Diversity of Phytoplasmas Infecting Plants and Insects in Iran Reveals Two Novel Ribosomal Subgroups

Phytoplasmas are obligate bacterial pathogens transmitted by phloem-feeding insects and responsible for severe diseases in numerous crops worldwide. In Iran, insect-associated phytoplasma transmission pathways remain poorly resolved, particularly at fine phylogenetic and vector-specific scales. Here, we investigated phytoplasma strains detected in four plant species, grapevine (<i>Vitis vinifera</i>), soybean (<i>Glycine max</i>), barberry (<i>Berberis vulgaris</i>), and the weed <i>Conyza canadensis</i>, and in three potential insect vectors (<i>Tropidocephala prasina</i>, <i>Eysarcoris ventralis</i>, and <i>Nysius graminicola</i>) collected from distinct agroecosystems across Iran. Phytoplasmas were characterized by using nearly full-length <i>16S rRNA</i> gene sequences and a multilocus dataset of protein-coding genes obtained through a targeted next-generation sequencing approach. Five phytoplasma strains belonging to ribosomal groups 16SrI, 16SrVI, 16SrIX, and 16SrXII were identified, including two novel ribosomal subgroups, 16SrI-AS and 16SrIX-K. Several previously unreported plant-phytoplasma and insect-phytoplasma associations were documented. Comparative phylogenetic analyses revealed that ribosomal and multilocus markers capture complementary evolutionary signals, with protein-coding genes providing additional resolution beyond 16S-based classification. These findings highlight the potential role of diverse hosts and polyphagous insects, not yet confirmed as vectors, in phytoplasma circulation and underscore how high-throughput next-generation sequencing and multilocus approaches advance our understanding of phytoplasma diversity and evolution.