Chrysanthemum Chlorotic Mottle Viroid

Chrysanthemum chlorotic mottle (CChM) disease was first reported 45 years ago and found to be caused by a graft-transmissible agent (Dimock et al., 1971). Because bioassays on indicator plants and examination by electron microscopy failed to reveal the presence of a virus associated with the affected plants, in which previous attempts to isolate bacteria or fungi had also failed (Dimock et al., 1971), a viroid etiology was suspected. In support of this view the infectious agent: (1) did not sediment after high speed centrifugation; (2) displayed in sucrose gradient centrifugation the sedimentation coefficient (6 14S) predicted for a small nucleic acid; and (3) was sensitive to RNase but not to DNase (Romaine and Horst, 1975).However, in contrast with previous results obtained with other viroids, the agent of CChM disease could not be identified as a differential band in polyacrylamide gel electrophoresis (PAGE) and, following fractionation with 2 M LiCl, it was preferentially found in the precipitate (Kawamoto et al., 1985). Moreover, while interference among chrysanthemum stunt viroid, citrus exocortis viroid, and a mild and a severe strain of potato spindle tuber viroid was observed in chrysanthemum plants coinoculated in a variety of combinations, this effect was not observed with the agent ofCChM disease, further supporting the existence of key differences between the latter and the other three known viroids (Niblett et al., 1978). To explore the possibility that the expected viroid-like RNA inciting the CChM disease could have been masked by comigrating host RNAs in the short gels initially used, preparations enriched in the pathogenic RNA were reexamined in long sequencing gels. With this modification, a distinct RNA was identified in the affected plants, which when eluted and inoculated mechanically, replicated and caused the typical CChM symptoms, thus fulfilling Koch's postulates (Navarro and Flores, 1997). The extremely low accumulation in vivo of the linear, and especially of the circular forms of chrysanthemum chlorotic mottle viroid (CChMVd), accounted in part for the negative results of the first tests.