Host-driven structuring shapes the landscape of phytoplasma diversity across Indian agroecosystems revealed by multilocus phylogenomics.

Phytoplasmas are phloem-limited, wall-less plant pathogens that impose significant biotic stress on host plants, resulting in substantial yield losses worldwide. However, their genetic diversity, host associations, and population structure in tropical agroecosystems remain poorly understood. This study presents a nationwide multilocus assessment of 'Ca. Phytoplasma' diversity, distribution, and population structure across Indian agroecosystems. A survey (2020-2023) across ten states identified 124 phytoplasma-positive samples from 42 host species and associated leafhopper insect vectors. Multilocus sequence analysis of 16S rRNA, secY, and rpl22 genes, supported by in silico RFLP, resolved the strains into five major ribosomal groups (16SrI, II, V, VI, and XI) encompassing 14 subgroups, with several strains (similarity coefficient ≤ 0.97) indicating candidate novel subgroups. Genetic diversity analysis revealed high haplotype diversity (Hd = 0.991) and substantial multilocus variation, with AMOVA showing > 50% of genetic variance structured by host association rather than geography. Haplotype network and neutrality analyses indicated demographically stable, host-structured populations. Network and multivariate analyses revealed non-random host associations, strong host specificity, and ecological modularity, with subgroup 16SrII-D acting as a central hub linking multiple hosts and regions. Correspondence analysis further confirmed significant host-associated structuring among major phytoplasma groups. Novel host associations, including Vitex negundo, V. altissima, and paper daisy, further expanded the known phytoplasma host range in India. Collectively, these findings provide important insights into phytoplasma diversity, host-pathogen interactions, and their implications for plant health and disease management, offering a framework for improved surveillance of emerging phytoplasma-associated diseases.