Apple dimple fruit viroid: Fulfillment of Koch's Postulates and Symptom Characteristics.

The previously reported properties of a small circular RNA isolated from plants of cultivar Starking Delicious with symptoms similar to those of the dapple apple disease induced by Apple scar skin viroid (ASSVd), strongly suggested that it is a distinct viroid species of genus Apscaviroid to which ASSVd also belongs. Nevertheless, the autonomous replication of this novel RNA, termed Apple dimple fruit viroid (ADFVd), and the nature of the symptoms incited when inoculated free of other pathogens that might be present in the original source, were not determined. We addressed these questions by mechanically inoculating purified ADFVd into young apple seedlings (Golden). Dot blot hybridization of leaf extracts collected ten months after inoculation showed the presence of ADFVd in most of the inoculated seedlings, thus proving the ability of this RNA to replicate independently. Material from these ADFVd-infected seedlings grafted onto the ASSVd indicator Starkrimson incited the appearance on the fruit skin of discolored spots more or less depressed which were particularly frequent around the calyx cavity. Apple cultivars such as Gala, Pink Lady, and Braeburn reacted to ADFVd with similar symptoms, occasionally accompanied by scar skin in Braeburn, whereas ADFVd replicated without eliciting any visible symptoms in other apple cultivars such as Golden, or in the pear indicator Fieud 37. No interference in symptom expression or viroid accumulation were observed in plants inoculated first with ASSVd and then with ADFVd.

Pear Blister Canker Viroid: Host Range and Improved Bioassay with Two New Pear Indicators, Fieud 37 and Fieud 110.

An investigation was conducted to improve the biological detection of pear blister canker viroid (PBCVd), which over a period of 2 to 3 years induces pear blister canker disease on the perry pear (Pyrus communis) cv. A20. PBCVd was not detected by dot blot hybridization or bioassay in any of the tested species of Amelanchier, Aronia, Cotoneaster, Crataegus, and Pyracantha. However, some species of Chaenomeles, Cydonia, and Sorbus, five out of 13 species of Malus, 15 Pyrus species, and 16 commercial pear cultivars were susceptible to PBCVd, although none developed symptoms. Only in perry pear seedlings did approximately 5% of the population react to infection with pure PBCVd strains by developing petiole, leaf, or bark necrosis 2 to 3 years (cv. A20) or 3 to 5 months (cv. Fieudière) after inoculation. The selected Fieud 37 and Fieud 110 clones are proposed as PBCVd indicators to replace A20. Results from bioassays on the indicators and from detection by a PBCVd-cRNA probe were essentially in agreement except for some Malus species.

Biological Properties of Apple Scar Skin Viroid: Isolates, Host Range, Different Sensitivity of Apple Cultivars, Elimination, and Natural Transmission.

Studies conducted over the last 10 years have revealed that the disease caused by the apple scar skin viroid (ASSVd) is extremely rare in Europe. ASSVd was detected by molecular hybridization and indexing in field plots on the apple indicators Starkrimson and Indo, which showed symptoms of dapple apple disease within 2 years, and rough scarred skin within 3 years, respectively. Results from both approaches were in agreement. In an attempt to improve the biological detection of ASSVd, the Japanese PK13 isolate was inoculated to 4 Prunus, 13 Malus, 17 Pyrus, and 17 other pomaceous species. All the species tested of the Malus, Pyrus, Sorbus, Chaenomeles, Cydonia, and Pyronia genera were susceptible to ASSVd based upon back indexing and hybridization, but none developed leaf or bark symptoms during a 2-year period. The viroid was not detected in the tested members of genera Amelanchier, Aronia, Cotoneaster, Crataegus, Prunus, and Pyracantha. Symptoms on fruit of 42 commercial apple cultivars experimentally inoculated with ASSVd fell into five groups ranging from inconspicuous spots to severely scarred skin and cracking. ASSVd was eliminated from most of the infected apple plants when they were subjected to a dormant stage followed by thermotherapy and shoot tip grafting. Analysis of more than 400 apple seedlings, originated from Starkrimson and Indo fruits with typical ASSVd symptoms, showed that there is little or no seed transmission of this viroid. However, ASSVd was transmitted at a low rate under field conditions to adjacent trees.

Genomic structure of three phenotypically different isolates of peach latent mosaic viroid: implications of the existence of constraints limiting the heterogeneity of viroid quasispecies.

The peach latent mosaic viroid (PLMVd) is used to study the interactions between a viroid containing hammerhead ribozymes and its natural host, peach. To gain insight into the molecular basis of the phenotypic effects observed upon viroid infection, sequence variants from three PLMVd isolates that differ in symptom expression on the peach indicator GF-305 have been characterized. Analysis of the primary structures of a total of 29 different sequence variants derived from a severe and two latent isolates has revealed a large number of polymorphic positions in the viroid molecule. The variability pattern indicates that preservation of the stability of both hammerhead structures and conservation of a branched secondary structure of the viroid molecule may be factors limiting sequence heterogeneity in PLMVd. Moreover, compensatory mutations in two hairpin loops of the proposed secondary structure, suggesting that a pseudoknot-like interaction may exist between them, have also been observed. Phylogenetic analysis has allowed the allocation of PLMVd molecules into three major groups. This clustering does not strictly correlate with the source isolate from which the variants were obtained, providing insights into the complex mixture of molecules which make up each isolate. Bioassays of individual PLMVd sequence variants on GF-305 peach seedlings have shown that the biological properties of the PLMVd isolates may be correlated with both the complexity of their viroid populations and the presence of specific sequence variants.

Pear blister canker viroid: sequence variability and causal role in pear blister canker disease.

The sequences of several cDNA clones of pear blister canker viroid (PBCVd) P1914T and P47A isolates have been determined. Seven out of eight P1914T clones analysed have a constant sequence which differs at six positions from that of the P2098T isolate reported previously. The remaining P1914T clone (8) has a single nucleotide substitution. The same six changes have been also observed in most of the ten P47A clones sequenced. However, some P47A clones show additional variability in positions on both strands of the central conserved region (CCR) and in another conserved sequence at the left-terminal region. This is the first report of a change affecting the upper strand of a viroid CCR. Reasons why such a change is tolerated are discussed. Infectivity bioassays have demonstrated that PBCVd is the causal agent of PBC disease.

Identification of a new viroid as the putative causal agent of pear blister canker disease.

A small RNA species with the structural and functional properties characteristic of viroids has been isolated from three different pear sources each of which induced symptoms of the pear blister canker (PBC) disease when indexed in the pear indicator A 20. A close association between this RNA and PBC disease was established, since two of the three studied sources were known to be affected by this malady only, and the viroid was not detected in healthy pear tissue. Moreover, the PBC-associated viroid (PBCVd) replicated when purified preparations were inoculated into cucumber and pear plants. PBCVd behaved in denaturing polyacrylamide gels as a circular RNA with a molecular size of approximately 315 nucleotide residues. Analysis by dot blot hybridization indicated that PBCVd shares similarities in sequence with peach latent mosaic viroid and hop stunt viroid, and to a lesser extent with apple scar skin viroid.

Some properties of the viroid inducing peach latent mosaic disease.

Analysis by polyacrylamide gel electrophoresis of nucleic acid extracts from different peach samples, healthy or infected with the peach latent mosaic (PLM) disease, demonstrated the association of this disease with an RNA exhibiting the electrophoretic properties typical of circular viroid molecules. This RNA was called peach latent mosaic viroid (PLMV), since a purified preparation of it, when inoculated into GF 305 peach seedlings induced characteristic symptoms of PLM disease. PLMV was estimated to have a molecular size in the range of 330-340 bases, by comparison of its electrophoretic mobility under denaturing conditions with those of several viroid RNA. Dot-blot analysis showed that PLMV had a sequence clearly different from other viroids, including citrus exocortis viroid, apple scar skin viroid (ASSV), hop stunt viroid (HSV) and avocado sunblotch viroid. The possible significance of the limited sequence homology shared by PLMV with HSV, and especially with ASSV, is discussed.