Incidence, molecular characterization and phylogenetic diversity of phytoplasmas associated with brinjal little leaf disease in Maharashtra and Karnataka, India

Brinjal little leaf disease (BLL), caused by phytoplasmas, represents a major constraint to eggplant (Solanum melongena L.) production in India, leading to substantial yield losses. The present study aimed to assess the incidence of BLL in selected eggplant-growing regions of Maharashtra and Karnataka and to molecularly characterize the associated phytoplasma isolates using PCR–RFLP and phylogenetic analyses. Field surveys conducted across twelve locations during 2022–2023 revealed a high mean disease incidence of 43.12%, with considerable spatial variation ranging from 26.6% to 61.9%. Symptomatic plants exhibited typical features of phytoplasma infection, including reduced leaf size, chlorosis, virescence, witches’ broom, and severe stunting. Molecular detection using nested PCR consistently amplified phytoplasma-specific fragments confirming the presence of phytoplasmas in all analysed samples. PCR–RFLP analysis revealed limited genetic variability among most isolates, although distinct restriction profiles were observed for selected samples, particularly BLL-11. Cluster analysis grouped the isolates into two main clusters, indicating the presence of genetically differentiated lineages. Sequence analysis of the 16S rRNA gene and phylogenetic reconstruction demonstrated that all isolates belong to the 16SrVI ribosomal group and are closely related to “Candidatus Phytoplasma trifolii”. However, the formation of distinct phylogenetic clusters and haplotypes suggests the occurrence of previously uncharacterised genetic variants within this group. The median-joining haplotype network further supported recent diversification and local evolution of phytoplasma populations. These findings contribute to a better understanding of the genetic diversity and epidemiology of BLL-associated phytoplasmas and provide a foundation for future studies aimed at improving disease management strategies.