1Apple Research Center, National Institute of Fruit Tree Science (NIFTS), Morioka 020-0123, Japan; 2Hirosaki University, Hirosaki 036-8224, Japan

Background and objectives
Apple fruit crinkle (AFC) disease is a graft-transmissible fruit and/or bark disorder of apple , occurring only in Japan so far. A viroid, which is now called apple fruit crinkle viroid (AFCVd), was detected in association with this disease [1] and proved to be the causal agent of AFC [2]. However, the nucleotide sequence of this viroid was unknown to date. So we tried to determine the nucleotide sequence of AFCVd to clarify the relationship with other known viroids.

Results and conclusions
AFCVd (isolate P-196) was a circular molecule of 371 nucleotides (nts) with a G + C content of 57.1% (103 C, 109 G, 76 A, 83 U), and its secondary structure of minimal free energy was rod-like typical of other viroids; 69.0% of all nucleotides were paired and the proportions of GC, AU and GU pairs were 60.9, 32.0 and 7.0%, respectively. The central region of AFCVd contained the core nucleotides of the central conserved region (CCR) of the apple scar skin viroid (ASSVd) group. When we compared the overall nucleotide sequence of AFCVd with those of other known viroids, the highest sequence similarity (85.3%) was obtained with Australian grapevine viroid (AGVd: 369 nts; Rezaian, 1990), a member of the ASSVd group, in contrast with less than 60% sequence similarity with other viroids. Thus, AFCVd differed from AGVd at 55 sites in the sequence; 27 base exchanges, 15 insertions, 13 deletions. On the other hand, nucleotide sequence analysis of another three AFCVd isolates (PK-10, PK-11, PK-48) showed more than 96% sequence similarity among the four AFCVd isolates. Consequently, we decided to use the name of apple fruit crinkle viroid (AFCVd) for the causal viroid of AFC, leaving the possibility that AFCVd is a distinct strain of AGVd. The reasons for this conclusion are as follows. (i) At present, about 90% sequence similarity between two viroid molecules are used as a border to discriminate a viroid species from a strain or a variant in a species (Keese et al., 1988; Koltunow and Rezaian,1989). (ii) Sequence variation of AGVd is unknown because sequence report of AGVd is only one to date. (iii) Both the pathogenicity of AGVd to apple and that of AFCVd to grapevine are unknown. (iv) AFCVd causes remarkable symptoms in apple, while AGVd is only latent in grapevine. Further investigations will clarify the relationship between AFCVd and AGVd.

1. Ito T, Kanematsu S, Koganezawa H, Tsuchizaki T, Yoshida K, 1993. Annals of the Phytopathological Society of Japan 59, 520-527.
2. Ito T, Yoshida K, 1996. Annals of the Phytopathological Society of Japan 62, 597 (Abstr. in Japanese).