Elimination of Chrysanthemum Chlorotic Mottle Viroid (CChMVd) Recently Detected in Japan by Leaf-primordia Free Shoot Apical Meristem Culture from Infected Cultivars

The disease symptoms of chrysanthemum chlorotic mottle viroid (CChMVd) that had not been observed in chrysanthemum (Dendranthema grandiflorum) plants grown in Japan were found recently. In our first experimental test to examine whether the disease symptoms, such as chlorosis of newly expanded leaves in Japanese chrysanthemum, confirmed that the disease was induced by CChMVd. The viroid was detected in cultivars with the disease symptoms by polymerase chain reaction (PCR). The nucleotide sequence of CChMVd extracted from an infected ‘Piato’ plant, almost completely agreed with that of the non-symptomatic CChMVd strain that was reported previously. In the second experiment from cultivars randomly collected from various areas in Japan, namely, Fukuoka, Hiroshima, Osaka, Shiga, Kyoto and Aichi prefectures, CChMVd was detected in many of the collected cultivars. Moreover, CChMVd was detected in several samples at low concentrations from cut chrysanthemum flowers sold in Kyoto prefecture shipped from various areas in Japan and the Netherlands. Hence, we concluded that CChMVd had already spread in Japan. In the third experiment to develop a CChMVd-free plant through a newly established method, that is, leaf primordia-free shoot apical meristem (LP-free SAM) culture was utilized to eliminate CChMVd from infected cultivars. These excised LP-free SAMs derived from CChMVd-infected ‘Piato’ and ‘Sttetsuman’ plants were attached to the root tip of in vitro-sown cabbage (Brassica oleracea) ‘Harunami’ and regenerated. One out of 29 regenerated ‘Piato’ plants and two out of 6 regenerated ‘Sttetsuman’ plants were ascertained to be CChMVd-free by nested PCR.