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

## TL;DR

This study identifies two new types of phytoplasmas in Iran, revealing complex transmission patterns between plants and insects.

## Contribution

The discovery of two novel phytoplasma ribosomal subgroups and new host-vector associations in Iran.

## Key findings

- Two novel phytoplasma ribosomal subgroups (16SrI-AS and 16SrIX-K) were identified in Iran.
- New plant–phytoplasma and insect–phytoplasma associations were documented.
- Multilocus analysis improved phytoplasma classification and revealed complementary evolutionary signals.

## Abstract

Phytoplasmas are bacteria that cause serious diseases in many crops and are transmitted by sap-feeding insects. Iran hosts diverse agricultural systems and insect communities, yet insect–phytoplasma associations remain poorly investigated and incompletely documented. In this study, we investigated phytoplasma infections in economically and ecologically important plants, including grapevine, soybean, barberry, and weeds associated with olive orchards, and in potential insect vectors collected from date palm groves in Iran. Using targeted next-generation sequencing, we characterized phytoplasma strains based on the 16S rRNA gene and multiple protein-coding genes. We detected five phytoplasma strains belonging to four ribosomal groups, including two previously undescribed subgroups. Several new plant–phytoplasma and insect–phytoplasma associations were identified, highlighting complex transmission pathways involving crops, weeds, and polyphagous insects. Our results show that combining ribosomal and multilocus analyses improves the resolution of phytoplasma diversity and provides insights into their ecology, evolution, and potential spread in agroecosystems.

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 (Vitis vinifera), soybean (Glycine max), barberry (Berberis vulgaris), and the weed Conyza canadensis, and in three potential insect vectors (Tropidocephala prasina, Eysarcoris ventralis, and Nysius graminicola) collected from distinct agroecosystems across Iran. Phytoplasmas were characterized by using nearly full-length 16S rRNA 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.