Molecular detection and identification of phytoplasmas associated with tomato diseases in Sri Lanka

Tomato cultivation in Sri Lanka is increasingly threatened by phytoplasma infections. In recent years, phytoplasma-like symptoms have been frequently observed in tomato fields across diverse agro-climatic regions of the country. This study assessed the incidence of these symptoms and identified the associated ‘Candidatus Phytoplasma’ species with tomato crops in selected districts. Field surveys conducted in seven districts revealed three distinct symptom types: little-leaf type I (small, wrinkled, and curled young leaves with dense apical leaf proliferation, shortened internodes and severe stunting), little leaf type II (milder leaf size reduction with upward curling, wrinkling, crinkled veins, and thickened, slightly curved shoots), and big-bud. The overall incidence of each symptom type remained around 10% during both the Maha (major) and Yala (minor) cultivation seasons. Total DNA was extracted from symptomatic plants and subjected to nested PCR using phytoplasma-specific primers targeting the 16 S rRNA gene, followed by sequencing and bioinformatic analyses. BLASTn, iPhyClassifier, and phylogenetic analyses revealed the association of ‘Ca. Phytoplasma australasiaticum’ (16SrII-D and 16SrII-A) with big bud and little-leaf type II disease. Subspecies-level analysis further confirmed the presence of ‘Ca. P. australasiaticum subsp. australasiaticum’ in big bud samples, and ‘Ca. P. australasiaticum subsp. taiwanense’ in little-leaf type II. Isolate associated with little-leaf type I were most closely related to, and likely represent a variant of ‘Ca. P. asiaticum’. To our knowledge, this is the first confirmed report of ‘Ca. P. australasiaticum’ and ‘Ca. P. asiaticum’ associated with tomato in Sri Lanka. These findings enhance current understanding of phytoplasma diversity in the region and provide a molecular basis for future epidemiological studies and the development of sustainable disease management strategies.