A viroid was isolated from symptomless Nematanthus wettsteinii plants using the return-PAGE method for analysis of low M(r) nucleic acids. The RNA was transmitted to tomato, three cultivars of potato, and Scopolia sinensis plants by mechanical inoculation or by grafting. Infected solanaceous plants developed symptoms similar to those caused by potato spindle tuber viroid (PSTVd). The Nematanthus viroid consists of 372 nucleotides, 214 G+C, 158 A+U, with a G+C/A+U ratio of 1.35. One of seven cDNA clones showed a sequence heterogeneity (G to A) at position 73. The most stable secondary structure of this viroid has 78 G:C, 37 A:U and 11 G:U base pairs with a minimum free energy of -456.9 kJ. The viroid is closely related to the 370 nucleotide Columnea latent viroid. The Nematanthus viroid possesses regions of 100% sequence identity with six viroids belonging to the PSTVd and apple scar skin viroid groups. The viroid also replicated in tomato plants when mixed with PSTVd. Tomato plants were cross-protected against PSTVd when preinfected with the viroid from N. wettsteinii.

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When nucleic acid extracts from leaves infected with various strains of potato spindle tuber viroid (PSTV) were electrophoresed by Return-polyacrylamide gel electrophoresis (R-PAGE), viroid RNA migrated more slowly than other nucleic acids in the extract. Electrophoretic mobility of viroid bands from samples containing a severe strain (S-PSTV) was 3–4 mm slower in the return direction than those containing mild strains (M-PSTV). Mixed preparations of severe and various mild strains separated into well-defined bands, which verified the differential migration of S-PSTV from M-PSTV strains. The origin of extracts from different organs of the potato (sprout, tuber, stolon, and leaf) or different plant species (tomato, potato, and Scopolia sinensis) did not affect the migration pattern of viroid strains. This method, if verified with more severe isolates, may enable separation and identification of mild and severe strains of PSTV within a few hours, as compared with several weeks required for the standard biological cross-protection test.

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Seventeen plant introductions (PI) of Solanum berthaultii were tested. Two clones (1726 and 1729) of S. berthaultii (PI 473340) were resistant to the mild and severe strains of potato spindle tuber viroid (PSTV). Both clones were resistant to PSTV by sap inoculation but were susceptible by graft inoculation. Even after graft inoculation, plants remained symptomless and PSTV was not detected by polyacrylamide gel electrophoresis. However, bioassay on Scopolia sinensis and dot-blot tests detected PSTV in such plants. Both clones were susceptible to potato viruses A, S, X, and Y by sap inoculation and to leafroll virus upon grafting. The importance of the PSTV resistance in these clones is discussed in connection with multiple insect resistance found in clones of S. berthaultii.

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5' Phosphorylation in vitro with gamma-32P-ATP and T4 phage induced polynucleotide kinase was used to obtain RNAase A and RNAase T1 fingerprints of three plant viroids: Potato spindle tuber viroid from tomato (PSTV-tom), chrysanthemum stunt viroid from cineraria (ChSV-cin) and citrus exocortis viroid from Gynura aurantiaca (CEV-gyn). These three viroids differ significantly from each other as judged from their oligonucleotide patterns. This supports the concept of individual viroid species.

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The existence of three infectious forms of potato spindle tuber viroid (PSTV) RNA from Scopolia sinensis was demonstrated by fractionation with high salt, by reverse phase and high pressure liquid chromatography, and by polyacrylamide gel electrophoresis. Purification of fraction II was achieved by the following steps: extraction of nucleic acid with phenol, precipitation of the RNA with cetyltrimethylammonium bromide, fractionation of the RNA with lithium chloride and isopropanol, and finally gel electrophoresis. A procedure using reverse phase chromatography was developed to obtain 70-90% recovery of RNA from polyacrylamide gels. Purified PSTV fraction II RNA was digested with ribonuclease A and T and labelled with [gamma-32P[ATP using polynucleotide kinase. The labelled digests were separated by the electrophoresis-homochromatography procedures of Sanger. About 20 and 30 spots were obtained with ribonuclease A and T, respectively.

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