First Report of Tobacco Ringspot Virus Infecting Pawpaw Orchard (Asimina triloba (L.) Dunal) in North America.

Pawpaw (Asimina triloba (L.) Dunal, Annonaceae) is a fruit tree native to eastern North America, increasingly grown for commercial production in the United States, Europe, and Western Asia. In 2012, virus-like symptoms were noticed in a 0.3 ha pawpaw orchard at Michigan State University Plant Pathology Research Station; ~30% of the trees presented symptoms first mistaken for nutrient (magnesium/zinc) deficiency. Preliminary testing using ImmunoStrips for cucumber mosaic virus, impatiens necrotic spot virus, tobacco mosaic virus, tomato spotted wilt virus and the genus Potyvirus were negative. However, icosahedral virus particles were observed by TEM. To establish virus identity, we deep-sequenced tissue from a symptomatic pawpaw obtained from same site in summer 2021. Two contigs of 7511 bp and 3924 bp showed 94% and 95% nt identities with tobacco ringspot virus (TRSV) RNA1 and RNA2 respectively. TRSV infection was confirmed on leaf extracts by RT-PCR and Sanger sequencing. We further screened for TRSV infection in leaves from four symptomatic and three non-symptomatic pawpaw trees collected from the same site in 2022. Our results confirm the presence of TRSV infection in symptomatic pawpaw trees and emphasize the importance of also monitoring non-symptomatic trees. We confirmed graft transmission with 100% transmission rate observed in 200 trees grafted from a TRSV-infected pawpaw (Shenandoah cultivar), and investigation of other transmission vectors is on going. Because of TRSV's wide host range, its broad transmission profile in other crops (via nematodes, thrips, seeds, sap inoculation, and grafting), and the notable decline observed in infected pawpaws from different cultivars, TRSV appears to pose a new threat to pawpaw orchards. To the best of our knowledge, this is the first report of TRSV infecting pawpaw in North America and the world.