BEYOND THE GROVE: UNVEILING THE ECOLOGY AND DIVERSITY OF X-DISEASE, ‘CANDIDADUS PHYTOPLASMA PRUNI’, STRAINS IN EXTRA-ORCHARD ENVIRONMENTS

Mason Chad Hoskins

Commercial cherry growers throughout North America are suffering losses to their crops over the last few decades due to an outbreak of X-disease, caused by ‘Candidatus Phytoplasma pruni’. In previous epidemics, this phytoplasma was thought to have originated in free-living Prunus spp. adjacent to the orchard environment. Taking advantage of recent research into the diversity of ‘Ca. P. pruni’, which identified seven distinct strains, five associated with commercial Prunus spp. and two specific to wild Prunus, we aimed to test that hypothesis by determining which strains were present incultivated vs. free-living Prunus across the western and mountain states of the United States during the growing seasons of 2023 and 2024. Chokecherry (Prunus virginiana) were sampled, 518 total, across 13 states, tested for ‘Ca. P. pruni’ presence using a species-specific real-time qPCR, followed by rapid identification of strains genotype using the imp gene. Multi-locus sequence analysis was performed using the 16S, secY, secA, imp, and Ef-Tu genes, for increased resolution of strain relations and diversity. Metagenomic analysis was conducted for 29 samples via Illumina sequences, then single-copy orthologs were identified as a final effort to understand the diversity of the 16SrIII group as a whole. MLSA revealed clear geographic segregation among strains found in wild Prunus spp., but not in commercial orchards in most states. This pattern suggests that strain introductions into orchards may occur independently of native wild Prunus populations. Additional novel strain populations were identified, indicating that this phytoplasma is more genetically diverse than previously recognized. Notably, the Rocky Mountains emerged as a potential barrier to strain movement, with increased strain diversification observed on the westside as compared to the east. Metagenomic analyses further distinguished traditional X-disease strains from X-related strains. X-related strains exhibited unique biological characteristics—including differences in primary host range, putative insect vectors, and geographic origins—that were not shared with classical X-disease strains. Current taxonomic standards for ‘Candidatus Phytoplasma’ rely primarily on genetic relatedness and do not incorporate biological traits in species delineation. In addition to the genetic divergence of X-related strains falling below the established threshold for taxonomic separation within the 16SrIII group, their distinct biological profiles highlight the need to reevaluate their classification and consider incorporating ecological and epidemiological criteria into taxonomic frameworks.

BEYOND THE GROVE: UNVEILING THE ECOLOGY AND DIVERSITY OF X-DISEASE, ‘CANDIDADUS PHYTOPLASMA PRUNI’, STRAINS IN EXTRA-ORCHARD ENVIRONMENTS

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