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Outbreaks of emerging and re-emerging diseases of both animals and plants are increasing due to climate change, globalization, land-use change, and agricultural intensification. While most pathogen monitoring programs focus on zoonotic threats, wildlife and other organisms in natural habitats can also serve as reservoirs and sentinels for pathogens of agricultural and ecological concern. Plant communities and the pathogens circulating within them are underrepresented in integrated disease monitoring frameworks. This study demonstrates how biodiversity and zoonosis monitoring programs conducted in protected habitats (tallgrass prairies and woodlands) across Illinois, together with insect specimens preserved in biorepositories, can be leveraged to improve knowledge of the identities and ecological associations of a wide range of potential pathogens. We developed an integrative workflow combining taxonomic identification, molecular screening, and epidemiological inference to detect vector-borne plant pathogens from archived insect material. Focusing on Hemiptera (Auchenorrhyncha), we screened specimens for phytoplasmas (Mollicutes), uncultured bacterial plant pathogens transmitted by sap-feeding insects, and characterized host-pathogen associations. At least three distinct phytoplasma strains were detected: ‘Candidatus Phytoplasma asteris’ (16SrI-B), ‘Candidatus Phytoplasma pruni’–related strains (16SrIII), and ‘Candidatus Phytoplasma sacchari’–related strains (16SrXI-H). The latter represents the first documented occurrence of a 16SrXI-H phytoplasma subgroup in Illinois. Overall, we identified five insect specimens harboring phytoplasmas across four preserved sites, all of them were previously unreported insect-phytoplasma associations. These findings demonstrate the value of existing biodiversity infrastructures for proactive surveillance of plant pathogens and extend the One Health paradigm to explicitly include natural ecosystems.