First report of ringspot and vein-clearing symptoms on Wasabia japonica plants associated with Wasabi mottle virus in North America

Wasabia japonica (Miq) Matsum. is cultivated for its rhizome which is traditionally consumed with Japanese cuisine. In 2017, ringspot and vein-clearing symptoms were observed in a commercial greenhouse in British Columbia. Symptomatic leaves were ground in phosphate buffer and used to mechanically inoculate Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. occidentalis, N. tabacum, Chenopodium quinoa, C. amaranticolor, Gomphrena globosa, Brassica perviridis and Tetragonia tetragonioides. After 4-11 days most hosts showed symptoms of necrotic lesions. N. benthamiana showed mild puckering, and B. perviridis showed mild puckering, vein banding, and chlorosis. Transmission electron microscopy revealed the presence of ~250-300 nm long rod-shaped virions in inoculated N. occidentalis and N. clevelandii. RNA was extracted and purified from symptomatic wasabi tissue, and RT-PCR was conducted with six degenerate broad-spectrum primer sets for detecting the genera Tobamovirus, Nepovirus, Potyvirus, Ilarvirus, and Cucumovirus, and the species Turnip ringspot virus and Alfalfa mosaic virus. One primer set, Ilar1F5 univ-F/Ilar1R7 univ-R, produced positive band sizes of ~300 and ~600 bp, which were sequenced. A MegaBLAST (NCBI) search identified Wasabi mottle virus (WMoV), a Tobamovirus, to be present. Two wasabi cultivars were inoculated as previously described: ‘Greenthumb’ developed ringspots and vein-clearing after 22-23 days in the greenhouse, while ‘Daruma’ were non-symptomatic. A PCR test confirmed the presence of WMoV in naturally infected and inoculated wasabi plants. A survey of a greenhouse facility conducted in June 2018 showed the frequency of diseased, typically non-symptomatic, plants to be 38%. Symptom development was more pronounced at higher temperatures (>30° C) than cooler temperatures (21-24° C). BC isolates showed 99% sequence identity to isolate ‘Alishan’ (GenBank accession no. KJ207375.1) and 98% identity to a Japanese strain (GenBank accession no. AB017504.1). The origin of WMoV is presumed to be through the importation of infected ‘Greenthumb’ plants from Taiwan, and subsequent propagation.