as ziziphines, the plant is crucial in Ayurvedic medicine. Despite its medicinal importance, Z. oenoplia faces susceptibility to little leaf and witches' broom diseases incited by phytoplasma, suspected as potentially playing a role in sandal spike disease epidemics as an alternate host in Kerala. Plant samples, both infected and healthy, were collected from Wadakkancherry forest area and the Kerala Forest Research Institute campus. The pathogenic agent responsible for wild jujube little leaf and witches’ broom disease was identified through symptomatology, microscopic observation (Scanning Electron Microscope), PCR, nested PCR, and phylogenetic analysis. Symptomatic features included a severe reduction of leaf size, yellowing, and numerous small leaves which appeared in clusters resembling witches’ broom. Scanning electron microscopic (SEM) assay confirmed the presence of phytoplasma bodies, which are pleomorphic in shape and absence of cell walls, were found in the phloem sieve tubes of diseased Ziziphus plants, but absent in healthy Ziziphus plants. Molecular analysis using PCR and nested PCR primers authenticated the existence of phytoplasma in the diseased plants. Results of the nucleotide sequence analysis of 16s ribosomal gene showed 100% nucleotide identity with 16SrII phytoplasma isolates, specifically Candidatus Phytoplasma balanitae isolate JWB_K15 (MN902087). The study underscores the molecular distinction between the phytoplasma strains infecting Ziziphus and Sandalwood, providing valuable insights for disease management and ecological preservation.