SUMMARYViroids have been isolated from plum trees (Prunus salicina Lindley) affected with plum dapple fruit disease and from peach trees (Prunus persica Batsch) showing dapple fruit symptoms. The viroids were inoculated mechanically to cucurbitaceous plants, in which symptoms typical of hop stunt viroid (HSV) infection appeared. The complete nucleotide sequences of an isolate from plum and an isolate from peach (AF isolate) were shown to be identical, consisting of 297 nucleotides with a 93-6~o sequence homology to HSV-hop. Another isolate from peach (A9 isolate) also consists of 297 nucleotides, but the sequence homology to HSV-hop is 99"7~o, showing only one nucleotide replacement. These results indicate that these three viroids are strains of HSV, which we designate HSV-plum, HSV-peach (AF) and HSV-peach (A9), respectively. Comparative analysis of the nucleotide sequences of HSV strains from hop, grapevine, citrus, cucumber, plum and peach revealed variable and conserved regions in the HSV molecule. In Japan, these viroids are closely related not only to dapple fruit disease in plum cv. Taiyo, but also to dapple fruit symptoms on peach cv. Asama-Hakutou.

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Introduction. Plum dapple disease is a newly described disease by Terai (1985) 3) in Japanese Plum (Prunes salicina Lindley cv. Taiyo) cultivated in Yamanashi Prefecture, Japan. The infected trees produce dapple fruits with faint reddish and chlorotic blotches, but no visible symptom is recognized on the leaves and stems. The disease is transmissible by grafting, however, the causal agent has not yet known. In this paper, we describe detection and some properties of a viroid-like RNA associated with plum dapple disease. Materials and methods. Restriction enzyme and DNA polymerase I were purchased from Takara Shuzo Co., Ltd. DNase I was from Cooper Biomedical, Inc. a-32P-dCTP (800 Ci/mmol) was from Muromachi Chemical Inc. Fruits and leaves of plum dapple disease (PDD) affected and of PDD free trees were collected from Yamanashi Prefecture. Low molecular weight RNAs were extracted from frozen leaves and skins of fruits by using previously described method,°) with some modifications. When leaves were homogenized in 1 M K2HPO4, the extracts became dark brown with high viscosity because of the high concentration of polyphenolic substances in plum leaves, and large quantities of low molecular weight RNAs were precipitated by 2 M LiCI precipitation together with high molecular weight ribosomal RNAs and contaminated-impurities. To avoid this loss, the low molecular weight RNAs were recovered from the precipitates by another cycles of 2 M LiCI solubilizations. Polyacrylamide gel electrophoresis (PAGE) under non-denaturing condition was carried out on a 15% gel (acrylamide : bis=30 : 0.8) in a buffer containing 40 mM tris, 20 mM sodium acetate and 2 mM EDTA, pH 7.2 (Loening, 1967).1) PAGE under fully denaturing condition was carried out on an 8 M urea 5 % gel (acrylamide : bis=30 : 0.8) in a buffer containing 25 mM tris, 25 mM bolic acid and 0.5 mM EDTA, pH 8.3. After electrophoresis, gels were stained with ethidium bromide (1 µg/ml) and observed on a trans-illuminator. Dot blot hybridization was performed according to the method described by Thomas (1980).J) A full length (1 unit) cDNA copy of hop stunt viroid-cucumber isolate (HSV-c) was excised by restriction endonuclease Xho I digestion from recombinant plasmid pCP 316, which containing 2 units copies of HSV-c cDNA. The 1 unit cDNA was labelled with a-32P-dCTP by nick-translation, specific activity was 2x107 cpm/ µg DNA, and used for hybridization probe. Two µg aliquote of the low molecular weight RNAs was spotted onto a nitrocellulose paper (Schleicher and Schuell) and hybridized with 32P-labelled cDNA of HSV-c

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