New genetically distinct phytoplasmas and insect carriers associated with pine tree disease revealed by a survey in Curonian Spit, Lithuania

Our previous studies reported that phytoplasma was the causative agent of the pine disease in Curonian spit, Lithuania. In this study, insects from diseased pine trees and their adjacent areas were collected from 2016 to 2019 to further identify potential insect vectors that spread phytoplasmas. A total of 1018 phloem-feeding insects (order Hemiptera) were identified, 98.62% of which were aphids (Aphididae), and no known phytoplasma vectors were found. Results from semi-nested PCR using phytoplasma-universal primers revealed that phytoplasmas were detected in scots pine aphids (Cinara pini), waxy grey pine needle aphids (Cinara pineti), and species-unknown aphids. Further sequence analysis and virtual RFLP analysis of aphid-harbored phytoplasma strains indicated that they were closely related to ‘Candidatus Phytoplasma pini’ (16SrXXI-A), but mainly 16SrXXI-A variants, which were also main strains identified in diseased pine trees. In addition, three new phytoplasma subgroups were delineated in the present study. Subgroups 16SrXXI-C and 16SrXXI-D were unveiled from previously identified (but classification was overlooked) Lithuanian pine phytoplasma strains. Subgroup 16SrXXI-E was discovered from the newly identified aphid-harbored phytoplasmas. Further transmission trial study on these aphids will provide insights into the epidemiology, and pathosystem of pine phytoplasma diseases, as well as the disease management.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author