Association of a viroid-like RNA from plum dapple disease occurring in Japan.

Abstract

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

Hop Stunt Viroid Strains from Dapple Fruit Disease of Plum and Peach in Japan

“…Taiyo, has an Mr similar to that of hop stunt viroid (HSV)-grapevine and has a relatively high sequence homology with the HSV group (Sano et aL, 1986b).…”

Peach red marbling and peach sooty ringspot, two new virus‐like degenerative diseases of Prunus

“…Only some P. salicina cultivars such as Angeleno and Taiyo displayed some transient discoloration on the fruits and these symptoms disappeared at maturity. It cannot yet be confirmed that HSVd is responsible for dapple plum, since both the original Taiyo PR72 cultivar from Japan in which this association was established (Sano et al, 1986), and the Angeleno 892 cultivar from Italy (Giunchedi et al, 1997) were doubly infected by HSVd and the agent of PRMa. The natural manner of transmission of HSVd in Prunus is unknown.…”

“…Hop stunt viroid (HSVd) was identified on Japanese plum (Prunus salicina) cv. Taiyo for the first time in Japan (Sano et al, 1986). The disease symptoms, ringshaped discolorations, are obvious only on fruits with a red skin.…”

Sequence analysis of minute amounts of viroid RNA using the polymerase chain reaction (PCR)

“…Thus far, viroids have only been found in higher plants [2,20]. Recent investigations have shown that in certain non-herbaceous crop plants such as citrus, grapefruit, plum, and grapevine, various viroids are present in such minute amounts [3,4,8,10,14,[16][17][18] that their isolation, purification and subsequent direct sequence analysis is rather cumbersome, if not practically impossible. If successful at all [3] the transmission of such viroids to and their propagation in herbaceous plants can be problematic because it may lead to the selection of sequence variants which differ from the viroid predominating in the original non-herbaceous host plant [ 12].…”

“…The resulting double-stranded cDNA is purified by PAGE and then sequenced chemically [11]. We applied this whole procedure to the sequence analysis of hop stunt viroid (HSVd) [6], which has been found to be present worldwide in many plant species [8,10,14,[16][17][18].…”